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Sample records for actin-based cytoskeletal network

  1. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design.

  2. Mechanics of composite cytoskeletal and extracellular networks

    NASA Astrophysics Data System (ADS)

    Das, Moumita

    2014-03-01

    Living cells sense and respond to mechanical forces in their surroundings. This mechanical response is mainly due to the cell cytoskeleton, and its interaction with the extracellular matrix (ECM). The cell cytoskeleton is a composite polymeric scaffold made of many different types of protein filaments and crosslinking proteins. Two major filament systems in the cytoskeleton are actin filaments (F-actin) and microtubules (MTs). Actin filaments are semiflexible, while the much stiffer MTs behave as rigid rods. I shall discuss theories that help understand how the direct coupling to the surrounding F-actin matrix allows intracellular MTs to bear large compressive forces and controls the range of force transmission along the MTs, and how the MTs not only enhance the stiffness of the cell cytoskeleton, but can also dramatically endow an initially nearly incompressible F-actin matrix with enhanced compressibility relative to its shear compliance. A second source of compositeness in the cytoskeleton is the presences of different types of crosslinkers that can interact cooperatively leading to enhanced mechanical rigidity and tunable response. Like the cytoskeleton, the ECM is also a polymeric composite. It is primarily composed of a mesh of fibrous proteins, mainly stiff collagen filaments, and a comparatively flexible gel of proteoglycans and hyaluronan. I shall discuss a model that shows how the interplay between the collagen network and the background elastic gel leads to a mechanically robust ECM.

  3. Hierarchical self-organization of cytoskeletal active networks

    NASA Astrophysics Data System (ADS)

    Gordon, Daniel; Bernheim-Groswasser, Anne; Keasar, Chen; Farago, Oded

    2012-04-01

    The structural reorganization of the actin cytoskeleton is facilitated through the action of motor proteins that crosslink the actin filaments and transport them relative to each other. Here, we present a combined experimental-computational study that probes the dynamic evolution of mixtures of actin filaments and clusters of myosin motors. While on small spatial and temporal scales the system behaves in a very noisy manner, on larger scales it evolves into several well distinct patterns such as bundles, asters and networks. These patterns are characterized by junctions with high connectivity, whose formation is possible due to the organization of the motors in ‘oligoclusters’ (intermediate-size aggregates). The simulations reveal that the self-organization process proceeds through a series of hierarchical steps, starting from local microscopic moves and ranging up to the macroscopic large scales where the steady-state structures are formed. Our results shed light on the mechanisms involved in processes such as cytokinesis and cellular contractility, where myosin motors organized in clusters operate cooperatively to induce the structural organization of cytoskeletal networks.

  4. Direct interaction of microtubule- and actin-based transport motors

    NASA Technical Reports Server (NTRS)

    Huang, J. D.; Brady, S. T.; Richards, B. W.; Stenolen, D.; Resau, J. H.; Copeland, N. G.; Jenkins, N. A.

    1999-01-01

    The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

  5. The dynamics of filament assembly define cytoskeletal network morphology

    PubMed Central

    Foffano, Giulia; Levernier, Nicolas; Lenz, Martin

    2016-01-01

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

  6. The dynamics of filament assembly define cytoskeletal network morphology

    NASA Astrophysics Data System (ADS)

    Foffano, Giulia; Levernier, Nicolas; Lenz, Martin

    2016-12-01

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

  7. Morphological Transformation and Force Generation of Active Cytoskeletal Networks

    PubMed Central

    Maruri, Daniel; Kamm, Roger D.

    2017-01-01

    Cells assemble numerous types of actomyosin bundles that generate contractile forces for biological processes, such as cytokinesis and cell migration. One example of contractile bundles is a transverse arc that forms via actomyosin-driven condensation of actin filaments in the lamellipodia of migrating cells and exerts significant forces on the surrounding environments. Structural reorganization of a network into a bundle facilitated by actomyosin contractility is a physiologically relevant and biophysically interesting process. Nevertheless, it remains elusive how actin filaments are reoriented, buckled, and bundled as well as undergo tension buildup during the structural reorganization. In this study, using an agent-based computational model, we demonstrated how the interplay between the density of myosin motors and cross-linking proteins and the rigidity, initial orientation, and turnover of actin filaments regulates the morphological transformation of a cross-linked actomyosin network into a bundle and the buildup of tension occurring during the transformation. PMID:28114384

  8. Microrheology of single microtubule filaments and synthesized cytoskeletal networks

    NASA Astrophysics Data System (ADS)

    Koch, Matthias; Rohrbach, Alexander

    2015-03-01

    The ability to sense and respond to external mechanical forces is crucial for cells in many processes such as cell growth and division. Common models on mechanotransduction rely on the conversion of mechanical stimuli to chemical signals in the cell periphery and their translocation by diffusion (passive) or molecular motors (active). These processes are rather slow (~ seconds) and it has been argued that the cytoskeleton itself might be able to transport a mechanical signal within microseconds via stress waves. Microtubules are the stiffest component of the cytoskeleton and thus ideal candidates for this purpose. We study the frequency dependent response of single microtubule filaments and small networks thereof in a bottom-up approach using several (N =2-10) time-multiplexed optical tweezers together with back focal plane interferometry. Small synthesized networks with a defined geometry are constructed using trapped Neutravidin beads as anchor points for biotinylated filaments. The network is then probed by a defined oscillation of one anchor (actor). The frequency dependent response of the remaining beads (sensors) is analyzed experimentally and modeled theoretically over a wide frequency range.

  9. Three-dimensional Organization of Layered Apical Cytoskeletal Networks Associated with Mouse Airway Tissue Development

    PubMed Central

    Tateishi, Kazuhiro; Nishida, Tomoki; Inoue, Kanako; Tsukita, Sachiko

    2017-01-01

    The cytoskeleton is an essential cellular component that enables various sophisticated functions of epithelial cells by forming specialized subcellular compartments. However, the functional and structural roles of cytoskeletons in subcellular compartmentalization are still not fully understood. Here we identified a novel network structure consisting of actin filaments, intermediate filaments, and microtubules directly beneath the apical membrane in mouse airway multiciliated cells and in cultured epithelial cells. Three-dimensional imaging by ultra-high voltage electron microscopy and immunofluorescence revealed that the morphological features of each network depended on the cell type and were spatiotemporally integrated in association with tissue development. Detailed analyses using Odf2 mutant mice, which lack ciliary basal feet and apical microtubules, suggested a novel contribution of the intermediate filaments to coordinated ciliary beating. These findings provide a new perspective for viewing epithelial cell differentiation and tissue morphogenesis through the structure and function of apical cytoskeletal networks. PMID:28272499

  10. Three-dimensional Organization of Layered Apical Cytoskeletal Networks Associated with Mouse Airway Tissue Development

    NASA Astrophysics Data System (ADS)

    Tateishi, Kazuhiro; Nishida, Tomoki; Inoue, Kanako; Tsukita, Sachiko

    2017-03-01

    The cytoskeleton is an essential cellular component that enables various sophisticated functions of epithelial cells by forming specialized subcellular compartments. However, the functional and structural roles of cytoskeletons in subcellular compartmentalization are still not fully understood. Here we identified a novel network structure consisting of actin filaments, intermediate filaments, and microtubules directly beneath the apical membrane in mouse airway multiciliated cells and in cultured epithelial cells. Three-dimensional imaging by ultra-high voltage electron microscopy and immunofluorescence revealed that the morphological features of each network depended on the cell type and were spatiotemporally integrated in association with tissue development. Detailed analyses using Odf2 mutant mice, which lack ciliary basal feet and apical microtubules, suggested a novel contribution of the intermediate filaments to coordinated ciliary beating. These findings provide a new perspective for viewing epithelial cell differentiation and tissue morphogenesis through the structure and function of apical cytoskeletal networks.

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

    PubMed

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

    2017-04-11

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

  12. Mechanical models of the cellular cytoskeletal network for the analysis of intracellular mechanical properties and force distributions: a review.

    PubMed

    Chen, Ting-Jung; Wu, Chia-Ching; Su, Fong-Chin

    2012-12-01

    The cytoskeleton, which is the major mechanical component of cells, supports the cell body and regulates the cellular motility to assist the cell in performing its biological functions. Several cytoskeletal network models have been proposed to investigate the mechanical properties of cells. This review paper summarizes these models with a focus on the prestressed cable network, the semi-flexible chain network, the open-cell foam, the tensegrity, and the granular models. The components, material parameters, types of connection joints, tension conditions, and the advantages and disadvantages of each model are evaluated from a structural and biological point of view. The underlying mechanisms that are associated with the morphological changes of spreading cells are expected to be simulated using a cytoskeletal model; however, it is still paid less attention most likely due to the lack of a suitable cytoskeletal model that can accurately model the spreading process. In this review article, the established cytoskeletal models are hoped to provide useful information for the development of future cytoskeletal models with different degrees of cell attachment for the study of the mechanical mechanisms underlying the cellular behaviors in response to external stimulations.

  13. The use of neural networks and texture analysis for rapid objective selection of regions of interest in cytoskeletal images.

    PubMed

    Derkacs, Amanda D Felder; Ward, Samuel R; Lieber, Richard L

    2012-02-01

    Understanding cytoskeletal dynamics in living tissue is prerequisite to understanding mechanisms of injury, mechanotransduction, and mechanical signaling. Real-time visualization is now possible using transfection with plasmids that encode fluorescent cytoskeletal proteins. Using this approach with the muscle-specific intermediate filament protein desmin, we found that a green fluorescent protein-desmin chimeric protein was unevenly distributed throughout the muscle fiber, resulting in some image areas that were saturated as well as others that lacked any signal. Our goal was to analyze the muscle fiber cytoskeletal network quantitatively in an unbiased fashion. To objectively select areas of the muscle fiber that are suitable for analysis, we devised a method that provides objective classification of regions of images of striated cytoskeletal structures into "usable" and "unusable" categories. This method consists of a combination of spatial analysis of the image using Fourier methods along with a boosted neural network that "decides" on the quality of the image based on previous training. We trained the neural network using the expert opinion of three scientists familiar with these types of images. We found that this method was over 300 times faster than manual classification and that it permitted objective and accurate classification of image regions.

  14. Proteomic and Microscopic Strategies towards the Analysis of the Cytoskeletal Networks in Major Neuropsychiatric Disorders

    PubMed Central

    Coumans, Joëlle V. F.; Palanisamy, Suresh K. A.; McFarlane, Jim; Moens, Pierre D. J.

    2016-01-01

    Mental health disorders have become worldwide health priorities. It is estimated that in the next 20 years they will account for a 16 trillion United State dollars (US$) loss. Up to now, the underlying pathophysiology of psychiatric disorders remains elusive. Altered cytoskeleton proteins expression that may influence the assembly, organization and maintenance of cytoskeletal integrity has been reported in major depressive disorders, schizophrenia and to some extent bipolar disorders. The use of quantitative proteomics, dynamic microscopy and super-resolution microscopy to investigate disease-specific protein signatures holds great promise to improve our understanding of these disorders. In this review, we present the currently available quantitative proteomic approaches use in neurology, gel-based, stable isotope-labelling and label-free methodologies and evaluate their strengths and limitations. We also reported on enrichment/subfractionation methods that target the cytoskeleton associated proteins and discuss the need of alternative methods for further characterization of the neurocytoskeletal proteome. Finally, we present live cell imaging approaches and emerging dynamic microscopy technology that will provide the tools necessary to investigate protein interactions and their dynamics in the whole cells. While these areas of research are still in their infancy, they offer huge potential towards the understanding of the neuronal network stability and its modification across neuropsychiatric disorders. PMID:27104521

  15. The cortical cytoskeletal network and cell-wall dynamics in the unicellular charophycean green alga Penium margaritaceum

    PubMed Central

    Ochs, Julie; LaRue, Therese; Tinaz, Berke; Yongue, Camille; Domozych, David S.

    2014-01-01

    Background and Aims Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division. Methods Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy. Key Results The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division. Conclusions The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre

  16. Initiation of Chondrocyte Self-Assembly Requires an Intact Cytoskeletal Network

    PubMed Central

    Lee, Jennifer K.; Hu, Jerry C.Y.

    2016-01-01

    Self-assembly and self-organization have recently emerged as robust scaffold-free tissue engineering methodologies that can be used to generate various tissues, including cartilage, vessel, and liver. Self-assembly, in particular, is a scaffold-free platform for tissue engineering that does not require the input of exogenous energy to the system. Although self-assembly can generate functional tissues, most notably neocartilage, the mechanisms of self-assembly remain unclear. To study the self-assembling process, we used articular chondrocytes as a model to identify parameters that can affect this process. Specifically, the roles of cell–cell and cell–matrix adhesion molecules, surface-bound collagen, and the actin cytoskeletal network were investigated. Using time-lapse imaging, we analyzed the early stages of chondrocyte self-assembly. Within hours, chondrocytes rapidly coalesced into cell clusters before compacting to form tight cellular structures. Chondrocyte self-assembly was found to depend primarily on integrin function and secondarily on cadherin function. In addition, actin or myosin II inhibitors prevented chondrocyte self-assembly, suggesting that cell adhesion alone is not sufficient, but rather the active contractile actin cytoskeleton is essential for proper chondrocyte self-assembly and the formation of neocartilage. Better understanding of the self-assembly mechanisms allows for the rational modulation of this process toward generating neocartilages with improved properties. These findings are germane to understanding self-assembly, an emerging platform for tissue engineering of a plethora of tissues, especially as these neotissues are poised for translation. PMID:26729374

  17. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

    Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

    2001-05-01

    Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

  18. Transcriptional networks regulating the costamere, sarcomere, and other cytoskeletal structures in striated muscle.

    PubMed

    Estrella, Nelsa L; Naya, Francisco J

    2014-05-01

    Structural abnormalities in striated muscle have been observed in numerous transcription factor gain- and loss-of-function phenotypes in animal and cell culture model systems, indicating that transcription is important in regulating the cytoarchitecture. While most characterized cytoarchitectural defects are largely indistinguishable by histological and ultrastructural criteria, analysis of dysregulated gene expression in each mutant phenotype has yielded valuable information regarding specific structural gene programs that may be uniquely controlled by each of these transcription factors. Linking the formation and maintenance of each subcellular structure or subset of proteins within a cytoskeletal compartment to an overlapping but distinct transcription factor cohort may enable striated muscle to control cytoarchitectural function in an efficient and specific manner. Here we summarize the available evidence that connects transcription factors, those with established roles in striated muscle such as MEF2 and SRF, as well as other non-muscle transcription factors, to the regulation of a defined cytoskeletal structure. The notion that genes encoding proteins localized to the same subcellular compartment are coordinately transcriptionally regulated may prompt rationally designed approaches that target specific transcription factor pathways to correct structural defects in muscle disease.

  19. Biomimetic systems for studying actin-based motility.

    PubMed

    Upadhyaya, Arpita; van Oudenaarden, Alexander

    2003-09-16

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

  20. Actin-based propulsion of a microswimmer.

    PubMed

    Leshansky, A M

    2006-07-01

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

  1. Cross-Linker Unbinding and Self-Similarity in Bundled Cytoskeletal Networks

    NASA Astrophysics Data System (ADS)

    Lieleg, O.; Bausch, A. R.

    2007-10-01

    The macromechanical properties of purely bundled in vitro actin networks are not only determined by the micromechanical properties of individual bundles but also by molecular unbinding events of the actin-binding protein (ABP) fascin. Under high mechanical load the network elasticity depends on the forced unbinding of individual ABPs in a rate dependent manner. Cross-linker unbinding in combination with the structural self-similarity of the network enables the introduction of a concentration-time superposition principle—broadening the mechanically accessible frequency range over 8 orders of magnitude.

  2. Mechanics model for actin-based motility

    NASA Astrophysics Data System (ADS)

    Lin, Yuan

    2009-02-01

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

  3. Mechanics model for actin-based motility.

    PubMed

    Lin, Yuan

    2009-02-01

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

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

    PubMed

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

    2011-08-23

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

  5. Cooperativity and redundancy in the mechanics of compositely crosslinked branched anisotropic cytoskeletal networks

    NASA Astrophysics Data System (ADS)

    Schwarz, J. M.; Zhang, Tao; Das, Moumita

    2013-03-01

    At the leading edge of a crawling cell, the actin cytoskeleton extends itself in a particular direction via a branched crosslinked network of actin filaments with some overall alignment. This network is known as the lamellipodium. Branching via the complex Arp2/3 occurs at a reasonably well-defined angle of 70 degrees from the plus end of the mother filament such that Arp2/3 can be modeled as an angle-constraining crosslinker. Freely-rotating crosslinkers, such as alpha-actinin, are also present in lamellipodia. Therefore, we study the interplay between these two types of crosslinkers, angle-constraining and free-rotating, both analytically and numerically, to begin to quantify the mechanics of lamellipodia. We also investigate how the orientational ordering of the filaments affects this interplay. Finally, while role of Arp2/3 as a nucleator for filaments along the leading edge of a crawling cell has been studied intensely, much less is known about its mechanical contribution. Our work seeks to fill in this important gap in modeling the mechanics of lamellipodia.

  6. Control of developmental networks by Rac/Rho small GTPases: How cytoskeletal changes during embryogenesis are orchestrated

    PubMed Central

    Sáenz‐Narciso, Beatriz; Gómez‐Orte, Eva; Zheleva, Angelina; Gastaca, Irene

    2016-01-01

    Small GTPases in the Rho family act as major nodes with functions beyond cytoskeletal rearrangements shaping the Caenorhabditis elegans embryo during development. These small GTPases are key signal transducers that integrate diverse developmental signals to produce a coordinated response in the cell. In C. elegans, the best studied members of these highly conserved Rho family small GTPases, RHO‐1/RhoA, CED‐10/Rac, and CDC‐42, are crucial in several cellular processes dealing with cytoskeletal reorganization. In this review, we update the functions described for the Rho family small GTPases in spindle orientation and cell division, engulfment, and cellular movements during C. elegans embryogenesis, focusing on the Rho subfamily Rac. Please also see the video abstract here PMID:27790724

  7. Disruption of the three cytoskeletal networks in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock.

    PubMed Central

    Welch, W J; Feramisco, J R

    1985-01-01

    Mammalian cells show a complex series of transcriptional and translational switching events in response to heat shock treatment which ultimately lead to the production and accumulation of a small number of proteins, the so-called heat shock (or stress) proteins. We investigated the heat shock response in both qualitative and quantitative ways in cells that were pretreated with drugs that specifically disrupt one or more of the three major cytoskeletal networks. (These drugs alone, cytochalasin E and colcemid, do not result in induction of the heat shock response.) Our results indicated that disruption of the actin microfilaments, the vimentin-containing intermediate filaments, or the microtubules in living cells does not hinder the ability of the cell to undergo an apparently normal heat shock response. Even when all three networks were simultaneously disrupted (resulting in a loose, baglike appearance of the cells), the cells still underwent a complete heat shock response as assayed by the appearance of the heat shock proteins. In addition, the major induced 72-kilodalton heat shock protein was efficiently translocated from the cytoplasm into its proper location in the nucleus and nucleolus irrespective of the condition of the three cytoskeletal elements. Images PMID:4040602

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

    PubMed

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

    2004-06-22

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

  9. Actin-Based Motility of Intracellular Microbial Pathogens

    PubMed Central

    Goldberg, Marcia B.

    2001-01-01

    A diverse group of intracellular microorganisms, including Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, utilize actin-based motility to move within and spread between mammalian host cells. These organisms have in common a pathogenic life cycle that involves a stage within the cytoplasm of mammalian host cells. Within the cytoplasm of host cells, these organisms activate components of the cellular actin assembly machinery to induce the formation of actin tails on the microbial surface. The assembly of these actin tails provides force that propels the organisms through the cell cytoplasm to the cell periphery or into adjacent cells. Each of these organisms utilizes preexisting mammalian pathways of actin rearrangement to induce its own actin-based motility. Particularly remarkable is that while all of these microbes use the same or overlapping pathways, each intercepts the pathway at a different step. In addition, the microbial molecules involved are each distinctly different from the others. Taken together, these observations suggest that each of these microbes separately and convergently evolved a mechanism to utilize the cellular actin assembly machinery. The current understanding of the molecular mechanisms of microbial actin-based motility is the subject of this review. PMID:11729265

  10. Vaccinia locomotion in host cells: evidence for the universal involvement of actin-based motility sequences ABM-1 and ABM-2.

    PubMed

    Zeile, W L; Condit, R C; Lewis, J I; Purich, D L; Southwick, F S

    1998-11-10

    Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in Listeria ActA and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 microM (intracellular) ABM-2 peptide (GPPPPP)3 blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of Listeria, Shigella, and now vaccinia.

  11. Bacillus anthracis Edema Toxin Impairs Neutrophil Actin-Based Motility▿

    PubMed Central

    Szarowicz, Sarah E.; During, Russell L.; Li, Wei; Quinn, Conrad P.; Tang, Wei-Jen; Southwick, Frederick S.

    2009-01-01

    Inhalation anthrax results in high-grade bacteremia and is accompanied by a delay in the rise of the peripheral polymorphonuclear neutrophil (PMN) count and a paucity of PMNs in the infected pleural fluid and mediastinum. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of the adenylate cyclase edema factor (EF) and protective antigen (PA). Relatively low concentrations of ET (100 to 500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and ability to polarize. These changes are accompanied by a reduction in chemoattractant-stimulated PMN actin assembly. ET also causes a significant decrease in Listeria monocytogenes intracellular actin-based motility within HeLa cells. These defects in actin assembly are accompanied by a >50-fold increase in intracellular cyclic AMP and a >4-fold increase in the phosphorylation of protein kinase A. We have previously shown that anthrax lethal toxin (LT) also impairs neutrophil actin-based motility (R. L. During, W. Li, B. Hao, J. M. Koenig, D. S. Stephens, C. P. Quinn, and F. S. Southwick, J. Infect. Dis. 192:837-845, 2005), and we now find that LT combined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP (N-formyl-met-leu-phe)-induced actin assembly. We conclude that ET alone or combined with LT impairs PMN actin assembly, resulting in paralysis of PMN chemotaxis. PMID:19349425

  12. Multiple actin-based motor genes in Dictyostelium.

    PubMed Central

    Titus, M A; Warrick, H M; Spudich, J A

    1989-01-01

    Dictyostelium cells, devoid of conventional myosin, display a variety of motile activities, consistent with the presence of other molecular motors. The Dictyostelium genome was probed at low stringency with a gene fragment containing the conserved conventional myosin head domain sequences to identify other actin-based motors that may play a role in the observed motility of these mutant cells. One gene (abmA) has been characterized and encodes a polypeptide of approximately 135 kDa with a head region homologous to other myosin head sequences and a tail region that is not predicted to form either an alpha-helical structure of coiled-coil interactions. Comparisons of the amino acid sequences of the tail regions of abmA, Dictyostelium myosin I, and Acanthamoeba myosins IB and IL reveal an area of sequence similarity in the amino terminal half of the tail that may be a membrane-binding domain. The abmA gene, however, does not contain an unusual Gly, Pro, Ala stretch typical of many of the previously described myosin Is. Two additional genes (abmB and abmC) were identified using this approach and also found to contain sequences that encode proteins with typical conserved myosin head sequences. The abm genes may be part of a large family of actin-based motors that play various roles in diverse aspects of cellular motility. Images PMID:2519618

  13. Regulatory mimicry in Listeria monocytogenes actin-based motility

    PubMed Central

    Chong, Ryan; Swiss, Rachel; Briones, Gabriel; Stone, Kathryn L.; Gulcicek, Erol E.; Agaisse, Hervé

    2009-01-01

    Summary The actin-based motility of the intracellular pathogen Listeria monocytogenes relies on ActA, a bacterial factor mimicking the activity of host cell nucleation-promoting factors of the WASP/WAVE family. The activity of WASP and WAVE is tightly regulated in cells. However, it is not known whether the activity of ActA is regulated upon L. monocytogenes infection. Here, we used an RNAi-based genetic approach in combination with computer-assisted image analysis to investigate the role of host factors in L. monocytogenes spread from cell to cell. We showed that the host cell serine/threonine kinase CK2 is required for efficient actin tail formation. We demonstrated that, similar to WASP and WAVE, the affinity of ActA for the ARP2/3 complex is regulated by CK2-mediated phosphorylation. We also demonstrated the importance of this regulatory mechanism in a mouse model of infection. Our work suggests that ActA is a bacterial virulence factor that not only displays a structural mimic of the VCA domain of WASP/WAVE family members, but also co-opted CK2 as the host cell factor regulating its activity, a form of mimicry that we refer to as regulatory mimicry. We present comparative evidence supporting the notion that unrelated pathogens displaying actin-based motility may have evolved a similar strategy. PMID:19748468

  14. Curved trajectories of actin-based motility in two dimensions

    NASA Astrophysics Data System (ADS)

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

    2012-05-01

    Recent experiments have reported fascinating geometrical trajectories for actin-based motility of bacteria Listeria monocytogenes and functionalized beads. To understand the physical mechanism for these trajectories, we constructed a phenomenological model to study the motion of an actin-propelled disk in two dimensions. In our model, the force and actin density on the surface of the disk are influenced by the translation and rotation of the disk, which in turn is induced by the asymmetric distributions of those densities. We show that this feedback can destabilize a straight trajectory, leading to circular, S-shape and other geometrical trajectories observed in the experiments through bifurcations in the distributions of the force and actin density. The relation between our model and the models for self-propelled deformable particles is emphasized and discussed.

  15. Villin severing activity enhances actin-based motility in vivo.

    PubMed

    Revenu, Céline; Courtois, Matthieu; Michelot, Alphée; Sykes, Cécile; Louvard, Daniel; Robine, Sylvie

    2007-03-01

    Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

  16. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  17. Villin Severing Activity Enhances Actin-based Motility In Vivo

    PubMed Central

    Revenu, Céline; Courtois, Matthieu; Michelot, Alphée; Sykes, Cécile; Louvard, Daniel

    2007-01-01

    Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition. PMID:17182858

  18. Cytoskeletal mechanics: Structure and Dynamics

    NASA Astrophysics Data System (ADS)

    Bausch, Andreas

    2008-03-01

    The actin cytoskeleton, a dynamic network of semiflexible filaments and associated regulatory proteins, is responsible for the extraordinary viscoelastic properties of cells. Especially for cellular motility the controlled self assembly to defined structures and the dynamic reorganization on different time scales are of outstanding importance. A prominent example for the controlled self assembly are actin bundles: in many cytoskeletal processes cells rely on the tight control of the structural and mechanical properties of the actin bundles. Using an in vitro model system we show that size control relies on a mismatch between the helical structure of individual actin filaments and the packing symmetry within bundles. While such self assembled structure may evoke the picture of a static network the contrary is the case: the cytoskeleton is highly dynamic and a constant remodeling takes place in vivo. Such dynamic reorganization of the cytoskeleton relies on the non-static nature of single actin/ABP bonds. Here, we study the thermal and forced unbinding events of individual ABP in such in vitro networks. The binding kinetics of the transient crosslinkers determines the mechanical response of such networks -- in the linear as well in the non-linear regime. These effects are important prerequisites for the high adaptability of cells and at the same time might be the molecular mechanism employed by them for mechanosensing.

  19. CHUP1 mediates actin-based light-induced chloroplast avoidance movement in the moss Physcomitrella patens.

    PubMed

    Usami, Hiroka; Maeda, Takuma; Fujii, Yusuke; Oikawa, Kazusato; Takahashi, Fumio; Kagawa, Takatoshi; Wada, Masamitsu; Kasahara, Masahiro

    2012-12-01

    Chloroplasts change their intracellular distribution in response to light intensity. CHUP1 (CHLOROPLAST UNUSUAL POSITIONING1) is indispensable for this response in Arabidopsis thaliana. However, involvement of CHUP1 in light-induced chloroplast movement is unknown in other plants. In this study, CHUP1 orthologues were isolated from a moss, Physcomitrella patens, and a fern, Adiantum capillus-veneris, by cDNA library screening and PCR cloning based on the P. patens genome sequence. Functional motifs found in CHUP1 of A. thaliana were conserved among the CHUP1 orthologues. In addition to the putative functional regions, the C-terminal regions (approximately 250 amino acids), which are unique in CHUP1s, were highly conserved. Green fluorescent protein (GFP) fusions of P. patens CHUP1s (PpCHUP1A, PpCHUP1B and PpCHUP1C) were transiently expressed in protoplast cells. All GFP fusions were localized on the chloroplasts. Light-induced chloroplast avoidance movement of chup1 disruptants of P. patens was examined in the presence of cytoskeletal inhibitors because of the utilization of both microtubules and actin filaments for the movement in P. patens. When actin filaments were disrupted by cytochalasin B, the wild type (WT) and all chup1 disruptants showed chloroplast avoidance movement. However, when microtubules were disrupted by Oryzalin, chloroplasts in ∆chup1A and ∆chup1A/B rarely moved and stayed in the strong light-irradiated area. On the other hand, WT, ∆chup1B and ∆chup1C showed chloroplast avoidance movement. These results suggest that PpCHUP1A predominantly mediates the actin-based light-induced chloroplast avoidance movement. This study reveals that CHUP1 functions on the chloroplasts and is involved in the actin-based light-induced chloroplast avoidance movement in P. patens.

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

    PubMed Central

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

    2009-01-01

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

  1. Actin-based motility propelled by molecular motors

    NASA Astrophysics Data System (ADS)

    Upadyayula, Sai Pramod; Rangarajan, Murali

    2012-09-01

    Actin-based motility of Listeria monocytogenes propelled by filament end-tracking molecular motors has been simulated. Such systems may act as potential nanoscale actuators and shuttles useful in sorting and sensing biomolecules. Filaments are modeled as three-dimensional elastic springs distributed on one end of the capsule and persistently attached to the motile bacterial surface through an end-tracking motor complex. Filament distribution is random, and monomer concentration decreases linearly as a function of position on the bacterial surface. Filament growth rate increases with monomer concentration but decreases with the extent of compression. The growing filaments exert push-pull forces on the bacterial surface. In addition to forces, torques arise due to two factors—distribution of motors on the bacterial surface, and coupling of torsion upon growth due to the right-handed helicity of F-actin—causing the motile object to undergo simultaneous translation and rotation. The trajectory of the bacterium is simulated by performing a force and torque balance on the bacterium. All simulations use a fixed value of torsion. Simulations show strong alignment of the filaments and the long axis of the bacterium along the direction of motion. In the absence of torsion, the bacterial surface essentially moves along the direction of the long axis. When a small amount of the torsion is applied to the bacterial surface, the bacterium is seen to move in right-handed helical trajectories, consistent with experimental observations.

  2. Cytoskeletal Proteins of Actinobacteria

    PubMed Central

    Letek, Michal; Fiuza, María; Villadangos, Almudena F.; Mateos, Luís M.; Gil, José A.

    2012-01-01

    Although bacteria are considered the simplest life forms, we are now slowly unraveling their cellular complexity. Surprisingly, not only do bacterial cells have a cytoskeleton but also the building blocks are not very different from the cytoskeleton that our own cells use to grow and divide. Nonetheless, despite important advances in our understanding of the basic physiology of certain bacterial models, little is known about Actinobacteria, an ancient group of Eubacteria. Here we review current knowledge on the cytoskeletal elements required for bacterial cell growth and cell division, focusing on actinobacterial genera such as Mycobacterium, Corynebacterium, and Streptomyces. These include some of the deadliest pathogens on earth but also some of the most prolific producers of antibiotics and antitumorals. PMID:22481946

  3. Cytoskeletal elements in the bacterium Mycoplasma pneumoniae

    NASA Astrophysics Data System (ADS)

    Hegermann, Jan; Herrmann, Richard; Mayer, Frank

    2002-09-01

    Mycoplasma pneumoniae is a pathogenic eubacterium lacking a cell wall. Three decades ago, a "rod", an intracellular cytoskeletal structure, was discovered that was assumed to define and stabilize the elongated cell shape. Later, by treatment with detergent, a "Triton shell" (i.e. a fraction of detergent-insoluble cell material) could be obtained, believed to contain additional cytoskeletal elements. Now, by application of a modified Triton X-100 treatment, we are able to demonstrate that M. pneumoniae possesses a cytoskeleton consisting of a blade-like rod and a peripheral lining located close to the inner face of the cytoplasmic membrane, exhibiting features of a highly regular network. Attached "stalks" may support the cytoplasmic membrane. The rod was connected to the cell periphery by "spokes" and showed a defined ultrastructure. Its proximal end was found to be attached to a wheel-like complex. Fibrils extended from the proximal end of the rod into the cytoplasm.

  4. Spontaneous symmetry breaking for geometrical trajectories of actin-based motility in three dimensions

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

    Actin-based motility is important for many cellular processes. In this article we extend our previous studies of an actin-propelled circular disk in two dimensions to an actin-propelled spherical bead in three dimensions. We find that for an achiral load the couplings between the motion of the load and the actin network induce a series of bifurcations, starting with a transition from rest to moving state, followed by a transition from straight to planar curves, and finally a further transition from motion in a plane to one with torsion. To address the intriguing, experimentally observed chiral motility of the bacterium Listeria monocytogenes, we also study the motility of a spherical load with a built-in chirality. For such a chiral load, stable circular trajectories are no longer found in numerical simulations. Instead, helical trajectories with handedness that depends on the chirality of the load are found. Our results reveal the relation between the symmetry of actin network and the trajectories of actin-propelled loads.

  5. Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells

    PubMed Central

    Boraas, Liana C.; Guidry, Julia B.; Pineda, Emma T.; Ahsan, Tabassum

    2016-01-01

    Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this

  6. Cell Forces and Cytoskeletal Order Parameters

    NASA Astrophysics Data System (ADS)

    Discher, Dennis

    2012-02-01

    Nematic, Smectic and Isotropic Order parameters have found wide-spread use in characterizing all manner of soft matter systems, but have not yet been applied to characterize and understand the structures within living cells, particularly cytoskeletal structures. Several examples will be used to illustrate the utility of such analyses, ranging from experiments on stem cells attached to or in various elastic matrices to embryonic heart tissue and simulations of membrane cytoskeletons under all manner of stressing. Recently developed theory will be shown to apply in general with account of cell contractility, matrix elasticity and dimensionality as well as cell shape and a newly defined ``cytoskeletal polarizability.'' The latter property of cells is likely different between different cell types due to different amounts of key cytoskeletal components with some types of stem cells being more polarizable than others. Evidence of coupling to the nucleus as a viscoelastic inclusion will also be presented. [4pt] References: (1) P. Dalhaimer, D.E. Discher, T. Lubensky. Crosslinked actin networks exhibit liquid crystal elastomer behavior, including soft-mode elasticity. Nature Physics 3: 354-360 (2007). (2) A. Zemel, F.Rehfeldt, A.E.X. Brown, D.E. Discher, and S.A. Safran. Optimal matrix rigidity in the self-polarization of stem cells. Nature Physics 6: 468 - 473 (2010).

  7. Extending the molecular clutch beyond actin-based cell motility

    NASA Astrophysics Data System (ADS)

    Havrylenko, Svitlana; Mezanges, Xavier; Batchelder, Ellen; Plastino, Julie

    2014-10-01

    Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the ‘molecular clutch’ description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of major sperm protein, which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton.

  8. Extending the molecular clutch beyond actin-based cell motility

    PubMed Central

    Havrylenko, Svitlana; Mezanges, Xavier; Batchelder, Ellen; Plastino, Julie

    2014-01-01

    Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the “molecular clutch” description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of Major Sperm Protein (MSP), which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton. PMID:25383039

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

    NASA Astrophysics Data System (ADS)

    Wang, Xinxin

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

  10. Kinetic Proofreading of Cytoskeletal Structures

    NASA Astrophysics Data System (ADS)

    Swanson, Douglas; Wingreen, Ned

    2010-03-01

    Cytoskeletal polymer dynamics play a role in cellular processes as varied as reproduction, locomotion, and intracellular transport. Microtubules are cytoskeletal biopolymers that grow by accumulating tubulin subunits bound to guanosine triphosphate (GTP). The subunits hydrolyze GTP to guanosine diphosphate (GDP), causing a conformational change in the protein that destabilizes the microtubule. GDP-bound subunits tend to depolymerize, leading to stochastic microtubule disassembly in a process known as dynamic instability. Over time polymerization and depolymerization come to steady state, leading to a local steady-state concentration of tubulin subunits. This may be viewed as a kind of ``kinetic proofreading,'' in which the system consumes energy actively to ``proofread'' the steady-state subunit concentration. We suggest that the same mechanism could also ``proofread'' between different cytoskeletal structures. For example, we show that a small free-energy difference between two polymer orientations, combined with dynamic instability, can strongly drive the system towards the lower free-energy state. This might help to explain the long-time stability of many cytoskeletal structures despite the short-time rapid turnover of the individual subunits.

  11. Disruption of the Rickettsia rickettsii Sca2 autotransporter inhibits actin-based motility.

    PubMed

    Kleba, Betsy; Clark, Tina R; Lutter, Erika I; Ellison, Damon W; Hackstadt, Ted

    2010-05-01

    Rickettsii rickettsii, the etiologic agent of Rocky Mountain spotted fever, replicates within the cytosol of infected cells and uses actin-based motility to spread inter- and intracellularly. Although the ultrastructure of the actin tail and host proteins associated with it are distinct from those of Listeria or Shigella, comparatively little is known regarding the rickettsial proteins involved in its organization. Here, we have used random transposon mutagenesis of R. rickettsii to generate a small-plaque mutant that is defective in actin-based motility and does not spread directly from cell to cell as is characteristic of spotted fever group rickettsiae. The transposon insertion site of this mutant strain was within Sca2, a member of a family of large autotransporter proteins. Sca2 exhibits several features suggestive of its apparent role in actin-based motility. It displays an N-terminal secretory signal peptide, a C-terminal predicted autotransporter domain, up to four predicted Wasp homology 2 (WH2) domains, and two proline-rich domains, one with similarity to eukaryotic formins. In a guinea pig model of infection, the Sca2 mutant did not elicit fever, suggesting that Sca2 and actin-based motility are virulence factors of spotted fever group rickettsiae.

  12. Purification of Tetrahymena cytoskeletal proteins.

    PubMed

    Honts, Jerry E

    2012-01-01

    Like all eukaryotic cells, Tetrahymena thermophila contains a rich array of cytoskeletal proteins, some familiar and some novel. A detailed analysis of the structure, function, and interactions of these proteins requires procedures for purifying the individual protein components. Procedures for the purification of actin and tubulin from Tetrahymena are reviewed, followed by a description of a procedure that yields proteins from the epiplasmic layer and associated structures, including the tetrins. Finally, the challenges and opportunities for future advances are assessed.

  13. Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells.

    PubMed

    Estes, Kathleen A; Szumowski, Suzannah C; Troemel, Emily R

    2011-09-01

    The intestine is a common site for invasion by intracellular pathogens, but little is known about how pathogens restructure and exit intestinal cells in vivo. The natural microsporidian parasite N. parisii invades intestinal cells of the nematode C. elegans, progresses through its life cycle, and then exits cells in a transmissible spore form. Here we show that N. parisii causes rearrangements of host actin inside intestinal cells as part of a novel parasite exit strategy. First, we show that N. parisii infection causes ectopic localization of the normally apical-restricted actin to the basolateral side of intestinal cells, where it often forms network-like structures. Soon after this actin relocalization, we find that gaps appear in the terminal web, a conserved cytoskeletal structure that could present a barrier to exit. Reducing actin expression creates terminal web gaps in the absence of infection, suggesting that infection-induced actin relocalization triggers gap formation. We show that terminal web gaps form at a distinct stage of infection, precisely timed to precede spore exit, and that all contagious animals exhibit gaps. Interestingly, we find that while perturbations in actin can create these gaps, actin is not required for infection progression or spore formation, but actin is required for spore exit. Finally, we show that despite large numbers of spores exiting intestinal cells, this exit does not cause cell lysis. These results provide insight into parasite manipulation of the host cytoskeleton and non-lytic escape from intestinal cells in vivo.

  14. 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

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

    PubMed

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

    2015-09-01

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

  16. Cytoskeletal prestress regulates nuclear shape and stiffness in cardiac myocytes.

    PubMed

    Lee, Hyungsuk; Adams, William J; Alford, Patrick W; McCain, Megan L; Feinberg, Adam W; Sheehy, Sean P; Goss, Josue A; Parker, Kevin Kit

    2015-11-01

    Mechanical stresses on the myocyte nucleus have been associated with several diseases and potentially transduce mechanical stimuli into cellular responses. Although a number of physical links between the nuclear envelope and cytoplasmic filaments have been identified, previous studies have focused on the mechanical properties of individual components of the nucleus, such as the nuclear envelope and lamin network. The mechanical interaction between the cytoskeleton and chromatin on nuclear deformability remains elusive. Here, we investigated how cytoskeletal and chromatin structures influence nuclear mechanics in cardiac myocytes. Rapid decondensation of chromatin and rupture of the nuclear membrane caused a sudden expansion of DNA, a consequence of prestress exerted on the nucleus. To characterize the prestress exerted on the nucleus, we measured the shape and the stiffness of isolated nuclei and nuclei in living myocytes during disruption of cytoskeletal, myofibrillar, and chromatin structure. We found that the nucleus in myocytes is subject to both tensional and compressional prestress and its deformability is determined by a balance of those opposing forces. By developing a computational model of the prestressed nucleus, we showed that cytoskeletal and chromatin prestresses create vulnerability in the nuclear envelope. Our studies suggest the cytoskeletal-nuclear-chromatin interconnectivity may play an important role in mechanics of myocyte contraction and in the development of laminopathies by lamin mutations.

  17. Cytoskeletal Mechanisms for Breaking Cellular Symmetry

    PubMed Central

    Mullins, R. Dyche

    2010-01-01

    Cytoskeletal systems are networks of polymers found in all eukaryotic and many prokaryotic cells. Their purpose is to transmit and integrate information across cellular dimensions and help turn a disorderly mob of macromolecules into a spatially organized, living cell. Information, in this context, includes physical and chemical properties relevant to cellular physiology, including: the number and activity of macromolecules, cell shape, and mechanical force. Most animal cells are 10–50 microns in diameter, whereas the macromolecules that comprise them are 10,000-fold smaller (2–20 nm). To establish long-range order over cellular length scales, individual molecules must, therefore, self-assemble into larger polymers, with lengths (0.1–20 m) comparable to the size of a cell. These polymers must then be cross-linked into organized networks that fill the cytoplasm. Such cell-spanning polymer networks enable different parts of the cytoplasm to communicate directly with each other, either by transmitting forces or by carrying cargo from one spot to another. PMID:20182610

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

    PubMed Central

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

    2011-01-01

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

  19. Alternative cytoskeletal landscapes: cytoskeletal novelty and evolution in basal excavate protists

    PubMed Central

    Dawson, Scott C.; Paredez, Alexander R.

    2016-01-01

    Microbial eukaryotes encompass the majority of eukaryotic evolutionary and cytoskeletal diversity. The cytoskeletal complexity observed in multicellular organisms appears to be an expansion of components present in genomes of diverse microbial eukaryotes such as the basal lineage of flagellates, the Excavata. Excavate protists have complex and diverse cytoskeletal architectures and life cycles – essentially alternative cytoskeletal “landscapes” – yet still possess conserved microtubule- and actin-associated proteins. Comparative genomic analyses have revealed that a subset of excavates, however, lack many canonical actin-binding proteins central to actin cytoskeleton function in other eukaryotes. Overall, excavates possess numerous uncharacterized and “hypothetical” genes, and may represent an undiscovered reservoir of novel cytoskeletal genes and cytoskeletal mechanisms. The continued development of molecular genetic tools in these complex microbial eukaryotes will undoubtedly contribute to our overall understanding of cytoskeletal diversity and evolution. PMID:23312067

  20. Structure of Cytoskeletal Supramolecular Assemblies in the Nerve Cell Axon

    NASA Astrophysics Data System (ADS)

    Ojeda-López, Miguel A.; Case, Ryan; Miller, Herb P.; Wilson, Leslie; Safinya, Cyrus R.

    2001-03-01

    The cytoskeleton of eucaryotic cells is an intricate network of supramolecular assemblies of protein filaments, e.g., actin, intermediate filaments (IFs), tubulin, and a multi-associated family of cross-linking proteins. Most of its multiple functions rely on its structural stability, which depends on a variety of specific interactions of the subunit proteins and its local physico-chemical environment. In neurodegenerative diseases the cytoskeletal supramolecular structure is almost universally altered. Preliminary results on the supramolecular structure of cytoskeletal filaments in isolated axons from bovine white matter will be presented as obtained using synchrotron small angle x-ray diffraction. These results will be compared to our ongoing cell-free studies on the structures formed by neurofilaments in vitro. Supported by NIH GM59288, NSF-DMR-9972246, and University of California Biotechnology Grant 99-14 & UC.

  1. Implementing cell contractility in filament-based cytoskeletal models.

    PubMed

    Fallqvist, B

    2016-02-01

    Cells are known to respond over time to mechanical stimuli, even actively generating force at longer times. In this paper, a microstructural filament-based cytoskeletal network model is extended to incorporate this active response, and a computational study to assess the influence on relaxation behaviour was performed. The incorporation of an active response was achieved by including a strain energy function of contractile activity from the cross-linked actin filaments. A four-state chemical model and strain energy function was adopted, and generalisation to three dimensions and the macroscopic deformation field was performed by integration over the unit sphere. Computational results in MATLAB and ABAQUS/Explicit indicated an active cellular response over various time-scales, dependent on contractile parameters. Important features such as force generation and increasing cell stiffness due to prestress are qualitatively predicted. The work in this paper can easily be extended to encompass other filament-based cytoskeletal models as well.

  2. Actin-based motility of Listeria: Right-handed helical trajectories

    NASA Astrophysics Data System (ADS)

    Rangarajan, Murali

    2012-06-01

    Bacteria such as Listeria monocytogenes recruit cellular machinery to move in and between cells. Understanding the mechanism of motility, including force and torque generation and the resultant displacements, holds keys to numerous applications in medicine and biosensing. In this work, a simple back-of-the-envelope calculation is presented to illustrate that a biomechanical model of actin-based motility of a rigid surface through persistently attached filaments propelled by affinity-modulated molecular motors can produce a right-handed helical trajectory consistent with experimental observations. The implications of the mechanism to bacterial motility are discussed.

  3. Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding.

    PubMed

    Fykerud, Tone A; Knudsen, Lars M; Totland, Max Z; Sørensen, Vigdis; Dahal-Koirala, Shiva; Lothe, Ragnhild A; Brech, Andreas; Leithe, Edward

    2016-11-01

    In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed "mitotic nanotubes," were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.

  4. Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding

    PubMed Central

    Fykerud, Tone A.; Knudsen, Lars M.; Totland, Max Z.; Dahal-Koirala, Shiva; Lothe, Ragnhild A.; Brech, Andreas; Leithe, Edward

    2016-01-01

    ABSTRACT In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed “mitotic nanotubes,” were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding. PMID:27625181

  5. Actin-based motility drives baculovirus transit to the nucleus and cell surface

    PubMed Central

    Ohkawa, Taro; Volkman, Loy E.

    2010-01-01

    Most viruses move intracellularly to and from their sites of replication using microtubule-based mechanisms. In this study, we show that nucleocapsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus undergo intracellular motility driven by actin polymerization. Motility requires the viral P78/83 capsid protein and the host Arp2/3 complex. Surprisingly, the virus directs two sequential and coordinated phases of actin-based motility. Immediately after cell entry, motility enables exploration of the cytoplasm and collision with the nuclear periphery, speeding nuclear entry and the initiation of viral gene expression. Nuclear entry itself requires transit through nuclear pore complexes. Later, after the onset of early gene expression, motility is required for accumulation of a subpopulation of nucleocapsids in the tips of actin-rich surface spikes. Temporal coordination of actin-based nuclear and surface translocation likely enables rapid transmission to neighboring cells during infection in insects and represents a distinctive evolutionary strategy for overcoming host defenses. PMID:20660627

  6. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

    NASA Astrophysics Data System (ADS)

    Loisel, Thomas P.; Boujemaa, Rajaa; Pantaloni, Dominique; Carlier, Marie-France

    1999-10-01

    Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

  7. Diffusion Rate Limitations in Actin-Based Propulsion of Hard and Deformable Particles

    PubMed Central

    Dickinson, Richard B.; Purich, Daniel L.

    2006-01-01

    The mechanism by which actin polymerization propels intracellular vesicles and invasive microorganisms remains an open question. Several recent quantitative studies have examined propulsion of biomimetic particles such as polystyrene microspheres, phospholipid vesicles, and oil droplets. In addition to allowing quantitative measurement of parameters such as the dependence of particle speed on its size, these systems have also revealed characteristic behaviors such a saltatory motion of hard particles and oscillatory deformation of soft particles. Such measurements and observations provide tests for proposed mechanisms of actin-based motility. In the actoclampin filament end-tracking motor model, particle-surface-bound filament end-tracking proteins are involved in load-insensitive processive insertion of actin subunits onto elongating filament plus-ends that are persistently tethered to the surface. In contrast, the tethered-ratchet model assumes working filaments are untethered and the free-ended filaments grow as thermal ratchets in a load-sensitive manner. This article presents a model for the diffusion and consumption of actin monomers during actin-based particle propulsion to predict the monomer concentration field around motile particles. The results suggest that the various behaviors of biomimetic particles, including dynamic saltatory motion of hard particles and oscillatory vesicle deformations, can be quantitatively and self-consistently explained by load-insensitive, diffusion-limited elongation of (+)-end-tethered actin filaments, consistent with predictions of the actoclampin filament-end tracking mechanism. PMID:16731556

  8. Cytoskeletal prestress regulates nuclear shape and stiffness in cardiac myocytes

    PubMed Central

    Lee, Hyungsuk; Adams, William J; Alford, Patrick W; McCain, Megan L; Feinberg, Adam W; Sheehy, Sean P; Goss, Josue A

    2015-01-01

    Mechanical stresses on the myocyte nucleus have been associated with several diseases and potentially transduce mechanical stimuli into cellular responses. Although a number of physical links between the nuclear envelope and cytoplasmic filaments have been identified, previous studies have focused on the mechanical properties of individual components of the nucleus, such as the nuclear envelope and lamin network. The mechanical interaction between the cytoskeleton and chromatin on nuclear deformability remains elusive. Here, we investigated how cytoskeletal and chromatin structures influence nuclear mechanics in cardiac myocytes. Rapid decondensation of chromatin and rupture of the nuclear membrane caused a sudden expansion of DNA, a consequence of prestress exerted on the nucleus. To characterize the prestress exerted on the nucleus, we measured the shape and the stiffness of isolated nuclei and nuclei in living myocytes during disruption of cytoskeletal, myofibrillar, and chromatin structure. We found that the nucleus in myocytes is subject to both tensional and compressional prestress and its deformability is determined by a balance of those opposing forces. By developing a computational model of the prestressed nucleus, we showed that cytoskeletal and chromatin prestresses create vulnerability in the nuclear envelope. Our studies suggest the cytoskeletal–nuclear–chromatin interconnectivity may play an important role in mechanics of myocyte contraction and in the development of laminopathies by lamin mutations. PMID:25908635

  9. The TRPM7 interactome defines a cytoskeletal complex linked to neuroblastoma progression.

    PubMed

    Middelbeek, Jeroen; Vrenken, Kirsten; Visser, Daan; Lasonder, Edwin; Koster, Jan; Jalink, Kees; Clark, Kristopher; van Leeuwen, Frank N

    2016-11-01

    Neuroblastoma is the second-most common solid tumor in children and originates from poorly differentiated neural crest-derived progenitors. Although most advanced stage metastatic neuroblastoma patients initially respond to treatment, a therapy resistant pool of poorly differentiated cells frequently arises, leading to refractory disease. A lack of insight into the molecular mechanisms that underlie neuroblastoma progression hampers the development of effective new therapies for these patients. Normal neural crest development and maturation is guided by physical interactions between the cell and its surroundings, in addition to soluble factors such as growth factors. This mechanical crosstalk is mediated by actin-based adhesion structures and cell protrusions that probe the cellular environment to modulate migration, proliferation, survival and differentiation. Whereas such signals preserve cellular quiescence in non-malignant cells, perturbed adhesion signaling promotes de-differentiation, uncontrolled cell proliferation, tissue invasion and therapy resistance. We previously reported that high expression levels of the channel-kinase TRPM7, a protein that maintains the progenitor state of embryonic neural crest cells, are closely associated with progenitor-like features of tumor cells, accompanied by extensive cytoskeletal reorganization and adhesion remodeling. To define mechanisms by which TRPM7 may contribute to neuroblastoma progression, we applied a proteomics approach to identify TRPM7 interacting proteins. We show that TRPM7 is part of a large complex of proteins, many of which function in cytoskeletal organization, cell protrusion formation and adhesion dynamics. Expression of a subset of these TRPM7 interacting proteins strongly correlates with neuroblastoma progression in independent neuroblastoma patient datasets. Thus, TRPM7 is part of a large cytoskeletal complex that may affect the malignant potential of tumor cells by regulating actomyosin dynamics

  10. Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix.

    PubMed

    Evans, S S; Schleider, D M; Bowman, L A; Francis, M L; Kansas, G S; Black, J D

    1999-03-15

    L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.

  11. Vinculin Proteolysis Unmasks an ActA Homolog for Actin-based Shigella Motility

    PubMed Central

    Laine, Roney O.; Zeile, William; Kang, Fan; Purich, Daniel L.; Southwick, Frederick S.

    1997-01-01

    To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular pathogens Shigella flexneri and Listeria monocytogenes use molecular mimicry to create their own actin-based motors. We raised an antibody (designated FS-1) against the FEFPPPPTDE sequence of Listeria ActA, and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited intracellular locomotion upon microinjection of Shigella-infected cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the head region of vinculin (Vin 11-5) localized to the back of motile bacteria (an identical staining pattern observed with the anti-ActA FS-1 antibody), indicating that motile bacteria attract a form of vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular motility, underscoring the functional importance of this sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1–3 h time frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected p90, but not full-length vinculin, accelerates rates of pathogen motility by a factor of 3 ± 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based Shigella motility, and that

  12. Nuclear envelope rupture is induced by actin-based nucleus confinement.

    PubMed

    Hatch, Emily M; Hetzer, Martin W

    2016-10-10

    Repeated rounds of nuclear envelope (NE) rupture and repair have been observed in laminopathy and cancer cells and result in intermittent loss of nucleus compartmentalization. Currently, the causes of NE rupture are unclear. Here, we show that NE rupture in cancer cells relies on the assembly of contractile actin bundles that interact with the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex. We found that the loss of actin bundles or the LINC complex did not rescue nuclear lamina defects, a previously identified determinant of nuclear membrane stability, but did decrease the number and size of chromatin hernias. Finally, NE rupture inhibition could be rescued in cells treated with actin-depolymerizing drugs by mechanically constraining nucleus height. These data suggest a model of NE rupture where weak membrane areas, caused by defects in lamina organization, rupture because of an increase in intranuclear pressure from actin-based nucleus confinement.

  13. Biotechnological aspects of cytoskeletal regulation in plants.

    PubMed

    Komis, George; Luptovciak, Ivan; Doskocilova, Anna; Samaj, Jozef

    2015-11-01

    The cytoskeleton is a protein-based intracellular superstructure that evolved early after the appearance of bacterial prokaryotes. Eventually cytoskeletal proteins and their macromolecular assemblies were established in eukaryotes and assumed critical roles in cell movements, intracellular organization, cell division and cell differentiation. In biomedicine the small-molecules targeting cytoskeletal elements are in the frontline of anticancer research with plant-derived cytoskeletal drugs such as Vinca alkaloids and toxoids, being routinely used in the clinical practice. Moreover, plants are also major material, food and energy resources for human activities ranging from agriculture, textile industry, carpentry, energy production and new material development to name some few. Most of these inheritable traits are associated with cell wall synthesis and chemical modification during primary and secondary plant growth and inevitably are associated with the dynamics, organization and interactions of the plant cytoskeleton. Taking into account the vast intracellular spread of microtubules and actin microfilaments the cytoskeleton collectively assumed central roles in plant growth and development, in determining the physical stance of plants against the forces of nature and becoming a battleground between pathogenic invaders and the defense mechanisms of plant cells. This review aims to address the role of the plant cytoskeleton in manageable features of plants including cellulose biosynthesis with implications in wood and fiber properties, in biofuel production and the contribution of plant cytoskeletal elements in plant defense responses against pathogens or detrimental environmental conditions. Ultimately the present work surveys the potential of cytoskeletal proteins as platforms of plant genetic engineering, nominating certain cytoskeletal proteins as vectors of favorable traits in crops and other economically important plants.

  14. Run-and-pause dynamics of cytoskeletal motor proteins

    PubMed Central

    Hafner, Anne E.; Santen, Ludger; Rieger, Heiko; Shaebani, M. Reza

    2016-01-01

    Cytoskeletal motor proteins are involved in major intracellular transport processes which are vital for maintaining appropriate cellular function. When attached to cytoskeletal filaments, the motor exhibits distinct states of motility: active motion along the filaments, and pause phase in which it remains stationary for a finite time interval. The transition probabilities between motion and pause phases are asymmetric in general, and considerably affected by changes in environmental conditions which influences the efficiency of cargo delivery to specific targets. By considering the motion of individual non-interacting molecular motors on a single filament as well as a dynamic filamentous network, we present an analytical model for the dynamics of self-propelled particles which undergo frequent pause phases. The interplay between motor processivity, structural properties of filamentous network, and transition probabilities between the two states of motility drastically changes the dynamics: multiple transitions between different types of anomalous diffusive dynamics occur and the crossover time to the asymptotic diffusive or ballistic motion varies by several orders of magnitude. We map out the phase diagrams in the space of transition probabilities, and address the role of initial conditions of motion on the resulting dynamics. PMID:27849013

  15. Run-and-pause dynamics of cytoskeletal motor proteins

    NASA Astrophysics Data System (ADS)

    Hafner, Anne E.; Santen, Ludger; Rieger, Heiko; Shaebani, M. Reza

    2016-11-01

    Cytoskeletal motor proteins are involved in major intracellular transport processes which are vital for maintaining appropriate cellular function. When attached to cytoskeletal filaments, the motor exhibits distinct states of motility: active motion along the filaments, and pause phase in which it remains stationary for a finite time interval. The transition probabilities between motion and pause phases are asymmetric in general, and considerably affected by changes in environmental conditions which influences the efficiency of cargo delivery to specific targets. By considering the motion of individual non-interacting molecular motors on a single filament as well as a dynamic filamentous network, we present an analytical model for the dynamics of self-propelled particles which undergo frequent pause phases. The interplay between motor processivity, structural properties of filamentous network, and transition probabilities between the two states of motility drastically changes the dynamics: multiple transitions between different types of anomalous diffusive dynamics occur and the crossover time to the asymptotic diffusive or ballistic motion varies by several orders of magnitude. We map out the phase diagrams in the space of transition probabilities, and address the role of initial conditions of motion on the resulting dynamics.

  16. Quasi-3D Cytoskeletal Dynamics of Osteocytes under Fluid Flow

    PubMed Central

    Baik, Andrew D.; Lu, X. Lucas; Qiu, Jun; Huo, Bo; Hillman, Elizabeth M.C.; Dong, Cheng; Guo, X. Edward

    2010-01-01

    Osteocytes respond to dynamic fluid shear loading by activating various biochemical pathways, mediating a dynamic process of bone formation and resorption. Whole-cell deformation and regional deformation of the cytoskeleton may be able to directly regulate this process. Attempts to image cellular deformation by conventional microscopy techniques have been hindered by low temporal or spatial resolution. In this study, we developed a quasi-three-dimensional microscopy technique that enabled us to simultaneously visualize an osteocyte's traditional bottom-view profile and a side-view profile at high temporal resolution. Quantitative analysis of the plasma membrane and either the intracellular actin or microtubule (MT) cytoskeletal networks provided characterization of their deformations over time. Although no volumetric dilatation of the whole cell was observed under flow, both the actin and MT networks experienced primarily tensile strains in all measured strain components. Regional heterogeneity in the strain field of normal strains was observed in the actin networks, especially in the leading edge to flow, but not in the MT networks. In contrast, side-view shear strains exhibited similar subcellular distribution patterns in both networks. Disruption of MT networks caused actin normal strains to decrease, whereas actin disruption had little effect on the MT network strains, highlighting the networks' mechanical interactions in osteocytes. PMID:21044578

  17. Biophysical models of length control of cytoskeletal structures

    NASA Astrophysics Data System (ADS)

    Mohapatra, Lishibanya

    Cells contain elaborate and interconnected networks of protein polymers which make up the cytoskeleton. The cytoskeleton governs the internal positioning and movement of vesicles and organelles, and controls dynamic changes in cell polarity, shape and movement. Many of these processes require tight control of the size and shape of these cytoskeletal structures. A key question in cell biology is how these structures maintain a particular size and shape despite the rapid turnover of their components. In this thesis I show that the emerging mechanisms by which cells control and regulate the size of filamentous cytoskeletal structures can be classified using key parameters related to their assembly and disassembly kinetics. First, I examine quantitative models based on these specific molecular mechanisms of length control and make experimentally testable predictions that can be used to distinguish different mechanisms of length-control. Second, I study the length control of actin cables in budding yeast cells. Inspired by recent experimental observations in cells, I propose a novel antenna mechanism for cable length control which involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. My results provide testable predictions of the antenna mechanism of actin-cable length control. Next I consider the question of how different sized structures can co-exist in the same cytoplasm while making use of the same building blocks. Using theory, I discover limitations imposed by physics on the finite monomer pool as a mechanism of size control and conclude that additional length control mechanisms are required if a cell is to maintain multiple structures. While the primary focus of this thesis is on cytoskeletal structures, the broader principles and mechanisms discussed herein will apply to a range of

  18. Tubules of plant reoviruses exploit tropomodulin to regulate actin-based tubule motility in insect vector

    PubMed Central

    Chen, Qian; Zhang, Linghua; Zhang, Yanshuang; Mao, Qianzhuo; Wei, Taiyun

    2017-01-01

    Plant reoviruses are known to exploit virion-packaging tubules formed by virus-encoding non-structural proteins for viral spread in insect vectors. Tubules are propelled by actin-based tubule motility (ABTM) to overcome membrane or tissue barriers in insect vectors. To further understand which insect factors mediate ABTM, we utilized yeast two-hybrid and bimolecular fluorescence complementation assays to test interactions between tubule protein Pns10 of rice dwarf virus (RDV), a plant reovirus, and proteins of its insect vector, the leafhopper Nephotettix cincticeps. Tropomodulin (Tmod), vitellogenin, and lipophorin precursor of N. cincticep displayed positive and strong interaction with Pns10, and actin-associated protein Tmod interacted with Pns10 in pull-down assay and the co-immunoprecipitation system. Further, we determined Pns10 tubules associated with Tmod in cultured cells and midgut of N. cincticep. The expression dynamic of Tmod was consistent with that of Pns10 and the fluctuation of RDV accumulation. Knockdown of Tmod inhibited the Pns10 expression and viral accumulation, thus decreasing the viruliferous rates of leafhopper. These results suggested that Tmod was involved in viral spread by directly interacting with Pns10 tubules, finally promoting RDV infection. This study provided direct evidence of plant reoviruses utilizing an actin-associated protein to manipulate ABTM in insect vectors, thus facilitating viral spread. PMID:28067229

  19. Molecular requirements for actin-based lamella formation in Drosophila S2 cells.

    PubMed

    Rogers, Stephen L; Wiedemann, Ursula; Stuurman, Nico; Vale, Ronald D

    2003-09-15

    Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

  20. Two kinesin-like proteins mediate actin-based chloroplast movement in Arabidopsis thaliana.

    PubMed

    Suetsugu, Noriyuki; Yamada, Noboru; Kagawa, Takatoshi; Yonekura, Hisashi; Uyeda, Taro Q P; Kadota, Akeo; Wada, Masamitsu

    2010-05-11

    Organelle movement is essential for efficient cellular function in eukaryotes. Chloroplast photorelocation movement is important for plant survival as well as for efficient photosynthesis. Chloroplast movement generally is actin dependent and mediated by blue light receptor phototropins. In Arabidopsis thaliana, phototropins mediate chloroplast movement by regulating short actin filaments on chloroplasts (cp-actin filaments), and the chloroplast outer envelope protein CHUP1 is necessary for cp-actin filament accumulation. However, other factors involved in cp-actin filament regulation during chloroplast movement remain to be determined. Here, we report that two kinesin-like proteins, KAC1 and KAC2, are essential for chloroplasts to move and anchor to the plasma membrane. A kac1 mutant showed severely impaired chloroplast accumulation and slow avoidance movement. A kac1kac2 double mutant completely lacked chloroplast photorelocation movement and showed detachment of chloroplasts from the plasma membrane. KAC motor domains are similar to those of the kinesin-14 subfamily (such as Ncd and Kar3) but do not have detectable microtubule-binding activity. The C-terminal domain of KAC1 could interact with F-actin in vitro. Instead of regulating microtubules, KAC proteins mediate chloroplast movement via cp-actin filaments. We conclude that plants have evolved a unique mechanism to regulate actin-based organelle movement using kinesin-like proteins.

  1. Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis.

    PubMed

    Kadota, Akeo; Yamada, Noboru; Suetsugu, Noriyuki; Hirose, Mana; Saito, Chieko; Shoda, Keiko; Ichikawa, Satoshi; Kagawa, Takatoshi; Nakano, Akihiko; Wada, Masamitsu

    2009-08-04

    Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement.

  2. Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis

    PubMed Central

    Kadota, Akeo; Yamada, Noboru; Suetsugu, Noriyuki; Hirose, Mana; Saito, Chieko; Shoda, Keiko; Ichikawa, Satoshi; Kagawa, Takatoshi; Nakano, Akihiko; Wada, Masamitsu

    2009-01-01

    Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement. PMID:19620714

  3. Bacterial Shape and ActA Distribution Affect Initiation of Listeria monocytogenes Actin-Based Motility

    PubMed Central

    Rafelski, Susanne M.; Theriot, Julie A.

    2005-01-01

    We have examined the process by which the intracellular bacterial pathogen Listeria monocytogenes initiates actin-based motility and determined the contribution of the variable surface distribution of the ActA protein to initiation and steady-state movement. To directly correlate ActA distributions to actin dynamics and motility of live bacteria, ActA was fused to a monomeric red fluorescent protein (mRFP1). Actin comet tail formation and steady-state bacterial movement rates both depended on ActA distribution, which in turn was tightly coupled to the bacterial cell cycle. Motility initiation was found to be a highly complex, multistep process for bacteria, in contrast to the simple symmetry breaking previously observed for ActA-coated spherical beads. F-actin initially accumulated along the sides of the bacterium and then slowly migrated to the bacterial pole expressing the highest density of ActA as a tail formed. Early movement was highly unstable with extreme changes in speed and frequent stops. Over time, saltatory motility and sensitivity to the immediate environment decreased as bacterial movement became robust at a constant steady-state speed. PMID:15980176

  4. HSF-1 mediated cytoskeletal integrity determines thermotolerance and lifespan

    PubMed Central

    Baird, Nathan A.; Douglas, Peter M.; Simic, Milos S.; Grant, Ana R.; Moresco, James J.; Wolff, Suzanne C.; Yates, John R.; Manning, Gerard; Dillin, Andrew

    2015-01-01

    The conserved transcription factor HSF-1 is essential to cellular stress resistance and organismal lifespan determination. The canonical function of HSF-1 is to regulate a network of molecular chaperones that maintain protein homeostasis during extrinsic environmental stresses or intrinsic age related deterioration. In the metazoan C. elegans, we engineered a modified HSF-1 strain that increases stress resistance and longevity without enhancing chaperone induction. This HSF-1 dependent health assurance acts through the regulation of pat-10. Upon heat stress pat-10 upregulation maintains a functional actin cytoskeleton and endocytic network. Loss of pat-10 causes a collapse of organismal health and failure of stress resistance. Furthermore, overexpression of pat-10 is sufficient to increase both thermotolerance and longevity by mechanisms that affect actin stability. Our findings indicate that in addition to chaperone induction, HSF-1 plays a prominent role in cytoskeletal integrity to ensure proper cellular function during times of stress and aging. PMID:25324391

  5. Modeling Cytoskeletal Active Matter Systems

    NASA Astrophysics Data System (ADS)

    Blackwell, Robert

    Active networks of filamentous proteins and crosslinking motor proteins play a critical role in many important cellular processes. One of the most important microtubule-motor protein assemblies is the mitotic spindle, a self-organized active liquid-crystalline structure that forms during cell division and that ultimately separates chromosomes into two daughter cells. Although the spindle has been intensively studied for decades, the physical principles that govern its self-organization and function remain mysterious. To evolve a better understanding of spindle formation, structure, and dynamics, I investigate course-grained models of active liquid-crystalline networks composed of microtubules, modeled as hard spherocylinders, in diffusive equilibrium with a reservoir of active crosslinks, modeled as hookean springs that can adsorb to microtubules and and translocate at finite velocity along the microtubule axis. This model is investigated using a combination of brownian dynamics and kinetic monte carlo simulation. I have further refined this model to simulate spindle formation and kinetochore capture in the fission yeast S. pombe. I then make predictions for experimentally realizable perturbations in motor protein presence and function in S. pombe.

  6. On the significance of microtubule flexural behavior in cytoskeletal mechanics.

    PubMed

    Mehrbod, Mehrdad; Mofrad, Mohammad R K

    2011-01-01

    Quantitative description of cell mechanics has challenged biological scientists for the past two decades. Various structural models have been attempted to analyze the structure of the cytoskeleton. One important aspect that has been largely ignored in all these modeling approaches is related to the flexural and buckling behavior of microtubular filaments. The objective of this paper is to explore the influence of this flexural and buckling behavior in cytoskeletal mechanics.In vitro the microtubules are observed to buckle in the first mode, reminiscent of a free, simply-supported beam. In vivo images of microtubules, however, indicate that the buckling mostly occurs in higher modes. This buckling mode switch takes place mostly because of the lateral support of microtubules via their connections to actin and intermediate filaments. These lateral loads are exerted throughout the microtubule length and yield a considerable bending behavior that, unless properly accounted for, would produce erroneous results in the modeling and analysis of the cytoskeletal mechanics.One of the promising attempts towards mechanical modeling of the cytoskeleton is the tensegrity model, which simplifies the complex network of cytoskeletal filaments into a combination merely of tension-bearing actin filaments and compression-bearing microtubules. Interestingly, this discrete model can qualitatively explain many experimental observations in cell mechanics. However, evidence suggests that the simplicity of this model may undermine the accuracy of its predictions, given the model's underlying assumption that "every single member bears solely either tensile or compressive behavior," i.e. neglecting the flexural behavior of the microtubule filaments. We invoke an anisotropic continuum model for microtubules and compare the bending energy stored in a single microtubule with its axial strain energy at the verge of buckling. Our results suggest that the bending energy can exceed the axial energy

  7. Actin-based motility of the intracellular pathogen Listeria monocytogenes: assessing the inhibitory specificity of ABM-1 peptide analogues.

    PubMed

    Purich, D L; Southwick, F S

    1999-06-01

    Actin-Based Motility motifs [ABM-1 sequence = (D/E)FPPPPX(D/E), where X = P or T, and ABM-2 sequence = XPPPPP, where X denotes G, A, L, P, and S] facilitate assembly of an activated motility complex. Potent inhibition of intracellular motility of pathogens by ABM-1 and ABM-2 peptide analogues has served as a criterion for investigating actin-based motility. To assess the specificity of ABM-1 peptide inhibitors, we microinjected proline-rich peptides into Listeria-infected PtK2 host cells. Use of a combinatorial ABM-1 peptide library (empirical formula = D1E2F2P4T1) demonstrated that high-potency inhibition requires a precise sequence, and not merely a particular amino acid composition. Calculated concentrations of specific sequences in this library indicate that the entire (D/E)FPPPPX(D/E) motif is needed to achieve high-affinity inhibition in living cells. The failure of the well known proline-rich SH3 binding antagonists VSL-12 or APP-12 to inhibit Listeria motility also indicates that SH3 interactions are unlikely to control actin-based motility directly.

  8. The cytoskeletal arrangements necessary to neurogenesis.

    PubMed

    Compagnucci, Claudia; Piemonte, Fiorella; Sferra, Antonella; Piermarini, Emanuela; Bertini, Enrico

    2016-04-12

    During the process of neurogenesis, the stem cell committed to the neuronal cell fate starts a series of molecular and morphological changes. The understanding of the physio-pathology of mechanisms controlling the molecular and morphological changes occurring during neuronal differentiation is fundamental to the development of effective therapies for many neurologic diseases. Unfortunately, our knowledge of the biological events occurring in the cell during neuronal differentiation is still poor. In this study, we focus preliminarily on the relevance of the cytoskeletal rearrangements, which earlier drive the morphology of the neuronal precursors, and later the migrating/mature neurons. In fact, neuritogenesis, neurite branching, outgrowth and retraction are seminal to the development of a fully functional nervous system. With this in mind, we highlight the importance of iPSC technology to study the processes of cytoskeletal-driven morphological changes during neuronal differentiation.

  9. The cytoskeletal arrangements necessary to neurogenesis

    PubMed Central

    Compagnucci, Claudia; Piemonte, Fiorella; Sferra, Antonella; Piermarini, Emanuela; Bertini, Enrico

    2016-01-01

    During the process of neurogenesis, the stem cell committed to the neuronal cell fate starts a series of molecular and morphological changes. The understanding of the physio-pathology of mechanisms controlling the molecular and morphological changes occurring during neuronal differentiation is fundamental to the development of effective therapies for many neurologic diseases. Unfortunately, our knowledge of the biological events occurring in the cell during neuronal differentiation is still poor. In this study, we focus preliminarily on the relevance of the cytoskeletal rearrangements, which earlier drive the morphology of the neuronal precursors, and later the migrating/mature neurons. In fact, neuritogenesis, neurite branching, outgrowth and retraction are seminal to the development of a fully functional nervous system. With this in mind, we highlight the importance of iPSC technology to study the processes of cytoskeletal-driven morphological changes during neuronal differentiation. PMID:26760504

  10. Intracellular cytoskeletal elements and cytoskeletons in bacteria.

    PubMed

    Madkour, Mohamed H F; Mayer, Frank

    2007-01-01

    Within a short period of time after the discovery of bacterial cytoskletons, major progress had been made in areas such as general spatial layout of cytoskeletons, their involvement in a variety of cellfunctions (shape control, cell division, chromosome segregation, cell motility). This progress was achieved by application of advanced investigation techniques. Homologs of eukaryotic actin, tubulin, and intermediate filaments were found in bacteria; cytoskeletal proteins not closely or not at all related to any of these major cytoskeletal proteins were discovered in a number of bacteria such as Mycoplasmas, Spiroplasmas, Spirochetes, Treponema, Caulobacter. A structural role for bacterial elongation factor Tu was indicated. On the basis of this new thinking, new approaches in biotechnology and new drugs are on the way.

  11. p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats.

    PubMed

    Kunimoto, M; Shibata, K; Miura, T

    1987-12-11

    Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.

  12. HSF-1-mediated cytoskeletal integrity determines thermotolerance and life span.

    PubMed

    Baird, Nathan A; Douglas, Peter M; Simic, Milos S; Grant, Ana R; Moresco, James J; Wolff, Suzanne C; Yates, John R; Manning, Gerard; Dillin, Andrew

    2014-10-17

    The conserved heat shock transcription factor-1 (HSF-1) is essential to cellular stress resistance and life-span determination. The canonical function of HSF-1 is to regulate a network of genes encoding molecular chaperones that protect proteins from damage caused by extrinsic environmental stress or intrinsic age-related deterioration. In Caenorhabditis elegans, we engineered a modified HSF-1 strain that increased stress resistance and longevity without enhanced chaperone induction. This health assurance acted through the regulation of the calcium-binding protein PAT-10. Loss of pat-10 caused a collapse of the actin cytoskeleton, stress resistance, and life span. Furthermore, overexpression of pat-10 increased actin filament stability, thermotolerance, and longevity, indicating that in addition to chaperone regulation, HSF-1 has a prominent role in cytoskeletal integrity, ensuring cellular function during stress and aging.

  13. Supramolecular Assembly in Cytoskeletal Filaments and their Associated Biomolecules

    NASA Astrophysics Data System (ADS)

    Safinya, Cyrus R.

    2002-03-01

    With the completion of the Human Genome Project and the emerging proteomics era, the biosciences community is beginning the daunting task of understanding the functions of a large number of interacting proteins. Cellular activity, which is usually tightly regulated, results from protein-protein and protein-nucleic acid interactions, which often lead to the formation of very large assemblies of biomolecules for distinct functions. Examples include DNA condensation states during the cell cycle, and bundle and network formation of filamentous proteins in cell attachment, motility, and cytokinesis. We present recent synchrotron x-ray diffraction and optical imaging data, in cell-free systems of cytoskeletal filaments and their associated biomolecules, which reveal novel supramolecular assemblies, spanning lengths from the nanometer to the micrometer scale. Supported by NSF DMR-9972246 and NIH GM59288.

  14. Temporal changes in cytoskeletal organisation within isolated chondrocytes quantified using a novel image analysis technique.

    PubMed

    Knight, M M; Idowu, B D; Lee, D A; Bader, D L

    2001-05-01

    This paper examines temporal changes in the organisation of the cytoskeleton within isolated articular chondrocytes cultured for up to 7 days in agarose constructs. Fluorescent labelling and confocal microscopy were employed to visualise microtubules (MT), vimentin intermediate filaments (VIF) and actin microfilaments (AMF). To quantify the degree of cytoskeletal organisation within populations of cells, a novel image analysis technique has been developed and fully characterised. Organisation was quantified in terms of an Edge Index, which reflects the density of 'edges' present within the confocal images as defined by a Sobel digital filter. This parameter was shown to be independent of image intensity and, for all three cytoskeletal components, was validated statistically against a visual assessment of organisation. Both MT and VIF exhibited fibrous networks extending throughout the cytoplasm, while AMF appeared as punctate units associated with the cell membrane. The use of the Edge Index parameter revealed statistical significant temporal variation, in particular associated with VIF and AMF. These findings indicate the possibility of cytoskeletal mediated temporal variation in many aspects of cell behaviour following isolation from the intact tissue. Furthermore, the image analysis techniques are likely to be useful for future studies aiming to quantify changes in cytoskeletal organisation.

  15. Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

    PubMed Central

    Nadakavukaren, K K; Chen, L B; Habliston, D L; Griffith, O H

    1983-01-01

    Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actin-containing stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures. Images PMID:6191327

  16. The degree of resistance of erythrocyte membrane cytoskeletal proteins to supra-physiologic concentrations of calcium: an in vitro study.

    PubMed

    Mostafavi, Ebrahim; Nargesi, Arash Aghajani; Ghazizadeh, Zaniar; Larry, Mehrdad; Farahani, Roya Horabad; Morteza, Afsaneh; Esteghamati, Alireza; Vigneron, Claude; Nakhjavani, Manouchehr

    2014-08-01

    Calcium is a key regulator of cell dynamics. Dysregulation of its cytosolic concentration is implicated in the pathophysiology of several diseases. This study aimed to assess the effects of calcium on the network of membrane cytoskeletal proteins. Erythrocyte membranes were obtained from eight healthy donors and incubated with 250 µM and 1.25 mM calcium solutions. Membrane cytoskeletal proteins were quantified using SDS-PAGE at baseline and after 3 and 5 days of incubation. Supra-physiologic concentrations of calcium (1.25 mM) induced a significant proteolysis in membrane cytoskeletal proteins, compared with magnesium (p < 0.001). Actin exhibited the highest sensitivity to calcium-induced proteolysis (6.8 ± 0.3 vs. 5.3 ± 0.6, p < 0.001), while spectrin (39.9 ± 1.0 vs. 40.3 ± 2.0, p = 0.393) and band-6 (6.3 ± 0.3 vs. 6.8 ± 0.8, p = 0.191) were more resistant to proteolysis after incubation with calcium in the range of endoplasmic reticulum concentrations (250 µM). Aggregation of membrane cytoskeletal proteins was determined after centrifugation and was significantly higher after incubation with calcium ions compared with control, EDTA and magnesium solutions (p < 0.001). In a supra-physiologic range of 1.25-10 mM of calcium ions, there was a nearly perfect linear relationship between calcium concentration and aggregation of erythrocyte membrane cytoskeletal proteins (R(2) = 0.971, p < 0.001). Our observation suggests a strong interaction between calcium ions and membrane cytoskeletal network. Cumulative effects of disrupted calcium homeostasis on cytoskeletal proteins need to be further investigated at extended periods of time in disease states.

  17. Cytoskeletal architecture and motility in a giant freshwater amoeba, Reticulomyxa.

    PubMed

    Koonce, M P; Euteneuer, U; McDonald, K L; Menzel, D; Schliwa, M

    1986-01-01

    Reticulomyxa is a large, multinucleated freshwater protozoan with striking intracellular transport. Cytoplasmic streaming and saltatory movements of individual organelles (at rates of up to 25 micron/sec) are observed within the naked cell body and the extensive reticulate peripheral network of fine cytoplasmic strands. As demonstrated by video-enhanced light microscopy, individual organelles move only when associated with cytoskeletal linear elements. The linear elements are composed of mixed colinear bundles of microtubules and actin filaments, which form the backbone of the reticulopodial network. The constant branching, sprouting, and fusion of network strands suggest unique membrane properties and an unusually dynamic cytoskeleton. The electrophoretic mobility of Reticulomyxa tubulins and the lack of crossreactivity with several antibodies known to react with many plant and animal tubulins suggest that they may differ from other tubulins more widely than might be expected. Reticulomyxa's large size, the rapidity and pervasiveness of the two forms of transport, and the simple and ordered cytoskeleton make the organism well suited for future studies on the mechanisms of intracellular transport.

  18. Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization.

    PubMed

    Gutiérrez-Venegas, Gloria; Contreras-Marmolejo, Luis Arturo; Román-Alvárez, Patricia; Barajas-Torres, Carolina

    2008-04-01

    The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

  19. Lamellipodin Is Important for Cell-to-Cell Spread and Actin-Based Motility in Listeria monocytogenes

    PubMed Central

    Wang, Jiahui; King, Jane E.; Goldrick, Marie; Lowe, Martin; Gertler, Frank B.

    2015-01-01

    Listeria monocytogenes is a foodborne pathogen capable of invading a broad range of cell types and replicating within the host cell cytoplasm. This paper describes the colocalization of host cell lamellipodin (Lpd) with intracellular L. monocytogenes detectable 6 h postinfection of epithelial cells. The association was mediated via interactions between both the peckstrin homology (PH) domain in Lpd and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] on the bacterial surface and by interactions between the C-terminal EVH1 (Ena/VASP [vasodilator-stimulated phosphoprotein] homology domain 1) binding domains of Lpd and the host VASP (vasodilator-stimulated phosphoprotein) recruited to the bacterial cell surface by the listerial ActA protein. Depletion of Lpd by short interfering RNA (siRNA) resulted in reduced plaque size and number, indicating a role for Lpd in cell-to-cell spread. In contrast, overexpression of Lpd resulted in an increase in the number of L. monocytogenes-containing protrusions (listeriopods). Manipulation of the levels of Lpd within the cell also affected the intracellular velocity of L. monocytogenes, with a reduction in Lpd corresponding to an increase in intracellular velocity. These data, together with the observation that Lpd accumulated at the interface between the bacteria and the developing actin tail at the initiation of actin-based movement, indicate a possible role for Lpd in the actin-based movement and the cell-to-cell spread of L. monocytogenes. PMID:26169271

  20. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    NASA Astrophysics Data System (ADS)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

  1. Listeria's right-handed helical rocket-tail trajectories: mechanistic implications for force generation in actin-based motility.

    PubMed

    Zeile, William L; Zhang, Fangliang; Dickinson, Richard B; Purich, Daniel L

    2005-02-01

    Listeria monocytogenes forms right-handed helical rocket tail trajectories during actin-based motility in cell-free extracts, and this stereochemical feature is consistent with actoclampin's affinity-modulated, clamped-filament elongation model [Dickinson and Purich, 2002: Biophys J 82:605-617]. In that mechanism, right-handed torque is generated by an end-tracking molecular motor, each comprised of a filament barbed end and clamping protein that processively traces the right-handed helix of its filament partner. By contrast, torque is not a predicted property of those models (e.g., elastic propulsion, elastic Brownian ratchet, tethered ratchet, and insertional polymerization models) requiring filament barbed ends to depart/detach from the motile object's surface during/after each monomer-addition step. Helical trajectories also explain why Listeria undergoes longitudinal-axis rotation on a length-scale matching the helical periodicity of Listeria's rocket tails.

  2. Reinforcement versus fluidization in cytoskeletal mechanoresponsiveness.

    PubMed

    Krishnan, Ramaswamy; Park, Chan Young; Lin, Yu-Chun; Mead, Jere; Jaspers, Richard T; Trepat, Xavier; Lenormand, Guillaume; Tambe, Dhananjay; Smolensky, Alexander V; Knoll, Andrew H; Butler, James P; Fredberg, Jeffrey J

    2009-01-01

    Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment.

  3. Simultaneous Visualization of Peroxisomes and Cytoskeletal Elements Reveals Actin and Not Microtubule-Based Peroxisome Motility in Plants1[w

    PubMed Central

    Mathur, Jaideep; Mathur, Neeta; Hülskamp, Martin

    2002-01-01

    Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect. PMID:11891258

  4. ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression

    PubMed Central

    Ahn, Young-Ho; Gibbons, Don L.; Chakravarti, Deepavali; Creighton, Chad J.; Rizvi, Zain H.; Adams, Henry P.; Pertsemlidis, Alexander; Gregory, Philip A.; Wright, Josephine A.; Goodall, Gregory J.; Flores, Elsa R.; Kurie, Jonathan M.

    2012-01-01

    Metastatic cancer is extremely difficult to treat, and the presence of metastases greatly reduces a cancer patient’s likelihood of long-term survival. The ZEB1 transcriptional repressor promotes metastasis through downregulation of microRNAs (miRs) that are strong inducers of epithelial differentiation and inhibitors of stem cell factors. Given that each miR can target multiple genes with diverse functions, we posited that the prometastatic network controlled by ZEB1 extends beyond these processes. We tested this hypothesis using a mouse model of human lung adenocarcinoma metastasis driven by ZEB1, human lung carcinoma cells, and human breast carcinoma cells. Transcriptional profiling studies revealed that ZEB1 controls the expression of numerous oncogenic and tumor-suppressive miRs, including miR-34a. Ectopic expression of miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature enriched in cytoskeletal functions and predictive of outcome in human lung adenocarcinomas. We identified several miR-34a target genes, including Arhgap1, which encodes a RHO GTPase activating protein that was required for tumor cell invasion. These findings demonstrate that ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression and provide a compelling rationale to develop miR-34a as a therapeutic agent in lung cancer patients. PMID:22850877

  5. ERK and phosphoinositide 3-kinase temporally coordinate different modes of actin-based motility during embryonic wound healing.

    PubMed

    Li, Jingjing; Zhang, Siwei; Soto, Ximena; Woolner, Sarah; Amaya, Enrique

    2013-11-01

    Embryonic wound healing provides a perfect example of efficient recovery of tissue integrity and homeostasis, which is vital for survival. Tissue movement in embryonic wound healing requires two functionally distinct actin structures: a contractile actomyosin cable and actin protrusions at the leading edge. Here, we report that the discrete formation and function of these two structures is achieved by the temporal segregation of two intracellular upstream signals and distinct downstream targets. The sequential activation of ERK and phosphoinositide 3-kinase (PI3K) signalling divides Xenopus embryonic wound healing into two phases. In the first phase, activated ERK suppresses PI3K activity, and is responsible for the activation of Rho and myosin-2, which drives actomyosin cable formation and constriction. The second phase is dominated by restored PI3K signalling, which enhances Rac and Cdc42 activity, leading to the formation of actin protrusions that drive migration and zippering. These findings reveal a new mechanism for coordinating different modes of actin-based motility in a complex tissue setting, namely embryonic wound healing.

  6. Interactions of Listeria monocytogenes with mammalian cells during entry and actin-based movement: bacterial factors, cellular ligands and signaling.

    PubMed Central

    Cossart, P; Lecuit, M

    1998-01-01

    Although <50 kb of its 3.3 megabase genome is known, Listeria monocytogenes has received much attention and an impressive amount of data has contributed in raising this bacterium among the best understood intracellular pathogens. The mechanisms that Listeria uses to enter cells, escape from the phagocytic vacuole and spread from one cell to another using an actin-based motility process have been analysed in detail. Several bacterial proteins contributing to these events have been identified, including the invasion proteins internalin A (InlA) and B (InlB), the secreted pore-forming toxin listeriolysin O (LLO) which promotes the escape from the phagocytic vacuole, and the surface protein ActA which is required for actin polymerization and bacterial movement. While LLO and ActA are critical for the infectious process and are not redundant with other listerial proteins, the precise role of InlA and InlB in vivo remains unclear. How InlA, InlB, LLO or ActA interact with the mammalian cells is beginning to be deciphered. The picture that emerges is that this bacterium uses general strategies also used by other invasive bacteria but has evolved a panel of specific tools and tricks to exploit mammalian cell functions. Their study may lead to a better understanding of important questions in cell biology such as ligand receptor signalling and dynamics of actin polymerization in mammalian cells. PMID:9669997

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

    PubMed Central

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

    2001-01-01

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

  8. Role of Proteins of the Ena/VASP Family in Actin-based Motility of Listeria monocytogenes

    PubMed Central

    Laurent, Valérie; Loisel, Thomas P.; Harbeck, Birgit; Wehman, Ann; Gröbe, Lothar; Jockusch, Brigitte M.; Wehland, Jürgen; Gertler, Frank B.; Carlier, Marie-France

    1999-01-01

    Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (−/−) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association–dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 × 108 M−1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment–detachment of VASP to F-actin allows its sliding along the growing filament. PMID:10087267

  9. The role of cytoskeletal elements in shaping bacterial cells.

    PubMed

    Cho, Hongbaek

    2015-03-01

    Beginning from the recognition of FtsZ as a bacterial tubulin homolog in the early 1990s, many bacterial cytoskeletal elements have been identified, including homologs to the major eukaryotic cytoskeletal elements (tubulin, actin, and intermediate filament) and the elements unique in prokaryotes (ParA/MinD family and bactofilins). The discovery and functional characterization of the bacterial cytoskeleton have revolutionized our understanding of bacterial cells, revealing their elaborate and dynamic subcellular organization. As in eukaryotic systems, the bacterial cytoskeleton participates in cell division, cell morphogenesis, DNA segregation, and other important cellular processes. However, in accordance with the vast difference between bacterial and eukaryotic cells, many bacterial cytoskeletal proteins play distinct roles from their eukaryotic counterparts; for example, control of cell wall synthesis for cell division and morphogenesis. This review is aimed at providing an overview of the bacterial cytoskeleton, and discussing the roles and assembly dynamics of bacterial cytoskeletal proteins in more detail in relation to their most widely conserved functions, DNA segregation and coordination of cell wall synthesis.

  10. Cytoskeletal organization by motor and polymerization forces

    NASA Astrophysics Data System (ADS)

    Koenderink, Gijsje

    2014-03-01

    Cells need to constantly change their change to perform vital functions, such as growth, division, and movement. Dysregulation of cell shape can have severe consequences such as cancer. Our goal is to resolve physical mechanisms that contribute to cell shape control. For this purpose, we study simplified experimental model systems reconstituted from purified cellular components. In this talk, I will give two examples of our recent work. The first example concerns active contractility of the actin cortex, which lies underneath the cell membrane and drives shape changes by means of myosin motors. Using in vitro models, we studied how myosin motors and actin filaments collectively self-organize into force-generating arrays. I will show that motors contract actin networks only above a sharp threshold in crosslink density. We discovered that right at this threshold, the motors rupture the network into clusters that exhibit a broad distribution of sizes, as expected in filamentous networks near a percolation threshold. The second example I will discuss concerns cell shape polarization directed by interactions between the actin and microtubule (MT) cytoskeletons. A prominent example is the guidance of MT growth along F-actin bundles towards specific targets, i.e. focal adhesions. It has been suggested that MT end-tracking proteins (+TIPs) that also bind F-actin are responsible for this process. We built an in vitro system involving a simplified actin-MT crosslinker molecule and could show that the interaction between MT ends and actin is sufficient to capture and re-direct MT growth along actin bundles. By keeping MT growth tightly coupled to F-actin, this mechanism allows linear arrays of actin bundles to act as templates for MT organization. Instead, when interacting with single actin filaments, MT ends become the dominant organizing factor, exerting forces that align, pull and even transport actin filaments in the direction of MT growth. We conclude that actin and MTs

  11. Thermally Driven and Cytoskeletal-Assisted Dynamics of the Mitochondrial Reticulum

    NASA Astrophysics Data System (ADS)

    Knowles, Michelle K.; Marcus, Andrew H.

    2003-05-01

    We report Fourier imaging correlation spectroscopy (FICS) and digital video fluorescence microscopy (DVFM) measurements of the dynamics of the mitochondrial reticulum in living osteosarcoma cells. Mitochondrial dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, which lead to complex multi-exponential relaxations that occur over a wide range of spatial and temporal scales. The cytoskeleton consists of an interconnected polymer network whose primary components are microfilaments (actin) and microtubules (tubulin). These filaments work with motor proteins to translate organelles through the cell. We studied the dynamics of osteosarcoma cells labeled with red fluorescent protein in the mitochondrial matrix space using DVFM and FICS. Cells were then treated with cytoskeletal destabilizing drugs. Analysis of microscopy data allows for us to determine whether dynamic processes are diffusive or driven (by the cytoskeleton or collective dynamics). In FICS experiments, the control cells exhibit a unique pattern of dynamics that are then simplified when the cytoskeleton is depolymerized. Upon depolymerization, the dynamics of the organelle appear primarily diffusive.

  12. Simulated Microgravity Induced Cytoskeletal Rearrangements are Modulated by Protooncogenes

    NASA Technical Reports Server (NTRS)

    Melhado, C. D.; Sanford, G. L.; Bosah, F.; Harris-Hooker, S.

    1998-01-01

    Microgravity is the environment living systems encounter during space flight and gravitational unloading is the effect of this environment on living systems. The cell, being a multiphasic chemical system, is a useful starting point to study the potential impact of gravity unloading on physiological function. In the absence of gravity, sedimentation of organelles including chromosomes, mitochondria, nuclei, the Golgi apparatus, vacuoles, and the endoplasmic reticulum may be affected. Most of these organelles, however, are somewhat held in place by cytoskeleton. Hansen and Igber suggest that intermediate filaments act to stabilize the nuleus against rotational movement, and integrate cell and nuclear structure. The tensegrity theory supports the idea that mechanical or physical forces alters the cytoskeletal structures of a cell resulting in the changes in cell: matrix interactions and receptor-signaling coupling. This type of stress to the cytoskeleton may be largely responsible regulating cell shape, growth, movement and metabolism. Mouse MC3T3 El cells under microgravity exhibited significant cytoskeletal changes and alterations in cell growth. The alterations in cytoskeleton architecture may be due to changes in the expression of actin related proteins or integrins. Philopott and coworkers reported on changes in the distribution of microtubule and cytoskeleton elements in the cells of heart tissue from space flight rats and those centrifuged at 1.7g. Other researchers have showed that microgravity reduced EGF-induced c-fos and c-jun expression compared to 1 g controls. Since c-fos and c-jun are known regulators of cell growth, it is likely that altered signal transduction involving protooncogenes may play a crucial role in the reduced growth and alterations in cytoskeletal arrangements found during space flight. It is clear that a microgravity environment induces a number of changes in cell shape, cell surface molecules, gene expression, and cytoskeletal

  13. Processive cytoskeletal motors studied with single-molecule fluorescence techniques.

    PubMed

    Belyy, Vladislav; Yildiz, Ahmet

    2014-10-01

    Processive cytoskeletal motors from the myosin, kinesin, and dynein families walk on actin filaments and microtubules to drive cellular transport and organization in eukaryotic cells. These remarkable molecular machines are able to take hundreds of successive steps at speeds of up to several microns per second, allowing them to effectively move vesicles and organelles throughout the cytoplasm. Here, we focus on single-molecule fluorescence techniques and discuss their wide-ranging applications to the field of cytoskeletal motor research. We cover both traditional fluorescence and sub-diffraction imaging of motors, providing examples of how fluorescence data can be used to measure biophysical parameters of motors such as coordination, stepping mechanism, gating, and processivity. We also outline some remaining challenges in the field and suggest future directions.

  14. Cytoskeletal and membrane dynamics during higher plant cytokinesis.

    PubMed

    McMichael, Colleen M; Bednarek, Sebastian Y

    2013-03-01

    Following mitosis, cytoplasm, organelles and genetic material are partitioned into daughter cells through the process of cytokinesis. In somatic cells of higher plants, two cytoskeletal arrays, the preprophase band and the phragmoplast, facilitate the positioning and de novo assembly of the plant-specific cytokinetic organelle, the cell plate, which develops across the division plane and fuses with the parental plasma membrane to yield distinct new cells. The coordination of cytoskeletal and membrane dynamics required to initiate, assemble and shape the cell plate as it grows toward the mother cell cortex is dependent upon a large array of proteins, including molecular motors, membrane tethering, fusion and restructuring factors and biosynthetic, structural and regulatory elements. This review focuses on the temporal and molecular requirements of cytokinesis in somatic cells of higher plants gleaned from recent studies using cell biology, genetics, pharmacology and biochemistry.

  15. Quantitative Evaluation of Plant Actin Cytoskeletal Organization During Immune Signaling.

    PubMed

    Lu, Yi-Ju; Day, Brad

    2017-01-01

    High spatial and temporal resolution microscopy-based methods are valuable tools for the precise real-time imaging of changes in cellular organization in response to stimulus perception. Here, we describe a quantitative method for the evaluation of the plant actin cytoskeleton during immune stimulus perception and the activation of defense signaling. As a measure of the biotic stress-induced changes in actin filament organization, we present methods for analyzing changes in actin filament organization following elicitation of pattern-triggered immunity and effector-triggered immunity. Using these methods, it is possible to not only quantitatively evaluate changes in actin cytoskeletal organization following biotic stress perception, but to also use these protocols to assess changes in actin filament organization following perception of a wide range of stimuli, including abiotic and developmental cues. As described herein, we present an example application of this method, designed to evaluate changes in actin cytoskeletal organization following pathogen perception and immune signaling.

  16. Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

    PubMed

    Szulc-Dabrowska, Lidia; Gregorczyk, Karolina P; Struzik, Justyna; Boratynska-Jasinska, Anna; Szczepanowska, Joanna; Wyzewski, Zbigniew; Toka, Felix N; Gierynska, Malgorzata; Ostrowska, Agnieszka; Niemialtowski, Marek G

    2016-08-01

    Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc.

  17. Cytoskeletal mechanics in pressure-overload cardiac hypertrophy

    NASA Technical Reports Server (NTRS)

    Tagawa, H.; Wang, N.; Narishige, T.; Ingber, D. E.; Zile, M. R.; Cooper, G. 4th

    1997-01-01

    We have shown that the cellular contractile dysfunction characteristic of pressure-overload cardiac hypertrophy results not from an abnormality intrinsic to the myofilament portion of the cardiocyte cytoskeleton but rather from an increased density of the microtubule component of the extramyofilament portion of the cardiocyte cytoskeleton. To determine how, in physical terms, this increased microtubule density mechanically overloads the contractile apparatus at the cellular level, we measured cytoskeletal stiffness and apparent viscosity in isolated cardiocytes via magnetic twisting cytometry, a technique by which magnetically induced force is applied directly to the cytoskeleton through integrin-coupled ferromagnetic beads coated with Arg-Gly-Asp (RGD) peptide. Measurements were made in two groups of cardiocytes from cats with right ventricular (RV) hypertrophy induced by pulmonary artery banding: (1) those from the pressure-overloaded RV and (2) those from the normally loaded same-animal control left ventricle (LV). Cytoskeletal stiffness increased almost twofold, from 8.53 +/- 0.77 dyne/cm2 in the normally loaded LV cardiocytes to 16.46 +/- 1.32 dyne/cm2 in the hypertrophied RV cardiocytes. Cytoskeletal apparent viscosity increased almost fourfold, from 20.97 +/- 1.92 poise in the normally loaded LV cardiocytes to 87.85 +/- 6.95 poise in the hypertrophied RV cardiocytes. In addition to these baseline data showing differing stiffness and, especially, apparent viscosity in the two groups of cardiocytes, microtubule depolymerization by colchicine was found to return both the stiffness and the apparent viscosity of the pressure overload-hypertrophied RV cells fully to normal. Conversely, microtubule hyperpolymerization by taxol increased the stiffness and apparent viscosity values of normally loaded LV cardiocytes to the abnormal values given above for pressure-hypertrophied RV cardiocytes. Thus, increased microtubule density constitutes primarily a viscous load on

  18. Coiled coils and SAH domains in cytoskeletal molecular motors.

    PubMed

    Peckham, Michelle

    2011-10-01

    Cytoskeletal motors include myosins, kinesins and dyneins. Myosins move along tracks of actin filaments, whereas kinesins and dyneins move along microtubules. Many of these motors are involved in trafficking cargo in cells. However, myosins are mostly monomeric, whereas kinesins are mostly dimeric, owing to the presence of a coiled coil. Some myosins (myosins 6, 7 and 10) contain an SAH (single α-helical) domain, which was originally thought to be a coiled coil. These myosins are now known to be monomers, not dimers. The differences between SAH domains and coiled coils are described and the potential roles of SAH domains in molecular motors are discussed.

  19. Effect of Cytoskeletal Reagents on Stretch Activated Ion Channels

    DTIC Science & Technology

    1992-11-12

    transduction. Biophys J59: 1143-1145, 1991. 23. SACHS, F., W. SIGURDSON, A. RUKNUDIN, AND C. BOWMAN. Single- channel mechanosensitive currents. Science 253: 800... mechanosensitive ion channels . In: Advances in Comparative and Environmental Physiology, v0C, edited by F. Ito. Berlin: Springer-Verlag, 1992, p. 55-77. Report of Inventions: None 4 ...EFFECT OF CYTOSKELETAL REAGENTS ON STRETCH ACTIVATED ION CHANNELS b lfli..3-f-I’- o0*’t 6. AUTHOR(S) Dr.-Frederick Sachs DI 7. PERFORMING ORGANIZATION NAME

  20. Building Blocks of Functioning Brain: Cytoskeletal Dynamics in Neuronal Development

    PubMed Central

    Menon, Shalini; Gupton, Stephanie L.

    2016-01-01

    Neural connectivity requires proper polarization of neurons, guidance to appropriate target locations, and establishment of synaptic connections. From when neurons are born to when they finally reach their synaptic partners, neurons undergo constant rearrangment of the cytoskeleton to achieve appropriate shape and polarity. Of particular importance to neuronal guidance to target locations is the growth cone at the tip of the axon. Growth-cone steering is also dictated by the underlying cytoskeleton. All these changes require spatiotemporal control of the cytoskeletal machinery. This review summarizes the proteins that are involved in modulating the actin and microtubule cytoskeleton during the various stages of neuronal development. PMID:26940519

  1. Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells.

    PubMed

    Laine, R O; Phaneuf, K L; Cunningham, C C; Kwiatkowski, D; Azuma, T; Southwick, F S

    1998-08-01

    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 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/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

  2. Internalization of multiple cells during C. elegans gastrulation depends on common cytoskeletal mechanisms but different cell polarity and cell fate regulators

    PubMed Central

    Harrell, Jessica R.; Goldstein, Bob

    2010-01-01

    Understanding the links between developmental patterning mechanisms and force-producing cytoskeletal mechanisms is a central goal in studies of morphogenesis. Gastrulation is the first morphogenetic event in the development of many organisms. Gastrulation involves the internalization of surface cells, often driven by the contraction of actomyosin networks that are deployed with spatial precision -- both in specific cells and in a polarized manner within each cell. These cytoskeletal mechanisms rely on different cell fate and cell polarity regulators in different organisms. C. elegans gastrulation presents an opportunity to examine the extent to which diverse mechanisms may be used by dozens of cells that are internalized at distinct times within a single organism. We identified 66 cells that are internalized in C. elegans gastrulation, many of which were not known previously to gastrulate. To gain mechanistic insights into how these cells internalize, we genetically manipulated cell fate, cell polarity and cytoskeletal regulators and determined the effects on cell internalization. We found that cells of distinct lineages depend on common actomyosin-based mechanisms to gastrulate, but different cell fate regulators, and, surprisingly, different cell polarity regulators. We conclude that diverse cell fate and cell polarity regulators control common mechanisms of morphogenesis in C. elegans. The results highlight the variety of developmental patterning mechanisms that can be associated with common cytoskeletal mechanisms in the morphogenesis of an animal embryo. PMID:20875815

  3. Cytoskeletal regulation of calcium-permeable cation channels in the human syncytiotrophoblast: role of gelsolin

    PubMed Central

    Montalbetti, Nicolás; Li, Qiang; Timpanaro, Gustavo A; González-Perrett, Silvia; Dai, Xiao-Qing; Chen, Xing-Zhen; Cantiello, Horacio F

    2005-01-01

    The human syncytiotrophoblast (hST) is the most apical epithelial barrier that covers the villous tree of the human placenta. An intricate and highly organized network of cytoskeletal structures supports the hST. Recently, polycystin-2 (PC2), a TRP-type nonselective cation channel, was functionally observed in hST, where it may be an important player to Ca2+ transport. Little is known, however, about channel regulation in hST. In this report, the regulatory role of actin dynamics on PC2 channels reconstituted from hST apical membranes was explored. Acute addition of cytochalasin D (CD, 5 μg ml−1) to reconstituted hST apical membranes transiently increased K+-permeable channel activity. The actin-binding proteins α-actinin and gelsolin, as well as PC2, were observed by Western blot and immunofluorescence analyses in hST vesicles. CD treatment of hST vesicles resulted in a re-distribution of actin filaments, in agreement with the effect of CD on K+ channel activity. In contrast, addition of exogenous monomeric actin, but not prepolymerized actin, induced a rapid inhibition of channel function in hST. This inhibition was obliterated by the presence of CD in the medium. The acute (<15 min) CD stimulation of K+ channel activity was mimicked by addition of the actin-severing protein gelsolin in the presence, but not in the absence, of micromolar Ca2+. Ca2+ transport through PC2 triggers a regulatory feedback mechanism, which is based on the severing and re-formation of filamentous actin near the channels. Cytoskeletal structures may thus be relevant to ion transport regulation in the human placenta. PMID:15845576

  4. Rho-kinase mediated cytoskeletal stiffness in skinned smooth muscle

    PubMed Central

    Lan, Bo; Wang, Lu; Zhang, Jenny; Pascoe, Chris D.; Norris, Brandon A.; Liu, Jeffrey C.-Y.; Solomon, Dennis; Paré, Peter D.; Deng, Linhong

    2013-01-01

    The structurally dynamic cytoskeleton is important in many cell functions. Large gaps still exist in our knowledge regarding what regulates cytoskeletal dynamics and what underlies the structural plasticity. Because Rho-kinase is an upstream regulator of signaling events leading to phosphorylation of many cytoskeletal proteins in many cell types, we have chosen this kinase as the focus of the present study. In detergent skinned tracheal smooth muscle preparations, we quantified the proteins eluted from the muscle cells over time and monitored the muscle's ability to respond to acetylcholine (ACh) stimulation to produce force and stiffness. In a partially skinned preparation not able to generate active force but could still stiffen upon ACh stimulation, we found that the ACh-induced stiffness was independent of calcium and myosin light chain phosphorylation. This indicates that the myosin light chain-dependent actively cycling crossbridges are not likely the source of the stiffness. The results also indicate that Rho-kinase is central to the ACh-induced stiffness, because inhibition of the kinase by H1152 (1 μM) abolished the stiffening. Furthermore, the rate of relaxation of calcium-induced stiffness in the skinned preparation was faster than that of ACh-induced stiffness, with or without calcium, suggesting that different signaling pathways lead to different means of maintenance of stiffness in the skinned preparation. PMID:24072407

  5. Visualization of Cytoskeletal Elements by the Atomic Force Microscope

    NASA Astrophysics Data System (ADS)

    Berdyyeva, Tamara; Woodworth, Craig; Sokolov, Igor

    2004-03-01

    We describe a novel application of atomic force microscopy (AFM) to directly visualize cytoskeletal fibers in human foreskin epithelial cells. The nonionic detergent Triton X-100 in a low concentration was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized in either liquid or air-dried ambient conditions. These two types of scanning provide complimentary information. Scanning in liquids visualizes the surface filaments of the cytoskeleton, whereas scanning in air shows both the surface filaments and the total volume of the cytoskeletal fibers. The smallest fibers observed were ca. 50 nm in diameter. The lateral resolution of this technique was ca.20 nm, which can be increased to a single nanometer level by choosing sharper AFM tips. Because the AFM is a true 3 dimensional technique, we are able to quantify the observed cytoskeleton by its density and volume. The types of fibers can be identified by their size, similar to electron microscopy.

  6. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    PubMed Central

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  7. Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling.

    PubMed

    Ray, Poulomi; Chapman, Susan C

    2015-01-01

    Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.

  8. Cdk6 contributes to cytoskeletal stability in erythroid cells.

    PubMed

    Uras, Iris Z; Scheicher, Ruth M; Kollmann, Karoline; Glösmann, Martin; Prchal-Murphy, Michaela; Tigan, Anca S; Fux, Daniela A; Altamura, Sandro; Neves, Joana; Muckenthaler, Martina; Bennett, Keiryn L; Kubicek, Stefan; Hinds, Philip W; von Lindern, Marieke; Sexl, Veronika

    2017-03-02

    Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo. However, Cdk6-/- erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in the cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeletal organization. Cdk6-/- erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We show further that Cdk6 regulates the transcription of a panel of genes involved in actin (de-) polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.

  9. Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface

    PubMed Central

    1995-01-01

    The thermodynamic basis for actin-based motility of Listeria monocytogenes has been investigated using cytoplasmic extracts of Xenopus eggs, initially developed by Theriot et al. (Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Cell. 76:505-517) as an in vitro cell-free system. A large proportion (75%) of actin was found unpolymerized in the extracts. The amount of unassembled actin (12 microM) is accounted for by the sequestering functions of T beta 4Xen (20 microM) and profilin (5 microM), the barbed ends being capped. Movement of Listeria was not abolished by depletion of over 99% of the endogenous profilin. The proline-rich sequences of ActA are unlikely to be the target of profilin. All data support the view that actin assembly at the rear of Listeria results from a local shift in steady state due to a factor, keeping filaments uncapped, bound to the surface of the bacterium, while barbed ends are capped in the bulk cytoplasm. Movement is controlled by the energetic difference (i.e., the difference in critical concentration) between the two ends of the filaments, hence a constant ATP supply and the presence of barbed end capped F-actin in the medium are required to buffer free G-actin at a high concentration. The role of membrane components is demonstrated by the facts that: (a) Listeria movement can be reconstituted in the resuspended pellets of high speed-centrifuged extracts that are enriched in membranes; (b) Actin-based motility of endogenous vesicles, exhibiting the same rocketing movement as Listeria, can be observed in the extracts. PMID:7615635

  10. Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

    NASA Technical Reports Server (NTRS)

    Ingber, D. E.; Prusty, D.; Sun, Z.; Betensky, H.; Wang, N.

    1995-01-01

    Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed

  11. Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization.

    PubMed

    Liu, Youtao; Lacal, Jesus; Firtel, Richard A; Kortholt, Arjan

    2016-10-07

    The directional movement towards extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

  12. Tristetraprolin functions in cytoskeletal organization during mouse oocyte maturation

    PubMed Central

    Liu, Xiaohui; Li, Xiaoyan; Ma, Rujun; Xiong, Bo; Sun, Shao-Chen; Liu, Honglin; Gu, Ling

    2016-01-01

    Tristetraprolin (TTP), a member of TIS11 family containing CCCH tandem zinc finger, is one of the best characterized RNA-binding proteins. However, to date, the role of TTP in mammalian oocytes remains completely unknown. In the present study, we report the altered maturational progression and cytokinesis, upon specific knockdown of TTP in mouse oocytes. Furthermore, by confocal scanning, we observe the failure to form cortical actin cap during meiosis of TTP-depleted oocytes. Loss of TTP in oocytes also results in disruption of meiotic spindle morphology and chromosome alignment. In support of these findings, incidence of aneuploidy is accordingly increased when TTP is abated in oocytes. Our results suggest that TTP as a novel cytoskeletal regulator is required for spindle morphology/chromosome alignment and actin polymerization in oocytes. PMID:27458159

  13. Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

    PubMed Central

    Vatta, Matteo; Faulkner, Georgine

    2009-01-01

    Summary The heart is a force-generating organ that responds to self-generated electrical stimuli from specialized cardiomyocytes. This function is modulated by sympathetic and parasympathetic activity. In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes depend on their highly evolved and specialized cytoskeletal apparatus. Defects in components of the cytoskeleton, in the long term, affect the ability of the cell to compensate at both functional and structural levels. In addition to the structural remodeling, the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis. The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here I will review the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels and, I will discuss the future impact of new data on molecular cardiology research and clinical practice. PMID:19774097

  14. Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

    SciTech Connect

    Kaslow, H.R.; Groppi, V.E.; Abood, M.E.; Bourne, H.R.

    1981-11-01

    Cholera toxin catalyzes transfer of radiolabel from (/sup 32/P)NAD/sup +/ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of M/sub r/ = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (M/sub r/ = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and (/sup 32/P)NAD/sup +/ caused radiolabeling of purified microtubule and intermediate filament proteins.

  15. Altered cytoskeletal organization characterized lethal but not surviving Brtl+/− mice: insight on phenotypic variability in osteogenesis imperfecta

    PubMed Central

    Bianchi, Laura; Gagliardi, Assunta; Maruelli, Silvia; Besio, Roberta; Landi, Claudia; Gioia, Roberta; Kozloff, Kenneth M.; Khoury, Basma M.; Coucke, Paul J.; Symoens, Sofie; Marini, Joan C.; Rossi, Antonio; Bini, Luca; Forlino, Antonella

    2015-01-01

    Osteogenesis imperfecta (OI) is a heritable bone disease with dominant and recessive transmission. It is characterized by a wide spectrum of clinical outcomes ranging from very mild to lethal in the perinatal period. The intra- and inter-familiar OI phenotypic variability in the presence of an identical molecular defect is still puzzling to the research field. We used the OI murine model Brtl+/− to investigate the molecular basis of OI phenotypic variability. Brtl+/− resembles classical dominant OI and shows either a moderately severe or a lethal outcome associated with the same Gly349Cys substitution in the α1 chain of type I collagen. A systems biology approach was used. We took advantage of proteomic pathway analysis to functionally link proteins differentially expressed in bone and skin of Brtl+/− mice with different outcomes to define possible phenotype modulators. The skin/bone and bone/skin hybrid networks highlighted three focal proteins: vimentin, stathmin and cofilin-1, belonging to or involved in cytoskeletal organization. Abnormal cytoskeleton was indeed demonstrated by immunohistochemistry to occur only in tissues from Brtl+/− lethal mice. The aberrant cytoskeleton affected osteoblast proliferation, collagen deposition, integrin and TGF-β signaling with impairment of bone structural properties. Finally, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not from non-lethal, OI patients carrying an identical glycine substitution. Our data demonstrated that compromised cytoskeletal assembly impaired both cell signaling and cellular trafficking in mutant lethal mice, altering bone properties. These results point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment. PMID:26264579

  16. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    SciTech Connect

    Muthukumaran, Padmalosini; Lim, Chwee Teck; Lee, Taeyong

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Estradiol induced stiffness changes of osteoblasts were quantified using AFM. Black-Right-Pointing-Pointer Estradiol causes significant decrease in the stiffness of osteoblasts. Black-Right-Pointing-Pointer Decreased stiffness was caused by decreased density of f-actin network. Black-Right-Pointing-Pointer Stiffness changes were not associated with mineralized matrix of osteoblasts. Black-Right-Pointing-Pointer Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E{sup Asterisk-Operator }) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with {beta}-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E{sup Asterisk-Operator }. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E{sup Asterisk-Operator} of osteoblasts significantly decreased by 43-46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness

  17. A Model for Stress Fiber Realignment Caused by Cytoskeletal Fluidization During Cyclic Stretching.

    PubMed

    Pirentis, Athanassios P; Peruski, Elizabeth; Iordan, Andreea L; Stamenović, Dimitrije

    2011-03-01

    Uniaxial cyclic substrate stretching results in a concerted change of cytoskeletal organization such that stress fibers (SFs) realign away from the direction of stretching. Recent experiments revealed that brief transient stretch promptly ablates cellular contractile stress by means of cytoskeletal fluidization, followed by a slow stress recovery by means of resolidification. This, in turn, suggests that fluidization, resolidification and SF realignment may be linked together during stretching. We propose a mathematical model that simulates the effects of fluidization and resolidification on cytoskeletal contractile stress in order to investigate how these phenomena affect cytoskeletal realignment in response to pure uniaxial stretching of the substrate. The model comprises of individual elastic SFs anchored at the endpoints to an elastic substrate. Employing the global stability convention, the model predicts that in response to repeated stretch-unstretch cycles, SFs tend to realign in the direction perpendicular to stretching, consistent with data from the literature. The model is used to develop a computational scheme for predicting changes in cell orientation and polarity during stretching and how they relate to the underlying alterations in the cytoskeletal organization. We conclude that depletion of cytoskeletal contractile stress by means of fluidization and subsequent stress recovery by means of resolidification may play a key role in reorganization of cytoskeletal SFs in response to uniaxial stretching of the substrate.

  18. Growth factors and steroid mediated regulation of cytoskeletal protein expression in serum-deprived primary astrocyte cultures.

    PubMed

    Bramanti, Vincenzo; Bronzi, Daniela; Tomassoni, Daniele; Costa, Antonino; Raciti, Giuseppina; Avitabile, Marcello; Amenta, Francesco; Avola, Roberto

    2008-12-01

    In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under different experimental conditions. Condition I: 24 h pretreatment with bFGF, subsequent 72 h switching in serum-free medium (SFM) and final addition of GFs, alone or by two in the last 24 h, after a prolonged (60 h) DEX treatment. Condition II: 36 h pretreatment with DEX (with bFGF in the last 24 h), followed by SFM for 60 h and final addition for 24 h with growth factors alone or two of them together. Western blot analysis data showed a marked GFAP expression in cultures submitted to Condition I comparing results to untreated or treated controls. VIM expression was instead significantly reduced after GFs addition in the last 24 h of 60 h DEX treatment, respect to control DEX-pretreated ones. Referring data to untreated controls, VIM expression was significantly enhanced after GFs addition. GFAP showed also a significant increase in astrocytes submitted to Condition II, respect to untreated or treated control cultures. VIM expression was up and down regulated under Condition II. Collectively, our findings evidence an interactive dialogue between GFs and DEX in astroglial cultures, co-pretreated with DEX and bFGF, regulating cytoskeletal network under stressful conditions.

  19. Purinoreceptor P2X7 Regulation of Ca2+ Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury

    PubMed Central

    Minns, Martin S.; Teicher, Gregory; Rich, Celeste B.; Trinkaus-Randall, Vickery

    2017-01-01

    The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injury-induced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratch-wounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent protein–labeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing. PMID:26683661

  20. Cytoskeletal protein binding kinetics at planar phospholipid membranes.

    PubMed Central

    Mc Kiernan, A E; MacDonald, R I; MacDonald, R C; Axelrod, D

    1997-01-01

    It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an

  1. β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR

    PubMed Central

    Vasa, Suresh; Lin, Lin; Shi, Chaowei; Habenstein, Birgit; Riedel, Dietmar; Kühn, Juliane; Thanbichler, Martin; Lange, Adam

    2015-01-01

    Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix. PMID:25550503

  2. Cytoskeletal architecture and immunocytochemical localization of fodrin in the terminal web of the ciliated epithelial cell.

    PubMed

    Kobayashi, N; Hirokawa, N

    1988-01-01

    In order to understand the cytoskeletal architecture at the terminal web of the ciliated cell, we examined chicken tracheal epithelium by quick-freeze deep-etch (QFDE) electron microscopy combined with immunocytochemistry of fodrin. At the terminal web, the cilia ended into the basal bodies and then to the rootlets. The rootlets were composed of several filaments and globular structures attached regularly to them. Decoration with myosin subfragment 1 (S1) revealed that some actin filaments ran parallel to the apical plasma membrane between the basal bodies, and other population traveled perpendicularly or obliquely, i.e., along the rootlets. Some actin filaments were connected to the surface of the basal bodies and the basal feet. Among the basal bodies and the rootlets there existed three kinds of fine crossbridges, which were not decorated with S1. In the deeper part of the terminal web, intermediate filaments were observed between the rootlets and were sometimes crosslinked with the rootlets. Immunocytochemistry combined with the QFDE method revealed that fodrin was a component of fine crossbridges associated with the basal bodies. We concluded that an extensive crosslinker system among the basal bodies and the rootlets along with networks of actin and intermediate filaments formed a structural basis for the effective beating of cilia.

  3. Mapping cytoskeletal protein function in cells by means of nanobodies.

    PubMed

    Van Audenhove, Isabel; Van Impe, Katrien; Ruano-Gallego, David; De Clercq, Sarah; De Muynck, Kevin; Vanloo, Berlinda; Verstraete, Hanne; Fernández, Luis Á; Gettemans, Jan

    2013-10-01

    Nanobodies or VHHs are single domain antigen binding fragments derived from heavy-chain antibodies naturally occurring in species of the Camelidae. Due to their ease of cloning, high solubility and intrinsic stability, they can be produced at low cost. Their small size, combined with high affinity and antigen specificity, enables recognition of a broad range of structural (undruggable) proteins and enzymes alike. Focusing on two actin binding proteins, gelsolin and CapG, we summarize a general protocol for the generation, cloning and production of nanobodies. Furthermore, we describe multiple ways to characterize antigen-nanobody binding in more detail and we shed light on some applications with recombinant nanobodies. The use of nanobodies as intrabodies is clarified through several case studies revealing new cytoskeletal protein properties and testifying to the utility of nanobodies as intracellular bona fide protein inhibitors. Moreover, as nanobodies can traverse the plasma membrane of eukaryotic cells by means of the enteropathogenic E. coli type III protein secretion system, we show that in this promising way of nanobody delivery, actin pedestal formation can be affected following nanobody injection.

  4. RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics.

    PubMed

    Salzer, Elisabeth; Cagdas, Deniz; Hons, Miroslav; Mace, Emily M; Garncarz, Wojciech; Petronczki, Özlem Yüce; Platzer, René; Pfajfer, Laurène; Bilic, Ivan; Ban, Sol A; Willmann, Katharina L; Mukherjee, Malini; Supper, Verena; Hsu, Hsiang Ting; Banerjee, Pinaki P; Sinha, Papiya; McClanahan, Fabienne; Zlabinger, Gerhard J; Pickl, Winfried F; Gribben, John G; Stockinger, Hannes; Bennett, Keiryn L; Huppa, Johannes B; Dupré, Loïc; Sanal, Özden; Jäger, Ulrich; Sixt, Michael; Tezcan, Ilhan; Orange, Jordan S; Boztug, Kaan

    2016-12-01

    RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.

  5. Cytoskeletal Dynamics and Fluid Flow in Drosophila Oocytes

    NASA Astrophysics Data System (ADS)

    de Canio, Gabriele; Goldstein, Raymond; Lauga, Eric

    2015-11-01

    The biological world includes a broad range of phenomena in which transport in a fluid plays a central role. Among these is the fundamental issue of cell polarity arising during development, studied historically using the model organism Drosophila melanogaster. The polarity of the oocyte is known to be induced by the translocation of mRNAs by kinesin motor proteins along a dense microtubule cytoskeleton, a process which also induces cytoplasmic streaming. Recent experimental observations have revealed the remarkable fluid-structure interactions that occur as the streaming flows back-react on the microtubules. In this work we use a combination of theory and simulations to address the interplay between the fluid flow and the configuration of cytoskeletal filaments leading to the directed motion inside the oocyte. We show in particular that the mechanical coupling between the fluid motion and the orientation of the microtubules can lead to a transition to coherent motion within the oocyte, as observed. Supported by EPSRC and ERC Advanced Investigator Grant 247333.

  6. Cytoskeletal Modulation of Lipid Interactions Regulates Lck Kinase Activity*

    PubMed Central

    Chichili, Gurunadh R.; Cail, Robert C.; Rodgers, William

    2012-01-01

    The actin cytoskeleton promotes clustering of proteins associated with cholesterol-dependent rafts, but its effect on lipid interactions that form and maintain rafts is not understood. We addressed this question by determining the effect of disrupting the cytoskeleton on co-clustering of dihexadecyl-(C16)-anchored DiO and DiI, which co-enrich in ordered lipid environments such as rafts. Co-clustering was assayed by fluorescence resonance energy transfer (FRET) in labeled T cells, where rafts function in the phosphoregulation of the Src family kinase Lck. Our results show that probe co-clustering was sensitive to depolymerization of actin filaments with latrunculin B (Lat B), inhibition of myosin II with blebbistatin, and treatment with neomycin to sequester phosphatidylinositol 4,5-bisphosphate. Cytoskeletal effects on lipid interactions were not restricted to order-preferring label because co-clustering of C16-anchored DiO with didodecyl (C12)-anchored DiI, which favors disordered lipids, was also reduced by Lat B and blebbistatin. Furthermore, conditions that disrupted probe co-clustering resulted in activation of Lck. These data show that the cytoskeleton globally modulates lipid interactions in the plasma membrane, and this property maintains rafts that function in Lck regulation. PMID:22613726

  7. Force profiles of protein pulling with or without cytoskeletal links studied by AFM

    SciTech Connect

    Afrin, Rehana; Ikai, Atsushi . E-mail: aikai@bio.titech.ac.jp

    2006-09-15

    To test the capability of the atomic force microscope for distinguishing membrane proteins with/without cytoskeletal associations, we studied the pull-out mechanics of lipid tethers from the red blood cell (RBC). When wheat germ agglutinin, a glycophorin A (GLA) specific lectin, was used to pull out tethers from RBC, characteristic force curves for tether elongation having a long plateau force were observed but without force peaks which are usually attributed to the forced unbinding of membrane components from the cytoskeleton. The result was in agreement with the reports that GLA is substantially free of cytoskeletal interactions. On the contrary, when the Band 3 specific lectin, concanavalin A, was used, the force peaks were indeed observed together with a plateau supporting its reported cytoskeletal association. Based on these observations, we postulate that the state of cytoskeletal association of particular membrane proteins can be identified from the force profiles of their pull-out mechanics.

  8. Changes in cytoskeletal dynamics and nonlinear rheology with metastatic ability in cancer cell lines

    NASA Astrophysics Data System (ADS)

    Coughlin, Mark F.; Fredberg, Jeffrey J.

    2013-12-01

    Metastatic outcome is impacted by the biophysical state of the primary tumor cell. To determine if changes in cancer cell biophysical properties facilitate metastasis, we quantified cytoskeletal biophysics in well-characterized human skin, bladder, prostate and kidney cell line pairs that differ in metastatic ability. Using magnetic twisting cytometry with optical detection, cytoskeletal dynamics was observed through spontaneous motion of surface bound marker beads and nonlinear rheology was characterized through large amplitude forced oscillations of probe beads. Measurements of cytoskeletal dynamics and nonlinear rheology differed between strongly and weakly metastatic cells. However, no set of biophysical parameters changed systematically with metastatic ability across all cell lines. Compared to their weakly metastatic counterparts, the strongly metastatic kidney cancer cells exhibited both increased cytoskeletal dynamics and stiffness at large deformation which are thought to facilitate the process of vascular invasion.

  9. c-myc mRNA in cytoskeletal-bound polysomes in fibroblasts.

    PubMed

    Hesketh, J E; Campbell, G P; Whitelaw, P F

    1991-03-01

    3T3 fibroblasts were treated sequentially with 25 mM-KCl/0.05% Nonidet P40, 130 mM-KCl/0.05% Nonidet P40 and finally with 1% Nonidet P40/1% deoxycholate in order to release free, cytoskeletal-bound and membrane-bound polysomes respectively. The membrane-bound fraction was enriched in the mRNA for the membrane protein beta 2-microglobulin, whereas the cytoskeletal-bound polysomes were enriched in c-myc mRNA. Actin mRNA was present in both free and cytoskeletal-bound polysomes. The results suggest that cytoskeletal-bound polysomes are involved in the translation of specific mRNA species.

  10. Exploring the roles of integrin binding and cytoskeletal reorganization during mesenchymal stem cell mechanotransduction in soft and stiff hydrogels subjected to dynamic compression.

    PubMed

    Steward, Andrew J; Wagner, Diane R; Kelly, Daniel J

    2014-10-01

    The objective of this study was to explore how the response of mesenchymal stem cells (MSCs) to dynamic compression (DC) depends on their pericellular environment and the development of their cytoskeleton. MSCs were first seeded into 3% agarose hydrogels, stimulated with the chondrogenic growth factor TGF-β3 and exposed to DC (~10% strain at 1Hz) for 1h on either day 7, 14, or 21 of culture. At each time point, the actin, vimentin and tubulin networks of the MSCs were assessed using confocal microscopy. Similar to previous results, MSCs displayed a temporal response to DC; however, no dramatic changes in gross cytoskeletal organization were observed with time in culture. Vinculin (a membrane-cytoskeletal protein in focal adhesions) staining appeared more intense with time in culture. We next aimed to explore how changes to the pericellular environment, independent of the duration of exposure to TGF-β3, would influence the response of MSCs to DC. To this end, MSCs were encapsulated into either 'soft' or 'stiff' agarose hydrogels that are known to differentially support pericellular matrix (PCM) development. The application of DC led to greater relative increases in the expression of chondrogenic marker genes in the stiffer hydrogels, where the MSCs were found to have a more well developed PCM. These increases in gene expression were not observed following the addition of RGDS, an integrin blocker, suggesting that integrin binding plays a role in determining the response of MSCs to DC. Microtubule organization in MSCs was found to adapt in response to DC, but this effect was not integrin mediated, as this cytoskeletal reorganization was also observed in the presence of RGDS. In conclusion, although the PCM, integrin binding, and cytoskeletal reorganization are all involved in mechanotransduction of DC, none of these factors in isolation was able to completely explain the temporal mechanosensitivity of MSCs to dynamic compression.

  11. Local cytoskeletal and organelle interactions impact molecular motor-driven early endosomal trafficking

    PubMed Central

    Zajac, Allison L.; Goldman, Yale E.; Holzbaur, Erika L.F.; Ostap, E. Michael

    2013-01-01

    SUMMARY Background In the intracellular environment, motor-driven cargo must navigate a dense cytoskeletal network among abundant organelles. Results We investigated the effects of the crowded intracellular environment on early endosomal trafficking. Live-cell imaging of an endosomal cargo (endocytosed epidermal growth factor-conjugated quantum dots) combined with high-resolution tracking was used to analyze the heterogeneous motion of individual endosomes. The motile population of endosomes moved towards the perinuclear region in directed bursts of microtubule-based, dynein-dependent transport interrupted by longer periods of diffusive motion. Actin network density did not affect motile endosomes during directed runs or diffusive interruptions. Simultaneous two-color imaging was used to correlate changes in endosomal movement with potential obstacles to directed runs. Termination of directed runs spatially correlated with microtubule-dense regions, encounters with other endosomes, and interactions with the endoplasmic reticulum. During a subset of run terminations, we also observed merging and splitting of endosomes, deformation of the endoplasmic reticulum, and directional reversals at speeds up to ten-fold greater than characteristic in vitro motor velocities. These observations suggest endosomal membrane tension is high during directed run termination. Conclusions Our results indicate that the crowded cellular environment significantly impacts the motor-driven motility of organelles. Rather than simply acting as impediments to movement, interactions of trafficking cargos with intracellular obstacles may facilitate communication between membrane-bound compartments or contribute to the generation of membrane tension necessary for fusion and fission of endosomal membranes or remodeling of the endoplasmic reticulum. PMID:23770188

  12. Semaphorin 4D/Plexin-B1-mediated M-Ras GAP activity regulates actin-based dendrite remodeling through Lamellipodin.

    PubMed

    Tasaka, Gen-Ichi; Negishi, Manabu; Oinuma, Izumi

    2012-06-13

    Semaphorins have been identified as repulsive guidance molecules in the developing nervous system. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 induces repulsion in axon and dendrites by functioning as a GTPase-activating protein (GAP) for R-Ras and M-Ras, respectively. In axons, Sema4D stimulation induces growth cone collapse, and downregulation of R-Ras activity by Plexin-B1-mediated GAP activity is required for the action. Axonal R-Ras GAP activity downregulates phosphatidylinositol 3-kinase signaling pathway, and thereby induces inactivation of a microtubule assembly promoter protein, CRMP-2. However, in contrast to the well studied roles of semaphorins and plexins in axonal guidance, signaling molecules linking M-Ras GAP to dendritic cytoskeleton remain obscure. Here we identified an Ena/VASP ligand, Lamellipodin (Lpd), as a novel effector of M-Ras in dendrites. Lpd was expressed in F-actin-rich distal dendritic processes and was required for both basal and M-Ras-mediated dendrite development. Subcellular fractionation showed M-Ras-dependent membrane translocation of Lpd, which was suppressed by Sema4D. Furthermore, the Ena/VASP-binding region within Lpd was required for dendrite development, and its membrane targeting was sufficient to overcome the Sema4D-mediated reduction of dendritic outgrowth and disappearance of F-actin from distal dendrites. Furthermore, in utero electroporation experiments also indicated that regulation of the M-Ras-Lpd system by the GAP activity of Plexin is involved in the normal development of cortical dendrites in vivo. Overall, our study sheds light on how repulsive guidance molecules regulate actin cytoskeleton in dendrites, revealing a novel mechanism that the M-Ras-Lpd system regulates actin-based dendrite remodeling by Sema/Plexin in rats or mice of either sex.

  13. Integrin β1 regulates leiomyoma cytoskeletal integrity and growth

    PubMed Central

    Malik, Minnie; Segars, James; Catherino, William H.

    2014-01-01

    Uterine leiomyomas are characterized by an excessive extracellular matrix, increased mechanical stress, and increased active RhoA. Previously, we observed that mechanical signaling was attenuated in leiomyoma, but the mechanisms responsible remain unclear. Integrins, especially integrin β1, are transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence cell proliferation and differentiation. Here we characterized integrin and laminin to signaling in leiomyoma cells. We observed a 2.25 ± 0.32 fold increased expression of integrin β1 in leiomyoma cells, compared to myometrial cells. Antibody-mediated inhibition of integrin β1 led to significant growth inhibition in leiomyoma cells and a loss of cytoskeletal integrity. Specifically, polymerization of actin filaments and formation of focal adhesions were reduced by inhibition of integrin p1. Inhibition of integrin β1 in leiomyoma cells led to 0.81 ± 0.02 fold decrease in active RhoA, and resembled levels found in serum-starved cells. Likewise, inhibition of integrin β1 was accompanied by a decrease in phospho-ERK. Compared to myometrial cells, leiomyoma cells demonstrated increased expression of integrin α6 subunit to laminin receptor (1.91 ± 0.11 fold), and increased expression of laminin 5α (1.52±0.02), laminin 5β (3.06±0.92), and laminin 5γ (1.66 ± 0.06). Of note, leiomyoma cells grown on laminin matrix appear to realign themselves. Taken together, the findings reveal that the attenuated mechanical signaling in leiomyoma cells is accompanied by an increased expression and a dependence on integrin β1 signaling in leiomyoma cells, compared to myometrial cells. PMID:23023061

  14. CONSERVED ROLES FOR CYTOSKELETAL COMPONENTS IN DETERMINING LATERALITY

    PubMed Central

    McDowell, Gary S.; Lemire, Joan M.; Paré, Jean-Francois; Cammarata, Garrett; Lowery, Laura Anne; Levin, Michael

    2016-01-01

    SUMMARY Consistently-biased left-right (LR) patterning is required for the proper placement of organs including the heart and viscera. The LR axis is especially fascinating as an example of multi-scale pattern formation, since here chiral events at the subcellular level are integrated and amplified into asymmetric transcriptional cascades and ultimately into the anatomical patterning of the entire body. In contrast to the other two body axes, there is considerable controversy about the earliest mechanisms of embryonic laterality. Many molecular components of asymmetry have not been widely tested among phyla with diverse bodyplans, and it is unknown whether parallel (redundant) pathways may exist that could reverse abnormal asymmetry states at specific checkpoints in development. To address conservation of the early steps of LR patterning, we used the Xenopus laevis (frog) embryo to functionally test a number of protein targets known to direct asymmetry in plants, fruit fly, and rodent. Using the same reagents that randomize asymmetry in Arabidopsis, Drosophila, and mouse embryos, we show that manipulation of the microtubule and actin cytoskeleton immediately post-fertilization, but not later, results in laterality defects in Xenopus embryos. Moreover, we observed organ-specific randomization effects and a striking dissociation of organ situs from effects on the expression of left side control genes, which parallel data from Drosophila and mouse. Remarkably, some early manipulations that disrupt laterality of transcriptional asymmetry determinants can be subsequently “rescued” by the embryo, resulting in normal organ situs. These data reveal the existence of novel corrective mechanisms, demonstrate that asymmetric expression of Nodal is not a definitive marker of laterality, and suggest the existence of amplification pathways that connect early cytoskeletal processes to control of organ situs bypassing Nodal. Counter to alternative models of symmetry breaking

  15. A Role for Cytoskeletal Elements in the Light-Driven Translocation of Proteins in Rod Photoreceptors

    PubMed Central

    Peterson, James J.; Orisme, Wilda; Fellows, Jonathan; McDowell, J. Hugh; Shelamer, Charles L.; Dugger, Donald R.; Clay Smith, W.

    2006-01-01

    PURPOSE. Light-driven protein translocation is responsible for the dramatic redistribution of some proteins in vertebrate rod photoreceptors. In this study, the involvement of microtubules and microfilaments in the light-driven translocation of arrestin and transducin was investigated. METHODS. Pharmacologic reagents were applied to native and transgenic Xenopus tadpoles, to disrupt the microtubules (thiabendazole) and microfilaments (cytochalasin D and latrunculin B) of the rod photoreceptors. Quantitative confocal imaging was used to assess the impact of these treatments on arrestin and transducin translocation. A series of transgenic tadpoles expressing arrestin truncations were also created to identify portions of arrestin that enable arrestin to translocate. RESULTS. Application of cytochalasin D or latrunculin B to disrupt the microfilament organization selectively slowed only transducin movement from the inner to the outer segments. Perturbation of the microtubule cytoskeleton with thiabendazole slowed the translocation of both arrestin and transducin, but only in moving from the outer to the inner segments. Transgenic Xenopus expressing fusions of green fluorescent protein (GFP) with portions of arrestin implicates the C terminus of arrestin as an important portion of the molecule for promoting translocation. This C-terminal region can be used independently to promote translocation of GFP in response to light. CONCLUSIONS. The results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motor-driven mechanism for complete movement of arrestin and transducin. PMID:16249472

  16. Biogenesis of actin-like bacterial cytoskeletal filaments destined for positioning prokaryotic magnetic organelles.

    PubMed

    Pradel, Nathalie; Santini, Claire-Lise; Bernadac, Alain; Fukumori, Yoshihiro; Wu, Long-Fei

    2006-11-14

    Magnetosomes comprise a magnetic nanocrystal surrounded by a lipid bilayer membrane. These unique prokaryotic organelles align inside magnetotactic bacterial cells and serve as an intracellular compass allowing the bacteria to navigate along the geomagnetic field in aquatic environments. Cryoelectron tomography of Magnetospirillum strains has revealed that the magnetosome chain is surrounded by a network of filaments that may be composed of MamK given that the filaments are absent in the mamK mutant cells. The process of the MamK filament assembly is unknown. Here we prove the authenticity of the MamK filaments and show that MamK exhibits linear distribution inside Magnetospirillum sp. cells even in the area without magnetosomes. The mamK gene alone is sufficient to direct the synthesis of straight filaments in Escherichia coli, and one extremity of the MamK filaments is located at the cellular pole. By using dual fluorescent labeling of MamK, we found that MamK nucleates at multiple sites and assembles into mosaic filaments. Time-lapse experiments reveal that the assembly of the MamK filaments is a highly dynamic and kinetically asymmetrical process. MamK bundles might initiate the formation of a new filament or associate to one preexistent filament. Our results demonstrate the mechanism of biogenesis of prokaryotic cytoskeletal filaments that are structurally and functionally distinct from the known MreB and ParM filaments. In addition to positioning magnetosomes, other hypothetical functions of the MamK filaments in magnetotaxis might include anchoring magnetosomes and being involved in magnetic reception.

  17. Corticosterone treatment results in enhanced release of peptidergic vesicles in astrocytes via cytoskeletal rearrangements.

    PubMed

    Chatterjee, Sreejata; Sikdar, Sujit K

    2013-12-01

    While the effect of stress on neuronal physiology is widely studied, its effect on the functionality of astrocytes is not well understood. We studied the effect of high doses of stress hormone corticosterone, on two physiological properties of astrocytes, i.e., gliotransmission and interastrocytic calcium waves. To study the release of peptidergic vesicles from astrocytes, hippocampal astrocyte cultures were transfected with a plasmid to express pro-atrial natriuretic peptide (ANP) fused with the emerald green fluorescent protein (ANP.emd). The rate of decrease in fluorescence of ANP.emd on application of ionomycin, a calcium ionophore was monitored. Significant increase in the rate of calcium-dependent exocytosis of ANP.emd was observed with the 100 nM and 1 μM corticosterone treatments for 3 h, which depended on the activation of the glucocorticoid receptor. ANP.emd tagged vesicles exhibited increased mobility in astrocyte culture upon corticosterone treatment. Increasing corticosterone concentrations also resulted in concomitant increase in the calcium wave propagation velocity, initiated by focal ATP application. Corticosterone treatment also resulted in increased GFAP expression and F-actin rearrangements. FITC-Phalloidin immunostaining revealed increased formation of cross linked F-actin networks with the 100 nM and 1 μM corticosterone treatment. Alternatively, blockade of actin polymerization and disruption of microtubules prevented the corticosterone-mediated increase in ANP.emd release kinetics. This study reports for the first time the effect of corticosterone on gliotransmission via modulation of cytoskeletal elements. As ANP acts on both neurons and blood vessels, modulation of its release could have functional implications in neurovascular coupling under pathophysiological conditions of stress.

  18. Inhibition of cytoskeletal protein carbonylation may protect against oxidative damage in traumatic brain injury

    PubMed Central

    Zhang, Qiusheng; Zhang, Meng; Huang, Xianjian; Liu, Xiaojia; Li, Weiping

    2016-01-01

    Oxidative stress is the principal factor in traumatic brain injury (TBI) that initiates protracted neuronal dysfunction and remodeling. Cytoskeletal proteins are known to be carbonylated under oxidative stress; however, the complex molecular and cellular mechanisms of cytoskeletal protein carbonylation remain poorly understood. In the present study, the expression levels of glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were investigated in PC12 cells treated with H2O2. Western blot analysis was used to monitor the carbonylation levels of β-actin and β-tubulin. The results indicated that oxidative stress was increased in PC12 cells that were treated with H2O2 for 24 or 48 h. In addition, increased carbonylation levels of β-actin and β-tubulin were detected in H2O2-treated cells. However, these carbonylation levels were reduced by pretreatment with aminoguanidine, a type of reactive carbonyl species chelating agent, and a similar trend was observed following overexpression of proteasome β5 via transgenic technology. In conclusion, the present study results suggested that the development of TBI may cause carbonylation of cytoskeletal proteins, which would then undermine the stability of cytoskeletal proteins. Thus, the development of TBI may be improved via the inhibition of cytoskeletal protein carbonylation. PMID:28101189

  19. Accumulation of Glucosylceramide in the Absence of the Beta-Glucosidase GBA2 Alters Cytoskeletal Dynamics

    PubMed Central

    Raju, Diana; Schonauer, Sophie; Hamzeh, Hussein; Flynn, Kevin C.; Bradke, Frank; vom Dorp, Katharina; Dörmann, Peter; Yildiz, Yildiz; Trötschel, Christian; Poetsch, Ansgar; Breiden, Bernadette; Sandhoff, Konrad; Körschen, Heinz G.; Wachten, Dagmar

    2015-01-01

    Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types. PMID:25803043

  20. Real space flight travel is associated with ultrastructural changes, cytoskeletal disruption and premature senescence of HUVEC.

    PubMed

    Kapitonova, M Y; Muid, S; Froemming, G R A; Yusoff, W N W; Othman, S; Ali, A M; Nawawi, H M

    2012-12-01

    Microgravity, hypergravity, vibration, ionizing radiation and temperature fluctuations are major factors of outer space flight affecting human organs and tissues. There are several reports on the effect of space flight on different human cell types of mesenchymal origin while information regarding changes to vascular endothelial cells is scarce. Ultrastructural and cytophysiological features of macrovascular endothelial cells in outer space flight and their persistence during subsequent culturing were demonstrated in the present investigation. At the end of the space flight, endothelial cells displayed profound changes indicating cytoskeletal lesions and increased cell membrane permeability. Readapted cells of subsequent passages exhibited persisting cytoskeletal changes, decreased metabolism and cell growth indicating cellular senescence.

  1. Visualization of Actin Cytoskeletal Dynamics in Fixed and Live Drosophila Egg Chambers.

    PubMed

    Groen, Christopher M; Tootle, Tina L

    2015-01-01

    Visualization of actin cytoskeletal dynamics is critical for understanding the spatial and temporal regulation of actin remodeling. Drosophila oogenesis provides an excellent model system for visualizing the actin cytoskeleton. Here, we present methods for imaging the actin cytoskeleton in Drosophila egg chambers in both fixed samples by phalloidin staining and in live egg chambers using transgenic actin labeling tools.

  2. Cocaine affects the dynamics of cytoskeletal proteins via sigma(1) receptors.

    PubMed

    Su, T P; Hayashi, T

    2001-09-01

    Cytoskeletal proteins are important in protein trafficking, membrane protein clustering, dendrite growth and the morphological maintenance of neurons. Sigma(1) receptors are unique endoplasmic reticular (ER) proteins that bind (+)benzomorphans, neurosteroids and psychotropic drugs such as cocaine. Cocaine, via sigma(1) receptors, can cause the dissociation of a cytoskeletal adaptor protein ankyrin from inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] receptors on the ER as a sigma(1)-receptor-ankyrin complex, which then translocates to the plasma membrane and nucleus. The dissociation of sigma(1)-receptor-ankyrin from Ins(1,4,5)P(3) receptors also increases the intracellular Ca(2+) concentration [[Ca(2+)](i)], which affects the activity of cytoskeletal proteins. Furthermore, cocaine might increase [Ca(2+)](i) via phospholipase C (PLC)-linked dopamine D1 receptors. We hypothesize that cocaine might cause life-long changes in neurons via cytoskeletal proteins by interacting with both D1 receptors and sigma(1) receptors.

  3. Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing

    NASA Astrophysics Data System (ADS)

    Wülfing, Christoph; Purtic, Bozidar; Klem, Jennifer; Schatzle, John D.

    2003-06-01

    Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.

  4. PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling.

    PubMed

    Mansour, Mariam; Nievergall, Eva; Gegenbauer, Kristina; Llerena, Carmen; Atapattu, Lakmali; Hallé, Maxime; Tremblay, Michel L; Janes, Peter W; Lackmann, Martin

    2016-01-15

    Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling.

  5. Morphometric analysis of Mauthner axon cytoskeletal components in adult and subadult fish.

    PubMed

    Alfei, L; Medolago-Albani, L; Zezze, G M; Stefanelli, A

    1992-01-01

    A previous cytoskeletal analysis on trout MA during developmental stages demonstrated, during the subadult stages, neurofilaments (NF) as main components as expressed by the high values of neurofilament to microtubules (MT) ratio which was found to be of the order of 300:1. Since the MA cytoskeletal composition is not known in the adult fish, the MA cytoskeletal composition has been compared to other axons of much smaller diameter of the fasciculus longitudinalis medialis (flm) among which the MA run in the ventral spinal cord. The following parameters were measured on conventional electron microscopy in MA and flm axons cross sections micrographs by means of a computer linked graphic tablet (Apple II): axonal caliber, number of microtubules (MT), microtubular (MT/microns2) and neurofilament (NF/microns2) densities. The analysis of these parameters demonstrated that neurofilaments are the main architectural components in the adult and subadult fish MA and flm axons. However, MA cytoskeletal composition differs from the other flm axons because of its particular very high ratio of neurofilaments to microtubules. The inverse relationship of axonal caliber to microtubular density, previously found in the trout during developmental stages and suggested also for many other vertebrate species, was further confirmed for flm axons which, with calibers 10 times smaller than MA, exhibit a microtubular density 10 times larger.

  6. Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis.

    PubMed Central

    Ornelles, D A; Fey, E G; Penman, S

    1986-01-01

    Cytochalasin D was shown to be a reversible inhibitor of protein synthesis in HeLa cells. The inhibition was detectable at drug levels typically used to perturb cell structure and increased in a dose-dependent manner. The drug also released mRNA from the cytoskeletal framework in direct proportion to the inhibition of protein synthesis. The released mRNA was unaltered in its translatability as measured in vitro but was no longer translated in the cytochalasin-treated HeLa cells. The residual protein synthesis occurred on polyribosomes that were reduced in amount but displayed a normal sedimentation distribution. The results support the hypothesis that mRNA binding to the cytoskeletal framework is necessary although not sufficient for translation. Analysis of the cytoskeletal framework, which binds the polyribosomes, revealed no alterations in composition or amount of protein as a result of treatment with cytochalasin D. Electron microscopy with embedment-free sections shows the framework in great detail. The micrographs revealed the profound reorganization effected by the drug but did not indicate substantial disaggregation of the cytoskeletal elements. Images PMID:3785175

  7. Glycoprotein Ib-IX-V Complex Transmits Cytoskeletal Forces That Enhance Platelet Adhesion.

    PubMed

    Feghhi, Shirin; Munday, Adam D; Tooley, Wes W; Rajsekar, Shreya; Fura, Adriane M; Kulman, John D; López, Jose A; Sniadecki, Nathan J

    2016-08-09

    Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbα and the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbα and filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbα are involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.

  8. Hippocampal damage and cytoskeletal disruption resulting from impaired energy metabolism. Implications for Alzheimer disease.

    PubMed

    Geddes, J W; Pang, Z; Wiley, D H

    1996-01-01

    To determine if impaired energy metabolism might contribute to some aspects of Alzheimer disease (AD), including the vulnerability of the CA1 region of the hippocampal formation and the altered cytoskeleton evident in neurofibrillary tangles, we examined the effects of metabolic poisons on neuronal damage and cytoskeletal disruption in the hippocampal formation. Intrahippocampal injection of 3-nitropropionic acid (3-NP) and malonic acid resulted in neuronal death, particularly in CA1. Cytoskeletal disruption included loss of dendritic MAP2, but sparing of axonal gamma. MK-801 (a noncompetitive NMDA receptor antagonist) did not atentuate the lesions produced by intrahippocampal injection of malonate. MK-801, however, was effective against intrastriatal malonate. Acute systemic 3-NP resulted in neuronal damage and cytoskeletal disruption in the CA1 region of the hippocampal formation, including an extensive loss of MAP2 immuno-reactivity, but sparing of gamma. The neuronal loss in CA1 was delayed as compared to striatum. Chronic intraventricular infusion of 3-NP produced a different pattern of neuronal damage. Loss of gamma-1 immuno-reactivity was observed in CA3 and CA1 s. orients, whereas MAP2 immunostaining was preserved. These results demonstrate that chronic and acute administration of metabolic inhibitors produce distinct patterns of neuronal damage and cytoskeletal disruption. The results further suggest a differential involvement of the NMDA receptor in malonate-induced neuronal damage in striatum as compared to the hippocampus. The pattern of neuronal damage and cytoskeletal disruption observed following acute metabolic impairment resembled some aspects of neurofibrillary pathology in AD, but did not result in gamma hyperphosphorylation.

  9. X-ray radiation promotes the metastatic potential of tongue squamous cell carcinoma cells via modulation of biomechanical and cytoskeletal properties.

    PubMed

    Zheng, Q; Liu, Y; Zhou, H J; Du, Y T; Zhang, B P; Zhang, J; Miao, G Y; Liu, B; Zhang, H

    2015-09-01

    This study investigated the metastatic potential of tongue squamous cell carcinoma (TSCC) cells after X-ray irradiation as well as radiation-induced changes in the biomechanical properties and cytoskeletal structure that are relevant to metastasis. Tca-8113 TSCC cells were X-ray-irradiated at increasing doses (0, 1, 2, or 4 Gy), and 24 h later, migration was evaluated with the wound healing and transwell migration assays, while invasion was assessed with the Matrigel invasion assay. Confocal and atomic force microscopy were used to examine changes in the structure of the actin cytoskeleton and Young's modulus (cell stiffness), respectively. X-ray radiation induced dose-dependent increases in invasive and migratory potentials of cells relative to unirradiated control cells (p < 0.05). The Young's modulus of irradiated cells was decreased by radiation exposure (p < 0.05), which was accompanied by alterations in the integrity and organization of the cytoskeletal network, as evidenced by a decrease in the signal intensity of actin fibers (p < 0.05). X-ray irradiation enhanced migration and invasiveness in Tca-8113 TSCC cells by altering their biomechanical properties and the organization of the actin cytoskeleton. A biomechanics-based analysis can provide an additional platform for assessing tumor response to radiation and optimization of cancer therapies.

  10. Cytoskeletal dynamics during in vitro neurogenesis of induced pluripotent stem cells (iPSCs).

    PubMed

    Compagnucci, Claudia; Piermarini, Emanuela; Sferra, Antonella; Borghi, Rossella; Niceforo, Alessia; Petrini, Stefania; Piemonte, Fiorella; Bertini, Enrico

    2016-12-01

    Patient-derived induced pluripotent stem cells (iPSCs) provide a novel tool to investigate the pathophysiology of poorly known diseases, in particular those affecting the nervous system, which has been difficult to study for its lack of accessibility. In this emerging and promising field, recent iPSCs studies are mostly used as "proof-of-principle" experiments that are confirmatory of previous findings obtained from animal models and postmortem human studies; its promise as a discovery tool is just beginning to be realized. A recent number of studies point to the functional similarities between in vitro neurogenesis and in vivo neuronal development, suggesting that similar morphogenetic and patterning events direct neuronal differentiation. In this context, neuronal adhesion, cytoskeletal organization and cell metabolism emerge as an integrated and unexplored processes of human neurogenesis, mediated by the lack of data due to the difficult accessibility of the human neural tissue. These observations raise the necessity to understand which are the players controlling cytoskeletal reorganization and remodeling. In particular, we investigated human in vitro neurogenesis using iPSCs of healthy subjects to unveil the underpinnings of the cytoskeletal dynamics with the aim to shed light on the physiologic events controlling the development and the functionality of neuronal cells. We validate the iPSCs system to better understand the development of the human nervous system in order to set the bases for the future understanding of pathologies including developmental disorders (i.e. intellectual disability), epilepsy but also neurodegenerative disorders (i.e. Friedreich's Ataxia). We investigate the changes of the cytoskeletal components during the 30days of neuronal differentiation and we demonstrate that human neuronal differentiation requires a (time-dependent) reorganization of actin filaments, intermediate filaments and microtubules; and that immature neurons present

  11. Networks.

    ERIC Educational Resources Information Center

    Maughan, George R.; Petitto, Karen R.; McLaughlin, Don

    2001-01-01

    Describes the connectivity features and options of modern campus communication and information system networks, including signal transmission (wire-based and wireless), signal switching, convergence of networks, and network assessment variables, to enable campus leaders to make sound future-oriented decisions. (EV)

  12. Membrane protein carboxyl methylation does not appear to be involved in the response of erythrocytes to cytoskeletal stress.

    PubMed

    Barber, J R; Clarke, S

    1984-08-30

    We have investigated the effect of changes of human erythrocyte cell shape on the degree of covalent modification by carboxyl methylation of membrane cytoskeletal proteins. The results indicate that the cell probably does not utilize carboxyl methylation to respond to cytoskeletal perturbations caused by such agents as A23187, 2,4-dinitrophenol, and chlorpromazine, all of which are known to cause large changes in cell shape. Protein carboxyl methylation also remained unchanged in the presence of cytochalasin B, which prevents such changes in cell shape. These results are not consistent with a cytoskeletal regulatory role for protein methylation reactions in the intact erythrocyte.

  13. The cytoskeletal framework of sea urchin eggs and embryos: developmental changes in the association of messenger RNA.

    PubMed

    Moon, R T; Nicosia, R F; Olsen, C; Hille, M B; Jeffery, W R

    1983-02-01

    Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only 10-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Triton X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.

  14. Probing Cytoskeletal Structures by Coupling Optical Superresolution and AFM Techniques for a Correlative Approach

    PubMed Central

    Chacko, Jenu Varghese; Zanacchi, Francesca Cella; Diaspro, Alberto

    2013-01-01

    In this article, we describe and show the application of some of the most advanced fluorescence superresolution techniques, STED AFM and STORM AFM microscopy towards imaging of cytoskeletal structures, such as microtubule filaments. Mechanical and structural properties can play a relevant role in the investigation of cytoskeletal structures of interest, such as microtubules, that provide support to the cell structure. In fact, the mechanical properties, such as the local stiffness and the elasticity, can be investigated by AFM force spectroscopy with tens of nanometers resolution. Force curves can be analyzed in order to obtain the local elasticity (and the Young's modulus calculation by fitting the force curves from every pixel of interest), and the combination with STED/STORM microscopy integrates the measurement with high specificity and yields superresolution structural information. This hybrid modality of superresolution-AFM working is a clear example of correlative multimodal microscopy. PMID:24027190

  15. Systematic perturbation of cytoskeletal function reveals a linear scaling relationship between cell geometry and fitness.

    PubMed

    Monds, Russell D; Lee, Timothy K; Colavin, Alexandre; Ursell, Tristan; Quan, Selwyn; Cooper, Tim F; Huang, Kerwyn Casey

    2014-11-20

    Diversification of cell size is hypothesized to have occurred through a process of evolutionary optimization, but direct demonstrations of causal relationships between cell geometry and fitness are lacking. Here, we identify a mutation from a laboratory-evolved bacterium that dramatically increases cell size through cytoskeletal perturbation and confers a large fitness advantage. We engineer a library of cytoskeletal mutants of different sizes and show that fitness scales linearly with respect to cell size over a wide physiological range. Quantification of the growth rates of single cells during the exit from stationary phase reveals that transitions between "feast-or-famine" growth regimes are a key determinant of cell-size-dependent fitness effects. We also uncover environments that suppress the fitness advantage of larger cells, indicating that cell-size-dependent fitness effects are subject to both biophysical and metabolic constraints. Together, our results highlight laboratory-based evolution as a powerful framework for studying the quantitative relationships between morphology and fitness.

  16. Cytoskeletal changes in Eimeria bovis-infected host endothelial cells during first merogony.

    PubMed

    Hermosilla, Carlos; Schröpfer, Elmar; Stowasser, Michael; Eckstein-Ludwig, Ursula; Behrendt, Jan Hillern; Zahner, Horst

    2008-10-01

    The first merogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macromeronts up to 250 microm. In this study, we investigated the host cell cytoskeleton (actin filaments, microtubules, spectrin, vimentin intermediate filaments) associated with parasitic development in vitro by confocal laser scanning microscopy (CLSM) using primary bovine umbilical vein endothelial cells (BUVEC) and bovine spleen lymphatic endothelial cells (BSLEC) as host cells. No prominent changes in the host cell cytoskeleton were detected 1-3 days after E. bovis sporozoite invasion. With ongoing meront maturation a significant increase in microtubules and actin filaments close to the parasitophorous vacuole (PV) was found. Mature macromeronts within the PV were completely enclosed by these cytoskeletal elements. Our findings suggest, that in order to guarantee the survival of the host cell on the enlargement of macromeronts, E. bovis needs not only to augment but also to rearrange its cytoskeletal system.

  17. Fragility of foot process morphology in kidney podocytes arises from chaotic spatial propagation of cytoskeletal instability

    PubMed Central

    Deerinck, Thomas J.; Chen, Yibang; He, John C.; Ellisman, Mark H.; Iyengar, Ravi

    2017-01-01

    Kidney podocytes’ function depends on fingerlike projections (foot processes) that interdigitate with those from neighboring cells to form the glomerular filtration barrier. The integrity of the barrier depends on spatial control of dynamics of actin cytoskeleton in the foot processes. We determined how imbalances in regulation of actin cytoskeletal dynamics could result in pathological morphology. We obtained 3-D electron microscopy images of podocytes and used quantitative features to build dynamical models to investigate how regulation of actin dynamics within foot processes controls local morphology. We find that imbalances in regulation of actin bundling lead to chaotic spatial patterns that could impair the foot process morphology. Simulation results are consistent with experimental observations for cytoskeletal reconfiguration through dysregulated RhoA or Rac1, and they predict compensatory mechanisms for biochemical stability. We conclude that podocyte morphology, optimized for filtration, is intrinsically fragile, whereby local transient biochemical imbalances may lead to permanent morphological changes associated with pathophysiology. PMID:28301477

  18. The connection of cytoskeletal network with plasma membrane and the cell wall

    PubMed Central

    Liu, Zengyu; Persson, Staffan; Zhang, Yi

    2015-01-01

    The cell wall provides external support of the plant cells, while the cytoskeletons including the microtubules and the actin filaments constitute an internal framework. The cytoskeletons contribute to the cell wall biosynthesis by spatially and temporarily regulating the transportation and deposition of cell wall components. This tight control is achieved by the dynamic behavior of the cytoskeletons, but also through the tethering of these structures to the plasma membrane. This tethering may also extend beyond the plasma membrane and impact on the cell wall, possibly in the form of a feedback loop. In this review, we discuss the linking components between the cytoskeletons and the plasma membrane, and/or the cell wall. We also discuss the prospective roles of these components in cell wall biosynthesis and modifications, and aim to provide a platform for further studies in this field. PMID:25693826

  19. EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells.

    PubMed Central

    Clore, A. M.; Dannenhoffer, J. M.; Larkins, B. A.

    1996-01-01

    By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei. PMID:12239373

  20. Identification of paralogous life-cycle stage specific cytoskeletal proteins in the parasite Trypanosoma brucei.

    PubMed

    Portman, Neil; Gull, Keith

    2014-01-01

    The life cycle of the African trypanosome Trypanosoma brucei, is characterised by a transition between insect and mammalian hosts representing very different environments that present the parasite with very different challenges. These challenges are met by the expression of life-cycle stage-specific cohorts of proteins, which function in systems such as metabolism and immune evasion. These life-cycle transitions are also accompanied by morphological rearrangements orchestrated by microtubule dynamics and associated proteins of the subpellicular microtubule array. Here we employed a gel-based comparative proteomic technique, Difference Gel Electrophoresis, to identify cytoskeletal proteins that are expressed differentially in mammalian infective and insect form trypanosomes. From this analysis we identified a pair of novel, paralogous proteins, one of which is expressed in the procyclic form and the other in the bloodstream form. We show that these proteins, CAP51 and CAP51V, localise to the subpellicular corset of microtubules and are essential for correct organisation of the cytoskeleton and successful cytokinesis in their respective life cycle stages. We demonstrate for the first time redundancy of function between life-cycle stage specific paralogous sets in the cytoskeleton and reveal modification of cytoskeletal components in situ prior to their removal during differentiation from the bloodstream form to the insect form. These specific results emphasise a more generic concept that the trypanosome genome encodes a cohort of cytoskeletal components that are present in at least two forms with life-cycle stage-specific expression.

  1. Cytoskeletal Configuration Modulates Mechanically Induced Changes in Mesenchymal Stem Cell Osteogenesis, Morphology, and Stiffness

    NASA Astrophysics Data System (ADS)

    Pongkitwitoon, Suphannee; Uzer, Gunes; Rubin, Janet; Judex, Stefan

    2016-10-01

    Mesenchymal stem cells (MSC) responding to mechanical cues generated by physical activity is critical for skeletal development and remodeling. Here, we utilized low intensity vibrations (LIV) as a physiologically relevant mechanical signal and hypothesized that the confined cytoskeletal configuration imposed by 2D culture will enable human bone marrow MSCs (hBMSC) to respond more robustly when LIV is applied in-plane (horizontal-LIV) rather than out-of-plane (vertical-LIV). All LIV signals enhanced hBMSC proliferation, osteogenic differentiation, and upregulated genes associated with cytoskeletal structure. The cellular response was more pronounced at higher frequencies (100 Hz vs 30 Hz) and when applied in the horizontal plane. Horizontal but not vertical LIV realigned the cell cytoskeleton, culminating in increased cell stiffness. Our results show that applying very small oscillatory motions within the primary cell attachment plane, rather than perpendicular to it, amplifies the cell’s response to LIV, ostensibly facilitating a more effective transfer of intracellular forces. Transcriptional and structural changes in particular with horizontal LIV, together with the strong frequency dependency of the signal, emphasize the importance of intracellular cytoskeletal configuration in sensing and responding to high-frequency mechanical signals at low intensities.

  2. Phosphocreatine as an energy source for actin cytoskeletal rearrangements during myoblast fusion.

    PubMed

    O'Connor, Roddy S; Steeds, Craig M; Wiseman, Robert W; Pavlath, Grace K

    2008-06-15

    Myoblast fusion is essential for muscle development, postnatal growth and muscle repair after injury. Recent studies have demonstrated roles for actin polymerization during myoblast fusion. Dynamic cytoskeletal assemblies directing cell-cell contact, membrane coalescence and ultimately fusion require substantial cellular energy demands. Various energy generating systems exist in cells but the partitioning of energy sources during myoblast fusion is unknown. Here, we demonstrate a novel role for phosphocreatine (PCr) as a spatiotemporal energy buffer during primary mouse myoblast fusion with nascent myotubes. Creatine treatment enhanced cell fusion in a creatine kinase (CK)-dependent manner suggesting that ATP-consuming reactions are replenished through the PCr/CK system. Furthermore, selective inhibition of actin polymerization prevented myonuclear addition following creatine treatment. As myotube formation is dependent on cytoskeletal reorganization, our findings suggest that PCr hydrolysis is coupled to actin dynamics during myoblast fusion. We conclude that myoblast fusion is a high-energy process, and can be enhanced by PCr buffering of energy demands during actin cytoskeletal rearrangements in myoblast fusion. These findings implicate roles for PCr as a high-energy phosphate buffer in the fusion of multiple cell types including sperm/oocyte, trophoblasts and macrophages. Furthermore, our results suggest the observed beneficial effects of oral creatine supplementation in humans may result in part from enhanced myoblast fusion.

  3. Early changes in Huntington's disease patient brains involve alterations in cytoskeletal and synaptic elements.

    PubMed

    DiProspero, Nicholas A; Chen, Er-Yun; Charles, Vinod; Plomann, Markus; Kordower, Jeffrey H; Tagle, Danilo A

    2004-09-01

    Huntington's disease (HD) is caused by a polyglutamine repeat expansion in the N-terminus of the huntingtin protein. Huntingtin is normally present in the cytoplasm where it may interact with structural and synaptic elements. The mechanism of HD pathogenesis remains unknown but studies indicate a toxic gain-of-function possibly through aberrant protein interactions. To investigate whether early degenerative changes in HD involve alterations of cytoskeletal and vesicular components, we examined early cellular changes in the frontal cortex of HD presymptomatic (PS), early pathological grade (grade 1) and late-stage (grade 3 and 4) patients as compared to age-matched controls. Morphologic analysis using silver impregnation revealed a progressive decrease in neuronal fiber density and organization in pyramidal cell layers beginning in presymptomatic HD cases. Immunocytochemical analyses for the cytoskeletal markers alpha -tubulin, microtubule-associated protein 2, and phosphorylated neurofilament demonstrated a concomitant loss of staining in early grade cases. Immunoblotting for synaptic proteins revealed a reduction in complexin 2, which was marked in some grade 1 HD cases and significantly reduced in all late stage cases. Interestingly, we demonstrate that two synaptic proteins, dynamin and PACSIN 1, which were unchanged by immunoblotting, showed a striking loss by immunocytochemistry beginning in early stage HD tissue suggesting abnormal distribution of these proteins. We propose that mutant huntingtin affects proteins involved in synaptic function and cytoskeletal integrity before symptoms develop which may influence early disease onset and/or progression.

  4. Regulation of cytoskeletal dynamics by redox signaling and oxidative stress: implications for neuronal development and trafficking

    PubMed Central

    Wilson, Carlos; González-Billault, Christian

    2015-01-01

    A proper balance between chemical reduction and oxidation (known as redox balance) is essential for normal cellular physiology. Deregulation in the production of oxidative species leads to DNA damage, lipid peroxidation and aberrant post-translational modification of proteins, which in most cases induces injury, cell death and disease. However, physiological concentrations of oxidative species are necessary to support important cell functions, such as chemotaxis, hormone synthesis, immune response, cytoskeletal remodeling, Ca2+ homeostasis and others. Recent evidence suggests that redox balance regulates actin and microtubule dynamics in both physiological and pathological contexts. Microtubules and actin microfilaments contain certain amino acid residues that are susceptible to oxidation, which reduces the ability of microtubules to polymerize and causes severing of actin microfilaments in neuronal and non-neuronal cells. In contrast, inhibited production of reactive oxygen species (ROS; e.g., due to NOXs) leads to aberrant actin polymerization, decreases neurite outgrowth and affects the normal development and polarization of neurons. In this review, we summarize emerging evidence suggesting that both general and specific enzymatic sources of redox species exert diverse effects on cytoskeletal dynamics. Considering the intimate relationship between cytoskeletal dynamics and trafficking, we also discuss the potential effects of redox balance on intracellular transport via regulation of the components of the microtubule and actin cytoskeleton as well as cytoskeleton-associated proteins, which may directly impact localization of proteins and vesicles across the soma, dendrites and axon of neurons. PMID:26483635

  5. Cytoskeletal to Nuclear Strain Transfer Regulates YAP Signaling in Mesenchymal Stem Cells

    PubMed Central

    Driscoll, Tristan P.; Cosgrove, Brian D.; Heo, Su-Jin; Shurden, Zach E.; Mauck, Robert L.

    2015-01-01

    Mechanical forces transduced to cells through the extracellular matrix are critical regulators of tissue development, growth, and homeostasis, and can play important roles in directing stem cell differentiation. In addition to force-sensing mechanisms that reside at the cell surface, there is growing evidence that forces transmitted through the cytoskeleton and to the nuclear envelope are important for mechanosensing, including activation of the Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) pathway. Moreover, nuclear shape, mechanics, and deformability change with differentiation state and have been likewise implicated in force sensing and differentiation. However, the significance of force transfer to the nucleus through the mechanosensing cytoskeletal machinery in the regulation of mesenchymal stem cell mechanobiologic response remains unclear. Here we report that actomyosin-generated cytoskeletal tension regulates nuclear shape and force transmission through the cytoskeleton and demonstrate the differential short- and long-term response of mesenchymal stem cells to dynamic tensile loading based on the contractility state, the patency of the actin cytoskeleton, and the connections it makes with the nucleus. Specifically, we show that while some mechanoactive signaling pathways (e.g., ERK signaling) can be activated in the absence of nuclear strain transfer, cytoskeletal strain transfer to the nucleus is essential for activation of the YAP/TAZ pathway with stretch. PMID:26083918

  6. Sporadic inclusion body myositis: morphology, regeneration, and cytoskeletal structure of muscle fibres

    PubMed Central

    Arnardottir, S; Borg, K; Ansved, T

    2004-01-01

    Methods: 14 muscle biopsies from 11 patients with s-IBM were characterised for morphological abnormalities and fibre type composition as well as muscle fibre regeneration and cytoskeletal structure, using histochemical and immunohistochemical techniques. Results: Morphological abnormalities included inflammatory infiltrates and "rimmed vacuoles," and pronounced variation in fibre size. There were no significant differences in fibre type composition between s-IBM patients and controls based on the myofibrillar ATPase staining. A differential effect on muscle fibre sizes was noted, type II fibres being smaller in the s-IBM patients than in the controls. Conversely, the mean type I muscle fibre diameter in the s-IBM patients was larger than in the controls, though this difference was not significant. An ongoing intense regeneration process was present in s-IBM muscle, as indicated by the expression of neonatal myosin heavy chain, vimentin, and CD56 (Leu-19) in most of the muscle fibres. The cytoskeletal proteins dystrophin and desmin were normally expressed in s-IBM muscle fibres that were not undergoing degeneration or regeneration. Conclusions: There are extensive morphological and morphometric alterations in s-IBM, affecting different muscle fibre types in different ways. The cytoskeletal structure of type I and II muscle fibres remains unaffected in different stages of the disease. PMID:15146016

  7. Magnetic phagosome motion in J774A.1 macrophages: influence of cytoskeletal drugs.

    PubMed Central

    Möller, W; Nemoto, I; Matsuzaki, T; Hofer, T; Heyder, J

    2000-01-01

    The role of the different cytoskeletal structures like microfilaments (MF), microtubuli (MT), and intermediate filaments (IF) in phagosome motion is unclear. These cytoskeletal units play an important role in macrophage function (migration, phagocytosis, phagosome transport). We investigated ferromagnetic phagosome motions by cell magnetometry. J774A.1 macrophages were incubated with 1.3-microm spherical magnetite particles for 24 h, after which more than 90% of the particles had been phagocytized. Phagosome motions can be caused either by the cell itself (relaxation) or by applying magnetic twisting forces, yielding cell stiffness and viscoelastic properties of the cytoskeleton. Apparent viscosity of the cytoplasm was non-Newtonian and showed a shear-rate-dependent power law behavior. Elastically stored energy does not force the magnetic phagosomes back to their initial orientation: 57% of the twisting shear was not recoverable. Cytoskeletal drugs, like Cytochalasin D (CyD, 2 - 4 microM), Colchicine (CoL, 10 microM), or Acrylamide (AcL, 40 mM) were added in order to disturb the different cytoskeletal structures. AcL disintegrates IF, but affected neither stochastic (relaxation) nor directed phagosome motions. CyD disrupts MF, resulting in a retarded stochastic phagosome motion (relative decay 0.53 +/- 0.01 after 5 min versus 0.34 +/- 0.01 in control), whereas phagosome twisting shows only a small response with a 9% increase of stiffness and a small reduction of recoverable strain. CoL depolymerizes the MT, inducing a moderately accelerated relaxation (relative decay 0.28 +/- 0.01 after 5 min) and a 10% increase of cell stiffness, where the pure viscous shear is increased and the viscoelastic recoil is inhibited by 40%. Combining the two drugs conserves both effects. After disintegrating either MF or MT, phagosome motion and cytoskeletal stiffness reflect the behavior of either MT or MF, respectively. The results verify that the dominant phagosome transport

  8. Down-regulation of Slit-Robo pathway mediating neuronal cytoskeletal remodeling processes facilitates the antidepressive-like activity of Gastrodia elata Blume.

    PubMed

    Lin, Shih-Hang; Chen, Wei-Cheng; Lu, Kuan-Hung; Chen, Pei-Ju; Hsieh, Shu-Chen; Pan, Tzu-Ming; Chen, Shui-Tein; Sheen, Lee-Yan

    2014-10-29

    Nowadays, depression is a serious psychological disorder that causes extreme economic loss and social problems. Previously, we discovered that the water extract of Gastrodia elata Blume (WGE) improved depressive-like behavior by influencing neurotransmitters in rats subjected to the forced swimming test. To elucidate possible mechanisms, in the present study, we performed a proteomics and bioinformatics analysis to identify the related pathways. Western blot-validated results indicated that the core protein network modulated by WGE administration was closely associated with down-regulation of the Slit-Robo pathway, which modulates neuronal cytoskeletal remodeling processes. Although Slit-Robo signaling has been well investigated in neuronal development, its relationship with depression is not fully understood. We provide a potential hint on the mechanism responsible for the antidepressive-like activity of WGE. In conclusion, we suggest that the Slit-Robo pathway and neuronal cytoskeleton remodeling are possibly one of the pathways associated with the antidepressive-like effects of WGE.

  9. Networking.

    ERIC Educational Resources Information Center

    Duvall, Betty

    Networking is an information giving and receiving system, a support system, and a means whereby women can get ahead in careers--either in new jobs or in current positions. Networking information can create many opportunities: women can talk about how other women handle situations and tasks, and previously established contacts can be used in…

  10. Coupled biopolymer networks

    NASA Astrophysics Data System (ADS)

    Schwarz, J. M.; Zhang, Tao

    2015-03-01

    The actin cytoskeleton provides the cell with structural integrity and allows it to change shape to crawl along a surface, for example. The actin cytoskeleton can be modeled as a semiflexible biopolymer network that modifies its morphology in response to both external and internal stimuli. Just inside the inner nuclear membrane of a cell exists a network of filamentous lamin that presumably protects the heart of the cell nucleus--the DNA. Lamins are intermediate filaments that can also be modeled as semiflexible biopolymers. It turns out that the actin cytoskeletal biopolymer network and the lamin biopolymer network are coupled via a sequence of proteins that bridge the outer and inner nuclear membranes. We, therefore, probe the consequences of such a coupling via numerical simulations to understand the resulting deformations in the lamin network in response to perturbations in the cytoskeletal network. Such study could have implications for mechanical mechanisms of the regulation of transcription, since DNA--yet another semiflexible polymer--contains lamin-binding domains, and, thus, widen the field of epigenetics.

  11. Involvement of oxidative stress and cytoskeletal disruption in microcystin-induced apoptosis in CIK cells.

    PubMed

    Huang, Xiao; Chen, Liang; Liu, Wanjing; Qiao, Qin; Wu, Kang; Wen, Jing; Huang, Cuihong; Tang, Rong; Zhang, Xuezhen

    2015-08-01

    The outbreak of cyanobacterial blooms induces the production and release of microcystins (MCs) into water, representing a health hazard to aquatic organisms and even humans. Some recent studies have suggested that kidney is another important target organ of MCs except liver, however, the potential toxicity mechanisms are still unclear. In this study, we first investigated the collaborative effect of oxidative stress and cytoskeletal disruption in microcystin-induced apoptosis in CIK (Ctenopharyngodon idellus kidney) cells in vitro. CIK cells were treated with 0, 1, 10, and 100μg/L microcystin-LR (MC-LR) for 24 and 48h. Cell viability was increased by MC-LR in 1μg/L group, while decreased in 100μg/L group at 48h. Cell cycle assay showed that 1 and 10μg/L MC-LR induced cell cycle through G1 into S and G2/M phases, while 100μg/L MC-LR reduced G2/M phase population. MC-LR markedly induced apoptosis in 10 and 100μg/L groups. Elevated reactive oxygen species (ROS) production, increased malondialdehyde (MDA) contents, decreased glutathione (GSH) levels, and modulated antioxidant enzymes including catalase (CAT) and superoxide dismutase (SOD) were observed in CIK cells exposed to MC-LR. These alterations were more pronounced at higher doses (10 and 100μg/L), indicating that oxidative stress was induced by MC-LR. Laser scanning confocal microscope observation showed aggregation and collapse of microfilaments (MFs) and microtubules (MTs) in CIK cells, and even loss of some cytoskeleton structure. Moreover, transcriptional changes of cytoskeletal genes (β-actin, lc3a, and keratin) were also determined, which have a high probability with cytoskeleton structure damage. Our data suggest that oxidative stress and cytoskeletal disruption may interact with each other and jointly lead to apoptosis and renal toxicity induced by MCs.

  12. Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth

    PubMed Central

    1987-01-01

    We have examined the movements, composition, and cellular origin of phase-dense varicosities in cultures of chick sympathetic and sensory neurons. These organelles are variable in diameter (typically between 0.2 and 2 microns) and undergo saltatory movements both towards and away from the neuronal cell body. Their mean velocities vary inversely with the size of the organelle and are greater in the retrograde than the anterograde direction. Organelles stain with the lipophilic dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine and with antibodies to cytoskeletal components. In cultures double-stained with antibodies to alpha-tubulin and 70-kD neurofilament protein (NF-L), approximately 40% of the organelles stain for tubulin, 30% stain for NF- L, 10% stain for both tubulin and NF-L, and 40% show no staining with either antibody. The association of cytoskeletal proteins with the organelles shows that these proteins are able to move by a form of rapid axonal transport. Under most culture conditions the predominant direction of movement is towards the cell body, suggesting that the organelles are produced at or near the growth cone. Retrograde movements continue in culture medium lacking protein or high molecular mass components and increase under conditions in which the advance of the growth cone is arrested. There is a fourfold increase in the number of organelles moving retrogradely in neurites that encounter a substratum-associated barrier to elongation; retrograde movements increase similarly in cultures exposed to cytochalasin at levels known to block growth cone advance. No previously described organelle shows behavior coordinated with axonal growth in this way. We propose that the organelles contain membrane and cytoskeletal components that have been delivered to the growth cone, by slow or fast anterograde transport, in excess of the amounts required to synthesize more axon. In view of their rapid mobility and variable contents, we suggest that they

  13. Induction of Plant Curvature by Magnetophoresis and Cytoskeletal Changes during Root Graviresponse

    NASA Technical Reports Server (NTRS)

    Hasenstein, Karl H.; Kuznetsov, Oleg A.; Blancaflor, Eilson B.

    1996-01-01

    High gradient magnetic fields (HGMF) induce curvature in roots and shoots. It is considered that this response is likely to be based on the intracellular displacement of bulk starch (amyloplasts) by the ponderomotive force generated by the HGMF. This process is called magnetophoresis. The differential elongation during the curvature along the concave and convex flanks of growing organs may be linked to the microtubular and/or microfilament cytoskeleton. The possible existence of an effect of the HGMF on the cytoskeleton was tested for, but none was found. The application of cytoskeletal stabilizers or depolymerizers showed that neither microtubules, nor microfilaments, are involved in the graviresponse.

  14. Messengers, motors and mysteries: sorting of eukaryotic mRNAs by cytoskeletal transport.

    PubMed

    Bullock, Simon L

    2011-10-01

    It has become increasingly apparent in recent years that the subcellular localization of specific mRNAs is a prevalent method for spatially controlling gene expression. In most cases, targeting of mRNAs is mediated by transport along cytoskeletal filaments by molecular motors. However, the means by which specific messages are recognized and linked to the motors are poorly understood. Here, I will provide an overview of recent progress in elucidating the molecular mechanisms and principles of mRNA transport, including several studies highlighting the co-operation of different motors during the localization process. Important outstanding questions will also be highlighted.

  15. The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid

    SciTech Connect

    Pierozan, Paula; Ferreira, Fernanda; Ortiz de Lima, Bárbara; Gonçalves Fernandes, Carolina; Totarelli Monteforte, Priscila; Castro Medaglia, Natalia de; Bincoletto, Claudia; Soubhi Smaili, Soraya; Pessoa-Pureur, Regina

    2014-04-01

    Quinolinic acid (QUIN) is a glutamate agonist which markedly enhances the vulnerability of neural cells to excitotoxicity. QUIN is produced from the amino acid tryptophan through the kynurenine pathway (KP). Dysregulation of this pathway is associated with neurodegenerative conditions. In this study we treated striatal astrocytes in culture with QUIN and assayed the endogenous phosphorylating system associated with glial fibrillary acidic protein (GFAP) and vimentin as well as cytoskeletal remodeling. After 24 h incubation with 100 µM QUIN, cells were exposed to {sup 32}P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca{sup 2+}/calmodulin II (PKCaMII) or protein kinase C (PKC) inhibitors, H89 (20 μM), KN93 (10 μM) and staurosporin (10 nM), respectively. Results showed that hyperphosphorylation was abrogated by PKA and PKC inhibitors but not by the PKCaMII inhibitor. The specific antagonists to ionotropic NMDA and non-NMDA (50 µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50 µM MCPG), mGLUR1 (100 µM MPEP) and mGLUR5 (10 µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca{sup 2+} quelators (1 mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca{sup 2+} influx through voltage-dependent Ca{sup 2+} channel type L (L-VDCC) (blocker: 10 µM verapamil) is not implicated in this effect. Morphological analysis showed dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork, supporting misregulation of actin cytoskeleton. Both hyperphosphorylation and cytoskeletal remodeling were reversed 24 h after QUIN removal. Astrocytes are highly plastic cells and the vulnerability of astrocyte cytoskeleton may have important implications for understanding the neurotoxicity of QUIN in neurodegenerative

  16. Active contraction of microtubule networks

    PubMed Central

    Foster, Peter J; Fürthauer, Sebastian; Shelley, Michael J; Needleman, Daniel J

    2015-01-01

    Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction. DOI: http://dx.doi.org/10.7554/eLife.10837.001 PMID:26701905

  17. Differential impact of REM sleep deprivation on cytoskeletal proteins of brain regions involved in sleep regulation.

    PubMed

    Rodríguez-Vázquez, Jennifer; Camacho-Arroyo, Ignacio; Velázquez-Moctezuma, Javier

    2012-01-01

    Rapid eye movement (REM) sleep is involved in memory consolidation, which implies synaptic plasticity. This process requires protein synthesis and the reorganization of the neural cytoskeleton. REM sleep deprivation (REMSD) has an impact on some neuronal proteins involved in synaptic plasticity, such as glutamate receptors and postsynaptic density protein 95, but its effects on cytoskeletal proteins is unknown. In this study, the effects of REMSD on the content of the cytoskeletal proteins MAP2 and TAU were analyzed. Adult female rats were submitted to selective REMSD by using the multiple platform technique. After 24, 48 or 72 h of REMSD, rats were decapitated and the following brain areas were dissected: pons, preoptic area, hippocampus and frontal cortex. Protein extraction and Western blot were performed. Results showed an increase in TAU content in the pons, preoptic area and hippocampus after 24 h of REMSD, while in the frontal cortex a significant increase in TAU content was observed after 72 h of REMSD. A TAU content decrease was observed in the hippocampus after 48 h of REMSD. Interestingly, a marked increase in TAU content was observed after 72 h of REMSD. MAP2 content only increased in the preoptic area at 24 h, and in the frontal cortex after 24 and 72 h of REMSD, without significant changes in the pons and hippocampus. These results support the idea that REM sleep plays an important role in the organization of neural cytoskeleton, and that this effect is tissue-specific.

  18. Actinobacillus actinomycetemcomitans adheres to human gingival fibroblasts and modifies cytoskeletal organization.

    PubMed

    Gutiérrez-Venegas, Gloria; Kawasaki-Cárdenas, Perla; Garcés, Carla Portillo; Román-Alvárez, Patricia; Barajas-Torres, Carolina; Contreras-Marmolejo, Luis Arturo

    2007-09-01

    Adherence of Actinobacillus actinomycetemcomitans to human gingival fibroblast cells induces cytoskeletal reorganization. A. actinomycetemcomitans is considered a pathogenic bacteria involved in localized aggressive periodontitis. Studies with epithelial cells have shown an adherent capacity of bacteria that is increased under anaerobic conditions. For adherence to take place, there is a need for interaction between extracellular vesicles and bacterial fimbriae. However, molecular events associated with the adherence process are still unknown. The aim of this study was to investigate whether A. actinomycetemcomitans adherence to human gingival fibroblasts promotes cytoskeletal reorganization. Adherence was determined with light microscopy and scanning electron microscopy. For F-actin visualization, cells were treated with fluorescein-isothiocyanate-phalloidin and samples were examined with epifluorescence optics. Fluorescent was recorded on Kodak T-Max 400 film. We showed that A. actinomycetemcomitans adheres to human gingival fibroblast primary cultures, this property stimulating an increase in the intracellular calcium levels. In human gingival fibroblast primary cultures, we observed that maximal A. actinomycetemcomitans adherence took place 1.5h after culture infection occurred and remained for 6h. The adherence was associated with morphologic alterations and an increased in the intracellular calcium levels. These experiments suggest that A. actinomycetemcomitans adherence cause morphological alterations, induce actin stress fibers and recruitment of intracellular calcium levels.

  19. Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging.

    PubMed

    Chazotte, Brad

    2010-05-01

    The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. The cytoskeleton is composed of a series of filamentous structures, including intermediate filaments, actin filaments, and microtubules. Immunofluorescent staining has been most frequently used to study cytoskeletal components. However, it is also possible to fluorescently label isolated cytoskeletal proteins and either microinject them back into the cell or add them to fixed, permeabilized cells. Alternatively, it is possible to use the mushroom-derived fluorescinated toxins, phalloidin or phallacidin, to label F-actin of the cytoskeleton, as is described in this article. Phalloidin is available labeled with different fluorophores. The choice of the specific fluorophore should depend on whether phalloidin labeling for actin is part of a double-label experiment. In most cells, the abundance of actin filaments should provide a very strong signal. In double-label experiments, the fluorophore should be chosen to take this into account. In general, rhodamine labels are more resistant to photobleaching and can be subjected to the longer exposures required for finer structures.

  20. Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces

    PubMed Central

    Bagchi, Sonchita; Tomenius, Henrik; Belova, Lyubov M; Ausmees, Nora

    2008-01-01

    Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks. PMID:18976278

  1. Podocyte-Specific Deletion of Dicer Alters Cytoskeletal Dynamics and Causes Glomerular Disease

    PubMed Central

    Harvey, Scott J.; Jarad, George; Cunningham, Jeanette; Goldberg, Seth; Schermer, Bernhard; Harfe, Brian D.; McManus, Michael T.; Benzing, Thomas; Miner, Jeffrey H.

    2008-01-01

    MicroRNAs (miRNAs) regulate gene expression by binding the 3′ untranslated region of mRNAs. To define their role in glomerular function, miRNA biogenesis was disrupted in mouse podocytes using a conditional Dicer allele. Mutant mice developed proteinuria by 3 wk after birth and progressed rapidly to end-stage kidney disease. Podocyte pathology included effacement, vacuolization, and hypertrophy with crescent formation. Despite normal expression of WT1, podocytes underwent dedifferentiation, exemplified by cytoskeletal disruption with early transcriptional downregulation of synaptopodin. These abnormalities differed from Cd2ap−/− mice, indicating they were not a general consequence of glomerular disease. Glomerular labeling of ezrin, moesin, and gelsolin was altered at 3 wk, but expression of nestin and α-actinin was unchanged. Abnormal cell proliferation or apoptosis was not responsible for the glomerular injury. Mutant podocytes were incapable of synthesizing mature miRNA, as revealed by their loss of miR-30a. In contrast, expression of glomerular endothelial and mesangial cell miRNAs (miR-126 and miR-145, respectively) was unchanged. These findings demonstrate a critical role for miRNA in glomerular function and suggest a pathway that may participate in the pathogenesis of kidney diseases of podocyte origin. The unique architecture of podocytes may make them especially susceptible to cytoskeletal alterations initiated by aberrant miRNA dynamics. PMID:18776121

  2. Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration.

    PubMed

    Li, Aiqing; Benoit, Joshua B; Lopez-Martinez, Giancarlo; Elnitsky, Michael A; Lee, Richard E; Denlinger, David L

    2009-05-01

    Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2-DE and nanoscale capillary LC/MS/MS. Twenty-four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration-regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.

  3. α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

    PubMed Central

    Sousa, Vítor L.; Bellani, Serena; Giannandrea, Maila; Yousuf, Malikmohamed; Valtorta, Flavia; Meldolesi, Jacopo

    2009-01-01

    The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration. PMID:19553474

  4. The Effect of Ultrasound Stimulation on the Cytoskeletal Organization of Chondrocytes Seeded In 3D Matrices

    PubMed Central

    Noriega, Sandra; Hasanova, Gulnara; Subramanian, Anuradha

    2013-01-01

    The impact of low intensity diffuse ultrasound (LIDUS) stimulation on the cytoskeletal organization of chondrocytes seeded in 3D scaffolds was evaluated. Chondrocytes seeded on 3D chitosan matrices were exposed to LIDUS at 5.0 MHz (~15kPa, 51-secs, 4-applications/day) in order to study the organization of actin, tubulin and vimentin. The results showed that actin presented a cytosolic punctuated distribution, tubulin presented a quasi parallel organization of microtubules whereas vimentin distribution was unaffected. Chondrocytes seeded on 3D scaffolds responded to US stimulation by the disruption of actin stress fibers and were sensitive to the presence of ROCK inhibitor (Y27632). The gene expression of ROCK-I, a key element in the formation of stress fibers and mDia1, was significantly up-regulated under the application of US. We conclude that the results of both the cytoskeletal analyses and gene expression support the argument that the presence of punctuated actin upon US stimulation was accompanied by the up-regulation of the RhoA/ROCK pathway. PMID:22987069

  5. Nesprin-3 regulates endothelial cell morphology, perinuclear cytoskeletal architecture, and flow-induced polarization

    PubMed Central

    Morgan, Joshua T.; Pfeiffer, Emily R.; Thirkill, Twanda L.; Kumar, Priyadarsini; Peng, Gordon; Fridolfsson, Heidi N.; Douglas, Gordon C.; Starr, Daniel A.; Barakat, Abdul I.

    2011-01-01

    Changes in blood flow regulate gene expression and protein synthesis in vascular endothelial cells, and this regulation is involved in the development of atherosclerosis. How mechanical stimuli are transmitted from the endothelial luminal surface to the nucleus is incompletely understood. The linker of nucleus and cytoskeleton (LINC) complexes have been proposed as part of a continuous physical link between the plasma membrane and subnuclear structures. LINC proteins nesprin-1, -2, and -4 have been shown to mediate nuclear positioning via microtubule motors and actin. Although nesprin-3 connects intermediate filaments to the nucleus, no functional consequences of nesprin-3 mutations on cellular processes have been described. Here we show that nesprin-3 is robustly expressed in human aortic endothelial cells (HAECs) and localizes to the nuclear envelope. Nesprin-3 regulates HAEC morpho­logy, with nesprin-3 knockdown inducing prominent cellular elongation. Nesprin-3 also organizes perinuclear cytoskeletal organization and is required to attach the centrosome to the nuclear envelope. Finally, nesprin-3 is required for flow-induced polarization of the centrosome and flow-induced migration in HAECs. These results represent the most complete description to date of nesprin-3 function and suggest that nesprin-3 regulates vascular endothelial cell shape, perinuclear cytoskeletal architecture, and important aspects of flow-mediated mechanotransduction. PMID:21937718

  6. Modulation of cytoskeletal dynamics by mammalian nucleoside diphosphate kinase (NDPK) proteins.

    PubMed

    Snider, Natasha T; Altshuler, Peter J; Omary, M Bishr

    2015-02-01

    Nucleoside diphosphate kinase (NDPK) proteins comprise a family of ten human isoforms that participate in the regulation of multiple cellular processes via enzymatic and nonenzymatic functions. The major enzymatic function of NDPKs is the generation of nucleoside triphosphates, such as guanosine triphosphate (GTP). Mechanisms behind the nonenzymatic NDPK functions are not clear but likely involve context-dependent signaling roles of NDPK within multi-protein complexes. This is most evident for NDPK-A, which is encoded by the human NME1 gene, the first tumor metastasis suppressor gene to be identified. Understanding which protein interactions are most relevant for the biological and metastasis-related functions of NDPK will be important in the potential utilization of NDPK as a disease target. Accumulating evidence suggests that NDPK interacts with and affects various components and regulators of the cytoskeleton, including actin-binding proteins, intermediate filaments, and cytoskeletal attachment structures (adherens junctions, desmosomes, and focal adhesions). We review the existing literature on this topic and highlight outstanding questions and potential future directions that should clarify the impact of NDPK on the different cytoskeletal systems.

  7. MIEN1 drives breast tumor cell migration by regulating cytoskeletal-focal adhesion dynamics

    PubMed Central

    Van Treuren, Timothy; Vishwanatha, Jamboor K.

    2016-01-01

    Migration and invasion enhancer 1 (MIEN1) is an important regulator of cell migration and invasion. MIEN1 overexpression represents an oncogenic event that promotes tumor cell dissemination and metastasis. The underlying mechanism by which MIEN1 regulates migration and invasion has yet to be deciphered. Here, we demonstrate that MIEN1 acts as a cytoskeletal-signaling adapter protein to drive breast cancer cell migration. MIEN1 localization is concentrated underneath the actin-enriched protrusive structures of the migrating breast cancer cells. Depletion of MIEN1 led to the loss of actin-protrusive structures whereas the over-expression of MIEN1 resulted in rich and thick membrane extensions. Knockdown of MIEN1 also decreased the cell-substratum adhesion, suggesting a role for MIEN1 in actin cytoskeletal dynamics. Our results show that MIEN1 supports the transition of G-actin to F-actin polymerization and stabilizes F-actin polymers. Additionally, MIEN1 promotes cellular adhesion and actin dynamics by inducing phosphorylation of FAK at Tyr-925 and reducing phosphorylation of cofilin at Ser-3, which results in breast cancer cell migration. Collectively, our data show that MIEN1 plays an essential role in maintaining the plasticity of the dynamic membrane-associated actin cytoskeleton, which leads to an increase in cell motility. Hence, targeting MIEN1 might represent a promising means to prevent breast tumor metastasis. PMID:27462783

  8. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure

    NASA Technical Reports Server (NTRS)

    Maniotis, A. J.; Chen, C. S.; Ingber, D. E.

    1997-01-01

    We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.

  9. Probing bilayer-cytoskeletal interactions in erythrocytes using a two-component dissipative particle dynamics model

    NASA Astrophysics Data System (ADS)

    Peng, Zhangli; Li, Xuejin; Pivkin, Igor; Dao, Ming; Karniadakis, George

    2013-11-01

    We develop a two-component dissipative particle dynamics (DPD) model of the red blood cell (RBC) membrane by modeling the lipid bilayer and the cytoskeleton separately. By applying this model to simulate four different experiments on RBCs, including micropipette aspiration, membrane fluctuations, tank-treading motions in shear flow and bilayer tethering in a flow channel, we validated our model and studied the mechanical properties of the bilayer-cytoskeletal interaction in a systematic and controlled manner, such as its elastic stiffness, viscous friction and strength. In the same time, we also resolved several controversies in RBC mechanics, e.g., the dependence of tank-treading frequency on shear rates and the possibility of bilayer-cytoskeletal slip. Furthermore, to investigate RBC dynamics in the microcirculation, we simulated the passages of RBCs through narrow channels of the flow cytometer in vitro and their passages through the splenic inter-endothelial slits in vivo. The effects of RBC geometry and membrane stiffness on the critical pressure gradient of passage were studied, and the simulation results agree well with experimental measurements. This work was supported by National Institutes of Health Grant R01HL094270 and the new Department of Energy Collaboratory on Mathematics for Mesoscopic Modeling of Materials (CM4).

  10. Analysis of the expression of cytoskeletal proteins of Taenia crassiceps ORF strain cysticerci (Cestoda).

    PubMed

    Reynoso-Ducoing, Olivia; Valverde-Islas, Laura; Paredes-Salomon, Cristina; Pérez-Reyes, América; Landa, Abraham; Robert, Lilia; Mendoza, Guillermo; Ambrosio, Javier R

    2014-05-01

    The Taenia crassiceps ORF strain is used to generate a murine model of cysticercosis, which is used for diagnosis, evaluation of drugs, and vaccination. This particular strain only exists as cysticerci, is easily maintained under in vivo and in vitro conditions, and offers an excellent model for studying the cytoskeletons of cestodes. In this study, several experimental approaches were used to determine the tissue expression of its cytoskeletal proteins. The techniques used were microscopy (video, confocal, and transmission electron), one-dimensional (1D) and two-dimensional (2D) electrophoresis, immunochemistry, and mass spectrometry. The tissue expression of actin, tubulin, and paramyosin was assessed using microscopy, and their protein isoforms were determined with 1D and 2D electrophoresis and immunochemistry. Nineteen spots were excised from a proteomic gel and identified by liquid chromatography-tandem mass spectrometry and immunochemistry. The proteins identified were classic cytoskeletal proteins, metabolic enzymes, and proteins with diverse biological functions, but mainly involved in detoxification activities. Research suggests that most noncytoskeletal proteins interact with actin or tubulin, and the results of the present study suggest that the proteins identified may be involved in supporting the dynamics and plasticity of the cytoskeleton of T. crassiceps cysticerci. These results contribute to our knowledge of the cellular biology and physiology of cestodes.

  11. GRIM-19 inhibits v-Src-induced cell motility by interfering with cytoskeletal restructuring

    PubMed Central

    Sun, Peng; Nallar, Shreeram C.; Kalakonda, Sudhakar; Lindner, Daniel J.; Martin, Stuart S.; Kalvakolanu, Dhananjaya V.

    2008-01-01

    GRIM-19 (Gene associated with Retinoid-Interferon-induced Mortality 19) is a novel tumor suppressor regulated by Interferon/retinoid combination. We have recently shown that GRIM-19 inhibits v-Src-induced oncogenic transformation and metastatic behavior of cells. Oncogenic v-Src induces cell motility by cytoskeletal remodeling especially the formation of podosomes and. Here we show that GRIM-19 inhibited the v-Src-induced cell motility by inhibiting cytoskeletal remodeling i.e., podosome formation. We also show that the N-terminus of GRIM-19 played a major role in this process and identified critical residues in this region. More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility. These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition. Lastly, tumor-associated GRIM-19 mutants significantly lost their ability to control v-Src-induced metastases in vivo, indicating the biological and pathological significance of these observations. PMID:19151760

  12. Epigenetic repression of ribosomal RNA transcription by ROCK-dependent aberrant cytoskeletal organization

    PubMed Central

    Wu, Tse-Hsiang; Kuo, Yuan-Yeh; Lee, Hsiao-Hui; Kuo, Jean-Cheng; Ou, Meng-Hsin; Chang, Zee-Fen

    2016-01-01

    It is known that ribosomal RNA (rRNA) synthesis is regulated by cellular energy and proliferation status. In this study, we investigated rRNA gene transcription in response to cytoskeletal stress. Our data revealed that the cell shape constrained by isotropic but not elongated micropatterns in HeLa cells led to a significant reduction in rRNA transcription dependent on ROCK. Expression of a dominant-active form of ROCK also repressed rRNA transcription. Isotropic constraint and ROCK over-activation led to different types of aberrant F-actin organization, but their suppression effects on rRNA transcription were similarly reversed by inhibition of histone deacetylase (HDAC) or overexpression of a dominant negative form of Nesprin, which shields the signal transmitted from actin filament to the nuclear interior. We further showed that the binding of HDAC1 to the active fraction of rDNA genes is increased by ROCK over-activation, thus reducing H3K9/14 acetylation and suppressing transcription. Our results demonstrate an epigenetic control of active rDNA genes that represses rRNA transcription in response to the cytoskeletal stress. PMID:27350000

  13. Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice

    PubMed Central

    Morikawa, Yuka; Zhang, Min; Heallen, Todd; Leach, John; Tao, Ge; Xiao, Yang; Bai, Yan; Li, Wei; Willerson, James T.; Martin, James F.

    2015-01-01

    The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signaling-deficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex (DGC), a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near scar tissue of injured Hippo signaling-deficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to the mechanical changes associated with heart injury to promote repair. PMID:25943351

  14. Atomic-resolution structure of cytoskeletal bactofilin by solid-state NMR.

    PubMed

    Shi, Chaowei; Fricke, Pascal; Lin, Lin; Chevelkov, Veniamin; Wegstroth, Melanie; Giller, Karin; Becker, Stefan; Thanbichler, Martin; Lange, Adam

    2015-12-01

    Bactofilins are a recently discovered class of cytoskeletal proteins of which no atomic-resolution structure has been reported thus far. The bacterial cytoskeleton plays an essential role in a wide range of processes, including morphogenesis, cell division, and motility. Among the cytoskeletal proteins, the bactofilins are bacteria-specific and do not have a eukaryotic counterpart. The bactofilin BacA of the species Caulobacter crescentus is not amenable to study by x-ray crystallography or solution nuclear magnetic resonance (NMR) because of its inherent noncrystallinity and insolubility. We present the atomic structure of BacA calculated from solid-state NMR-derived distance restraints. We show that the core domain of BacA forms a right-handed β helix with six windings and a triangular hydrophobic core. The BacA structure was determined to 1.0 Å precision (heavy-atom root mean square deviation) on the basis of unambiguous restraints derived from four-dimensional (4D) HN-HN and 2D C-C NMR spectra.

  15. Cytoskeletal stability and heat shock-mediated thermoprotection of central pattern generation in Locusta migratoria.

    PubMed

    Garlick, Kristopher M; Robertson, R Meldrum

    2007-06-01

    Prior exposure to extreme temperatures can induce thermoprotection in migratory locusts, which is important for survival in their natural environment. An important motor activity that needs to be protected is ventilation. The mechanism underlying heat shock is not fully understood, and our goal was to test the idea that cytoskeletal stability is critical for such thermoprotection. Cytoskeletal stabilizers (concanavalin A) and destabilizers (colchicine) were bath-applied in semi-intact locust preparations in both control (C) and pre-treated heat-shocked (3 h, 45 degrees C) animals. We measured parameters of the ventilatory motor pattern during maintained high temperature (43 degrees C) and recorded the times taken for motor pattern generation to fail and then recover on returning to room temperature. We found that concanavalin A mimicked the effects of a prior heat stress in control animals by increasing time to failure and decreasing time to recovery of motor pattern generation. However, colchicine destroyed protection in heat-shocked animals by decreasing time to failure and increasing time to recovery. Our findings confirm that the cytoskeleton has a mechanistic role in preserving neural function at high temperatures, possibly through stabilizing ion channels and other integral membrane proteins (e.g. Na(+)/K(+) ATPase) and their interactions with heat shock proteins.

  16. Cytoskeletal Drugs Prevent Posterior Capsular Opacification in Human Lens Capsule in vitro

    PubMed Central

    Sureshkumar, Jeyalakshmi; Haripriya, Aravind; Muthukkaruppan, Veerappan; Kaufman, Paul L.; Tian, Baohe

    2012-01-01

    Background To determine whether the cytoskeletal drugs H-7 and Latrunculin B (LAT-B) inhibit posterior capsular opacification (PCO) in the cultured human lens capsular bag. Methods Following extracapsular cataract (lens) extraction in human donor eyes, the capsular bag was prepared and cultured by standard techniques. Forty-eight capsular bags were studied, in which 13 were treated with H-7 (50, 100 or 300μM), 12 with 1% BSS (vehicle of H-7), 11 with LAT-B (2, 5 or 10μM), and 12 with 0.25% DMSO (vehicle of LAT-B). Forty out of the 48 capsular bags were from paired eyes of 20 donors with 1 bag being treated with H-7/LAT-B and the other with BSS/DMSO for each pair, including 20 for the H-7-BSS protocol and 20 for the LAT-B-DMSO protocol. The medium with the cytoskeletal drug/vehicle was replaced every 3–4 days for 4 weeks. PCO was assessed daily using inverted phase-contrast microscopy and scored on a 4-point scale. Results In all cultures with BSS or DMSO, residual lens epithelial cells (LECs) on the anterior capsule migrated to and proliferated on the posterior capsule by 3–7 days and apparent LEC growth on the posterior capsule with severe capsular wrinkling (PCO Grade 3) was seen by 2-3weeks. When treated continuously with H-7 or LAT-B, the migration and proliferation of LECs and the capsular wrinkling were inhibited in a dose-dependent manner, with the inhibition being complete (PCO Grade 0) in the 300μM H-7 (n=8, p<0.001) or 10μM LAT-B culture (n=3, p=0.002). Conclusion H-7 and LAT-B dose-dependently inhibited PCO formation in the cultured human lens capsular bags, suggesting that cytoskeletal drugs might prevent PCO formation after surgery in the human eye. PMID:22138731

  17. A photoactivable phospholipid analogue that specifically labels membrane cytoskeletal proteins of intact erythrocytes

    SciTech Connect

    Pradhan, D.; Williamson, P.; Schlegel, R.A. )

    1989-08-22

    A radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho({sup 14}C)ethanolamine(({sup 14}C)AzPE), was synthesized. Upon incubation with erythrocytes in the dark, about 90% of ({sup 14}C)AzPE spontaneously incorporated into the cells; of this fraction, about 90% associated with the membrane, all of it noncovalently. Upon photoactivation, 3-4% of the membrane-associated probe was incorporated into protein. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as extraction of labeled membranes with alkali or detergent, showed that the probe preferentially labeled cytoskeletal proteins. ({sup 14}C)AzPE appears to be a useful tool for the study of lipid-protein interactions at the cytoplasmic face of the plasma membrane of intact cells.

  18. Exploiting molecular motors as nanomachines: the mechanisms of de novo and re-engineered cytoskeletal motors.

    PubMed

    DelRosso, Nicole V; Derr, Nathan D

    2017-01-11

    Cytoskeletal molecular motors provide exciting proof that nanoscale transporters can be highly efficient, moving for microns along filamentous tracks by hydrolyzing ATP to fuel nanometer-size steps. For nanotechnology, such conversion of chemical energy into productive work serves as an enticing platform for re-purposing and re-engineering. It also provides a roadmap for successful molecular mechanisms that can be mimicked to create de novo molecular motors for nanotechnology applications. Here we focus specifically on how the mechanisms of molecular motors are being re-engineered for greater control over their transport parameters. We then discuss mechanistic work to create fully synthetic motors de novo and conclude with future directions in creating novel motor systems.

  19. Cytoskeletal components of Beta vulgaris root hairs in altered gravity fields

    NASA Astrophysics Data System (ADS)

    Shevchenko, G. V.

    Root hairs of Beta vulgaris are protrusions from rhizodermal cells and are characterised by plagiotropic growth. The roles of the cytoskeleton and of gravity in this growth process are being studied with the help of a clinostat. Through the use of immunocytochemical and fluorescent staining methods which reveal microtubules (MTs) and microfilaments (MFs), it was found that these cytoskeletal components of the root hairs of 4-day-old seedlings of B. vulgaris were affected by clinorotation at 2 r.p.m. In control conditions, MTs were found to be distributed evenly throughout the root hair, and an intense fluorescence due to MFs was observed at the tip of the hairs. With clinorotation, however, MTs became distributed at random, though no redistribution of MFs was observed. The latter finding conforms to the idea that MFs are responsible for tip growth. That MTs are more sensitive to altered gravity conditions is presently being tested.

  20. Metabolic and cytoskeletal modulation of transferrin receptor mobility in mitogen-activated human lymphocytes.

    PubMed Central

    Galbraith, G M; Galbraith, R M

    1980-01-01

    The transferrin receptors which appear on mitogen-activated human peripheral blood lymphocytes were found by the use of immunofluorescence techniques to display temperature-dependent patching and capping reactions upon binding of transferrin. Lateral mobility of ligand-occupied membrane sites was accompanied by both shedding and endocytosis of receptor-transferrin complexes. In the presence of sodium azide or the microfilament inhibitor cytochalasin B, cap formation and shedding were markedly inhibited. In contrast, endocytosis of patched receptor-ligand complexes was inhibited by azide and microtubule inhibitors, including colchicine, vinblastine and vincristine. Co-capping experiments performed to elucidate further the alterations in membrane configuration involved in these reactions failed to reveal any topographical relationship between transferrin receptors and lectin-binding sites in these cells. These studied indicate that temperature-dependent mobility of transferrin receptors upon mitogen-activated peripheral blood lymphocytes is dependent upon the integrity of the cytoskeletal system and metabolic function of the cell. PMID:6258830

  1. Remote Actuation of Magnetic Nanoparticles For Cancer Cell Selective Treatment Through Cytoskeletal Disruption.

    PubMed

    Master, Alyssa M; Williams, Philise N; Pothayee, Nikorn; Pothayee, Nipon; Zhang, Rui; Vishwasrao, Hemant M; Golovin, Yuri I; Riffle, Judy S; Sokolsky, Marina; Kabanov, Alexander V

    2016-09-20

    Motion of micron and sub-micron size magnetic particles in alternating magnetic fields can activate mechanosensitive cellular functions or physically destruct cancer cells. However, such effects are usually observed with relatively large magnetic particles (>250 nm) that would be difficult if at all possible to deliver to remote sites in the body to treat disease. Here we show a completely new mechanism of selective toxicity of superparamagnetic nanoparticles (SMNP) of 7 to 8 nm in diameter to cancer cells. These particles are coated by block copolymers, which facilitates their entry into the cells and clustering in the lysosomes, where they are then magneto-mechanically actuated by remotely applied alternating current (AC) magnetic fields of very low frequency (50 Hz). Such fields and treatments are safe for surrounding tissues but produce cytoskeletal disruption and subsequent death of cancer cells while leaving healthy cells intact.

  2. Remote Actuation of Magnetic Nanoparticles For Cancer Cell Selective Treatment Through Cytoskeletal Disruption

    PubMed Central

    Master, Alyssa M.; Williams, Philise N.; Pothayee, Nikorn; Pothayee, Nipon; Zhang, Rui; Vishwasrao, Hemant M.; Golovin, Yuri I.; Riffle, Judy S.; Sokolsky, Marina; Kabanov, Alexander V.

    2016-01-01

    Motion of micron and sub-micron size magnetic particles in alternating magnetic fields can activate mechanosensitive cellular functions or physically destruct cancer cells. However, such effects are usually observed with relatively large magnetic particles (>250 nm) that would be difficult if at all possible to deliver to remote sites in the body to treat disease. Here we show a completely new mechanism of selective toxicity of superparamagnetic nanoparticles (SMNP) of 7 to 8 nm in diameter to cancer cells. These particles are coated by block copolymers, which facilitates their entry into the cells and clustering in the lysosomes, where they are then magneto-mechanically actuated by remotely applied alternating current (AC) magnetic fields of very low frequency (50 Hz). Such fields and treatments are safe for surrounding tissues but produce cytoskeletal disruption and subsequent death of cancer cells while leaving healthy cells intact. PMID:27644858

  3. Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization

    PubMed Central

    Meddens, Marjolein B. M.; Pandzic, Elvis; Slotman, Johan A.; Guillet, Dominique; Joosten, Ben; Mennens, Svenja; Paardekooper, Laurent M.; Houtsmuller, Adriaan B.; van den Dries, Koen; Wiseman, Paul W.; Cambi, Alessandra

    2016-01-01

    Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area. PMID:27721497

  4. Cytoskeletal Regulation of Inflammation and Its Impact on Skin Blistering Disease Epidermolysis Bullosa Acquisita

    PubMed Central

    Kopecki, Zlatko; Ludwig, Ralf J.; Cowin, Allison J.

    2016-01-01

    Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune skin blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA. PMID:27420054

  5. Cytoskeletal Components of an Invasion Machine—The Apical Complex of Toxoplasma gondii

    PubMed Central

    Hu, Ke; Johnson, Jeff; Florens, Laurence; Fraunholz, Martin; Suravajjala, Sapna; DiLullo, Camille; Yates, John; Roos, David S; Murray, John M

    2006-01-01

    The apical complex of Toxoplasma gondii is widely believed to serve essential functions in both invasion of its host cells (including human cells), and in replication of the parasite. The understanding of apical complex function, the basis for its novel structure, and the mechanism for its motility are greatly impeded by lack of knowledge of its molecular composition. We have partially purified the conoid/apical complex, identified ~200 proteins that represent 70% of its cytoskeletal protein components, characterized seven novel proteins, and determined the sequence of recruitment of five of these proteins into the cytoskeleton during cell division. Our results provide new markers for the different subcompartments within the apical complex, and revealed previously unknown cellular compartments, which facilitate our understanding of how the invasion machinery is built. Surprisingly, the extreme apical and extreme basal structures of this highly polarized cell originate in the same location and at the same time very early during parasite replication. PMID:16518471

  6. Four-dimensional imaging of cytoskeletal dynamics in Xenopus oocytes and eggs.

    PubMed

    Bement, William M; Sokac, Anna M; Mandato, Craig A

    2003-12-01

    The Xenopus laevis (African clawed frog) system has long been popular for studies of both developmental and cell biology, based on a variety of its intrinsic features including the large size of Xenopus oocytes, eggs, and embryos, and the relative ease of manipulation. Unfortunately, the large size has also been considered a serious impediment for high-resolution light microscopy, as has the heavy pigmentation. However, the recent development and exploitation of 4D imaging approaches, and the fact that much of what is of most interest to cell and developmental biologists takes place near the cell surface, indicates that such concerns are no longer valid. Consequently, the Xenopus system in many respects is now as good as other model systems considered to be ideal for microscopy-based studies. Here, 4D imaging and its recent applications to cytoskeletal imaging in Xenopus oocytes and eggs are discussed.

  7. Cytoskeletal Rearrangements in Synovial Fibroblasts as a Novel Pathophysiological Determinant of Modeled Rheumatoid Arthritis

    PubMed Central

    Aidinis, Vassilis; Carninci, Piero; Armaka, Maria; Witke, Walter; Harokopos, Vaggelis; Pavelka, Norman; Koczan, Dirk; Argyropoulos, Christos; Thwin, Maung-Maung; Möller, Steffen; Kazunori, Waki; Gopalakrishnakone, Ponnampalam; Ricciardi-Castagnoli, Paola; Thiesen, Hans-Jürgen; Hayashizaki, Yoshihide; Kollias, George

    2005-01-01

    Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology–assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease. PMID:16254600

  8. Newly Designed Tensile Test System for in vitro Measurement of Mechanical Properties of Cytoskeletal Filaments

    NASA Astrophysics Data System (ADS)

    Deguchi, Shinji; Ohashi, Toshiro; Sato, Masaaki

    A tensile test system for isolated cytoskeletal filaments, which enables to control strain rate, was newly designed. A pair of piezo-driven cantilevers were used to manipulate the specimen and to measure tensile load from the deflection of one of the cantilevers. The displacements of the cantilevers were optically and electrically detected. The specimen strain, determined from the cantilever displacements, was used as a feedback signal. We proposed a servo-system for strain rate control in which a desired path for the strain transition was designated. The path was chosen as a triangular-shape waveform against time, along which the strain rate is kept constant. We measured tensile properties of a single stress fiber isolated from a smooth muscle cell with this system to obtain a stretching stiffness of 45nN per strain. Performance evaluation and the tensile test demonstrated that the system enabled to carry out strain rate-controlled tensile test.

  9. Cytoskeletal dynamics of the teleostean fin ray during fin epimorphic regeneration.

    PubMed

    Santos-Ruiz, Leonor; Santamaría, Jesús Alberto; Becerra, José

    2005-04-01

    Teleost fishes can regenerate their fins by epimorphic regeneration, a process that involves the transition of the formerly quiescent tissues of the stump to an active, growing state. This involves dynamic modifications of cell phenotype and behavior that must rely on alterations of the cytoskeleton. We have studied the spatial and temporal distribution of three main components of the cytoskeleton (actin, keratin and vimentin) in the regenerating fin, in order to establish putative relationships between cell cytoskeleton and cell behavior. According to our results, the massive rearrangement undergone by the epidermis right after injury, which takes place by cell migration, correlates with a transient down-regulation of keratin and a strong up-regulation of actin in the epidermal cells. During the subsequent epidermal growth, based on cell proliferation, keratin normal pattern is recovered while actin is down-regulated, although not to normal (quiescent) levels. The epidermal basal layer in contact with the blastema displays a particular cytoskeletal profile, different to that of the rest of the epidermal cells, which reflects its special features. In the connective tissue compartment, somatic cells do not contain vimentin, but keratin, as intermediate filament. Proliferative and migrative activation of these cells after injury correlates with actin up-regulation. Although this initial activation does not involve keratin down-regulation, blastemal cells were later observed to lack keratin, suggesting that such cytoskeletal modification might be needed for connective tissue cells to dedifferentiate and form the blastema. Cell differentiation in the newly formed, regenerated ray is accompanied by actin down-regulation and keratin up-regulation.

  10. Quinolinic acid induces disrupts cytoskeletal homeostasis in striatal neurons. Protective role of astrocyte-neuron interaction.

    PubMed

    Pierozan, Paula; Ferreira, Fernanda; de Lima, Bárbara Ortiz; Pessoa-Pureur, Regina

    2015-02-01

    Quinolinic acid (QUIN) is an endogenous metabolite of the kynurenine pathway involved in several neurological disorders. Among the several mechanisms involved in QUIN-mediated toxicity, disruption of the cytoskeleton has been demonstrated in striatally injected rats and in striatal slices. The present work searched for the actions of QUIN in primary striatal neurons. Neurons exposed to 10 µM QUIN presented hyperphosphorylated neurofilament (NF) subunits (NFL, NFM, and NFH). Hyperphosphorylation was abrogated in the presence of protein kinase A and protein kinase C inhibitors H89 (20 μM) and staurosporine (10 nM), respectively, as well as by specific antagonists to N-methyl-D-aspartate (50 µM DL-AP5) and metabotropic glutamate receptor 1 (100 µM MPEP). Also, intra- and extracellular Ca(2+) chelators (10 µM BAPTA-AM and 1 mM EGTA, respectively) and Ca(2+) influx through L-type voltage-dependent Ca(2+) channel (10 µM verapamil) are implicated in QUIN-mediated effects. Cells immunostained for the neuronal markers βIII-tubulin and microtubule-associated protein 2 showed altered neurite/neuron ratios and neurite outgrowth. NF hyperphosphorylation and morphological alterations were totally prevented by conditioned medium from QUIN-treated astrocytes. Cocultured astrocytes and neurons interacted with one another reciprocally, protecting them against QUIN injury. Cocultured cells preserved their cytoskeletal organization and cell morphology together with unaltered activity of the phosphorylating system associated with the cytoskeleton. This article describes cytoskeletal disruption as one of the most relevant actions of QUIN toxicity in striatal neurons in culture with soluble factors secreted by astrocytes, with neuron-astrocyte interaction playing a role in neuroprotection.

  11. The secretory mechanisms in equine platelets are independent of cytoskeletal polymerization and occur through membrane fusion.

    PubMed

    Brunso, L; Segura, D; Monreal, L; Escolar, G; White, J G; Diaz-Ricart, M

    2010-01-01

    Studies in animal models are useful to understand the basic mechanisms involved in hemostasis and the functional differences among species. Ultrastructural observations led us to predict differences in the activation and secretion mechanisms between equine and human platelets. The potential mechanisms involved have been comparatively explored in the present study. Equine and human platelets were activated with thrombin (0.5 U/ml) and collagen (20 µg/ml), for 90 seconds, and samples processed to evaluate: i) ultrastructural changes, by electron microscopy, ii) actin polymerization and cytoskeletal assembly, by polyacrylamide gel electrophoresis, and iii) specific molecules involved in activation and secretion, by western blot. In activated human platelets, centralization of granules, cytoskeletal assembly and fusion of granules with the open canalicular system were observed. In activated equine platelets, granules fused together forming an organelle chain that fused with the surface membrane and released its content directly outside the platelets. Human platelets responded to activation with actin polymerization and the assembly of other contractile proteins to the cytoskeleton. These events were almost undetectable in equine platelets. When exploring the involvement of the synaptosomal-associated protein-23 (SNAP-23), a known regulator of secretory granule/plasma membrane fusion events, it was present in both human and equine platelets. SNAP-23 was shown to be more activated in equine platelets than human platelets in response to activation, especially with collagen. Thus, there are significant differences in the secretion mechanisms between human and equine platelets. While in human platelets, activation and secretion of granules depend on mechanisms of internal contraction and membrane fusion, in equine platelets the fusion mechanisms seem to be predominant.

  12. Gamma-diketone axonopathy: analyses of cytoskeletal motors and highways in CNS myelinated axons.

    PubMed

    Zhang, Lihai; Gavin, Terrence; DeCaprio, Anthony P; LoPachin, Richard M

    2010-09-01

    2,5-Hexanedione (HD) intoxication is associated with axon atrophy that might be responsible for the characteristic gait abnormalities, hindlimb skeletal muscle weakness and other neurological deficits that accompany neurotoxicity. Although previous mechanistic research focused on neurofilament triplet proteins (NFL, NFM, NFH), other cytoskeletal targets are possible. Therefore, to identify potential non-NF protein targets, we characterized the effects of HD on protein-protein interactions in cosedimentation assays using microtubules and NFs prepared from spinal cord of rats intoxicated at different daily dose rates (175 and 400 mg/kg/day). Results indicate that HD did not alter the presence of alpha- or beta-tubulins in these preparations, nor were changes noted in the distribution of either anterograde (KIF1A, KIF3, KIF5) or retrograde (dynein) molecular motors. The cosedimentation of dynactin, a dynein-associated protein, also was not affected. Immunoblot analysis of microtubule-associated proteins (MAPs) in microtubule preparations revealed substantial reductions (45-80%) in MAP1A, MAP1B heavy chain, MAP2, and tau regardless of HD dose rate. MAP1B light chain content was not altered. Finally, HD intoxication did not influence native NF protein content in either preparation. As per previous research, microtubule and NF preparations were enriched in high-molecular weight NF species. However, these NF derivatives were common to both HD and control samples, suggesting a lack of pathognomonic relevance. These data indicate that, although motor proteins were not affected, HD selectively impaired MAP-microtubule binding, presumably through adduction of lysine residues that mediate such interactions. Given their critical role in cytoskeletal physiology, MAPs could represent a relevant target for the induction of gamma-diketone axonopathy.

  13. Pathways of Ca2+ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle

    PubMed Central

    Zhang, Bao-Ting; Whitehead, Nicholas P.; Gervasio, Othon L.; Reardon, Trent F.; Vale, Molly; Fatkin, Diane; Dietrich, Alexander; Yeung, Ella W.

    2012-01-01

    Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca2+ entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca2+ entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca2+ entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1. PMID:22461447

  14. Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

    PubMed Central

    Bonin, Sawyer R.; Gibeault, Sabrina; De Repentigny, Yves; Kothary, Rashmi

    2016-01-01

    Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as was myelin basic

  15. Magnolol inhibits migration of vascular smooth muscle cells via cytoskeletal remodeling pathway to attenuate neointima formation

    SciTech Connect

    Karki, Rajendra; Kim, Seong-Bin; Kim, Dong-Wook

    2013-12-10

    Background: Increased proliferation and migration of vascular smooth muscle cells (VSMCs) contribute importantly to the formation of both atherosclerotic and restenotic lesions. The objective of this study was to investigate the effect of magnolol on VSMC migration. Methods: The proteolytic activity of matrix metalloproteinases (MMPs) in tumor necrosis factor alpha (TNF-α) stimulated VSMCs was performed by gelatin zymography. VSMC migration was assessed by wound healing and Boyden chamber methods. Collagen induced VSMC adhesion was determined by spectrofluorimeter and stress fibers formation was evaluated by fluorescence microscope. The expression of signaling molecules involved in stress fibers formation was determined by western blot. The phosphorylation of myosin light chain (MLC20) was determined by urea-glycerol polyacrylamide gel electrophoresis. Immunohistochemistry was performed to determine the expression of β1-integrin and collagen type I in the injured carotid arteries of rats on day 35 after vascular injury. Results: VSMC migration was strongly inhibited by magnolol without affecting MMPs expression. Also, magnolol inhibited β1-integrin expression, FAK phosphorylation and RhoA and Cdc42 activation to inhibit the collagen induced stress fibers formation. Moreover, magnolol inhibited the phosphorylation of MLC20. Our in vivo results showed that magnolol inhibited β1-integrin expression, collagen type I deposition and FAK phosphorylation in injured carotid arteries without affecting MMP-2 activity. Conclusions: Magnolol inhibited VSMC migration via inhibition of cytoskeletal remodeling pathway to attenuate neointima formation. General significance: This study provides a rationale for further evaluation of magnolol for the management of atherosclerosis and restenosis. - Highlights: • Magnolol strongly inhibited migration of VSMCs. • Magnolol inhibited stress fibers formation. • MLC20 phosphorylation was also inhibited by magnolol. • Anti

  16. Stress and strain in the contractile and cytoskeletal filaments of airway smooth muscle.

    PubMed

    Deng, Linhong; Bosse, Ynuk; Brown, Nathan; Chin, Leslie Y M; Connolly, Sarah C; Fairbank, Nigel J; King, Greg G; Maksym, Geoffrey N; Paré, Peter D; Seow, Chun Y; Stephen, Newman L

    2009-10-01

    Stress and strain are omnipresent in the lung due to constant lung volume fluctuation associated with respiration, and they modulate the phenotype and function of all cells residing in the airways including the airway smooth muscle (ASM) cell. There is ample evidence that the ASM cell is very sensitive to its physical environment, and can alter its structure and/or function accordingly, resulting in either desired or undesired consequences. The forces that are either conferred to the ASM cell due to external stretching or generated inside the cell must be borne and transmitted inside the cytoskeleton (CSK). Thus, maintaining appropriate levels of stress and strain within the CSK is essential for maintaining normal function. Despite the importance, the mechanisms regulating/dysregulating ASM cytoskeletal filaments in response to stress and strain remained poorly understood until only recently. For example, it is now understood that ASM length and force are dynamically regulated, and both can adapt over a wide range of length, rendering ASM one of the most malleable living tissues. The malleability reflects the CSK's dynamic mechanical properties and plasticity, both of which strongly interact with the loading on the CSK, and all together ultimately determines airway narrowing in pathology. Here we review the latest advances in our understanding of stress and strain in ASM cells, including the organization of contractile and cytoskeletal filaments, range and adaptation of functional length, structural and functional changes of the cell in response to mechanical perturbation, ASM tone as a mediator of strain-induced responses, and the novel glassy dynamic behaviors of the CSK in relation to asthma pathophysiology.

  17. Electron Tomographic Analysis of Cytoskeletal Cross-Bridges in the Paranodal Region of the Node of Ranvier in Peripheral Nerves

    PubMed Central

    Perkins, Guy A.; Sosinsky, Gina E.; Ghassemzadeh, Sassan; Perez, Alex; Jones, Ying; Ellisman, Mark H.

    2008-01-01

    The node of Ranvier is a site for ionic conductances along myelinated nerves and governs the saltatory transmission of action potentials. Defects in the cross-bridging and spacing of the cytoskeleton is a prominent pathologic feature in diseases of the peripheral nerve. Electron tomography was used to examine cytoskeletal-cytoskeletal, membrane-cytoskeletal, and heterologous cell connections in the paranodal region of the node of Ranvier in peripheral nerves. Focal attachment of cytoskeletal filaments to each other and to the axolemma and paranodal membranes of the Schwann cell via narrow cross-bridges was visualized in both neuronal and glial cytoplasms. A subset of intermediate filaments associates with the cytoplasmic surfaces of supramolecular complexes of transmembrane structures that are presumed to include known and unknown junctional proteins. Mitochondria were linked to both microtubules and neurofilaments in the axoplasm and to neighboring smooth endoplasmic reticulum by narrow cross-bridges. Tubular cisternae in the glial cytoplasm were also linked to the paranodal glial cytoplasmic loop juxtanodal membrane by short cross-bridges. In the extracellular matrix between axon and Schwann cell, junctional bridges formed long cylinders inking the two membranes. Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region is likely critical not only for scaffolding, but also for intracellular and extracellular communication. PMID:18096402

  18. Genetic study of interactions between the cytoskeletal assembly protein sla1 and prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces cerevisiae.

    PubMed Central

    Bailleul, P A; Newnam, G P; Steenbergen, J N; Chernoff, Y O

    1999-01-01

    Striking similarities between cytoskeletal assembly and the "nucleated polymerization" model of prion propagation suggest that similar or overlapping sets of proteins may assist in both processes. We show that the C-terminal domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release factor Sup35 (eRF3) in the two-hybrid system. Sla1C and several other Sup35N-interacting proteins also exhibit two-hybrid interactions with the poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes Huntington disease-associated aggregation. The Sup35N-Sla1C interaction is inhibited by Sup35N alterations that make Sup35 unable to propagate the [PSI(+)] state and by the absence of the chaperone protein Hsp104, which is essential for [PSI] propagation. In a Sla1(-) background, [PSI] curing by dimethylsulfoxide or excess Hsp104 is increased, while translational readthrough and de novo [PSI] formation induced by excess Sup35 or Sup35N are decreased. These data show that, in agreement with the proposed function of Sla1 during cytoskeletal formation, Sla1 assists in [PSI] formation and propagation, but is not required for these processes. Sla1(-) strains are sensitive to some translational inhibitors, and some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1, suggesting that the interaction between Sla1 and Sup35 proteins may play a role in the normal function of the translational apparatus. We hypothesize that Sup35N is involved in regulatory interactions with intracellular structural networks, and [PSI] prion may be formed as a by-product of this process. PMID:10471702

  19. Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein.

    PubMed

    Hirokawa, N; Takemura, R; Hisanaga, S

    1985-11-01

    We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three-dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button-like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule-associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75-kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic

  20. Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

    SciTech Connect

    Huang, Xionggao; Wei, Yantao; Ma, Haizhi; Zhang, Shaochong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

  1. TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

    PubMed Central

    Schwingshackl, Andreas; Roan, Esra; Teng, Bin; Waters, Christopher M.

    2015-01-01

    Background TREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements. Methods We determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA. Results Cytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells. Conclusion Although cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the

  2. Espin cytoskeletal proteins in the sensory cells of rodent taste buds.

    PubMed

    Sekerková, Gabriella; Freeman, David; Mugnaini, Enrico; Bartles, James R

    2005-09-01

    Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 +/- 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 +/- 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP(3)R(3))(,) alpha-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP(3)R(3), PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP(3)R(3), alpha-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a

  3. Coordination of individual and ensemble cytoskeletal motors studied using tools from DNA nanotechnology

    NASA Astrophysics Data System (ADS)

    Derr, Nathan Dickson

    The cytoskeletal molecular motors kinesin-1 and cytoplasmic dynein drive many diverse functions within eukaryotic cells. They are responsible for numerous spatially and temporally dependent intracellular processes crucial for cellular activity, including cytokinesis, maintenance of sub-cellular organization and the transport of myriad cargos along microtubule tracks. Cytoplasmic dynein and kinesin-1 are processive, but opposite polarity, homodimeric motors; they each can take hundreds of thousands of consecutive steps, but do so in opposite directions along their microtubule tracks. These steps are fueled by the binding and hydrolysis of ATP within the homodimer's two identical protomers. Individual motors achieve their processivity by maintaining asynchrony between the stepping cycles of each protomer, insuring that at least one protomer always maintains contact with the track. How dynein coordinates the asynchronous stepping activity of its protomers is unknown. We developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers, using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we found that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use "hand-over-hand" mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying that a tension-based mechanism governs these steps. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of both cytoplasmic dynein and kinesin-1. To investigate the mechanisms that coordinate the interactions between motors within an ensemble, we constructed programmable synthetic cargos using three-dimensional DNA origami. This system

  4. The Drosophila TIPE family member Sigmar interacts with the Ste20-like kinase Misshapen and modulates JNK signaling, cytoskeletal remodeling and autophagy.

    PubMed

    Chittaranjan, Suganthi; Xu, Jing; Kuzyk, Michael; Dullat, Harpreet K; Wilton, James; DeVorkin, Lindsay; Lebovitz, Chandra; Morin, Gregg B; Marra, Marco A; Gorski, Sharon M

    2015-04-02

    TNFAIP8 and other mammalian TIPE family proteins have attracted increased interest due to their associations with disease-related processes including oncogenic transformation, metastasis, and inflammation. The molecular and cellular functions of TIPE family proteins are still not well understood. Here we report the molecular and genetic characterization of the Drosophila TNFAIP8 homolog, CG4091/sigmar. Previous gene expression studies revealed dynamic expression of sigmar in larval salivary glands prior to histolysis. Here we demonstrate that in sigmar loss-of-function mutants, the salivary glands are morphologically abnormal with defects in the tubulin network and decreased autophagic flux. Sigmar localizes subcellularly to microtubule-containing projections in Drosophila S2 cells, and co-immunoprecipitates with the Ste20-like kinase Misshapen, a regulator of the JNK pathway. Further, the Drosophila TNF ligand Eiger can induce sigmar expression, and sigmar loss-of-function leads to altered localization of pDJNK in salivary glands. Together, these findings link Sigmar to the JNK pathway, cytoskeletal remodeling and autophagy activity during salivary gland development, and provide new insights into TIPE family member function.

  5. The Drosophila TIPE family member Sigmar interacts with the Ste20-like kinase Misshapen and modulates JNK signaling, cytoskeletal remodeling and autophagy

    PubMed Central

    Chittaranjan, Suganthi; Xu, Jing; Kuzyk, Michael; Dullat, Harpreet K.; Wilton, James; DeVorkin, Lindsay; Lebovitz, Chandra; Morin, Gregg B.; Marra, Marco A.; Gorski, Sharon M.

    2015-01-01

    TNFAIP8 and other mammalian TIPE family proteins have attracted increased interest due to their associations with disease-related processes including oncogenic transformation, metastasis, and inflammation. The molecular and cellular functions of TIPE family proteins are still not well understood. Here we report the molecular and genetic characterization of the Drosophila TNFAIP8 homolog, CG4091/sigmar. Previous gene expression studies revealed dynamic expression of sigmar in larval salivary glands prior to histolysis. Here we demonstrate that in sigmar loss-of-function mutants, the salivary glands are morphologically abnormal with defects in the tubulin network and decreased autophagic flux. Sigmar localizes subcellularly to microtubule-containing projections in Drosophila S2 cells, and co-immunoprecipitates with the Ste20-like kinase Misshapen, a regulator of the JNK pathway. Further, the Drosophila TNF ligand Eiger can induce sigmar expression, and sigmar loss-of-function leads to altered localization of pDJNK in salivary glands. Together, these findings link Sigmar to the JNK pathway, cytoskeletal remodeling and autophagy activity during salivary gland development, and provide new insights into TIPE family member function. PMID:25836674

  6. The Ovary of Tubifex tubifex (Clitellata, Naididae, Tubificinae) Is Composed of One, Huge Germ-Line Cyst that Is Enriched with Cytoskeletal Components.

    PubMed

    Urbisz, Anna Z; Chajec, Łukasz; Świątek, Piotr

    2015-01-01

    Recent studies on the ovary organization and oogenesis in Tubificinae have revealed that their ovaries are small polarized structures that are composed of germ cells in subsequent stages of oogenesis that are associated with somatic cells. In syncytial cysts, as a rule, each germ cell is connected to the central cytoplasmic mass, the cytophore, via only one stable intercellular bridge (ring canal). In this paper we present detailed data about the composition of germ-line cysts in Tubifex tubifex with special emphasis on the occurrence and distribution of the cytoskeletal elements. Using fixed material and live cell imaging techniques, we found that the entire ovary of T. tubifex is composed of only one, huge multicellular germ-line cyst, which may contain up to 2,600 cells. Its architecture is broadly similar to the cysts that are found in other clitellate annelids, i.e. a common, anuclear cytoplasmic mass in the center of the cyst and germ cells that are connected to it via intercellular bridges. The cytophore in the T. tubifex cyst extends along the long axis of the ovary in the form of elongated and branched cytoplasmic strands. Rhodamine-coupled phalloidin staining revealed that the prominent strands of actin filaments occur inside the cytophore. Similar to the cytophore, F-actin strands are branched and they are especially well developed in the middle and outermost parts of the ovary. Microfilaments are also present in the ring canals that connect the germ cells with the cytophore in the narrow end of the ovary. Using TubulinTracker, we found that the microtubules form a prominent network of loosely and evenly distributed tubules inside the cytophore as well as in every germ cell. The well-developed cytoskeletal elements in T. tubifex ovary seem to ensure the integrity of such a huge germ-line cyst of complex (germ cells-ring canals-cytophore) organization. A comparison between the cysts that are described here and other well-known female germ-line cysts is

  7. Cytoskeletal reorganization by mycophenolic acid alters mesangial cell migration and contractility.

    PubMed

    Dubus, Isabelle; L'Azou, Beatrice; Gordien, Myriam; Delmas, Yahsou; Labouyrie, Jean-Pierre; Bonnet, Jacques; Combe, Christian

    2003-11-01

    Cytoskeleton alterations are a hallmark of mesangial cell activation during glomerulosclerosis. The aim of this study was to investigate whether mycophenolic acid (MPA) affects cytoskeletal organization and motility of human mesangial cells. Using the IP15 cell line, we found that treatment with 1 micromol/L MPA inhibited both receptor-dependent (angiotensin II) and receptor-independent (KCl) contractile responses, as well as serum-induced migration activity, suggesting alterations in the intracellular mechanisms that control mesangial cell motility. Immunofluorescence studies of MPA-treated cells provided evidence for decreased membrane disassembly/reassembly of alpha-smooth muscle actin and F-actin fibers, which was correlated with sustained quantitative and qualitative modifications of actin-associated proteins: calponin was overexpressed and became associated with actin fibers, whereas phosphorylation levels of cofilin and myosin light chain increased, suggesting both an activation of the mechanisms responsible for actin polymerization and an inhibition of actin-depolymerizing processes. These observations support a stabilizing effect of MPA on the mesangial actin cytoskeleton, which constitutes an additive action by which MPA, beyond its anti-inflammatory, antiproliferative and antifibrotic properties, might protect against excessive mesangial activation in the context of various glomerulopathies and kidney transplantation.

  8. Effects of fixation protocol and gravistimulation on cytoskeletal organization in Brassica rapa roots

    NASA Astrophysics Data System (ADS)

    Edge, Andrea; Hasenstein, Karl H.

    2012-07-01

    In preparation for a flight experiment we have studied the optimization of the staining protocols for microtubules and actin filaments in Brassica rapa seedlings. Microtubules (MT) were stained with monoclonal antibody (mAb) YOL 1/34. F-actin (FA) staining was achieved with C4 mAb antibody. Fixative prepared more than three weeks before use produces specimens that stained poorly. Storage in fixative for more than four weeks resulted in noticeably poorer staining. Staining was best in cortical cells but more difficult and less consistent in cap cells, especially for FA. In addition, the quality of staining of root cap cells was dependent on the age of the formaldehyde. The organization of the MTs corresponded with previously published descriptions; FA was prominent in the stele with thick and numerous parallel bundles; cortical cells showed less dense and less directional organization of mostly thinner filaments. FA organization was determined by tissue rather than by differential elongation. The organization of MTs in cortical cells of curving roots was uniformly circular and perpendicular to the long cell axis despite different cell length. The effect of clinorotation around the horizontal axis and centrifugation on the cytoskeletal organization was inconsistent. (Supported by NASA grant NNX10AP91G)

  9. Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ.

    PubMed

    Mateos-Gil, Pablo; Paez, Alfonso; Hörger, Ines; Rivas, Germán; Vicente, Miguel; Tarazona, Pedro; Vélez, Marisela

    2012-05-22

    We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (T(b) ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer-monomer interactions, regardless of the nucleotide present, can adopt a curved configuration.

  10. p21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics

    PubMed Central

    Allen, Jayme D.; Jaffer, Zahara M.; Park, Su-Jung; Burgin, Sarah; Hofmann, Clemens; Sells, Mary Ann; Chen, Shi; Derr-Yellin, Ethel; Michels, Elizabeth G.; McDaniel, Andrew; Bessler, Waylan K.; Ingram, David A.; Atkinson, Simon J.; Travers, Jeffrey B.

    2009-01-01

    Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcϵRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, Wsh/Wsh mast cell–deficient mice locally reconstituted with Pak1−/− bone marrow–derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1−/− BMMCs exhibited a reduction in FcϵRI-induced degranulation. Further, Pak1−/− BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcϵRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases. PMID:19124833

  11. Oxytocin Increases Neurite Length and Expression of Cytoskeletal Proteins Associated with Neuronal Growth.

    PubMed

    Lestanova, Z; Bacova, Z; Kiss, A; Havranek, T; Strbak, V; Bakos, J

    2016-06-01

    Neuropeptide oxytocin acts as a growth and differentiation factor; however, its effects on neurite growth are poorly understood. The aims of the present study were (1) to evaluate time effects of oxytocin on expression of nestin and MAP2; (2) to measure the effect of oxytocin on gene expression of β-actin, vimentin, cofilin, and drebrin; and (3) to measure changes in neurite length and number in response to oxytocin/oxytocin receptor antagonist L-371,257. Exposure of SH-SY5Y cells to 1 μM oxytocin resulted in a significant increase in gene expression and protein levels of nestin after 12, 24, and 48 h. Oxytocin treatment induced no changes in gene expression of MAP2; however, a decrease of protein levels was observed in all time intervals. Gene expression of β-actin, vimentin, and drebrin increased in response to oxytocin. Oxytocin induced significant elongation of neurites after 12, 24, and 48 h. No change in neurite length was observed in the presence of the combination of retinoic acid and oxytocin receptor antagonist L-371,257. Oxytocin treatment for 12 h increased the number of neurites. Overall, the present data suggest that oxytocin contributes to the regulation of expression of cytoskeletal proteins associated with growth of neuronal cones and induces neurite elongation mediated by oxytocin receptors at least in certain types of neuronal cells.

  12. Sex hormones and expression pattern of cytoskeletal proteins in the rat brain throughout pregnancy.

    PubMed

    González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; González-Flores, Oscar; Galván-Rosas, Agustín; Porfirio Gómora-Arrati; Camacho-Arroyo, Ignacio

    2014-01-01

    Pregnancy involves diverse changes in brain function that implicate a re-organization in neuronal cytoskeleton. In this physiological state, the brain is in contact with several hormones that it has never been exposed, as well as with very high levels of hormones that the brain has been in touch throughout life. Among the latter hormones are progesterone and estradiol which regulate several brain functions, including learning, memory, neuroprotection, and the display of sexual and maternal behavior. These functions involve changes in the structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity is regulated by estradiol and progesterone. We have found that the expression pattern of Microtubule Associated Protein 2, Tau, and Glial Fibrillary Acidic Protein changes in a tissue-specific manner in the brain of the rat throughout gestation and the start of lactation, suggesting that these proteins participate in the plastic changes observed in the brain during pregnancy. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

  13. Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ

    PubMed Central

    Mateos-Gil, Pablo; Paez, Alfonso; Hörger, Ines; Rivas, Germán; Vicente, Miguel; Tarazona, Pedro; Vélez, Marisela

    2012-01-01

    We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (Tb ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer–monomer interactions, regardless of the nucleotide present, can adopt a curved configuration. PMID:22566654

  14. Immunohistochemical Studies of Cytoskeletal and Extracellular Matrix Components in Dogfish Scyliorhinus canicula L. Notochordal Cells.

    PubMed

    Restović, Ivana; Vukojević, Katarina; Paladin, Antonela; Saraga-Babić, Mirna; Bočina, Ivana

    2015-10-01

    Immunofluorescence and immunohistochemical techniques were used to define the distribution of cytoskeletal (cytokeratin 8, vimentin) and extracellular matrix components (collagen type I, collagen type II, hyaluronic acid, and aggrecan) and bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) in the notochord of the lesser spotted dogfish Scyliorhinus canicula L. Immunolocalization of hyaluronic acid was observed in the notochord, vertebral centrum, and neural and hemal arches, while positive labeling to aggrecan was observed in the ossified centrum, notochord, and the perichondrium of the hyaline cartilage. Type I collagen was observed in the mineralized cartilage of the vertebral bodies, the notochord, the fibrocartilage of intervertebral disc, and the perichondrium. A positive labeling to type II collagen was observed in the inner part of the cartilaginous vertebral centrum and the notochord, as well as in the neural arch and muscle tissue, but there was no appreciable labeling of the hyaline cartilage. The presence of both BMP4 and BMP7 was seen in the mineralized vertebral centrum, notochordal cells, and neural arch. The notochordal cells expressed both cytokeratin 8 and vimentin, but predominantly vimentin. Hyaluronic acid, collagen type I, and collagen type II expression confirmed the presence of a mixture of notochordal and fibrocartilaginous tissue in the intervertebral disc, while BMPs confirmed the presence of an ossification in the cartilaginous skeleton of the spotted dogfish.

  15. Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial lateral membrane.

    PubMed

    Wu, H; Reuver, S M; Kuhlendahl, S; Chung, W J; Garner, C C

    1998-08-01

    The synapse-associated protein SAP97 is a member of a novel family of cortical cytoskeletal proteins involved in the localization of ion channels at such membrane specializations as synaptic junctions. These multidomain proteins have binding sites for protein 4.1, GKAPs/SAPAPs, voltage- and ligand-gated ion channels and cell-adhesion molecules containing C-terminal T/SXV motifs. In this study, we evaluated the contribution of individual domains in SAP97 to its selective recruitment and attachment to the cortical cytoskeleton in epithelial cells. We find that the PDZ, SH3 and GK domains, as well as the I3 insert in SAP97, are not essential for subcellular targeting, though both PDZ1-2 domains and the I3 insert affect the efficiency of localization. Instead, we show that the first 65 amino acid residues in SAP97, which are absent from SAP90/PSD-95 and SAP102, direct the selective subcellular localization and can mediate at least one point of attachment of SAP97 to the cytoskeleton assembled at sites of cell-cell contact. Our data demonstrate that it is the sequences unique to SAP97 that direct its subcellular targeting to the epithelial lateral membrane.

  16. [Contractile properties of fibers and cytoskeletal proteins of gerbil's hindlimb muscles after space flight].

    PubMed

    Lipets, E N; Ponomareva, E V; Ogneva, I V; Vikhliantsev, I M; Karaduleva, E V; Kratashkina, N L; Kuznetsov, S L; Podlubnaia, Z A; Shenkman, B S

    2009-01-01

    The work had the goal to compare the microgravity effects on gerbil's muscles-antagonists, m. soleus and m. tibialis anterior. The animals were exposed in 12-d space microgravity aboard Earth's artificial satellite "Foton-M3". Findings of the analysis of single skinned fibers contractility are 19.7% diminution of the diameter and 21.8% loss of the total contractive force of m. soleus fibers post flight. However, there was no significant difference in calcium sensitivity which agrees with the absence of changes in the relative content of several major cytoskeletal proteins (titin and nebulin ratios to heavy chains of myosin were identical in the flight and control groups) and a slight shifting of the myosin phenotype toward the "fast type" (9%, p < 0.05). These parameters were mostly unaffected by the space flight in m. tibialis anterior. To sum up, the decline of contractility and diminution of gerbil's myofibers after the space flight were less significant as compared with rats and did not impact the sytoskeletal protein ratios.

  17. Cytoskeletal architecture and its evolutionary significance in amoeboid eukaryotes and their mode of locomotion

    PubMed Central

    Williams, Jessica R.

    2016-01-01

    The cytoskeleton is the hallmark of eukaryotic evolution. The molecular and architectural aspects of the cytoskeleton have been playing a prominent role in our understanding of the origin and evolution of eukaryotes. In this study, we seek to investigate the cytoskeleton architecture and its evolutionary significance in understudied amoeboid lineages belonging to Amoebozoa. These amoebae primarily use cytoplasmic extensions supported by the cytoskeleton to perform important cellular processes such as movement and feeding. Amoeboid structure has important taxonomic significance, but, owing to techniques used, its potential significance in understanding diversity of the group has been seriously compromised, leading to an under-appreciation of its value. Here, we used immunocytochemistry and confocal microscopy to study the architecture of microtubules (MTs) and F-actin in diverse groups of amoebae. Our results demonstrate that all Amoebozoa examined are characterized by a complex cytoskeletal array, unlike what has been previously thought to exist. Our results not only conclusively demonstrate that all amoebozoans possess complex cytoplasmic MTs, but also provide, for the first time, a potential synapomorphy for the molecularly defined Amoebozoa clade. Based on this evidence, the last common ancestor of amoebozoans is hypothesized to have had a complex interwoven MT architecture limited within the granular cell body. We also generate several cytoskeleton characters related to MT and F-actin, which are found to be robust for defining groups in deep and shallow nodes of Amoebozoa. PMID:27703691

  18. Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells.

    PubMed

    Prentice-Mott, Harrison V; Meroz, Yasmine; Carlson, Andreas; Levine, Michael A; Davidson, Michael W; Irimia, Daniel; Charras, Guillaume T; Mahadevan, L; Shah, Jagesh V

    2016-02-02

    Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations associated with low chemical concentrations and dynamically changing gradients as well as high saturating chemical concentrations. Although a number of reports have identified cellular behavior consistent with a directional memory that could account for behavior in these complex environments, the quantitative and molecular details of such a memory process remain unknown. Using microfluidics to confine cellular motion to a 1D channel and control chemoattractant exposure, we observed directional memory in chemotactic neutrophil-like cells. We modeled this directional memory as a long-lived intracellular asymmetry that decays slower than observed membrane phospholipid signaling. Measurements of intracellular dynamics revealed that moesin at the cell rear is a long-lived element that when inhibited, results in a reduction of memory. Inhibition of ROCK (Rho-associated protein kinase), downstream of RhoA (Ras homolog gene family, member A), stabilized moesin and directional memory while depolymerization of microtubules (MTs) disoriented moesin deposition and also reduced directional memory. Our study reveals that long-lived polarized cytoskeletal structures, specifically moesin, actomyosin, and MTs, provide a directional memory in neutrophil-like cells even as they respond on short time scales to external chemical cues.

  19. TRPV4 regulates calcium homeostasis, cytoskeletal remodeling, conventional outflow and intraocular pressure in the mammalian eye

    PubMed Central

    Ryskamp, Daniel A.; Frye, Amber M.; Phuong, Tam T. T.; Yarishkin, Oleg; Jo, Andrew O.; Xu, Yong; Lakk, Monika; Iuso, Anthony; Redmon, Sarah N.; Ambati, Balamurali; Hageman, Gregory; Prestwich, Glenn D.; Torrejon, Karen Y.; Križaj, David

    2016-01-01

    An intractable challenge in glaucoma treatment has been to identify druggable targets within the conventional aqueous humor outflow pathway, which is thought to be regulated/dysregulated by elusive mechanosensitive protein(s). Here, biochemical and functional analyses localized the putative mechanosensitive cation channel TRPV4 to the plasma membrane of primary and immortalized human TM (hTM) cells, and to human and mouse TM tissue. Selective TRPV4 agonists and substrate stretch evoked TRPV4-dependent cation/Ca2+ influx, thickening of F-actin stress fibers and reinforcement of focal adhesion contacts. TRPV4 inhibition enhanced the outflow facility and lowered perfusate pressure in biomimetic TM scaffolds populated with primary hTM cells. Systemic delivery, intraocular injection or topical application of putative TRPV4 antagonist prodrug analogs lowered IOP in glaucomatous mouse eyes and protected retinal neurons from IOP-induced death. Together, these findings indicate that TRPV4 channels function as a critical component of mechanosensitive, Ca2+-signaling machinery within the TM, and that TRPV4-dependent cytoskeletal remodeling regulates TM stiffness and outflow. Thus, TRPV4 is a potential IOP sensor within the conventional outflow pathway and a novel target for treating ocular hypertension. PMID:27510430

  20. Cytoskeletal and functional changes in bioreactor assembled thyroid tissue organoids exposed to gamma radiation

    NASA Technical Reports Server (NTRS)

    Green, Lora M.; Patel, Zarana; Murray, Deborah K.; Rightnar, Steven; Burell, Cheryl G.; Gridley, Daila S.; Nelson, Gregory A.

    2002-01-01

    Fischer rat thyroid cells were grown under low-shear stress in a bioreactor to a stage of organization composed of integrated follicles resembling small thyroid glands prior to exposure to 3 Gray-gamma radiation. Bioreactor tissues and controls (both irradiated and non-irradiated) were harvested at 24, 48, 96 and 144 hours post-exposure. Tissue samples were fixed and fluorescently labeled for actin and microtubules. Tissues were assessed for changes in cytoskeletal components induced by radiation and quantified by laser scanning cytometry. ELISA's were used to quantify transforming growth factor-beta and thyroxin released from cells to the culture supernatant. Tissue architecture was disrupted by exposure to radiation with the structural organization of actin and loss of follicular content the most obviously affected. With time post-irradiation the actin appeared disordered and the levels of fluorescence associated with filamentous-actin and microtubules cycled in the tissue analogs, but not in the flask-grown cultures. Active transforming growth factor-beta was higher in supernatants from the irradiated bioreactor tissue. Thyroxin release paralleled cell survival in the bioreactors and control cultures. Thus, the engineered tissue responses to radiation differed from those of conventional tissue culture making it a potentially better mimic of the in vivo situation.

  1. Organization of cytoskeletal elements and organelles preceding growth cone emergence from an identified neuron in situ

    PubMed Central

    1989-01-01

    The purpose of this study was to investigate the arrangement of cytoskeletal elements and organelles in an identified neuron in situ at the site of emergence of its growth cone just before and concurrent with the onset of axonogenesis. The Ti1 pioneer neurons are the first pair of afferent neurons to differentiate in embryonic grasshopper limbs. They arise at the distal tip of the limb bud epithelium, the daughter cells of a single precursor cell, the Pioneer Mother Cell (PMC). Using immunohistochemical markers, we characterized the organization of microtubules, centrosomes, Golgi apparatus, midbody, actin filaments, and chromatin from mitosis in the PMC through axonogenesis in the Tils. Just before and concurrent with the onset of axonogenesis, a characteristic arrangement of tubulin, actin filaments, and Golgi apparatus is localized at the proximal pole of the proximal pioneer neuron. The growth cone of the proximal cell stereotypically arises from this site. Although the distal cell's axon generally grows proximally, occasionally it arises from its distal pole; in such limbs, the axons from the sister cells extend from mirror symmetric locations on their somata. In the presence of cytochalasin D, the PMC undergoes nuclear division but not cytokinesis and although other neuronal phenotypes are expressed, axongenesis is inhibited. Our data suggest that intrinsic information determines the site of growth cone emergence of an identified neuron in situ. PMID:2654140

  2. Compare and contrast the reaction coordinate diagrams for chemical reactions and cytoskeletal force generators

    PubMed Central

    Scholey, Jonathan M.

    2013-01-01

    Reaction coordinate diagrams are used to relate the free energy changes that occur during the progress of chemical processes to the rate and equilibrium constants of the process. Here I briefly review the application of these diagrams to the thermodynamics and kinetics of the generation of force and motion by cytoskeletal motors and polymer ratchets as they mediate intracellular transport, organelle dynamics, cell locomotion, and cell division. To provide a familiar biochemical context for discussing these subcellular force generators, I first review the application of reaction coordinate diagrams to the mechanisms of simple chemical and enzyme-catalyzed reactions. My description of reaction coordinate diagrams of motors and polymer ratchets is simplified relative to the rigorous biophysical treatment found in many of the references that I use and cite, but I hope that the essay provides a valuable qualitative representation of the physical chemical parameters that underlie the generation of force and motility at molecular scales. In any case, I have found that this approach represents a useful interdisciplinary framework for understanding, researching, and teaching the basic molecular mechanisms by which motors contribute to fundamental cell biological processes. PMID:23408787

  3. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    PubMed

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.

  4. A useful programme in BASIC for axonal morphometry with introduction of new cytoskeletal parameters.

    PubMed

    Fernández, E; Cuenca, N; De Juan, J

    1991-10-01

    Interest in the structure of axons and quantification of their components has been growing over the last years. However, the existing literature contains few reports of available computer programmes to facilitate such studies. This paper presents a fully comprehensive BASIC programme for the morphometric analysis of electron micrographs of cross-sectional nerve fibres. From drawings of fibre and axonal contours and dots of the microtubules and neurofilaments, the programme calculates the following parameters: area, diameter and form factor of the fibres and axons, number and density of microtubules and neurofilaments, proportion between microtubules and neurofilaments (R-proportion), myelin thickness and the diameter of the axon relative to its sheath (g-ratio). The programme also introduces three new parameters to analyse the degree of uniformity of microtubule and neurofilament distribution: distances between microtubules and between neurofilaments, equilateral index and cytoskeletal intermingling index. The programme is written in Microsoft BASIC Interpreter for Apple Macintosh (Microsoft Corporation) but can be used on other computers. Although the programme has been tested on adult rat optic nerve fibres, it can be used for different projects concerning axonal morphometry.

  5. The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption.

    PubMed

    Murphy, Kaitlin C; Morgan, Joshua T; Wood, Joshua A; Sadeli, Adeline; Murphy, Christopher J; Russell, Paul

    2014-10-15

    The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 min. We hypothesize that changes in actin morphology correlate with alterations in cell stiffness induced by Lat-B but this is not a general consequence of other cytoskeletal disrupting agents such as Rho kinase inhibitors. We treated HTM cells with 2 µM Lat-B or 100 µM Y-27632 and allowed the cells to recover for 30-270 min. While examining actin morphology in Lat-B treated cells, we observed striking cortical actin arrays (CAAs). The percentage of CAA positive cells (CPCs) was time dependent and exceeded 30% at 90 min and decreased after 270 min. Y-27632 treated cells exhibited few CAAs and no changes in cell stiffness. Together, these data suggest that the increase in cell stiffness after Lat-B treatment is correlated with CAAs.

  6. Coronin1 Proteins Dictate Rac1 Intracellular Dynamics and Cytoskeletal Output

    PubMed Central

    Ojeda, Virginia; Castro-Castro, Antonio

    2014-01-01

    Rac1 regulates lamellipodium formation, myosin II-dependent contractility, and focal adhesions during cell migration. While the spatiotemporal assembly of those processes is well characterized, the signaling mechanisms involved remain obscure. We report here that the cytoskeleton-related Coronin1A and -1B proteins control a myosin II inactivation-dependent step that dictates the intracellular dynamics and cytoskeletal output of active Rac1. This step is signaling-branch specific, since it affects the functional competence of active Rac1 only when forming complexes with downstream ArhGEF7 and Pak proteins in actomyosin-rich structures. The pathway is used by default unless Rac1 is actively rerouted away from the structures by upstream activators and signals from other Rho GTPases. These results indicate that Coronin1 proteins are at the center of a regulatory hub that coordinates Rac1 activation, effector exchange, and the F-actin organization state during cell signaling. Targeting this route could be useful to hamper migration of cancer cells harboring oncogenic RAC1 mutations. PMID:24980436

  7. Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16

    PubMed Central

    Maruthappu, Thiviyani; Chikh, Anissa; Fell, Benjamin; Delaney, Paul J.; Brooke, Matthew A.; Levet, Clemence; Moncada-Pazos, Angela; Ishida-Yamamoto, Akemi; Blaydon, Diana; Waseem, Ahmad; Leigh, Irene M.; Freeman, Matthew; Kelsell, David P.

    2017-01-01

    Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2−/− mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this ‘stress' keratin is regulated. PMID:28128203

  8. Mechanical diagnosis of human erythrocytes by ultra-high speed manipulation unraveled critical time window for global cytoskeletal remodeling.

    PubMed

    Ito, Hiroaki; Murakami, Ryo; Sakuma, Shinya; Tsai, Chia-Hung Dylan; Gutsmann, Thomas; Brandenburg, Klaus; Pöschl, Johannes M B; Arai, Fumihito; Kaneko, Makoto; Tanaka, Motomu

    2017-02-24

    Large deformability of erythrocytes in microvasculature is a prerequisite to realize smooth circulation. We develop a novel tool for the three-step "Catch-Load-Launch" manipulation of a human erythrocyte based on an ultra-high speed position control by a microfluidic "robotic pump". Quantification of the erythrocyte shape recovery as a function of loading time uncovered the critical time window for the transition between fast and slow recoveries. The comparison with erythrocytes under depletion of adenosine triphosphate revealed that the cytoskeletal remodeling over a whole cell occurs in 3 orders of magnitude longer timescale than the local dissociation-reassociation of a single spectrin node. Finally, we modeled septic conditions by incubating erythrocytes with endotoxin, and found that the exposure to endotoxin results in a significant delay in the characteristic transition time for cytoskeletal remodeling. The high speed manipulation of erythrocytes with a robotic pump technique allows for high throughput mechanical diagnosis of blood-related diseases.

  9. Mechanical diagnosis of human erythrocytes by ultra-high speed manipulation unraveled critical time window for global cytoskeletal remodeling

    NASA Astrophysics Data System (ADS)

    Ito, Hiroaki; Murakami, Ryo; Sakuma, Shinya; Tsai, Chia-Hung Dylan; Gutsmann, Thomas; Brandenburg, Klaus; Pöschl, Johannes M. B.; Arai, Fumihito; Kaneko, Makoto; Tanaka, Motomu

    2017-02-01

    Large deformability of erythrocytes in microvasculature is a prerequisite to realize smooth circulation. We develop a novel tool for the three-step “Catch-Load-Launch” manipulation of a human erythrocyte based on an ultra-high speed position control by a microfluidic “robotic pump”. Quantification of the erythrocyte shape recovery as a function of loading time uncovered the critical time window for the transition between fast and slow recoveries. The comparison with erythrocytes under depletion of adenosine triphosphate revealed that the cytoskeletal remodeling over a whole cell occurs in 3 orders of magnitude longer timescale than the local dissociation-reassociation of a single spectrin node. Finally, we modeled septic conditions by incubating erythrocytes with endotoxin, and found that the exposure to endotoxin results in a significant delay in the characteristic transition time for cytoskeletal remodeling. The high speed manipulation of erythrocytes with a robotic pump technique allows for high throughput mechanical diagnosis of blood-related diseases.

  10. Linking TGF-beta-mediated Cdc25A inhibition and cytoskeletal regulation through RhoA/p160(ROCK) signaling.

    PubMed

    Brown, Kimberly; Bhowmick, Neil A

    2004-04-01

    Transforming growth factor-beta (TGF-beta) can mediate G(1)/S cell-cycle inhibition and changes in the cytoskeletal organization through multiple parallel downstream signaling pathways. Recent findings regarding TGF-beta-mediated cell-cycle checkpoint control and epithelial to mesenchymal transition have converged to the RhoA/p160(ROCK) signaling pathway. The activation of TGF-beta-mediated p160(ROCK)rapidly inhibits the Cdc25A phosphatase as a component of the G(1)/S checkpoint control at the time cytoskeletal re-organization occurs. This can be likened to the ability to preserve genomic integrity in circumstances of genotoxic stress. The inactivation of the RhoA/p160(ROCK) pathway may be a mechanism by which cancer cells bypass growth inhibition even in the presence of TGF-beta.

  11. Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture.

    PubMed

    Ciesielski-Treska, J; Ulrich, G; Mensch, C; Aunis, D

    1984-01-01

    One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [(35)S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.

  12. Ginsenoside Rg1 Protects against Oxidative Stress-induced Neuronal Apoptosis through Myosin IIA-actin Related Cytoskeletal Reorganization

    PubMed Central

    Wang, Yan; Liu, Qian; Xu, Yingqiong; Zhang, Yuanyuan; Lv, Yanni; Tan, Yisha; Jiang, Nan; Cao, Guosheng; Ma, Xiaonan; Wang, Jingrong; Cao, Zhengyu; Yu, Boyang; Kou, Junping

    2016-01-01

    Oxidative stress-induced cytoskeletal dysfunction of neurons has been implicated as a crucial cause of cell apoptosis or death in the central nervous system (CNS) diseases, such as neurodegenerative and psychiatric diseases. The application of neuroprotectants rescuing the neurons from cytoskeletal damage and apoptosis can be a potential treatment for these CNS diseases. Ginsenoside Rg1 (Rg1), one of the major active components of ginseng, has been reported possessing notable neuroprotective activities. However, there is rare report about its effect on cytoskeleton and its undergoing mechanism. The current study is to reveal the regulatory effects of Rg1 on cytoskeletal and morphological lesion in oxidative stress-induced neuronal apoptosis. The results demonstrated that pre-treatment with Rg1 (0.1-10 μM) attenuated hydrogen peroxide (H2O2)-induced neuronal apoptosis and oxidative stress through reducing the intracellular reactive oxygen species (ROS) production and methane dicarboxylic aldehyde (MDA) level. The Rg1 treatment also abolished H2O2-induced morphological changes, including cell rounding, membrane blebbing, neurite retraction and nuclei condensation, which were generated by myosin IIA-actin interaction. These effects were mediated via the down-regulation of caspase-3, ROCK1 (Rho-associated kinase1) activation and myosin light chain (MLC, Ser-19) phosphorylation. Furthermore, inhibiting myosin II activity with blebbistatin partly blocked the neuroprotective effects of Rg1. The computer-aided homology modelling revealed that Rg1 preferentially positioned in the actin binding cleft of myosin IIA and might block the binding of myosin IIA to actin filaments. Accordingly, the neuroprotective mechanism of Rg1 is related to the activity that inhibits myosin IIA-actin interaction and the caspase-3/ROCK1/MLC signaling pathway. These findings put some insights into the unique neuroprotective properties of Rg1 associated with the regulation of myosin IIA

  13. Cytoskeletal polarization of T cells is regulated by an immunoreceptor tyrosine-based activation motif-dependent mechanism.

    PubMed

    Lowin-Kropf, B; Shapiro, V S; Weiss, A

    1998-02-23

    Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell-cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex-dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-zeta and CD3epsilon, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-zeta and once in CD3epsilon, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling

  14. Heterogeneous Porphyromonas gingivalis LPS modulates immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in human gingival fibroblasts

    PubMed Central

    Herath, Thanuja D. K.; Darveau, Richard P.; Seneviratne, Chaminda J.; Wang, Cun-Yu; Wang, Yu; Jin, Lijian

    2016-01-01

    Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis. PMID:27538450

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

    PubMed

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

    2009-01-01

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

  16. PDZD8 is a novel moesin-interacting cytoskeletal regulatory protein that suppresses infection by herpes simplex virus type 1.

    PubMed

    Henning, Matthew S; Stiedl, Patricia; Barry, Denis S; McMahon, Robert; Morham, Scott G; Walsh, Derek; Naghavi, Mojgan H

    2011-07-05

    The host cytoskeleton plays a central role in the life cycle of many viruses yet our knowledge of cytoskeletal regulators and their role in viral infection remains limited. Recently, moesin and ezrin, two members of the ERM (Ezrin/Radixin/Moesin) family of proteins that regulate actin and plasma membrane cross-linking and microtubule (MT) stability, have been shown to inhibit retroviral infection. To further understand how ERM proteins function and whether they also influence infection by other viruses, we identified PDZD8 as a novel moesin-interacting protein. PDZD8 is a poorly understood protein whose function is unknown. Exogenous expression of either moesin or PDZD8 reduced the levels of stable MTs, suggesting that these proteins functioned as part of a cytoskeletal regulatory complex. Additionally, exogenous expression or siRNA-mediated knockdown of either factor affected Herpes Simplex Virus type 1 (HSV-1) infection, identifying a cellular function for PDZD8 and novel antiviral properties for these two cytoskeletal regulatory proteins.

  17. Progressive supranuclear palsy: neuronal and glial cytoskeletal pathology in the higher order processing autonomic nuclei of the lower brainstem.

    PubMed

    Rüb, U; Del Tredici, K; Schultz, C; de Vos, R A I; Jansen Steur, E N H; Arai, K; Braak, H

    2002-02-01

    The medial and lateral parabrachial nuclei (MPB, LPB), the gigantocellular reticular nucleus (GI), the raphes magnus (RMG) and raphes obscurus nuclei (ROB), as well as the intermediate reticular zone (IRZ) represent pivotal subordinate brainstem centres, all of which control autonomic functions. In this study, we investigated the occurrence and severity of the neuronal and glial cytoskeletal pathology in these six brainstem nuclei from 17 individuals with clinically diagnosed and neuropathologically confirmed progressive supranuclear palsy (PSP). The association between the severity of the pathology and the duration of the disease was investigated by means of correlation analysis. The brainstem nuclei in all of the PSP cases were affected by the neuronal cytoskeletal pathology, with the IRZ and GI regularly showing severe involvement, the MPB, RMG, and ROB marked involvement, and the LPB mild involvement. In the six nuclear greys studied, glial cells undergo alterations of their cytoskeleton on an irregular basis, whereby diseased oligodendrocytes predominantly presented as coiled bodies and affected astrocytes as thorn-shaped astrocytes. In all six nuclei, the severity of the neuronal or glial cytoskeletal pathology showed no correlation with the duration of PSP. In view of their functional role, the neuronal pathology in the nuclei studied offers a possible explanation for the autonomic dysfunctions that eventually develop in the course of PSP.

  18. Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture.

    PubMed

    Jorgens, Danielle M; Inman, Jamie L; Wojcik, Michal; Robertson, Claire; Palsdottir, Hildur; Tsai, Wen-Ting; Huang, Haina; Bruni-Cardoso, Alexandre; López, Claudia S; Bissell, Mina J; Xu, Ke; Auer, Manfred

    2017-01-01

    The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.

  19. Cytoskeletal disruption activates the DLK/JNK pathway, which promotes axonal regeneration and mimics a preconditioning injury

    PubMed Central

    Valakh, Vera; Frey, Erin; Babetto, Elisabetta; Walker, Lauren J; DiAntonio, Aaron

    2015-01-01

    Nerve injury can lead to axonal regeneration, axonal degeneration, and/or neuronal cell death. Remarkably, the MAP3K dual leucine zipper kinase, DLK, promotes each of these responses, suggesting that DLK is a sensor of axon injury. In Drosophila, mutations in proteins that stabilize the actin and microtubule cytoskeletons activate the DLK pathway, suggesting that DLK may be activated by cytoskeletal disruption. Here we test this model in mammalian sensory neurons. We find that pharmacological agents designed to disrupt either the actin or microtubule cytoskeleton activate the DLK pathway, and that activation is independent of calcium influx or induction of the axon degeneration program. Moreover, activation of the DLK pathway by targeting the cytoskeleton induces a pro-regenerative state, enhancing axon regeneration in response to a subsequent injury in a process akin to preconditioning. This highlights the potential utility of activating the DLK pathway as a method to improve axon regeneration. Moreover, DLK is required for these responses to cytoskeletal perturbations, suggesting that DLK functions as a key neuronal sensor of cytoskeletal damage. PMID:25726747

  20. Flat cells come full sphere: Are mutant cytoskeletal-related proteins oncoprotein-monsters or useful immunogens?

    PubMed

    Parry, Michele L; Blanck, George

    2016-01-01

    Osteogenesis imperfecta is inherited as a dominant disease because if one allele is mutated, it contributes a mutant, destructive subunit polypeptide to collagen, which requires many subunits to form normal, polymeric, collagenous structures. Recent cancer genome atlas (TCGA) data indicate that cytoskeletal-related proteins are among the most commonly mutated proteins in human cancers, in distinct mutation frequency groups, i.e., including low mutation frequency groups. Part of the explanation for this observation is likely to be the fact that many of the coding regions for these proteins are very large, and indeed, it is likely these coding regions are mutated in many cells that never become cancerous. However, it would not be surprising if mutations in cytoskeletal proteins, when combined with oncoprotein or tumor suppressor protein mutations, had significant impacts on cancer development, for a number of reasons, including results obtained almost 5 decades ago indicating that well-spread cells in tissue culture, with well-formed cytoskeletons, were less tumorigenic than spherical cells with disrupted cytoskeletons. This raises the question, are mutant cytoskeletal proteins, which would likely interfere with polymer formation, a new class of oncoproteins, in particular, dominant negative oncoproteins? If these proteins are so commonly mutant, could they be the bases for common cancer vaccines?

  1. [Cortical cytoskeletal ring in prophase II leads to correction of abnormalities of the first meiotic division and to meiotic restitution of pollen mother cell nucleus].

    PubMed

    Shamina, N V; Zaporozhchenko, I A; Maksiutova, Iu R; Shatskaia, O A

    2007-01-01

    The deviation of prophase cytoskeletal ring formation was determined during meiotic division in 50% of pollen mother cells (PMCs) in maize haploid No 1498 (Zea mays). At prophase in both meiotic divisions the cytoskeletal ring is formed in cortical region of cytoplasm instead of perinuclear. Sometimes formation of both perinuclear and cortical rings is observed in the same cell. It has been shown that in multinucleate PMCs the cortical ring leads to the consolidation of chromosomes into common spindle and to meiotic restitution.

  2. Neuroprotective effects of hypothermia on synaptic actin cytoskeletal changes induced by perinatal asphyxia.

    PubMed

    Muñiz, Javier; Romero, Juan; Holubiec, Mariana; Barreto, George; González, Janneth; Saint-Martin, Madeleine; Blanco, Eduardo; Carlos Cavicchia, Juan; Castilla, Rocío; Capani, Francisco

    2014-05-14

    Cerebral hypoxia-ischemia damages synaptic proteins, resulting in cytoskeletal alterations, protein aggregation and neuronal death. In the previous works, we have shown neuronal and synaptic changes in rat neostriatum subjected to hypoxia that leads to ubi-protein accumulation. Recently, we also showed that, changes in F-actin organization could be related to early alterations induced by hypoxia in the Central Nervous System. However, little is known about effective treatment to diminish the damage. The main aim of this work is to study the effects of birth hypothermia on the actin cytoskeleton of neostriatal post-synaptic densities (PSD) in 60 days olds rats by immunohistochemistry, photooxidation and western blot. We used 2 different protocols of hypothermia: (a) intrahypoxic hypothermia at 15°C and (b) post-hypoxia hypothermia at 32°C. Consistent with previous data at 30 days, staining with phalloidin-Alexa(488) followed by confocal microscopy analysis showed an increase of F-actin fluorescent staining in the neostriatum of hypoxic animals. Correlative photooxidation electron microscopy confirmed these observations showing an increment in the number of mushroom-shaped F-actin staining spines in neostriatal excitatory synapses in rats subjected to hypoxia. In addition, western blot revealed β-actin increase in PSDs in hypoxic animals. The optic relative density measurement showed a significant difference between controls and hypoxic animals. When hypoxia was induced under hypothermic conditions, the changes observed in actin cytoskeleton were blocked. Post-hypoxic hypothermia showed similar answer but actin cytoskeleton modifications were not totally reverted as we observed at 15°C. These data suggest that the decrease of the body temperature decreases the actin modifications in dendritic spines preventing the neuronal death.

  3. Abl2/Arg controls dendritic spine and dendrite arbor stability via distinct cytoskeletal control pathways.

    PubMed

    Lin, Yu-Chih; Yeckel, Mark F; Koleske, Anthony J

    2013-01-30

    Rho family GTPases coordinate cytoskeletal rearrangements in neurons, and mutations in their regulators are associated with mental retardation and other neurodevelopmental disorders (Billuart et al., 1998; Kutsche et al., 2000; Newey et al., 2005; Benarroch, 2007). Chromosomal microdeletions encompassing p190RhoGAP or its upstream regulator, the Abl2/Arg tyrosine kinase, have been observed in cases of mental retardation associated with developmental defects (Scarbrough et al., 1988; James et al., 1996; Takano et al., 1997; Chaabouni et al., 2006; Leal et al., 2009). Genetic knock-out of Arg in mice leads to synapse, dendritic spine, and dendrite arbor loss accompanied by behavioral deficits (Moresco et al., 2005; Sfakianos et al., 2007). To elucidate the cell-autonomous mechanisms by which Arg regulates neuronal stability, we knocked down Arg in mouse hippocampal neuronal cultures. We find that Arg knockdown significantly destabilizes dendrite arbors and reduces dendritic spine density by compromising dendritic spine stability. Inhibiting RhoA prevents dendrite arbor loss following Arg knockdown in neurons, but does not block spine loss. Interestingly, Arg-deficient neurons exhibit increased miniature EPSC amplitudes, and their remaining spines exhibit larger heads deficient in the actin stabilizing protein cortactin. Spine destabilization in Arg knockdown neurons is prevented by blocking NMDA receptor-dependent relocalization of cortactin from spines, or by forcing cortactin into spines via fusion to an actin-binding region of Arg. Thus, Arg employs distinct mechanisms to selectively regulate spine and dendrite stability: Arg dampens activity-dependent disruption of cortactin localization to stabilize spines and attenuates Rho activity to stabilize dendrite arbors.

  4. Effect of different methods of cryopreservation on the cytoskeletal integrity of dromedary camel (Camelus dromedarius) embryos.

    PubMed

    Skidmore, J A; Schoevers, E; Stout, T A E

    2009-07-01

    This study examined the effect of different methods of cryopreservation on the cytoskeletal integrity of camel embryos. A total of 32 embryos were recovered on Days 6 and 7 after ovulation and measured before being frozen using either a conventional slow-cooling technique (n=12: six Day 6 and six Day 7 embryos) or vitrification (n=12: four Day 6 and eight Day 7). The remaining 8 'control' embryos (four Day 6 and four Day 7) were not cryopreserved but instead incubated in holding medium for 30 min. After thawing, warming or incubation, the embryos were stained with 4,6-diamino-2-phenylindole dihydrochloride (DAPI) to identify dead cells. Subsequently, the embryos were fixed in 4% paraformaldehyde, permeabilized and labelled with Alexa Fluor 488-Phalloidin to enable assessment of cytoskeleton integrity. Vitrified-warmed embryos contained a significantly higher percentage of dead cells than either conventionally frozen embryos or controls (P<0.05). Although the proportion of dead cells in conventionally frozen embryos tended to be higher than in controls, the difference was not significant (P> or =0.07). Whereas embryo size did not affect the number of dead cells in conventionally frozen embryos, vitrified-warmed embryos >300 microm in diameter had a significantly higher percentage of dead cells than embryos < or =300 microm (P=0.01). Cytoskeleton integrity was also affected by both freezing method and embryo diameter. All 8 control embryos had a Grade I cytoskeleton, compared with only 2/24 (8.3%) frozen or vitrified embryos. Of the 8 slow-frozen or vitrified embryos with a Grade III cytoskeleton post-thaw, 7 had been vitrified and 6 were larger (Day 7) embryos. These results indicate that while both slow-freezing and vitrification of camel embryos lead to cytoskeleton disruption and cell death, embryo quality is better preserved by slow-freezing.

  5. Fisetin antagonizes cell fusion, cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages.

    PubMed

    Kim, Yun-Ho; Kim, Jung-Lye; Lee, Eun-Jung; Park, Sin-Hye; Han, Seon-Young; Kang, Soon Ah; Kang, Young-Hee

    2014-03-01

    Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10 μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone.

  6. Centrosomal Localization of the Psoriasis Candidate Gene Product, CCHCR1, Supports a Role in Cytoskeletal Organization

    PubMed Central

    Tervaniemi, Mari H.; Siitonen, H. Annika; Söderhäll, Cilla; Minhas, Gurinder; Vuola, Jyrki; Tiala, Inkeri; Sormunen, Raija; Samuelsson, Lena; Suomela, Sari; Kere, Juha; Elomaa, Outi

    2012-01-01

    CCHCR1 (Coiled-Coil α-Helical Rod protein 1), within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene with the psoriasis associated risk allele CCHCR1*WWCC. Although its expression pattern in psoriatic skin differs from healthy skin and its overexpression influences cell proliferation in transgenic mice, its role as a psoriasis effector gene has remained unsettled. The 5′-region of the gene contains a SNP (rs3130453) that controls a 5′-extended open reading frame and thus the translation of alternative isoforms. We have now compared the function of two CCHCR1 isoforms: the novel longer isoform 1 and the previously studied isoform 3. In samples of Finnish and Swedish families, the allele generating only isoform 3 shows association with psoriasis (P<10−7). Both isoforms localize at the centrosome, a cell organelle playing a role in cell division. In stably transfected cells the isoform 3 affects cell proliferation and with the CCHCR1*WWCC allele, also apoptosis. Furthermore, cells overexpressing CCHCR1 show isoform- and haplotype-specific influences in the cell size and shape and alterations in the organization and expression of the cytoskeletal proteins actin, vimentin, and cytokeratins. The isoform 1 with the non-risk allele induces the expression of keratin 17, a hallmark for psoriasis; the silencing of CCHCR1 reduces its expression in HEK293 cells. CCHCR1 also regulates EGF-induced STAT3 activation in an isoform-specific manner: the tyrosine phosphorylation of STAT3 is disturbed in isoform 3-transfected cells. The centrosomal localization of CCHCR1 provides a connection to the abnormal cell proliferation and offers a link to possible cellular pathways altered in psoriasis. PMID:23189171

  7. Graphene Oxide Induced Perturbation to Plasma Membrane and Cytoskeletal Meshwork Sensitize Cancer Cells to Chemotherapeutic Agents.

    PubMed

    Zhu, Jianqiang; Xu, Ming; Gao, Ming; Zhang, Zhihong; Xu, Yong; Xia, Tian; Liu, Sijin

    2017-03-28

    The outstanding physicochemical properties endow graphene materials (e.g., graphene oxide, GO) with beneficial potentials in diverse biomedical fields such as bioimaging, drug delivery, and biomolecular detection. GO recently emerged as a chemosensitizer; however, the detailed molecular basis underlying GO-conducted sensitization and corresponding biological effects are still elusive. Based on our recent findings that GO treatment at sublethal concentrations could impair the general cellular priming state, including disorders of plasma membrane and cytoskeleton construction, we aimed here to explore the mechanism of GO as a sensitizer to make cancer cells more susceptible to chemotherapeutic agents. We discovered that GO could not only compromise plasma membrane and cytoskeleton in J774A.1 macrophages and A549 lung cancer cells at sublethal concentrations without incurring significant cell death but also dampen a number of biological processes. Using the toxicogenomics approaches, we laid out the gene expression signature affected by GO and further defined those genes involved in membrane and cytoskeletal impairments responding to GO. The mechanistic investigation uncovered that the interactions of GO-integrin occurred on the plasma membrane and consequently activated the integrin-FAK-Rho-ROCK pathway and suppressed the expression of integrin, resulting in compromised cell membrane and cytoskeleton and a subsequent cellular priming state. By making use of this mechanism, the efficacy of chemotherapeutic agents (e.g., doxorubicin and cisplatin) could be enhanced by GO pretreatment in killing cancer cells. This study unveiled a feature of GO in cancer therapeutics: sensitizing cancer cells to chemotherapeutic agents by undermining the resistance capability of tumor cells against chemotherapeutic agents, at least partially, by compromising plasma membrane and cytoskeleton meshwork.

  8. Moesin is activated in cardiomyocytes in experimental autoimmune myocarditis and mediates cytoskeletal reorganization with protrusion formation.

    PubMed

    Miyawaki, Akimitsu; Mitsuhara, Yusuke; Orimoto, Aya; Nakayasu, Yusuke; Tsunoda, Shin-Ichi; Obana, Masanori; Maeda, Makiko; Nakayama, Hiroyuki; Yoshioka, Yasuo; Tsutsumi, Yasuo; Fujio, Yasushi

    2016-08-01

    Acute myocarditis is a self-limiting disease. Most patients with myocarditis recover without cardiac dysfunction in spite of limited capacity of myocardial regeneration. Therefore, to address intrinsic reparative machinery of inflamed hearts, we investigated the cellular dynamics of cardiomyocytes in response to inflammation using experimental autoimmune myocarditis (EAM) model. EAM was induced by immunization of BALB/c mice with α-myosin heavy chain peptides twice. The inflammatory reaction was evoked with myocardial damage with the peak at 3 wk after the first immunization (EAM3w). Morphological and functional restoration started from EAM3w, when active protrusion formation, a critical process of myocardial healing, was observed in cardiomyocytes. Shotgun proteomics revealed that cytoskeletal proteins were preferentially increased in cardiomyocytes at EAM3w, compared with preimmunized (EAM0w) hearts, and that moesin was the most remarkably upregulated among them. Immunoblot analyses demonstrated that the expression of both total and phosphorylated moesin was upregulated in isolated cardiomyocytes from EAM3w hearts. Immunofluorescence staining showed that moesin was localized at cardiomyocyte protrusions at EAM3w. Adenoviral vectors expressing wild-type, constitutively active and inactive form of moesin (wtMoesin, caMoesin, and iaMoesin, respectively) were transfected in neonatal rat cardiomyocytes. The overexpression of wtMoesin and caMoesin resulted in protrusion formation, while not iaMoesin. Finally, we found that cardiomyocyte protrusions were accompanied by cell-cell contact formation. The expression of moesin was upregulated in cardiomyocytes under inflammation, inducing protrusion formation in a phosphorylation-dependent fashion. Moesin signal could be a novel therapeutic target that stimulates myocardial repair by promoting contact formation of cardiomyocytes.

  9. Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture.

    PubMed

    Odintsova, Nelly A; Ageenko, Natalya V; Kipryushina, Yulia O; Maiorova, Mariia A; Boroda, Andrey V

    2015-08-01

    This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%).

  10. Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion

    PubMed Central

    Berens, Eric B.; Sharif, Ghada M.; Schmidt, Marcel O.; Yan, Gai; Shuptrine, Casey W.; Weiner, Louis M.; Glasgow, Eric; Riegel, Anna T.; Wellstein, Anton

    2016-01-01

    Cancer cell vascular invasion is a crucial step in the malignant progression towards metastasis. Here we used a genome-wide RNAi screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these keratin-associated proteins have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/β4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a keratin-associated protein can serve as a switch towards malignant progression. PMID:27375028

  11. Impact of Acrylamide on Calcium Signaling and Cytoskeletal Filaments in Testes From F344 Rat.

    PubMed

    Recio, Leslie; Friedman, Marvin; Marroni, Dennis; Maynor, Timothy; Chepelev, Nikolai L

    Acrylamide (AA) at high exposure levels is neurotoxic, induces testicular toxicity, and increases dominant lethal mutations in rats. RNA-sequencing in testes was used to identify differentially expressed genes (DEG), explore AA-induced pathway perturbations that could contribute to AA-induced testicular toxicity and then used to derive a benchmark dose (BMD). Male F344/DuCrl rats were administered 0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw/d in drinking water for 5, 15, or 31 days. The experimental design used exposure levels that spanned and exceeded the exposure levels used in the rat dominant lethal, 2-generation reproductive toxicology, and cancer bioassays. The time of sample collection was based on previous studies that developed gene expression-based BMD. At 12.0 mg/kg, there were 38, 33, and 65 DEG ( P value <.005; fold change >1.5) in the testes after 5, 15, or 31 days of exposure, respectively. At 31 days, there was a dose-dependent increase in the number of DEG, and at 12.0 mg/kg/d the top three functional clusters affected by AA exposure were actin filament organization, response to calcium ion, and regulation of cell proliferation. The BMD lower 95% confidence limit using DEG ranged from 1.8 to 6.8 mg/kg compared to a no-observed-adverse-effect-level of 2.0 mg/kg/d for male reproductive toxicity. These results are consistent with the known effects of AA on calcium signaling and cytoskeletal actin filaments leading to neurotoxicity and suggest that AA can cause rat dominant lethal mutations by these same mechanisms leading to impaired chromosome segregation during cell division.

  12. Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape

    NASA Technical Reports Server (NTRS)

    Sims, J. R.; Karp, S.; Ingber, D. E.

    1992-01-01

    Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS).

  13. Cytoskeletal Signaling: Is Memory Encoded in Microtubule Lattices by CaMKII Phosphorylation?

    PubMed Central

    Craddock, Travis J. A.; Tuszynski, Jack A.; Hameroff, Stuart

    2012-01-01

    Memory is attributed to strengthened synaptic connections among particular brain neurons, yet synaptic membrane components are transient, whereas memories can endure. This suggests synaptic information is encoded and ‘hard-wired’ elsewhere, e.g. at molecular levels within the post-synaptic neuron. In long-term potentiation (LTP), a cellular and molecular model for memory, post-synaptic calcium ion (Ca2+) flux activates the hexagonal Ca2+-calmodulin dependent kinase II (CaMKII), a dodacameric holoenzyme containing 2 hexagonal sets of 6 kinase domains. Each kinase domain can either phosphorylate substrate proteins, or not (i.e. encoding one bit). Thus each set of extended CaMKII kinases can potentially encode synaptic Ca2+ information via phosphorylation as ordered arrays of binary ‘bits’. Candidate sites for CaMKII phosphorylation-encoded molecular memory include microtubules (MTs), cylindrical organelles whose surfaces represent a regular lattice with a pattern of hexagonal polymers of the protein tubulin. Using molecular mechanics modeling and electrostatic profiling, we find that spatial dimensions and geometry of the extended CaMKII kinase domains precisely match those of MT hexagonal lattices. This suggests sets of six CaMKII kinase domains phosphorylate hexagonal MT lattice neighborhoods collectively, e.g. conveying synaptic information as ordered arrays of six “bits”, and thus “bytes”, with 64 to 5,281 possible bit states per CaMKII-MT byte. Signaling and encoding in MTs and other cytoskeletal structures offer rapid, robust solid-state information processing which may reflect a general code for MT-based memory and information processing within neurons and other eukaryotic cells. PMID:22412364

  14. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells

    PubMed Central

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L

    2015-01-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell–cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell–cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett’s, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin–catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells. PMID:26603452

  15. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells.

    PubMed

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L

    2015-11-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell-cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell-cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett's, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin-catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells.

  16. Modeling cytoskeletal traffic: an interplay between passive diffusion and active transport.

    PubMed

    Neri, Izaak; Kern, Norbert; Parmeggiani, Andrea

    2013-03-01

    We introduce the totally asymmetric simple exclusion process with Langmuir kinetics on a network as a microscopic model for active motor protein transport on the cytoskeleton, immersed in the diffusive cytoplasm. We discuss how the interplay between active transport along a network and infinite diffusion in a bulk reservoir leads to a heterogeneous matter distribution on various scales: we find three regimes for steady state transport, corresponding to the scale of the network, of individual segments, or local to sites. At low exchange rates strong density heterogeneities develop between different segments in the network. In this regime one has to consider the topological complexity of the whole network to describe transport. In contrast, at moderate exchange rates the transport through the network decouples, and the physics is determined by single segments and the local topology. At last, for very high exchange rates the homogeneous Langmuir process dominates the stationary state. We introduce effective rate diagrams for the network to identify these different regimes. Based on this method we develop an intuitive but generic picture of how the stationary state of excluded volume processes on complex networks can be understood in terms of the single-segment phase diagram.

  17. Autothixotropy of Water and its Possible Importance for the Cytoskeletal Structures

    NASA Astrophysics Data System (ADS)

    Vybíral, Bohumil

    2011-12-01

    an organization of water in cytoskeletal structures are outlined.

  18. Interplay between cytoskeletal polymerization and the chondrogenic phenotype in chondrocytes passaged in monolayer culture.

    PubMed

    Parreno, Justin; Nabavi Niaki, Mortah; Andrejevic, Katarina; Jiang, Amy; Wu, Po-Han; Kandel, Rita A

    2017-02-01

    Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non-chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly (P < 0.05) increased gene expression of proliferation-associated molecules (cyclin D1 and ki67), as well as significantly (P < 0.05) decreased cartilage matrix (type II collagen and aggrecan) and increased fibroblast-like matrix, type I collagen (COL1), gene expression by passage 2 (P2). Although tubulin polymerization status was not significantly (P > 0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL1 gene expression. To confirm that actin status regulated COL1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL1 gene expression in human passaged chondrocytes. Furthermore

  19. Identification and immunolocalization of actin cytoskeletal components in light- and dark-adapted octopus retinas.

    PubMed

    De Velasco, B; Martinez, J M; Ochoa, G H; Miller, A M; Clark, Y M; Matsumoto, B; Robles, L J

    1999-06-01

    speculate that these proteins and actin remain associated with an avillar membrane that connects opposing sets of rhabdomeres in light-adapted retinas. Association of these cytoskeletal proteins with the avillar membrane would constitute a pool of proteins that could be recruited for rapid microvillus formation from the previously avillar region.

  20. Cytoskeletal regulation dominates temperature-sensitive proteomic changes of hibernation in forebrain of 13-lined ground squirrels.

    PubMed

    Hindle, Allyson G; Martin, Sandra L

    2013-01-01

    13-lined ground squirrels, Ictidomys tridecemlineatus, are obligate hibernators that transition annually between summer homeothermy and winter heterothermy - wherein they exploit episodic torpor bouts. Despite cerebral ischemia during torpor and rapid reperfusion during arousal, hibernator brains resist damage and the animals emerge neurologically intact each spring. We hypothesized that protein changes in the brain underlie winter neuroprotection. To identify candidate proteins, we applied a sensitive 2D gel electrophoresis method to quantify protein differences among forebrain extracts prepared from ground squirrels in two summer, four winter and fall transition states. Proteins that differed among groups were identified using LC-MS/MS. Only 84 protein spots varied significantly among the defined states of hibernation. Protein changes in the forebrain proteome fell largely into two reciprocal patterns with a strong body temperature dependence. The importance of body temperature was tested in animals from the fall; these fall animals use torpor sporadically with body temperatures mirroring ambient temperatures between 4 and 21°C as they navigate the transition between summer homeothermy and winter heterothermy. Unlike cold-torpid fall ground squirrels, warm-torpid individuals strongly resembled the homeotherms, indicating that the changes observed in torpid hibernators are defined by body temperature, not torpor per se. Metabolic enzymes were largely unchanged despite varied metabolic activity across annual and torpor-arousal cycles. Instead, the majority of the observed changes were cytoskeletal proteins and their regulators. While cytoskeletal structural proteins tended to differ seasonally, i.e., between summer homeothermy and winter heterothermy, their regulatory proteins were more strongly affected by body temperature. Changes in the abundance of various isoforms of the microtubule assembly and disassembly regulatory proteins dihydropyrimidinase

  1. Neuronal loss and cytoskeletal disruption following intrahippocampal administration of the metabolic inhibitor malonate: lack of protection by MK-801.

    PubMed

    Pang, Z; Umberger, G H; Geddes, J W

    1996-02-01

    Impaired energy metabolism may contribute to the pathogenesis of late-onset neurodegenerative disorders such as Alzheimer's disease by increasing neuronal vulnerability to excitotoxic damage through the NMDA receptor. The effects of metabolic impairment on the striatum have been extensively examined, but relatively little is known regarding the vulnerability of the hippocampus. To examine the effect of metabolic impairment on the hippocampal formation, malonate (0.25-2.5 mumol), a reversible inhibitor of succinate dehydrogenase, was administered by stereotaxic injection into the hippocampus of male Sprague-Dawley rats. Neuronal loss was assessed by Nissl stain, and immunocytochemistry was used to examine cytoskeletal disruption. Malonate produced a dose-dependent lesion in which CA1 pyramidal neurons were most vulnerable, followed by CA3 and dentate gyrus. Cytoskeletal alterations included the loss of microtubule-associated protein 2 (MAP2) and dendritic MAP1B immunoreactivity, whereas axonal MAP1B and tau proteins were relatively spared. Spatially and temporally correlated with the loss of MAP2 was an increase in the immunoreactivity of calpain-cleaved spectrin. A similar pattern of neuronal damage and cytoskeletal disruption was produced by intrahippocampal injection of quinolinate (0.1 mumol), an NMDA agonist. Although these results are consistent with the hypothesis that metabolic impairment results in excitotoxic death, MK-801 (dizocilipine maleate), a noncompetitive NMDA receptor antagonist, did not attenuate the lesions produced by malonate but was effective against quinolinate. The results suggest that NMDA receptor activation is not required for malonate-induced damage in the hippocampal formation.

  2. Effects of transforming growth factor type beta on expression of cytoskeletal proteins in endosteal mouse osteoblastic cells

    SciTech Connect

    Lomri, A.; Marie, P.J. )

    1990-01-01

    Transforming growth factor beta (TGF beta) has been shown to influence the growth and differentiation of many cell types in vitro. We have examined the effects of TGF beta on cell morphology and cytoskeletal organization in relation to parameters of cell proliferation and differentiation in endosteal osteoblastic cells isolated from mouse caudal vertebrae. Treatment of mouse osteoblastic cells cultured in serum free medium for 24 hours with TGF beta (1.5-30 ng/mL) slightly (-23%) inhibited alkaline phosphatase activity. In parallel, TGF beta (0.5-30 ng/mL, 24 hours) greatly increased cell replication as evaluated by (3H)-thymidine incorporation into DNA (157% to 325% of controls). At a median dose (1.5 ng/mL) that affected both alkaline phosphatase and DNA synthesis (235% of controls) TGF beta induced rapid (six hours) cell respreading of quiescent mouse osteoblastic cells. This effect was associated with increased polymerization of actin, alpha actinin, and tubulins, as evaluated by both biochemical and immunofluorescence methods. In addition, TGF beta (1.5 ng/mL) increased the de novo biosynthesis of actin, alpha actinin, vimentin, and tubulins, as determined by {sup 35}S methionine labeling and fractionation of cytoskeletal proteins using two-dimensional gel electrophoresis. These effects were rapid and transient, as they occurred at six hours and were reversed after 24 hours of TGF beta exposure. The results indicate that the stimulatory effect of TGF beta on DNA synthesis in endosteal mouse osteoblastic cells is associated with a transient increase in cell spreading associated with enhanced polymerization and synthesis of cytoskeletal proteins.

  3. The Ig Superfamily Cell Adhesion Molecule, apCAM, Mediates Growth Cone Steering by Substrate–Cytoskeletal Coupling

    PubMed Central

    Suter, Daniel M.; Errante, Laura D.; Belotserkovsky, Victoria; Forscher, Paul

    1998-01-01

    Dynamic cytoskeletal rearrangements are involved in neuronal growth cone motility and guidance. To investigate how cell surface receptors translate guidance cue recognition into these cytoskeletal changes, we developed a novel in vitro assay where beads, coated with antibodies to the immunoglobulin superfamily cell adhesion molecule apCAM or with purified native apCAM, replaced cellular substrates. These beads associated with retrograde F-actin flow, but in contrast to previous studies, were then physically restrained with a microneedle to simulate interactions with noncompliant cellular substrates. After a latency period of ∼10 min, we observed an abrupt increase in bead-restraining tension accompanied by direct extension of the microtubule-rich central domain toward sites of apCAM bead binding. Most importantly, we found that retrograde F-actin flow was attenuated only after restraining tension had increased and only in the bead interaction axis where preferential microtubule extension occurred. These cytoskeletal and structural changes are very similar to those reported for growth cone interactions with physiological targets. Immunolocalization using an antibody against the cytoplasmic domain of apCAM revealed accumulation of the transmembrane isoform of apCAM around bead-binding sites. Our results provide direct evidence for a mechanical continuum from apCAM bead substrates through the peripheral domain to the central cytoplasmic domain. By modulating functional linkage to the underlying actin cytoskeleton, cell surface receptors such as apCAM appear to enable the application of tensioning forces to extracellular substrates, providing a mechanism for transducing retrograde flow into guided growth cone movement. PMID:9531561

  4. Early Cytoskeletal Protein Modifications Precede Overt Structural Degeneration in the DBA/2J Mouse Model of Glaucoma

    PubMed Central

    Wilson, Gina N.; Smith, Matthew A.; Inman, Denise M.; Dengler-Crish, Christine M.; Crish, Samuel D.

    2016-01-01

    Axonal transport deficits precede structural loss in glaucoma and other neurodegenerations. Impairments in structural support, including modified cytoskeletal proteins, and microtubule-destabilizing elements, could be initiating factors in glaucoma pathogenesis. We investigated the time course of changes in protein levels and post-translational modifications in the DBA/2J mouse model of glaucoma. Using anterograde tract tracing of the retinal projection, we assessed major cytoskeletal and transported elements as a function of transport integrity in different stages of pathological progression. Using capillary-based electrophoresis, single- and multiplex immunosorbent assays, and immunofluorescence, we quantified hyperphosphorylated neurofilament-heavy chain, phosphorylated tau (ptau), calpain-mediated spectrin breakdown product (145/150 kDa), β–tubulin, and amyloid-β42 proteins based on age and transport outcome to the superior colliculus (SC; the main retinal target in mice). Phosphorylated neurofilament-heavy chain (pNF-H) was elevated within the optic nerve (ON) and SC of 8–10 month-old DBA/2J mice, but was not evident in the retina until 12–15 months, suggesting that cytoskeletal modifications first appear in the distal retinal projection. As expected, higher pNF-H levels in the SC and retina were correlated with axonal transport deficits. Elevations in hyperphosphorylated tau (ptau) occurred in ON and SC between 3 and 8 month of age while retinal ptau accumulations occurred at 12–15 months in DBA/2J mice. In vitro co-immunoprecipitation experiments suggested increased affinity of ptau for the retrograde motor complex protein dynactin. We observed a transport-related decrease of β-tubulin in ON of 10–12 month-old DBA/2J mice, suggesting destabilized microtubule array. Elevations in calpain-mediated spectrin breakdown product were seen in ON and SC at the earliest age examined, well before axonal transport loss is evident. Finally, transport

  5. The interaction network of the chaperonin CCT.

    PubMed

    Dekker, Carien; Stirling, Peter C; McCormack, Elizabeth A; Filmore, Heather; Paul, Angela; Brost, Renee L; Costanzo, Michael; Boone, Charles; Leroux, Michel R; Willison, Keith R

    2008-07-09

    The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) has an important function in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin. Yet the nature of the proteins and cellular pathways dependent on CCT function has not been established globally. Here, we use proteomic and genomic approaches to define CCT interaction networks involving 136 proteins/genes that include links to the nuclear pore complex, chromatin remodelling, and protein degradation. Our study also identifies a third eukaryotic cytoskeletal system connected with CCT: the septin ring complex, which is essential for cytokinesis. CCT interactions with septins are ATP dependent, and disrupting the function of the chaperonin in yeast leads to loss of CCT-septin interaction and aberrant septin ring assembly. Our results therefore provide a rich framework for understanding the function of CCT in several essential cellular processes, including epigenetics and cell division.

  6. Sequence Analysis of LRPPRC and Its SEC1 Domain Interaction Partners Suggests Roles in Cytoskeletal Organization, Vesicular Trafficking, Nucleocytosolic Shuttling and Chromosome Activity

    PubMed Central

    Liu, Leyuan; McKeehan, Wallace L.

    2011-01-01

    LRPPRC (originally called LRP130) is an intracellular 130-kDa leucine-rich protein that co-purifies with the FGF receptor from liver cell extracts and has been detected in diverse multi-protein complexes from the cell membrane, cytoskeleton and nucleus. Here we report results of a sequence homology analysis of LRPPRC and its SEC1 domain interactive partners. Twenty-three copies of tandem repeats that are similar to PPR, TPR and HEAT repeats characterize the LRPPRC sequence. The N-terminus exhibits multiple copies of leucine-rich nuclear transport signals followed by ENTH, DUF28 and SEC1 homology domains. We used the SEC1 domain to trap interactive partners expressed from a human liver cDNA library. Interactive C19ORF5 (XP_038600) exhibited a strong homology to microtubule-associated proteins (MAP) and a potential arginine-rich mRNA binding motif. UXT (XP_033860) exhibited α-helical properties homologous to the actin-associated spectrin repeat and L/I heptad repeats in mobile transcription factors. C6ORF34 (XP_004305) was homologous to the non-DNA binding C-terminus of the E. coli Rob transcription factor. CECR2 (AAK15343) exhibited a transcription factor AT-hook motif next to two bromodomains and a homology to guanylate-binding protein 1. Taken together these features suggest a regulatory role of LRPPRC and its SEC1 domain-interactive partners in integration of cytoskeletal networks with vesicular trafficking, nucleocytosolic shuttling, chromosome remodeling and transcription. PMID:11827465

  7. Connective Tissue Growth Factor in Regulation of RhoA Mediated Cytoskeletal Tension Associated Osteogenesis of Mouse Adipose-Derived Stromal Cells

    PubMed Central

    Xu, Yue; Wagner, Diane R.; Bekerman, Elena; Chiou, Michael; James, Aaron W.; Carter, Dennis; Longaker, Michael T.

    2010-01-01

    Background Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways. Methods/Principal Findings Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm2), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm2) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis. Conclusions/Significance We conclude that CTGF is important in the regulation of cytoskeletal tension mediated ASC osteogenic differentiation. PMID:20585662

  8. Antibody-based analysis reveals “filamentous vs. non-filamentous” and “cytoplasmic vs. nuclear” crosstalk of cytoskeletal proteins

    SciTech Connect

    Kumeta, Masahiro; Hirai, Yuya; Yoshimura, Shige H.; Horigome, Tsuneyoshi; Takeyasu, Kunio

    2013-12-10

    To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus. - Highlights: • A set of monoclonal antibodies were raised against nuclear scaffold proteins. • Helix-based cytoskeletal proteins were involved in nuclear scaffold. • Many cytoskeletal components shuttle into the nucleus in a CRM1-dependent manner. • Sets of antibodies distinguished distinct subcellular localization of a single isoform. • Nuclear keratin is soluble and does not form an obvious filamentous structure.

  9. The effect of ultrasound stimulation on the cytoskeletal organization of chondrocytes seeded in three-dimensional matrices.

    PubMed

    Noriega, Sandra; Hasanova, Gulnara; Subramanian, Anuradha

    2013-01-01

    The impact of low-intensity diffuse ultrasound (LIDUS) stimulation on the cytoskeletal organization of chondrocytes seeded in three-dimensional (3D) scaffolds was evaluated. Chondrocytes seeded on 3D chitosan matrices were exposed to LIDUS at 5.0 MHz (approx. 15 kPa, 51 s, 4 applications/day) in order to study the organization of actin, tubulin and vimentin. The results showed that actin presented a punctate cytosolic distribution and tubulin presented a quasiparallel organization of microtubules, whereas vimentin distribution was unaffected. Chondrocytes seeded on 3D scaffolds responded to US stimulation by the disruption of actin stress fibers and were sensitive to the presence of Rho-activated kinase (ROCK) inhibitor (Y27632). The gene expression of ROCK-I, a key element in the formation of stress fibers and mDia1, was significantly upregulated under the application of US. We conclude that the results of both the cytoskeletal analyses and gene expression support the argument that the presence of punctate actin upon US stimulation was accompanied by the upregulation of the RhoA/ROCK pathway.

  10. Mechanical diagnosis of human erythrocytes by ultra-high speed manipulation unraveled critical time window for global cytoskeletal remodeling

    PubMed Central

    Ito, Hiroaki; Murakami, Ryo; Sakuma, Shinya; Tsai, Chia-Hung Dylan; Gutsmann, Thomas; Brandenburg, Klaus; Pöschl, Johannes M. B.; Arai, Fumihito; Kaneko, Makoto; Tanaka, Motomu

    2017-01-01

    Large deformability of erythrocytes in microvasculature is a prerequisite to realize smooth circulation. We develop a novel tool for the three-step “Catch-Load-Launch” manipulation of a human erythrocyte based on an ultra-high speed position control by a microfluidic “robotic pump”. Quantification of the erythrocyte shape recovery as a function of loading time uncovered the critical time window for the transition between fast and slow recoveries. The comparison with erythrocytes under depletion of adenosine triphosphate revealed that the cytoskeletal remodeling over a whole cell occurs in 3 orders of magnitude longer timescale than the local dissociation-reassociation of a single spectrin node. Finally, we modeled septic conditions by incubating erythrocytes with endotoxin, and found that the exposure to endotoxin results in a significant delay in the characteristic transition time for cytoskeletal remodeling. The high speed manipulation of erythrocytes with a robotic pump technique allows for high throughput mechanical diagnosis of blood-related diseases. PMID:28233788

  11. The Cytoskeletal Connection to Ion Channels as a Potential Mechanosensory Mechanism: Lessons from Polycystin-2 (TRPP2).

    PubMed

    Cantiello, Horacio F; Montalbetti, Nicolás; Li, Qiang; Chen, Xing-Zhen

    2007-01-01

    Mechanosensitivity of ion channels, or the ability to transfer mechanical forces into a gating mechanism of channel regulation, is split into two main working (not mutually exclusive) hypotheses. One is that elastic and/or structural changes in membrane properties act as a transducing mechanism of channel regulation. The other hypothesis involves tertiary elements, such as the cytoskeleton which, itself by dynamic interactions with the ion channel, may convey conformational changes, including those ascribed to mechanical forces. This hypothesis is supported by numerous instances of regulatory changes in channel behavior by alterations in cytoskeletal structures/interactions. However, only recently, the molecular nature of these interactions has slowly emerged. Recently, a surge of evidence has emerged to indicate that transient receptor potential (TRP) channels are key elements in the transduction of a variety of environmental signals. This chapter describes the molecular linkage and regulatory elements of polycystin-2 (PC2), a TRP-type (TRPP2) nonselective cation channel whose mutations cause autosomal dominant polycystic kidney disease (ADPKD). The chapter focuses on the involvement of cytoskeletal structures in the regulation of PC2 and discusses how these connections are the transducing mechanism of environmental signals to its channel function.

  12. A theoretical model of cytokinesis implicates feedback between membrane curvature and cytoskeletal organization in asymmetric cytokinetic furrowing

    PubMed Central

    Dorn, Jonas F.; Zhang, Li; Phi, Tan-Trao; Lacroix, Benjamin; Maddox, Paul S.; Liu, Jian; Maddox, Amy Shaub

    2016-01-01

    During cytokinesis, the cell undergoes a dramatic shape change as it divides into two daughter cells. Cell shape changes in cytokinesis are driven by a cortical ring rich in actin filaments and nonmuscle myosin II. The ring closes via actomyosin contraction coupled with actin depolymerization. Of interest, ring closure and hence the furrow ingression are nonconcentric (asymmetric) within the division plane across Metazoa. This nonconcentricity can occur and persist even without preexisting asymmetric cues, such as spindle placement or cellular adhesions. Cell-autonomous asymmetry is not explained by current models. We combined quantitative high-resolution live-cell microscopy with theoretical modeling to explore the mechanistic basis for asymmetric cytokinesis in the Caenorhabditis elegans zygote, with the goal of uncovering basic principles of ring closure. Our theoretical model suggests that feedback among membrane curvature, cytoskeletal alignment, and contractility is responsible for asymmetric cytokinetic furrowing. It also accurately predicts experimental perturbations of conserved ring proteins. The model further suggests that curvature-mediated filament alignment speeds up furrow closure while promoting energy efficiency. Collectively our work underscores the importance of membrane–cytoskeletal anchoring and suggests conserved molecular mechanisms for this activity. PMID:26912796

  13. Actin Cytoskeletal Organization in Drosophila Germline Ring Canals Depends on Kelch Function in a Cullin-RING E3 Ligase

    PubMed Central

    Hudson, Andrew M.; Mannix, Katelynn M.; Cooley, Lynn

    2015-01-01

    The Drosophila Kelch protein is required to organize the ovarian ring canal cytoskeleton. Kelch binds and cross-links F-actin in vitro, and it also functions with Cullin 3 (Cul3) as a component of a ubiquitin E3 ligase. How these two activities contribute to cytoskeletal remodeling in vivo is not known. We used targeted mutagenesis to investigate the mechanism of Kelch function. We tested a model in which Cul3-dependent degradation of Kelch is required for its function, but we found no evidence to support this hypothesis. However, we found that mutant Kelch deficient in its ability to interact with Cul3 failed to rescue the kelch cytoskeletal defects, suggesting that ubiquitin ligase activity is the principal activity required in vivo. We also determined that the proteasome is required with Kelch to promote the ordered growth of the ring canal cytoskeleton. These results indicate that Kelch organizes the cytoskeleton in vivo by targeting a protein substrate for degradation by the proteasome. PMID:26384358

  14. Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

    PubMed Central

    Stout, A. L.; Axelrod, D.

    1994-01-01

    removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites. These rest suggest that 1) protein 4.1 binds to membrane or submembrane sites at least in part reversibly ; 2) the most reversible sites are probably not proteinaceous and not glycophorin C, but possibly are phospholipids (especially phosphatidylserine); and 3) TIWRFRAP can successfully examine the fast reversible dynamics of cytoskeletal components binding to biological membranes. Images FIGURE 2 FIGURE 3 FIGURE 4 PMID:7811947

  15. E-4-hydroxy-2-nonenal is cytotoxic and cross-links cytoskeletal proteins in P19 neuroglial cultures.

    PubMed Central

    Montine, T. J.; Amarnath, V.; Martin, M. E.; Strittmatter, W. J.; Graham, D. G.

    1996-01-01

    Lipid peroxidation increases with age in brain and is elevated further in Alzheimer's disease. E-4-hydroxy-2-nonenal and malondialdehyde are products of lipid peroxidation that can adduct and cross-link protein. Neurofibrillary tangles, a feature of Alzheimer's disease composed mostly of tau protein, contain cross-linked and ubiquitin-conjugated protein. In P19 neuroglial cultures, E-4-hydroxy-2-nonenal was a potent cytotoxin that cross-linked cytoskeletal proteins, including tau into high molecular weight species that were conjugated with ubiquitin. Malondialdehyde formed monoadducts with cell protein but did not cross-link and was not cytotoxic. A non-crosslinking analogue of E-4-hydroxy-2-nonenal was not cytotoxic. E-4-Hydroxy-2-nonenal may contribute to neurodegeneration and neurofibrillary tangle formation in Alzheimer's disease. Images Figure 2 Figure 3 PMID:8546230

  16. Clinicopathological Analysis and Multipronged Quantitative Proteomics Reveal Oxidative Stress and Cytoskeletal Proteins as Possible Markers for Severe Vivax Malaria.

    PubMed

    Ray, Sandipan; Patel, Sandip K; Venkatesh, Apoorva; Bhave, Amruta; Kumar, Vipin; Singh, Vaidhvi; Chatterjee, Gangadhar; Shah, Veenita G; Sharma, Sarthak; Renu, Durairaj; Nafis, Naziya; Gandhe, Prajakta; Gogtay, Nithya; Thatte, Urmila; Sehgal, Kunal; Verma, Sumit; Karak, Avik; Khanra, Dibbendhu; Talukdar, Arunansu; Kochar, Sanjay K; S B, Vijeth; Kochar, Dhanpat K; Rojh, Dharmendra; Varma, Santosh G; Gandhi, Mayuri N; Srikanth, Rapole; Patankar, Swati; Srivastava, Sanjeeva

    2016-04-19

    In Plasmodium vivax malaria, mechanisms that trigger transition from uncomplicated to fatal severe infections are obscure. In this multi-disciplinary study we have performed a comprehensive analysis of clinicopathological parameters and serum proteome profiles of vivax malaria patients with different severity levels of infection to investigate pathogenesis of severe malaria and identify surrogate markers of severity. Clinicopathological analysis and proteomics profiling has provided evidences for the modulation of diverse physiological pathways including oxidative stress, cytoskeletal regulation, lipid metabolism and complement cascades in severe malaria. Strikingly, unlike severe falciparum malaria the blood coagulation cascade was not found to be affected adversely in acute P. vivax infection. To the best of our knowledge, this is the first comprehensive proteomics study, which identified some possible cues for severe P. vivax infection. Our results suggest that Superoxide dismutase, Vitronectin, Titin, Apolipoprotein E, Serum amyloid A, and Haptoglobin are potential predictive markers for malaria severity.

  17. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chin-Mei Chang-Liu

    1995-06-01

    Experiments examined the effects of radiation dose-rate and protein synthesis inhibition expression of cytoskeletal and matrix elements in Syrian hamster embryo cells. Results demonstrated little effect of dose-rate for neutrons when comparing expression of {alpha}-tubulin and fibronectin genes. Cycloheximide repressed accumulation of {alpha}-tubulin-mRNA following exposure to high dose-rate neutrons or {gamma} rays. Cycloheximide did not affect accumulation of actin mRNA. Cycloheximide abrogated induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to radiation. 24 refs., 3 tabs.

  18. Association of blebbing with assembly of cytoskeletal proteins in ATP-depleted EL-4 ascites tumour cells.

    PubMed

    Gabai, V L; Kabakov, A E; Mosin, A F

    1992-01-01

    ATP depletion in EL-4 ascites tumour cells rapidly induced the changes in cell morphology (blebbing), cytoskeletal protein assembly and finally resulted in cell death. After 1 hr of incubation with 2 microM rotenone (inhibitor of respiration) in glucose-free medium, when ATP level was 4% of the initial level, there were increases in triton-insoluble actin and vinculin levels (2.5-fold and 2.8-fold, respectively) and 44% of cells showed blebs; such treatment damaged cells irreversibly. Ca2+ removal did not diminish the effect of ATP depletion on cytoskeleton, blebbing and cell death, although the elevation of free intracellular Ca2+ in rotenone-treated cells was prevented. The role of ATP in maintaining cytoskeleton and cell shape is discussed.

  19. Alteration of a yeast SH3 protein leads to conditional viability with defects in cytoskeletal and budding patterns.

    PubMed

    Bauer, F; Urdaci, M; Aigle, M; Crouzet, M

    1993-08-01

    Mutations in genes necessary for survival in stationary phase were isolated to understand the ability of wild-type Saccharomyces cerevisiae to remain viable during prolonged periods of nutritional deprivation. Here we report results concerning one of these mutants, rvs167, which shows reduced viability and abnormal cell morphology upon carbon and nitrogen starvation. The mutant exhibits the same response when cells are grown in high salt concentrations and other unfavorable growth conditions. The RVS167 gene product displays significant homology with the Rvs161 protein and contains a SH3 domain at the C-terminal end. Abnormal actin distribution is associated with the mutant phenotype. In addition, while the budding pattern of haploid strains remains axial in standard growth conditions, the budding pattern of diploid mutant strains is random. The gene RVS167 therefore could be implicated in cytoskeletal reorganization in response to environmental stresses and could act in the budding site selection mechanism.

  20. Alteration of a yeast SH3 protein leads to conditional viability with defects in cytoskeletal and budding patterns.

    PubMed Central

    Bauer, F; Urdaci, M; Aigle, M; Crouzet, M

    1993-01-01

    Mutations in genes necessary for survival in stationary phase were isolated to understand the ability of wild-type Saccharomyces cerevisiae to remain viable during prolonged periods of nutritional deprivation. Here we report results concerning one of these mutants, rvs167, which shows reduced viability and abnormal cell morphology upon carbon and nitrogen starvation. The mutant exhibits the same response when cells are grown in high salt concentrations and other unfavorable growth conditions. The RVS167 gene product displays significant homology with the Rvs161 protein and contains a SH3 domain at the C-terminal end. Abnormal actin distribution is associated with the mutant phenotype. In addition, while the budding pattern of haploid strains remains axial in standard growth conditions, the budding pattern of diploid mutant strains is random. The gene RVS167 therefore could be implicated in cytoskeletal reorganization in response to environmental stresses and could act in the budding site selection mechanism. Images PMID:8336735

  1. Hic-5 Regulates Actin Cytoskeletal Reorganization and Expression of Fibrogenic Markers and Myocilin in Trabecular Meshwork Cells

    PubMed Central

    Pattabiraman, Padmanabhan Paranji; Rao, Ponugoti Vasantha

    2015-01-01

    Purpose To explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression of myocilin in trabecular meshwork (TM) cells. Methods Using primary cultures of human TM (HTM) cells, the effects of various external factors on Hic-5 protein levels, as well as the effects of recombinant Hic-5 and Hic-5 small interfering RNA (siRNA) on actin cytoskeleton, FAs, myocilin, α-smooth muscle actin (αSMA), and collagen-1 were determined by immunofluorescence and immunoblot analyses. Results Hic-5 distributes discretely to the FAs in HTM cells and throughout the TM and Schlemm's canal of the human aqueous humor (AH) outflow pathway. Transforming growth factor-β2 (TGF-β2), endothelin-1, lysophosphatidic acid, hydrogen peroxide, and RhoA significantly increased Hic-5 protein levels in HTM cells in association with reorganization of actin cytoskeleton and FAs. While recombinant Hic-5 induced actin stress fibers, FAs, αv integrin redistribution to the FAs, increased levels of αSMA, collagen-1, and myocilin, Hic-5 siRNA suppressed most of these responses in HTM cells. Hic-5 siRNA also suppressed TGF-β2-induced fibrogenic activity and dexamethasone-induced myocilin expression in HTM cells. Conclusions Taken together, these results reveal that Hic-5, whose levels were increased by various external factors implicated in elevated intraocular pressure, induces actin cytoskeletal reorganization, FAs, expression of fibrogenic markers, and myocilin in HTM cells. These characteristics of Hic-5 in TM cells indicate its importance in regulation of AH outflow through the TM in both normal and glaucomatous eyes. PMID:26313302

  2. Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements

    PubMed Central

    1987-01-01

    The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000- 74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane. PMID:3308904

  3. Pathways of Ca²⁺ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle.

    PubMed

    Zhang, Bao-Ting; Whitehead, Nicholas P; Gervasio, Othon L; Reardon, Trent F; Vale, Molly; Fatkin, Diane; Dietrich, Alexander; Yeung, Ella W; Allen, David G

    2012-06-01

    Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.

  4. Studies on the function of cell surface glycoproteins. II. Possible role of surface glycoproteins in the control of cytoskeletal organization and surface morphology

    PubMed Central

    1979-01-01

    Immunoglobulin from goat antiserum directed against purified surface membranes from transformed BHK21/C13 cells (anti-M) has been shown to cause both control and transformed hamster cells to round and detach from the substrate (see accompanying paper). This paper documents the effects of the antiserum on the cytoskeletal organization and cell surface morphology of control BHK21/C13 cells examined by scanning and transmission electron microscopy. As a result of antiserum-induced rounding, the normally smooth cell surface becomes covered with filopodia and blebs, and the organization of all three components of the filamentous cytoskeleton is altered. In terms of cell surface morphology and cytoskeletal organization, the cells resemble rounded, postmitotic or trypsinized BHK cells rather than cells treated with either anticytoskeletal drugs or lectins. Immunocytochemical and radioimmune assay experiments support the suggestion that the rounding reaction induced by anti-M serum results from the specific interaction of antibodies with molecules on the cell surface. It is suggested that anti-M serum induces alterations in cytoskeletal organization via a transmembrane signal and that cytoskeletal reorganization is a fundamental part of the rounding and detachment process. PMID:457749

  5. Visualization of antigens attached to cytoskeletal framework in animal cells: colocalization of simian virus 40 Vp1 polypeptide and actin in TC7 cells.

    PubMed Central

    Kasamatsu, H; Lin, W; Edens, J; Revel, J P

    1983-01-01

    Actin and the simian virus 40 viral structural polypeptide Vp1 are observed to be present on cytoskeletal fibers of virus-infected TC7 cells, when these antigens in detergent-extracted whole cell mounts were labeled by specific antibodies and colloidal gold particles coated with a second antibody. In both cases, actin and Vp1 were found associated with fibers and fiber-associated electron-dense materials. Patches or clusters of colloidal gold particles denoting the presence of either Vp1 or actin were found on fibers uniformly distributed throughout the cytoplasm. By using simultaneous decoration of the two antigens with colloidal gold particles of different diameters, it was shown that the majority of Vp1 appears attached to cytoskeletal fibers in association with cellular actin. When Vp1 and actin were decorated with Imposil and ferritin simultaneously in infected cells that were fixed first and then permeabilized with saponin, both labels were found in the same spatial domain of the cell cytoplasm. Thus, the colocalization of Vp1 and actin on the cytoskeletal framework seems to reflect their actual state in the living cells. The electron-dense material to which colloidal gold particles localize in our cytoskeletal preparations may be the remnants of subcellular structures with which actin and Vp1 are both associated in intact cells. Images PMID:6308616

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  8. Reorganization of the actin cytoskeleton via transcriptional regulation of cytoskeletal/focal adhesion genes by myocardin-related transcription factors (MRTFs/MAL/MKLs)

    SciTech Connect

    Morita, Tsuyoshi; Mayanagi, Taira; Sobue, Kenji

    2007-10-01

    RhoA is a crucial regulator of stress fiber and focal adhesion formation through the activation of actin nucleation and polymerization. It also regulates the nuclear translocation of myocardin-related transcription factor-A and -B (MRTF-A/B, MAL or MKL 1/2), which are co-activators of serum response factor (SRF). In dominant-negative MRTF-A (DN-MRTF-A)-expressing NIH 3T3 cell lines, the expressions of several cytoskeletal/focal adhesion genes were down-regulated, and the formation of stress fiber and focal adhesion was severely diminished. MRTF-A/B-knockdown cells also exhibited such cytoskeletal defects. In reporter assays, both RhoA and MRTF-A enhanced promoter activities of these genes in a CArG-box-dependent manner, and DN-MRTF-A inhibited the RhoA-mediated activation of these promoters. In dominant-negative RhoA (RhoA-N19)-expressing NIH 3T3 cell lines, the nuclear translocation of MRTF-A/B was predominantly prevented, resulting in the reduced expression of cytoskeletal/focal adhesion proteins. Further, constitutive-active MRTF-A/B increased the expression of endogenous cytoskeletal/focal adhesion proteins, and thereby rescued the defective phenotype of stress fibers and focal adhesions in RhoA-N19 expressing cells. These results indicate that MRTF-A/B act as pivotal mediators of stress fiber and focal adhesion formation via the transcriptional regulation of a subset of cytoskeletal/focal adhesion genes.

  9. NADPH oxidases regulate septin-mediated cytoskeletal remodeling during plant infection by the rice blast fungus

    PubMed Central

    Ryder, Lauren S.; Dagdas, Yasin F.; Mentlak, Thomas A.; Kershaw, Michael J.; Thornton, Christopher R.; Schuster, Martin; Chen, Jisheng; Wang, Zonghua; Talbot, Nicholas J.

    2013-01-01

    The rice blast fungus Magnaporthe oryzae infects plants with a specialized cell called an appressorium, which uses turgor to drive a rigid penetration peg through the rice leaf cuticle. Here, we show that NADPH oxidases (Nox) are necessary for septin-mediated reorientation of the F-actin cytoskeleton to facilitate cuticle rupture and plant cell invasion. We report that the Nox2–NoxR complex spatially organizes a heteroligomeric septin ring at the appressorium pore, required for assembly of a toroidal F-actin network at the point of penetration peg emergence. Maintenance of the cortical F-actin network during plant infection independently requires Nox1, a second NADPH oxidase, which is necessary for penetration hypha elongation. Organization of F-actin in appressoria is disrupted by application of antioxidants, whereas latrunculin-mediated depolymerization of appressorial F-actin is competitively inhibited by reactive oxygen species, providing evidence that regulated synthesis of reactive oxygen species by fungal NADPH oxidases directly controls septin and F-actin dynamics. PMID:23382235

  10. Nuclear–cytoskeletal linkages facilitate cross talk between the nucleus and intercellular adhesions

    PubMed Central

    Stewart, Rachel M.; Zubek, Amanda E.; Rosowski, Kathryn A.; Schreiner, Sarah M.

    2015-01-01

    The linker of nucleoskeleton and cytoskeleton (LINC) complex allows cells to actively control nuclear position by coupling the nucleus to the cytoplasmic cytoskeleton. Nuclear position responds to the formation of intercellular adhesions through coordination with the cytoskeleton, but it is not known whether this response impacts adhesion function. In this paper, we demonstrate that the LINC complex component SUN2 contributes to the mechanical integrity of intercellular adhesions between mammalian epidermal keratinocytes. Mice deficient for Sun2 exhibited irregular hair follicle intercellular adhesions, defective follicle structure, and alopecia. Primary mouse keratinocytes lacking Sun2 displayed aberrant nuclear position in response to adhesion formation, altered desmosome distribution, and mechanically defective adhesions. This dysfunction appeared rooted in a failure of Sun2-null cells to reorganize their microtubule network to support coordinated intercellular adhesion. Together, these results suggest that cross talk between the nucleus, cytoskeleton, and intercellular adhesions is important for epidermal tissue integrity. PMID:25963820

  11. Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study.

    PubMed Central

    Rotsch, C; Radmacher, M

    2000-01-01

    The effect of various drugs affecting the integrity of different components of the cytoskeleton on the elasticity of two fibroblast cell lines was investigated by elasticity measurements with an atomic force microscope (AFM). Disaggregation of actin filaments always resulted in a distinct decrease in the cell's average elastic modulus indicating the crucial importance of the actin network for the mechanical stability of living cells. Disruption or chemical stabilization of microtubules did not affect cell elasticity. For the f-actin-disrupting drugs different mechanisms of drug action were observed. Cytochalasins B and D and Latrunculin A disassembled stress fibers. For Cytochalasin D this was accompanied by an aggregation of actin within the cytosol. Jasplakinolide disaggregated actin filaments but did not disassemble stress fibers. Fibrous structures found in AFM images and elasticity maps of fibroblasts could be identified as stress fibers by correlation of AFM data and fluorescence images. PMID:10620315

  12. Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways

    PubMed Central

    2014-01-01

    Background Mechanical stimulation is necessary for regulating correct formation of the skeleton. Here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. We use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle (Splotch-delayed) where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. We used Microarray and RNA sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic (TS23) humerus tissue. Results We found that 680 independent genes were down-regulated and 452 genes up-regulated in humeri from muscle-less Spd embryos compared to littermate controls (at least 2-fold; corrected p-value ≤0.05). We analysed the resulting differentially expressed gene sets using Gene Ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. Among cell signalling pathways, the most strongly disturbed was Wnt signalling, with 34 genes including 19 pathway target genes affected. Spatial gene expression analysis showed that both a Wnt ligand encoding gene (Wnt4) and a pathway antagonist (Sfrp2) are up-regulated specifically in the developing joint line, while the expression of a Wnt target gene, Cd44, is no longer detectable in muscle-less embryos. The identification of 84 genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a

  13. Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells

    SciTech Connect

    Docheva, Denitsa; Padula, Daniela; Schieker, Matthias; Clausen-Schaumann, Hauke

    2010-11-12

    Research highlights: {yields} Depending on the metastatic origin, prostate cancer cells differ in their affinity to COL1. {yields} COL1 affects specifically the F-actin and cell elasticity of bone-derived prostate cancer cells. {yields} Cell elasticity can be used as a biomarker for cancer cells from different metastases. -- Abstract: Despite of intensive research efforts, the precise mechanism of prostate cancer metastasis in bone is still not fully understood. Several studies have suggested that specific matrix production by the bone cells, such as collagen I, supports cancer cell invasion. The aim of this study was to investigate the effect of collagen I (COL1) and fibronectin (FN) on cell adhesion, cell elasticity and cytoskeletal organization of prostate cancer cells. Two cell lines, bone marrow- (PC3) and lymph node-derived (LNCaP) were cultivated on COL1 and FN (control protein). By using a quantitative adhesion assay and time-lapse analysis, it was found that PC3, but not LNCaP, adhered strongly and were more spread on COL1. Next, PC3 and LNCaP were evaluated by atomic force microscopy (AFM) and flatness shape factor and cellular Young's modulus were calculated. The shape analysis revealed that PC3 were significantly flatter when grown on COL1 in comparison to LNCaP. In general, PC3 were also significantly stiffer than LNCaP and furthermore, their stiffness increased upon interaction with COL1. Since cell stiffness is strongly dependent on actin organization, phalloidin-based actin staining was performed and revealed that, of the two cell types as well as the two different matrix proteins, only PC3 grown on COL1 formed robust actin cytoskeleton. In conclusion, our study showed that PC3 cells have a strong affinity towards COL1. On this matrix protein, the cells adhered strongly and underwent a specific cell flattening. Moreover, with the establishment of PC3 contact to COL1 a significant increase of PC3 stiffness was observed due to a profound cytoskeletal

  14. Methylphenidate regulates activity regulated cytoskeletal associated but not brain-derived neurotrophic factor gene expression in the developing rat striatum.

    PubMed

    Chase, T; Carrey, N; Soo, E; Wilkinson, M

    2007-02-09

    Methylphenidate (MPH) is a psychostimulant drug used to treat attention deficit hyperactivity disorder in children. To explore the central effects of chronic MPH, we investigated the expression of an effector immediate early gene, activity regulated cytoskeletal associated (arc), and the neurotrophin, brain-derived neurotrophic factor (bdnf) in the brain of immature and adult rats following repeated MPH. Prepubertal (postnatal day (PD) 25-38) and adult (PD 53-66) male rats were injected once daily for: a) 14 days with saline or MPH (2 or 10 mg/kg; s.c.) or b) 13 days with saline followed by a single dose of MPH (2 or 10 mg/kg; s.c.). To determine possible long-term effects of MPH, prepubertal rats were allowed a drug-free period of 4 weeks following the 14 days of treatment, and then were given a challenge dose of MPH. We demonstrated, for the first time, that an acute injection of MPH increased levels of activity-regulated cytoskeletal protein (ARC) and arc mRNA in the prepubertal rat striatum and cingulate/frontal cortex. This response was significantly attenuated by chronic MPH. The desensitization in arc expression observed in prepubertal rats persisted in the adult striatum following a later MPH challenge. In contrast to these data we observed little effect of MPH on bdnf expression. We also developed an effective, non-stressful technique to treat freely moving immature rats with oral MPH. Consistent with the results described above, we observed that oral MPH (7.5 and 10 mg/kg) also increased arc expression in the prepubertal rat striatum. However, unlike the effects of injected MPH, repeated oral MPH (7.5 mg/kg) did not alter the normal arc response. This result raises the important possibility that oral doses of MPH that reproduce clinically relevant blood levels of MPH may not down-regulate gene expression, at least in the short term (14 days). We confirmed, using mass spectrometry, that the oral doses of MPH used in our experiments yielded blood levels

  15. Nano-ZnO leads to tubulin macrotube assembly and actin bundling, triggering cytoskeletal catastrophe and cell necrosis

    NASA Astrophysics Data System (ADS)

    García-Hevia, Lorena; Valiente, Rafael; Martín-Rodríguez, Rosa; Renero-Lecuna, Carlos; González, Jesús; Rodríguez-Fernández, Lidia; Aguado, Fernando; Villegas, Juan C.; Fanarraga, Mónica L.

    2016-05-01

    Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin filament bundling and structural changes in microtubules, transforming these highly dynamic 25 nm diameter polymers into rigid macrotubes of tubulin, severely affecting cell proliferation and survival. Our results demonstrate that nano-ZnO causes acute cytoskeletal collapse that triggers necrosis, followed by a late reactive oxygen species (ROS)-dependent apoptotic process.Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin

  16. The integrin alphav beta3 increases cellular stiffness and cytoskeletal remodeling dynamics to facilitate cancer cell invasion

    NASA Astrophysics Data System (ADS)

    Mierke, Claudia Tanja

    2013-01-01

    The process of cancer cell invasion through the extracellular matrix (ECM) of connective tissue plays a prominent role in tumor progression and is based fundamentally on biomechanics. Cancer cell invasion usually requires cell adhesion to the ECM through the cell-matrix adhesion receptors integrins. The expression of the αvβ3 integrin is increased in several tumor types and is consistently associated with increased metastasis formation in patients. The hypothesis was that the αvβ3 integrin expression increases the invasiveness of cancer cells through increased cellular stiffness, and increased cytoskeletal remodeling dynamics. Here, the invasion of cancer cells with different αvβ3 integrin expression levels into dense three-dimensional (3D) ECMs has been studied. Using a cell sorter, two subcell lines expressing either high or low amounts of αvβ3 integrins (αvβ3high or αvβ3low cells, respectively) have been isolated from parental MDA-MB-231 breast cancer cells. αvβ3high cells showed a threefold increased cell invasion compared to αvβ3low cells. Similar results were obtained for A375 melanoma, 786-O kidney and T24 bladder carcinoma cells, and cells in which the β3 integrin subunit was knocked down using specific siRNA. To investigate whether contractile forces are essential for αvβ3 integrin-mediated increased cellular stiffness and subsequently enhanced cancer cell invasion, invasion assays were performed in the presence of myosin light chain kinase inhibitor ML-7 and Rho kinase inhibitor Y27632. Indeed, cancer cell invasiveness was reduced after addition of ML-7 and Y27632 in αvβ3high cells but not in αvβ3low cells. Moreover, after addition of the contractility enhancer calyculin A, an increase in pre-stress in αvβ3low cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase, STAT3 or Rac1 strongly reduced the invasiveness of αvβ3high cells, whereas the invasiveness of β3 specific knock

  17. Analysis of cellular behavior and cytoskeletal dynamics reveal a constriction mechanism driving optic cup morphogenesis

    PubMed Central

    Nicolás-Pérez, María; Kuchling, Franz; Letelier, Joaquín; Polvillo, Rocío; Wittbrodt, Jochen; Martínez-Morales, Juan R

    2016-01-01

    Contractile actomyosin networks have been shown to power tissue morphogenesis. Although the basic cellular machinery generating mechanical tension appears largely conserved, tensions propagate in unique ways within each tissue. Here we use the vertebrate eye as a paradigm to investigate how tensions are generated and transmitted during the folding of a neuroepithelial layer. We record membrane pulsatile behavior and actomyosin dynamics during zebrafish optic cup morphogenesis by live imaging. We show that retinal neuroblasts undergo fast oscillations and that myosin condensation correlates with episodic contractions that progressively reduce basal feet area. Interference with lamc1 function impairs basal contractility and optic cup folding. Mapping of tensile forces by laser cutting uncover a developmental window in which local ablations trigger the displacement of the entire tissue. Our work shows that optic cup morphogenesis is driven by a constriction mechanism and indicates that supra-cellular transmission of mechanical tension depends on ECM attachment. DOI: http://dx.doi.org/10.7554/eLife.15797.001 PMID:27797321

  18. Neuroactive molecules and growth factors modulate cytoskeletal protein expression during astroglial cell proliferation and differentiation in culture.

    PubMed

    Bramanti, Vincenzo; Grasso, Sonia; Tibullo, Daniele; Giallongo, Cesarina; Pappa, Rita; Brundo, Maria Violetta; Tomassoni, Daniele; Viola, Maria; Amenta, Francesco; Avola, Roberto

    2016-01-01

    Steroid hormones and neurotrophic factors regulate astroglial cell survival, proliferation, and differentiation in culture. The present study examines the interaction between glucocorticoids and growth factors (GFs) on cytoskeletal proteins and extracellular signal-regulated kinase 2 (ERK2) expression in stressed astroglial cultures at 25 days in vitro, according to the following experimental condition. Pretreatment with basic fibroblast growth factor alone or in combination with dexamethasone 10(-9) M for 48 hr induced an enhancement of glial fibrillary acidic protein, vimetin, and ERK2 expression. Treatment with "progression" GFs alone and in the last 12 hr significantly increased the above-mentioned markers' expression. The present study shows that glucocorticoids may cooperate with GFs or may abrogate their effects, depending on the experimental culture conditions used as well as the exposure time and the types of GFs added. Our findings provide evidence of interactive dialogue between GFs and neurosteroids in cultured astrocytes. This may have implications in the therapeutic approach to neurologic disorders associated with astrogliosis.

  19. Composite polymer systems with control of local substrate elasticity and their effect on cytoskeletal and morphological characteristics of adherent cells.

    PubMed

    Chou, Szu-Yuan; Cheng, Chao-Min; LeDuc, Philip R

    2009-06-01

    At the interface between extracellular substrates and biological materials, substrate elasticity strongly influences cell morphology and function. The associated biological ramifications comprise a diversity of critical responses including apoptosis, differentiation, and motility, which can affect medical devices such as stents. The interactions of the extracellular environment with the substrate are also affected by local properties wherein cells sense and respond to different physical inputs. To investigate the effects of having localized elasticity control of substrate microenvironments on cell response, we have developed a method to control material interface interactions with cells by dictating local substrate elasticity. This system is created by generating a composite material system with alternating, linear regions of polymers that have distinct stiffness characteristics. This approach was used to examine cytoskeletal and morphological changes in NIH 3T3 fibroblasts with emphasis on both local and global properties, noting that cells sense and respond to distinct material elasticities. Isolated cells sense and respond to these local differences in substrate elasticity by extending processes along the interface. Also, cells grown on softer elastic regions at higher densities (in contact with each other) have a higher projected area than isolated cells. Furthermore, when using chemical agents such as cytochalasin-D to disrupt the actin cytoskeleton, there is a significant increase in projected area for cells cultured on softer elastic regions This method has the potential to promote understanding of biomaterial-affected responses in a diversity of areas including morphogenesis, mechanotransduction, stents, and stem cell differentiation.

  20. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase

    PubMed Central

    Bhargava, Amol; Cotton, James A.; Dixon, Brent R.; Gedamu, Lashitew; Yates, Robin M.; Buret, Andre G.

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections. PMID:26334299

  1. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    PubMed

    Bhargava, Amol; Cotton, James A; Dixon, Brent R; Gedamu, Lashitew; Yates, Robin M; Buret, Andre G

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  2. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chang-Liu, Chin-Mei

    1994-05-01

    Experiments were designed to examine the effects Of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either {alpha}-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide revealed that cycloheximide repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposure. (2) Cycloheximide did not affect accumulation of MRNA for actin genes; and that cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin MRNA accumulation following exposure to ionizing radiation. in addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.

  3. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chang-Liu, Chin-Mei

    1992-12-31

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either {alpha}-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide, however, revealed several interesting and novel findings: (1) Cycloheximide repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposure (2) Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation. In addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.

  4. Mitochondrial and cytoskeletal alterations are involved in the pathogenesis of hydronephrosis in ICR/Mlac-hydro mice.

    PubMed

    Isarangkul, Duangnate; Wiyakrutta, Suthep; Kengkoom, Kanchana; Reamtong, Onrapak; Ampawong, Sumate

    2015-01-01

    The pathogenesis of congenital hydronephrosis in laboratory animals has been studied for many years, yet little is known about the underlying mechanism of this disease. In this study, we investigated a MS-based comparative proteomics approach to characterize the differently expressed proteins between kidney tissue samples of ICR/Mlac-hydro and wild-type mice. Interestingly, proteomic results exhibited several mitochondrial protein alterations especially the up-regulation of 60 kDa heat shock protein (Hsp60), stress-70 protein (GRP75) dysfunction, and down-regulation of voltage-dependent anion-selective channel protein 1 (VDAC-1). The results demonstrated that mitochondrial alteration may lead to inadequate energy-supply to maintain normal water reabsorption from the renal tubule, causing hydronephrosis. Moreover, the alteration of cytoskeleton proteins in the renal tubule, in particular the up-regulation of tubulin beta-4B chain (Tb4B) and N-myc downstream-regulated gene 1 protein (Ndr-1) may also be related due to their fundamental roles in maintaining cell morphology and tissue stability. In addition, cytoskeletal alterations may consequence to the reduction of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), cytoplasmic enzyme, which modulates the capacity of structural proteins. Our findings highlight a number of target proteins that may play a crucial role in congenital hydronephrosis and emphasize that the disorder of mitochondria and cytoskeleton proteins may be involved.

  5. Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization

    PubMed Central

    Pecorelli, Alessandra; Belmonte, Giuseppe; Signorini, Cinzia; Leoncini, Silvia; Zollo, Gloria; Capone, Antonietta; Giovampaola, Cinzia Della; Sticozzi, Claudia; Valacchi, Giuseppe; Ciccoli, Lucia; Guerranti, Roberto; Hayek, Joussef

    2014-01-01

    Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenance of cell shape to transcription regulation, was evaluated in the erythrocyte membranes from patients with typical Rett syndrome (RTT) and methyl CpG binding protein 2 (MECP2) gene mutations. RTT, affecting almost exclusively females with an average frequency of 1∶10,000 female live births, is considered the second commonest cause of severe cognitive impairment in the female gender. Evaluation of beta-actin was carried out in a comparative cohort study on red blood cells (RBCs), drawn from healthy control subjects and RTT patients using mass spectrometry-based quantitative analysis. We observed a decreased expression of the beta-actin isoforms (relative fold changes for spots 1, 2 and 3: −1.82±0.15, −2.15±0.06, and −2.59±0.48, respectively) in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, beta-actin from RTT patients also showed a dramatic increase in oxidative posttranslational modifications (PTMs) as the result of its binding with the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE). Our findings demonstrate, for the first time, a beta-actin down-regulation and oxidative PTMs for RBCs of RTT patients, thus indicating an altered cytoskeletal organization. PMID:24671107

  6. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    SciTech Connect

    Woloschak, G.E. |; Felcher, P.; Chang-Liu, Chin-Mei

    1993-12-31

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Results demonstrated little effect of dose-rate for JANUS fission-spectrum neutrons when comparing expression of either a-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Cycloheximide, however, repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposures. Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation and that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.

  7. Cytoskeletal proteins in gastric H/sup +/ secretion: cAMP dependent phosphorylation, immunolocalization, and protein blotting

    SciTech Connect

    Cuppoletti, J.; Sachs, G.; Malinowska, D.H.

    1986-05-01

    The rabbit gastric parietal cell is an excellent model for the study of regulation of secretion and the role of cytoskeleton in secretion. Changes in morphology (appearance of expanded secretory canaliculi lined with microvilli) accompany H/sup +/ secretion stimulated by histamine (cAMP mediated). Parietal cells contain immunoreactive tubulin and are highly enriched in F-actin at secretory canaliculi, detected with fluorescently labelled phallacidin. They have previously shown increased protein phosphorylation in histamine-stimulated purified parietal cells concommitant with increases in H/sup +/ secretion. They report here possible functions of the phosphoproteins. Four of these proteins of apparent size on SDS PAGE of 24, 30, 48 and 130 Kd were membrane associated. /sup 125/I-actin binding to three proteins (24, 30 and 48 Kd) was shown using overlays. A 130 Kd protein reacted with anti-vinculin monoclonal antibody on immunoblots, and was immunolocalized at secretory canaliculi. As a working hypothesis, parietal cells possess membrane-associated proteins which change their state of phosphorylation upon stimulation of H/sup +/. These proteins may be cytoskeletal elements involved in regulation of H/sup +/ secretion. The 130 Kd vinculin-like protein may serve a microfilament-membrane linking role.

  8. Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence.

    PubMed

    Pich, Oscar Q; Burgos, Raul; Ferrer-Navarro, Mario; Querol, Enrique; Piñol, Jaume

    2008-10-01

    The terminal organelle is a differentiated structure that plays a key role in mycoplasma cytadherence and locomotion. For this reason, the analysis of Mycoplasma genitalium mutants displaying anomalous terminal organelles could improve our knowledge regarding the structural elements required for proper locomotion. In this study, we isolated several M. genitalium mutants having transposon insertions within the mg218 or mg317 genes, which encode the orthologues of Mycoplasma pneumoniae HMW2 and HMW3 cytoskeletal proteins, respectively. As expected, mg218(-) and mg317(-) mutants exhibit a reduced gliding motility, although their ability to attach to solid surfaces was not completely abolished. Interestingly, most of the mg218(-) mutants expressed N-terminal MG218 derivatives and showed the presence of short terminal organelles retaining many of the functions displayed by this structure in the wild-type strain, suggesting that the N-terminal region of this protein is an essential element in the architecture of the terminal organelle. Separately, the analysis of mg317(-) mutants indicates that MG317 protein is involved in the formation of the terminal button and contributes to anchoring the electron-dense core to the cell membrane. The results presented here clearly show that MG218 and MG317 proteins are implicated in the maintenance of gliding motility and cytadherence in M. genitalium.

  9. Nano-ZnO leads to tubulin macrotube assembly and actin bundling, triggering cytoskeletal catastrophe and cell necrosis.

    PubMed

    García-Hevia, Lorena; Valiente, Rafael; Martín-Rodríguez, Rosa; Renero-Lecuna, Carlos; González, Jesús; Rodríguez-Fernández, Lidia; Aguado, Fernando; Villegas, Juan C; Fanarraga, Mónica L

    2016-06-07

    Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin filament bundling and structural changes in microtubules, transforming these highly dynamic 25 nm diameter polymers into rigid macrotubes of tubulin, severely affecting cell proliferation and survival. Our results demonstrate that nano-ZnO causes acute cytoskeletal collapse that triggers necrosis, followed by a late reactive oxygen species (ROS)-dependent apoptotic process.

  10. Characterization and phylogeny of two beta-cytoskeletal actins from Hemibarbus mylodon (Cyprinidae, Cypriniformes), a threatened fish species in Korea.

    PubMed

    Kim, Keun-Yong; Lee, Sang Yoon; Cho, Young Sun; Bang, In Chul; Kim, Dong Soo; Nam, Yoon Kwon

    2008-04-01

    Complementary DNA and genomic sequences representing two different beta-actins were isolated from a threatened freshwater fish species Hemibarbus mylodon. The beta-actin 1 and 2 encoded an identical number of amino acids (375 aa), and shared 88.8 and 99.7% of identity at coding nucleotide and amino acid levels, respectively. Genomic open reading frame (ORF) sequences of both isoforms contained five translated exons interrupted by four introns with conserved GT/AG exon/intron boundary rule. Semi-quantitative RT-PCR showed that the two isoform mRNAs were ubiquitously detected in all tissues tested, but transcript levels were variable across tissues. Phylogenetic analysis showed that H. mylodon beta-actin 1 and 2 were clustered into two distinct major and minor branches of Cypriniformes, respectively. Comparisons of the 5'-upstream region and 3'-UTR of H. mylodon beta-actin 1 also showed a high degree of homology with those of the major teleost beta-actins and warmblooded vertebrate beta-cytoskeletal actins, suggesting their more recent common origin.

  11. Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg2+ homeostasis and cytoskeletal architecture

    PubMed Central

    Stritt, Simon; Nurden, Paquita; Favier, Remi; Favier, Marie; Ferioli, Silvia; Gotru, Sanjeev K.; van Eeuwijk, Judith M M.; Schulze, Harald; Nurden, Alan T.; Lambert, Michele P.; Turro, Ernest; Burger-Stritt, Stephanie; Matsushita, Masayuki; Mittermeier, Lorenz; Ballerini, Paola; Zierler, Susanna; Laffan, Michael A.; Chubanov, Vladimir; Gudermann, Thomas; Nieswandt, Bernhard; Braun, Attila

    2016-01-01

    Mg2+ plays a vital role in platelet function, but despite implications for life-threatening conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg2+]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic α-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7fl/fl-Pf4Cre) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7fl/fl-Pf4Cre MKs, which is rescued by Mg2+ supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice. PMID:27020697

  12. Presynaptic calcium channels and α3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components.

    PubMed

    Carlson, Steven S; Valdez, Gregorio; Sanes, Joshua R

    2010-11-01

    At chemical synapses, synaptic cleft components interact with elements of the nerve terminal membrane to promote differentiation and regulate function. Laminins containing the β2 subunit are key cleft components, and they act in part by binding the pore-forming subunit of a pre-synaptic voltage-gated calcium channel (Ca(v)α) (Nishimune et al. 2004). In this study, we identify Ca(v)α-associated intracellular proteins that may couple channel-anchoring to assembly or stabilization of neurotransmitter release sites called active zones. Using Ca(v)α-antibodies, we isolated a protein complex from Torpedo electric organ synapses, which resemble neuromuscular junctions but are easier to isolate in bulk. We identified 10 components of the complex: six cytoskeletal proteins (α2/β2 spectrins, plectin 1, AHNAK/desmoyokin, dystrophin, and myosin 1), two active zone components (bassoon and piccolo), synaptic laminin, and a calcium channel β subunit. Immunocytochemistry confirmed these proteins in electric organ synapses, and PCR analysis revealed their expression by developing mammalian motor neurons. Finally, we show that synaptic laminins also interact with pre-synaptic integrins containing the α3 subunit. Together with our previous finding that a distinct synaptic laminin interacts with SV2 on nerve terminals (Son et al. 2000), our results identify three paths by which synaptic cleft laminins can send developmentally important signals to nerve terminals.

  13. The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns.

    PubMed

    Loose, Martin; Mitchison, Timothy J

    2014-01-01

    Bacterial cytokinesis is commonly initiated by the Z-ring, a cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin superfamily GTPase, which is recruited to the membrane by the actin-related protein FtsA. Both proteins are required for the formation of the Z-ring, but if and how they influence each other's assembly dynamics is not known. Here, we reconstituted FtsA-dependent recruitment of FtsZ polymers to supported membranes, where both proteins self-organize into complex patterns, such as fast-moving filament bundles and chirally rotating rings. Using fluorescence microscopy and biochemical perturbations, we found that these large-scale rearrangements of FtsZ emerge from its polymerization dynamics and a dual, antagonistic role of FtsA: recruitment of FtsZ filaments to the membrane and negative regulation of FtsZ organization. Our findings provide a model for the initial steps of bacterial cell division and illustrate how dynamic polymers can self-organize into large-scale structures.

  14. Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation

    PubMed Central

    Liu, Ching-Yi; Lin, Hsi-Hui; Tang, Ming-Jer; Wang, Yang-Kao

    2015-01-01

    Modulations of cytoskeletal organization and focal adhesion turnover correlate to tumorigenesis and epithelial-mesenchymal transition (EMT), the latter process accompanied by the loss of epithelial markers and the gain of mesenchymal markers (e.g., vimentin). Clinical microarray results demonstrated that increased levels of vimentin mRNA after chemotherapy correlated to a poor prognosis of breast cancer patients. We hypothesized that vimentin mediated the reorganization of cytoskeletons to maintain the mechanical integrity in EMT cancer cells. By using knockdown strategy, the results showed reduced cell proliferation, impaired wound healing, loss of directional migration, and increased large membrane extension in MDA-MB 231 cells. Vimentin depletion also induced reorganization of cytoskeletons and reduced focal adhesions, which resulted in impaired mechanical strength because of reduced cell stiffness and contractile force. In addition, overexpressing vimentin in MCF7 cells increased cell stiffness, elevated cell motility and directional migration, reoriented microtubule polarity, and increased EMT phenotypes due to the increased β1-integrin and the loss of junction protein E-cadherin. The EMT-related transcription factor slug was also mediated by vimentin. The current study demonstrated that vimentin serves as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the balance of cell force generation in EMT cancer cells. PMID:25965826

  15. Cytoskeletal abnormalities in relation with meiotic competence and ageing in porcine and bovine oocytes during in vitro maturation.

    PubMed

    Somfai, T; Kikuchi, K; Kaneda, M; Akagi, S; Watanabe, S; Mizutani, E; Haraguchi, S; Dang-Nguyen, T Q; Inaba, Y; Geshi, M; Nagai, T

    2011-10-01

    We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.

  16. Slit2N Inhibits Transmission of HIV-1 from Dendritic Cells to T-cells by Modulating Novel Cytoskeletal Elements

    PubMed Central

    Shrivastava, Ashutosh; Prasad, Anil; Kuzontkoski, Paula M.; Yu, Jinlong; Groopman, Jerome E.

    2015-01-01

    Dendritic cells are among the first cells to encounter sexually acquired human immunodeficiency virus (HIV-1), in the mucosa, and they can transmit HIV-1 to CD4+ T-cells via an infectious synapse. Recent studies reveal that actin-rich membrane extensions establish direct contact between cells at this synapse and facilitate virus transmission. Genesis of these contacts involves signaling through c-Src and Cdc42, which modulate actin polymerization and filopodia formation via the Arp2/3 complex and Diaphanous 2 (Diaph2). We found that Slit2N, a ligand for the Roundabout (Robo) receptors, blocked HIV-1-induced signaling through Arp2/3 and Diaph2, decreased filopodial extensions on dendritic cells, and inhibited cell-to-cell transmission of HIV-1 in a Robo1-dependent manner. Employing proteomic analysis, we identified Flightless-1 as a novel, Robo1-interacting protein. Treatment with shRNAs reduced levels of Flightless-1 and demonstrated its role in efficient cell-to-cell transfer of HIV-1. These results suggest a novel strategy to limit viral infection in the host by targeting the Slit/Robo pathway with modulation of cytoskeletal elements previously unrecognized in HIV-1 transmission. PMID:26582347

  17. The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

    PubMed Central

    Satchwell, Timothy J; Hawley, Bethan R; Bell, Amanda J; Ribeiro, M. Leticia; Toye, Ashley M

    2015-01-01

    Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation. PMID:25344524

  18. Cell protrusions induced by hyaluronan synthase 3 (HAS3) resemble mesothelial microvilli and share cytoskeletal features of filopodia.

    PubMed

    Koistinen, Ville; Kärnä, Riikka; Koistinen, Arto; Arjonen, Antti; Tammi, Markku; Rilla, Kirsi

    2015-10-01

    Previous studies have shown that overexpression of enzymatically active GFP-HAS induces the growth of long, slender protrusions that share many features of both filopodia and microvilli. These protrusions are dependent on continuing hyaluronan synthesis, and disrupt upon digestion of hyaluronan by hyaluronidase. However, complete understanding of their nature is still missing. This work shows that the protrusions on rat peritoneal surface are ultrastructurally indistinguishable from those induced by GFP-HAS3 in MCF-7 cells. Analysis of the actin-associated proteins villin, ezrin, espin, fascin, and Myo10 indicated that the HAS3-induced protrusions share most cytoskeletal features with filopodia, but they do not require adherence to the substratum like traditional filopodia. GFP-HAS3 overexpression was found to markedly enhance filamentous actin in the protrusions and their cortical basis. Analysis of the protrusion dynamics after enzymatic digestion of hyaluronan revealed that while GFP-HAS3 escape from the protrusions and the protrusion collapse takes place immediately, the complete retraction of the protrusions occurs more slowly. This finding also suggests that hyaluronan chain maintains HAS3 in the plasma membrane. The results of this work suggest that protrusions similar to those of HAS3 overexpressing cells in vitro exist also in cells with active hyaluronan synthesis in vivo. These protrusions are similar to common filopodia but are independent of substratum attachment due to the extracellular scaffolding by the hyaluronan coat that accounts for the growth and maintenance of these structures, previously associated to invasion, adhesion and multidrug resistance.

  19. Dematin, a human erythrocyte cytoskeletal protein, is a substrate for a recombinant FIKK kinase from Plasmodium falciparum.

    PubMed

    Brandt, Gabriel S; Bailey, Scott

    2013-09-01

    P. falciparum causes the most deadly form of malaria, resulting from the adherence of infected red blood cells to blood vessels. During the blood stage of infection, the parasite secretes a large number of proteins into the host erythrocyte. The secretion of a 20-member family of protein kinases known as FIKK kinases, after a conserved Phe-Ile-Lys-Lys sequence motif, is unique to P. falciparum. Identification of physiological substrates of these kinases may provide perspective on the importance of FIKK kinase activity to P. falciparum virulence. We demonstrate, for the first time, the heterologous expression and purification of a FIKK kinase (PfFk4.1, PFD1165w). The recombinant kinase is active against general substrates and phosphorylates itself. Having demonstrated kinase activity, we incubated recombinant Fk4.1 with parasite and human erythrocyte lysates. No parasite-derived substrates were identified. However, treatment of erythrocyte ghosts shows that the FIKK kinase Fk4.1 phosphorylates dematin, a cytoskeletal protein found at the red blood cell spectrin-actin junction.

  20. Brain-Specific Cytoskeletal Damage Markers in Cerebrospinal Fluid: Is There a Common Pattern between Amyotrophic Lateral Sclerosis and Primary Progressive Multiple Sclerosis?

    PubMed Central

    Abdelhak, Ahmed; Junker, Andreas; Brettschneider, Johannes; Kassubek, Jan; Ludolph, Albert C.; Otto, Markus; Tumani, Hayrettin

    2015-01-01

    Many neurodegenerative disorders share a common pathophysiological pathway involving axonal degeneration despite different etiological triggers. Analysis of cytoskeletal markers such as neurofilaments, protein tau and tubulin in cerebrospinal fluid (CSF) may be a useful approach to detect the process of axonal damage and its severity during disease course. In this article, we review the published literature regarding brain-specific CSF markers for cytoskeletal damage in primary progressive multiple sclerosis and amyotrophic lateral sclerosis in order to evaluate their utility as a biomarker for disease progression in conjunction with imaging and histological markers which might also be useful in other neurodegenerative diseases associated with affection of the upper motor neurons. A long-term benefit of such an approach could be facilitating early diagnostic and prognostic tools and assessment of treatment efficacy of disease modifying drugs. PMID:26263977

  1. Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation.

    PubMed Central

    Tetzlaff, M T; Jäckle, H; Pankratz, M J

    1996-01-01

    Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. We have identified a lack-of-function mutation in the cytoskeletal isoform (cTmII). Zygotic mutant embryos show a defect in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton. Images PMID:8635457

  2. Use of Nanobodies to Localize Endogenous Cytoskeletal Proteins and to Determine Their Contribution to Cancer Cell Invasion by Using an ECM Degradation Assay.

    PubMed

    Van Audenhove, Isabel; Gettemans, Jan

    2016-01-01

    There are numerous ways to study actin cytoskeletal structures, and thereby identify the underlying mechanisms of organization and their regulating proteins. Traditional approaches make use of protein overexpression or siRNA. However to study or modulate resident endogenous proteins, complementary methods are required. Since the discovery of nanobodies in 1993, they have proven to represent interesting tools in a variety of applications due to their high affinity, solubility, and stability. Especially their intracellular functionality makes them ideally suited for the study of actin cytoskeletal regulation. Here we provide a protocol to clone nanobody cDNAs in frame with an EGFP or mCherry fluorescent tag. We explain how to transfect this fusion protein in eukaryotic (cancer) cells and how to perform immunofluorescence. This allows microscopic analysis of endogenous (cytoskeletal) proteins and gives insight into their endogenous localization. Moreover, we outline an extracellular matrix (ECM) degradation assay as an application of the general protocol. By seeding cells onto a fluorescently labeled gelatin matrix, degradation can be quantified by means of a matrix degradation index. This assay demonstrates the contribution of a protein during cancer cell invasiveness in vitro and the potential of a nanobody to inhibit this degradation through modulation of its target.

  3. Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells.

    PubMed

    Higaki, Takumi; Kutsuna, Natsumaro; Sano, Toshio; Kondo, Noriaki; Hasezawa, Seiichiro

    2010-01-01

    Manual evaluation of cellular structures is a popular approach in cell biological studies. However, such approaches are laborious and are prone to error, especially when large quantities of image data need to be analyzed. Here, we introduce an image analysis framework that overcomes these limitations by semi-automatic quantification and clustering of cytoskeletal structures. In our framework, cytoskeletal orientation, bundling and density are quantified by measurement of newly-developed, robust metric parameters from microscopic images. Thereafter, the microscopic images are classified without supervision by clustering based on the metric patterns. Clustering allows us to collectively investigate the large number of cytoskeletal structure images without laborious inspection. Application of this framework to images of GFP-actin binding domain 2 (GFP-ABD2)-labeled actin cytoskeletons in Arabidopsis guard cells determined that microfilaments (MFs) are radially oriented and transiently bundled in the process of diurnal stomatal opening. The framework also revealed that the expression of mouse talin GFP-ABD (GFP-mTn) continuously induced MF bundling and suppressed the diurnal patterns of stomatal opening, suggesting that changes in the level of MF bundling are crucial for promoting stomatal opening. These results clearly demonstrate the utility of our image analysis framework.

  4. The integrin adhesome network at a glance

    PubMed Central

    James, Jenny; Jones, Matthew C.; Askari, Janet A.

    2016-01-01

    ABSTRACT The adhesion nexus is the site at which integrin receptors bridge intracellular cytoskeletal and extracellular matrix networks. The connection between integrins and the cytoskeleton is mediated by a dynamic integrin adhesion complex (IAC), the components of which transduce chemical and mechanical signals to control a multitude of cellular functions. In this Cell Science at a Glance article and the accompanying poster, we integrate the consensus adhesome, a set of 60 proteins that have been most commonly identified in isolated IAC proteomes, with the literature-curated adhesome, a theoretical network that has been assembled through scholarly analysis of proteins that localise to IACs. The resulting IAC network, which comprises four broad signalling and actin-bridging axes, provides a platform for future studies of the regulation and function of the adhesion nexus in health and disease. PMID:27799358

  5. 3D actin network centerline extraction with multiple active contours.

    PubMed

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2014-02-01

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

  6. Protein Kinase CK2 Regulates Cytoskeletal Reorganization during Ionizing Radiation-Induced Senescence of Human Mesenchymal Stem Cells

    SciTech Connect

    Wang, Daojing; Jang, Deok-Jin

    2009-08-21

    Human mesenchymal stem cells (hMSC) are critical for tissue regeneration. How hMSC respond to genotoxic stresses and potentially contribute to aging and cancer remain underexplored. We demonstrated that ionizing radiation induced cellular senescence of hMSC over a period of 10 days, showing a critical transition between day 3 and day 6. This was confirmed by senescence-associated beta-galactosidase (SA-{beta}-gal) staining, protein expression profiles of key cell cycle regulators (retinoblastoma (Rb) protein, p53, p21{sup waf1/Cip1}, and p16{sup INK4A}), and senescence-associated secretory phenotypes (SASPs) (IL-8, IL-12, GRO, and MDC). We observed dramatic cytoskeletal reorganization of hMSC through reduction of myosin-10, redistribution of myosin-9, and secretion of profilin-1. Using a SILAC-based phosphoproteomics method, we detected significant reduction of myosin-9 phosphorylation at Ser1943, coinciding with its redistribution. Importantly, through treatment with cell permeable inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT)), and gene knockdown using RNA interference, we identified CK2, a kinase responsible for myosin-9 phosphorylation at Ser1943, as a key factor contributing to the radiation-induced senescence of hMSC. We showed that individual knockdown of CK2 catalytic subunits CK2{alpha} and CK2{alpha}{prime} induced hMSC senescence. However, only knockdown of CK2{alpha} resulted in morphological phenotypes resembling those of radiation-induced senescence. These results suggest that CK2{alpha} and CK2{alpha}{prime} play differential roles in hMSC senescence progression, and their relative expression might represent a novel regulatory mechanism for CK2 activity.

  7. Asparagine and Aspartate Hydroxylation of the Cytoskeletal Ankyrin Family Is Catalyzed by Factor-inhibiting Hypoxia-inducible Factor*

    PubMed Central

    Yang, Ming; Ge, Wei; Chowdhury, Rasheduzzaman; Claridge, Timothy D. W.; Kramer, Holger B.; Schmierer, Bernhard; McDonough, Michael A.; Gong, Lingzhi; Kessler, Benedikt M.; Ratcliffe, Peter J.; Coleman, Mathew L.; Schofield, Christopher J.

    2011-01-01

    Factor-inhibiting hypoxia-inducible factor (FIH) catalyzes the β-hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain of the hypoxia inducible factor (HIF), a modification that negatively regulates HIF transcriptional activity. FIH also catalyzes the hydroxylation of highly conserved Asn residues within the ubiquitous ankyrin repeat domain (ARD)-containing proteins. Hydroxylation has been shown to stabilize localized regions of the ARD fold in the case of a three-repeat consensus ankyrin protein, but this phenomenon has not been demonstrated for the extensive naturally occurring ARDs. Here we report that the cytoskeletal ankyrin family are substrates for FIH-catalyzed hydroxylations. We show that the ARD of ankyrinR is multiply hydroxylated by FIH both in vitro and in endogenous proteins purified from human and mouse erythrocytes. Hydroxylation of the D34 region of ankyrinR ARD (ankyrin repeats 13–24) increases its conformational stability and leads to a reduction in its interaction with the cytoplasmic domain of band 3 (CDB3), demonstrating the potential for FIH-catalyzed hydroxylation to modulate protein-protein interactions. Unexpectedly we found that aspartate residues in ankyrinR and ankyrinB are hydroxylated and that FIH-catalyzed aspartate hydroxylation also occurs in other naturally occurring AR sequences. The crystal structure of an FIH variant in complex with an Asp-substrate peptide together with NMR analyses of the hydroxylation product identifies the 3S regio- and stereoselectivity of the FIH-catalyzed Asp hydroxylation, revealing a previously unprecedented posttranslational modification. PMID:21177872

  8. Cytoskeletal rearrangement and Src and PI-3K-dependent Akt activation control GABA(B)R-mediated chemotaxis.

    PubMed

    Barati, Michelle T; Lukenbill, Janice; Wu, Rui; Rane, Madhavi J; Klein, Jon B

    2015-06-01

    The γ-amino butyric acid (GABA) type B receptors (GABA(B)R) function as chemoattractant receptors in response to GABA(B)R agonists in human neutrophils. The goal of this study was to define signaling mechanisms regulating GABA(B)R-mediated chemotaxis and cytoskeletal rearrangement. In a proteomic study we identified serine/threonine kinase Akt, tyrosine kinases Src and Pyk2, microtubule regulator kinesin and microtubule affinity-regulating kinase (MARK) co-immunoprecipitating with GABA(B)R. To define the contributions of these candidate signaling events in GABA(B)R-mediated chemotaxis, we used rat basophilic leukemic cells (RBL-2H3 cells) stably transfected with human GABA(B1b) and GABA(B2) receptors. The GABA(B)R agonist baclofen induced Akt phosphorylation and chemotaxis by binding to its specific GABA(B)R since pretreatment of cells with CGP52432, a GABA(B)R antagonist, blocked such effects. Moreover, baclofen induced Akt phosphorylation was shown to be dependent upon PI-3K and Src kinases. Baclofen failed to stimulate actin polymerization in suspended RBL cells unless exposed to a baclofen gradient. However, baclofen stimulated both actin and tubulin polymerization in adherent RBL-GABA(B)R cells. Blockade of actin and tubulin polymerization by treatment of cells with cytochalasin D or nocodazole respectively, abolished baclofen-mediated chemotaxis. Furthermore, baclofen stimulated Pyk2 and STAT3 phosphorylation, both known regulators of cell migration. In conclusion, GABA(B)R stimulation promotes chemotaxis in RBL cells which is dependent on signaling via PI3-K/Akt, Src kinases and on rearrangement of both microtubules and actin cytoskeleton. These data define mechanisms of GABA(B)R-mediated chemotaxis which may potentially be used to therapeutically regulate cellular response to injury and disease.

  9. Development of the electromotor system of Torpedo marmorata: distribution of extracellular matrix and cytoskeletal components during acetylcholine receptor focalization.

    PubMed

    Richardson, G P; Fiedler, W; Fox, G Q

    1987-03-01

    A combination of direct fluorescence and indirect immunofluorescence microscopy has been used to compare the distribution of the acetylcholine receptor with the distribution of major cytoskeletal and extracellular matrix components during electrocyte differentiation in the electric organs of Torpedo marmorata. Laminin, fibronectin and extracellular matrix proteoglycan are always more extensively distributed around the differentiating cell than the acetylcholine receptor-rich patch that forms on the ventral surface of the cell. The distribution of acetylcholinesterase within the ventral surface of the differentiating electrocyte closely resembles the distribution of the acetylcholine receptor. Areas of apparently high acetylcholine receptor density within the ventrally forming acetylcholine receptor-rich patch are always areas of apparently high extracellular matrix proteoglycan density but are not always areas of high laminin or fibronectin density. Desmin levels appear to increase at the onset of differentiation and desmin initially accumulates in the ventral pole of each myotube as it begins to form an electrocyte. During differentiation F-actin-positive filament bundles are observed that extend from the nuclei down to the ventrally forming acetylcholine receptor-rich patch. Most filament bundles terminate in the acetylcholine receptor-rich region of the cell membrane. Electron-microscopic autoradiography suggests that the filament bundles attach to the membrane at sites where small acetylcholine receptor clusters are found. The results of this study suggest that, out of the four extracellular matrix components studied, only the distribution of acetylcholinesterase (which may be both matrix- and membrane-bound at this stage) closely parallels that of the acetylcholine receptor, and that F-actin filament bundles terminate in a region of the cell that is becoming an area of high acetylcholine receptor density.

  10. DAMTC regulates cytoskeletal reorganization and cell motility in human lung adenocarcinoma cell line: an integrated proteomics and transcriptomics approach.

    PubMed

    Goel, A; Chhabra, R; Ahmad, S; Prasad, A K; Parmar, V S; Ghosh, B; Saini, N

    2012-10-11

    DAMTC (7,8-diacetoxy-4-methylcoumarin) is a thioderivative of 4-methyl coumarin, and previously we have shown that DAMTC is a potent inhibitor of cell growth and an inducer of apoptosis in non-small cell lung cancer (A549) cells. It induces apoptosis through mitochondrial pathway by modulating NF-κB, mitogen-activated protein kinase (MAPK) and p53 pathways. Herein, we explored the genome-wide effects of DAMTC in A549 cells using the concerted approach of transcriptomics and proteomics. In addition to apoptotic pathways, which have been validated earlier, the bioinformatic analysis of microarray data identified small GTPase-mediated signal transduction among the significantly altered biological processes. Interestingly, we observed significant downregulation of some members of the Rho family GTPases in the proteomics data too. Downregulation of Rho GTPases (RhoGDIα (Rho GDP dissociation inhibitor-α, also known as ARHGDIA), Ras homolog family member A, Ras-related C3 botulinum toxin substrate 1 and cell division cycle 42) was validated by western blotting. The Rho protein family is implicated in maintaining the actin filament assembly and cell motility, and we also observed that DAMTC treatment causes actin cytoskeletal reorganization, promotes filopodia formation and inhibits cell motility in A549 cells. The effect of DAMTC treatment on cytoskeleton was reversed after the overexpression of RhoGDIα. In addition, DAMTC augmented the apoptotic effect of etoposide, a proapoptotic chemotherapeutic drug. This elucidation of the mechanism behind DAMTC-induced apoptosis and inhibition of cell motility in A549 cells may make it a potential therapeutic for lung cancer.

  11. A model for mammalian cochlear hair cell differentiation in vitro: effects of retinoic acid on cytoskeletal proteins and potassium conductances.

    PubMed

    Helyer, R; Cacciabue-Rivolta, D; Davies, D; Rivolta, M N; Kros, C J; Holley, M C

    2007-02-01

    We have established a model for the in-vitro differentiation of mouse cochlear hair cells and have used it to explore the influence of retinoic acid on proliferation, cytoskeletal proteins and voltage-gated potassium conductances. The model is based on the conditionally immortal cell line University of Sheffield/ventral otocyst-epithelial cell line clone 36 (US/VOT-E36), derived from ventral otic epithelial cells of the mouse at embryonic day 10.5 and transfected with a reporter for myosin VIIa. Retinoic acid did not increase cell proliferation but led to up-regulation of myosin VIIa and formation of prominent actin rings that gave rise to numerous large, linear actin bundles. Cells expressing myosin VIIa had larger potassium conductances and did not express the cyclin-dependent kinase inhibitor p27(kip1). US/VOT-E36 endogenously expressed the voltage-gated potassium channel alpha-subunits Kv1.3 and Kv2.1, which we subsequently identified in embryonic and neonatal hair cells in both auditory and vestibular sensory epithelia in vivo. These subunits could underlie the embryonic and neonatal delayed-rectifiers recorded in nascent hair cells in vivo. Kv2.1 was particularly prominent on the basolateral membrane of cochlear inner hair cells. Kv1.3 was distributed throughout all hair cells but tended to be localized to the cuticular plates. US/VOT-E36 recapitulates a coherent pattern of cell differentiation under the influence of retinoic acid and will provide a convenient model for screening the effects of other extrinsic factors on the differentiation of cochlear epithelial cell types in vitro.

  12. Multiple cytoskeletal pathways and PI3K signaling mediate CDC-42-induced neuronal protrusion in C. elegans.

    PubMed

    Alan, Jamie K; Struckhoff, Eric C; Lundquist, Erik A

    2013-01-01

    Rho GTPases are key regulators of cellular protrusion and are involved in many developmental events including axon guidance during nervous system development. Rho GTPase pathways display functional redundancy in developmental events, including axon guidance. Therefore, their roles can often be masked when using simple loss-of-function genetic approaches. As a complement to loss-of-function genetics, we constructed a constitutively activated CDC-42(G12V) expressed in C. elegans neurons. CDC-42(G12V) drove the formation of ectopic lamellipodial and filopodial protrusions in the PDE neurons, which resembled protrusions normally found on migrating growth cones of axons. We then used a candidate gene approach to identify molecules that mediate CDC-42(G12V)-induced ectopic protrusions by determining if loss of function of the genes could suppress CDC-42(G12V). Using this approach, we identified 3 cytoskeletal pathways previously implicated in axon guidance, the Arp2/3 complex, UNC-115/abLIM, and UNC-43/Ena. We also identified the Nck-interacting kinase MIG-15/NIK and p21-activated kinases (PAKs), also implicated in axon guidance. Finally, PI3K signaling was required, specifically the Rictor/mTORC2 branch but not the mTORC1 branch that has been implicated in other aspects of PI3K signaling including stress and aging. Our results indicate that multiple pathways can mediate CDC-42-induced neuronal protrusions that might be relevant to growth cone protrusions during axon pathfinding. Each of these pathways involves Rac GTPases, which might serve to integrate the pathways and coordinate the multiple CDC-42 pathways. These pathways might be relevant to developmental events such as axon pathfinding as well as disease states such as metastatic melanoma.

  13. Utilizing Ultrasound to Transiently Increase Blood-Brain Barrier Permeability, Modulate of the Tight Junction Proteins, and Alter Cytoskeletal Structure.

    PubMed

    Bae, Mi Jung; Lee, Young Mi; Kim, Yeoun Hee; Han, Hyung Soo; Lee, Hak Jong

    2015-01-01

    The central nervous system is protected by the blood-brain barrier (BBB). The tight junction (TJ) proteins claudin-5 and zonula occludens-1 (ZO-1) as well as the cytoskeletal component F-actin play key roles in maintaining homeostasis of the BBB. Increases in BBB permeability may be beneficial for the delivery of pharmacological substances into the brain. Therefore, here, we assessed the use of ultrasound to induce transient enhancement of BBB permeability. We used fluorescein isothiocyanate (FITC)-dextran 40 to detect changes in the membrane permeability of bEnd.3 cells during ultrasound treatment. Ultrasound increased FITC-dextran 40 uptake into bEnd.3 cells for 2-6 h after treatment; however, normal levels returned after 24 h. An insignificant increase in lactate dehydrogenase (LDH) leakage also occurred 3 and 6 h after ultrasound treatment, whereas at 24 h, LDH leakage was indistinguishable between the control and treatment groups. Expression of claudin-5, ZO-1, and F-actin at the messenger RNA (mRNA) and protein levels was assessed with real-time polymerase chain reaction and western blotting. Ultrasound induced a transient decrease in claudin-5 mRNA and protein expression within 2 h of treatment; however, no significant changes in ZO-1 and F-actin expression were observed. Claudin-5, ZO-1, and F-actin immunofluorescence demonstrated that the cellular structures incorporating these proteins were transiently impaired by ultrasound. In conclusion, our ultrasound technique can temporarily increase BBB permeability without cytotoxicity to exposed cells, and the method can be exploited in the delivery of drugs to the brain with minimal damage.

  14. Recurrent viral infections associated with a homozygous CORO1A mutation that disrupts oligomerization and cytoskeletal association

    PubMed Central

    Yee, Christina S.; Massaad, Michel J.; Bainter, Wayne; Ohsumi, Toshiro K.; Föger, Niko; Chan, Andrew C.; Akarsu, Nurten A.; Aytekin, Caner; Ayvaz, Deniz Çagdas; Tezcan, Ilhan; Sanal, Özden; Geha, Raif S.; Chou, Janet

    2015-01-01

    Background Coronin-1A (CORO1A) is a regulator of actin dynamics important for T cell homeostasis. CORO1A deficiency causes T−B+NK+ severe combined immunodeficiency or T cell lymphopenia with severe viral infections. However, since all known human mutations in CORO1A abrogate protein expression, the role of the protein’s functional domains in host immunity is unknown. Objective To identify the cause of the primary immunodeficiency in two young adult siblings with a history of disseminated varicella, cutaneous warts, and CD4+ T cell lymphopenia. Methods We performed immunologic, genetic, and biochemical studies in the patients, family members, and healthy controls. Results Both patients had CD4+ T cell lymphopenia and decreased lymphocyte proliferation to mitogens. IgG, IgM, IgA and specific antibody responses were normal. Whole genome sequencing identified a homozygous frameshift mutation in CORO1A disrupting the last two C-terminal domains by replacing 61 a.a. with a novel 91 a.a. sequence. The CORO1AS401fs mutant was expressed in the patients’ lymphocytes at a level comparable with that of wild-type CORO1A in normal lymphocytes, but failed to oligomerize and had impaired cytoskeletal association. CORO1AS401fs was associated with increased F-actin accumulation in T cells, severely defective thymic output, and impaired T cell survival, but normal calcium flux and cytotoxicity, demonstrating the importance of CORO1A oliogomerization and subcellular localization in T cell homeostasis. Conclusions We describe a truncating mutation in CORO1A that permits protein expression and survival into young adulthood. Our studies demonstrate the importance of intact CORO1A C-terminal domains in thymic egress and T cell survival as well as in the defense against viral pathogens. PMID:26476480

  15. Disruption of cytoskeletal structures mediates shear stress-induced endothelin-1 gene expression in cultured porcine aortic endothelial cells.

    PubMed Central

    Morita, T; Kurihara, H; Maemura, K; Yoshizumi, M; Yazaki, Y

    1993-01-01

    Hemodynamic shear stress alters the architecture and functions of vascular endothelial cells. We have previously shown that the synthesis of endothelin-1 (ET-1) in endothelial cells is increased by exposure to shear stress. Here we examined whether shear stress-induced alterations in cytoskeletal structures are responsible for increases in ET-1 synthesis in cultured porcine aortic endothelial cells. Exposure of endothelial cells to 5 dyn/cm2 of low shear stress rapidly increased monomeric G-actin contents within 5 min without changing total actin contents. The ratio of G- to total actin, 54 +/- 0.8% in quiescent endothelial cells, increased to 87 +/- 4.2% at 6 h and then decreased. Following the disruption of filamentous (F)-actin into G-actin, ET-1 mRNA levels in endothelial cells also increased within 30 min and reached a peak at 6 h. The F-actin stabilizer, phalloidin, abolished shear stress-induced increases in ET-1 mRNA; however, it failed to inhibit increases in ET-1 mRNA secondary to other stimulants. This indicates that shear stress-induced increases in ET-1 mRNA levels may be mediated by the disruption of actin fibers. Furthermore, increases in ET-1 gene expression can be induced by actin-disrupting agents, cytochalasin B and D. Another cytoskeleton-disrupting agent, colchicine, which inhibits dimerization of tubulin, did not affect the basal level of ET-1 mRNA. However, colchicine completely inhibited shear stress- and cytochalasin B-induced increases in ET-1 mRNA levels. These results suggest that shear stress-induced ET-1 gene expression in endothelial cells is mediated by the disruption of actin cytoskeleton and this induction is dependent on the integrity of microtubules. Images PMID:8408624

  16. Activated ADF/cofilin sequesters phosphorylated microtubule-associated-protein during the assembly of Alzheimer-like neuritic cytoskeletal striations

    PubMed Central

    Whiteman, Ineka T.; Gervasio, Othon L.; Cullen, Karen M.; Guillemin, Gilles J.; Jeong, Erica V.; Witting, Paul K.; Antao, Shane T.; Minamide, Laurie S.; Bamburg, James R.; Goldsbury, Claire

    2009-01-01

    In Alzheimer disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads) and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence Resonance Energy Transfer (FRET) analysis revealed co-localization of cofilin-GFP and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and siRNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous Aβ peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction or Aβ peptides, all suspected initiators of synaptic loss and neurodegeneration in AD. PMID:19828813

  17. Disrupted cytoskeletal homeostasis, astrogliosis and apoptotic cell death in the cerebellum of preweaning rats injected with diphenyl ditelluride.

    PubMed

    Heimfarth, Luana; Loureiro, Samanta Oliveira; Dutra, Márcio Ferreira; Petenuzzo, Letícia; de Lima, Bárbara Ortiz; Fernandes, Carolina Gonçalves; da Rocha, João Batista Teixeira; Pessoa-Pureur, Regina

    2013-01-01

    In the present report 15 day-old rats were injected with 0.3μmol of diphenyl ditelluride (PhTe)(2)/kg body weight and parameters of neurodegeneration were analyzed in slices from cerebellum 3 and 6 days afterwards. The earlier responses, at day 3 after injection, included hyperphosphorylation of intermediate filament (IF) proteins from astrocyte (glial fibrillary acidic protein - GFAP - and vimentin) and neuron (low-, medium- and high molecular weight neurofilament subunits: NF-L, NF-M and NF-H); increased mitogen-activated protein kinase (MAPK) (Erk and p38MAPK) and cAMP-dependent protein kinase (PKA) activities. Also, reactive astrogliosis takes part of the early responses to the insult with (PhTe)(2), evidenced by upregulated GFAP in Western blot, PCR and immunofluorescence analysis. Six days after (PhTe)(2) injection we found persistent astrogliosis, increased propidium iodide (PI) positive cells in NeuN positive population evidenced by flow cytometry and reduced immunofluorescence for NeuN, suggesting that the in vivo exposure to (PhTe)(2) progressed to neuronal death. Moreover, activated caspase 3 suggested apoptotic neuronal death. Neurodegeneration was related with decreased [(3)H]glutamate uptake and decreased Akt immunoreactivity, however phospho-GSK-3-β (Ser9) was not altered in (PhTe)(2) injected rat. Therefore, the present results show that the earlier cerebellar responses to (PhTe)(2) include disruption of cytoskeletal homeostasis that could be related with MAPK and PKA activation and reactive astrogliosis. Akt inhibition observed at this time could also play a role in the neuronal death evidenced afterwards. The later events of the neurodegenerative process are characterized by persistent astrogliosis and activation of apoptotic neuronal death through caspase 3 mediated mechanisms, which could be related with glutamate excitotoxicity. The progression of these responses are therefore likely to be critical for the outcome of the neurodegeneration

  18. Cytoskeletal role in the transition from compensated to decompensated hypertrophy during adult canine left ventricular pressure overloading

    NASA Technical Reports Server (NTRS)

    Tagawa, H.; Koide, M.; Sato, H.; Zile, M. R.; Carabello, B. A.; Cooper, G. 4th

    1998-01-01

    Increased microtubule density causes cardiocyte contractile dysfunction in right ventricular (RV) pressure-overload hypertrophy, and these linked phenotypic and contractile abnormalities persist and progress during the transition to failure. Although more severe in cells from failing than hypertrophied RVs, the mechanical defects are normalized in each case by microtubule depolymerization. To define the role of increased microtubule density in left ventricular (LV) pressure-overload hypertrophy and failure, in a given LV we examined ventricular mechanics, sarcomere mechanics, and free tubulin and microtubule levels in control dogs and in dogs with aortic stenosis both with LV hypertrophy alone and with initially compensated hypertrophy that had progressed to LV muscle failure. In comparing initial values with those at study 8 weeks later, dogs with hypertrophy alone had a very substantial increase in LV mass but preservation of a normal ejection fraction and mean systolic wall stress. Dogs with hypertrophy and associated failure had a substantial but lesser increase in LV mass and a reduction in ejection fraction, as well as a marked increase in mean systolic wall stress. Cardiocyte contractile function was equivalent, and unaffected by microtubule depolymerization, in cells from control LVs and those with compensated hypertrophy. In contrast, cardiocyte contractile function in cells from failing LVs was quite depressed but was normalized by microtubule depolymerization. Microtubules were increased only in failing LVs. These contractile and cytoskeletal changes, when assayed longitudinally in a given dog by biopsy, appeared in failing ventricles only when wall stress began to increase and function began to decrease. Thus, the microtubule-based cardiocyte contractile dysfunction characteristic of pressure-hypertrophied myocardium, originally described in the RV, obtains equally in the LV but is shown here to have a specific association with increased wall stress.

  19. Muscular dystrophy-associated SUN1 and SUN2 variants disrupt nuclear-cytoskeletal connections and myonuclear organization.

    PubMed

    Meinke, Peter; Mattioli, Elisabetta; Haque, Farhana; Antoku, Susumu; Columbaro, Marta; Straatman, Kees R; Worman, Howard J; Gundersen, Gregg G; Lattanzi, Giovanna; Wehnert, Manfred; Shackleton, Sue

    2014-09-01

    Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning.

  20. Muscular Dystrophy-Associated SUN1 and SUN2 Variants Disrupt Nuclear-Cytoskeletal Connections and Myonuclear Organization

    PubMed Central

    Antoku, Susumu; Columbaro, Marta; Straatman, Kees R.; Worman, Howard J.; Gundersen, Gregg G.; Lattanzi, Giovanna; Wehnert, Manfred; Shackleton, Sue

    2014-01-01

    Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning. PMID:25210889

  1. Effects of novel ethacrynic acid derivatives on human trabecular meshwork cell shape, actin cytoskeletal organization, and transcellular fluid flow.

    PubMed

    Rao, Ponugoti Vasantha; Shimazaki, Atsushi; Ichikawa, Masaki; Franse-Carman, Linda; Alvarado, Jorge A; Epstein, David L

    2005-12-01

    To determine efficacy and therapeutic index in the context of ocular hypotensive activity of the new ethacrynic acid (ECA) derivatives of the series (SA8,248 and SA8,389), 9,000 series (SA9,000, SA9,622 and SA9,995) and ticrynafen, we undertook a comparative evaluation of the dose-dependent effects of these compounds on human trabecular meshwork (HTM) cell shape, actin cytoskeletal organization, focal adhesions and transcellular fluid flow. Responses were either scored using an arbitrary scale of 1-5 or quantified. Compounds of the 9000 series (SA9,995>SA9,000>SA9,622) were found to be 14- to 20-fold more potent than ECA, ticrynafen or analogs from the 8,000 series (SA8,389>SA8,248) in terms of ability to induce cell shape alterations in HTM cells. Similarly, compounds of the 9,000 series (SA9,995>SA9,622>SA9,000) were found to be much stronger (2 to 20 fold) than ECA, ticrynafen or analogs of the 8000 series in terms of affecting decreases in actin stress fiber content in HTM cells. Analogs of the 9000 series (SA9,622>SA9,995>SA9,000) were also observed to be 8 to 10 fold more potent than ECA (SA8,389>ECA>SA8,248>ticrynafen) at eliciting decreases in cellular focal adhesions. Interestingly, analogs of the 9000 series (SA9,000>SA9,622>SA9,995) and SA8,248 demonstrated a huge increase (by many folds) in transcellular fluid flow of HTM cell monolayers as compared to ECA and ticrynafen. Collectively, these analyses revealed that the structural modification of ECA improves its ocular hypotensive efficacy, indicating that the SA9,000 series compounds might be promising novel ocular hypotensive drugs.

  2. Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps.

    PubMed

    Chu, Haiyan; Puchulu-Campanella, Estela; Galan, Jacob A; Tao, W Andy; Low, Philip S; Hoffman, Joseph F

    2012-07-31

    The type of metabolic compartmentalization that occurs in red blood cells differs from the types that exist in most eukaryotic cells, such as intracellular organelles. In red blood cells (ghosts), ATP is sequestered within the cytoskeletal-membrane complex. These pools of ATP are known to directly fuel both the Na(+)/K(+) and Ca(2+) pumps. ATP can be entrapped within these pools either by incubation with bulk ATP or by operation of the phosphoglycerate kinase and pyruvate kinase reactions to enzymatically generate ATP. When the pool is filled with nascent ATP, metabolic labeling of the Na(+)/K(+) or Ca(2+) pump phosphoproteins (E(Na)-P and E(Ca)-P, respectively) from bulk [γ-(32)P]-ATP is prevented until the pool is emptied by various means. Importantly, the pool also can be filled with the fluorescent ATP analog trinitrophenol ATP, as well as with a photoactivatable ATP analog, 8-azido-ATP (N(3)-ATP). Using the fluorescent ATP, we show that ATP accumulates and then disappears from the membrane as the ATP pools are filled and subsequently emptied, respectively. By loading N(3)-ATP into the membrane pool, we demonstrate that membrane proteins that contribute to the pool's architecture can be photolabeled. With the aid of an antibody to N(3)-ATP, we identify these labeled proteins by immunoblotting and characterize their derived peptides by mass spectrometry. These analyses show that the specific peptides that corral the entrapped ATP derive from sequences within β-spectrin, ankyrin, band 3, and GAPDH.

  3. Stepwise morphological changes and cytoskeletal reorganization of human mesenchymal stem cells treated by short-time cyclic uniaxial stretch.

    PubMed

    Parandakh, Azim; Tafazzoli-Shadpour, Mohammad; Khani, Mohammad-Mehdi

    2017-02-15

    This study aimed to investigate stepwise remodeling of human mesenchymal stem cells (hMSCs) in response to cyclic stretch through rearrangement and alignment of cells and cytoskeleton regulation toward smooth muscle cell (SMC) fate in different time spans. Image analysis techniques were utilized to calculate morphological parameters. Cytoskeletal reorganization was observed by investigating F-actin filaments using immunofluorescence staining, and expression level of contractile SMC markers was followed by a quantitative polymerase chain reaction method. Applying cyclic uniaxial stretch on cultured hMSCs, utilizing a costume-made device, led to alteration in fractal dimension (FD) and cytoskeleton structure toward continuous alignment and elongation of cells by elevation of strain duration. Actin filaments became more aligned perpendicular to the axis of mechanical stretch by increasing uniaxial loading duration. At first, FD met a significant decrease in 4 h loading duration then increased significantly by further loading up to 16 h, followed by another decrease up to 1 d of uniaxial stretching. HMSCs subjected to 24 h cyclic uniaxial stretching significantly expressed early and intermediate contractile SM markers. It was hypothesized that the increase in FD after 4 h while cells continuously became more aligned and elongated was due to initiation of change in phenotype that influenced arrangement of cells. At this point, change in cell phenotype started leading to change in morphology while mechanical loading still caused cell alignment and rearrangement. Results can be helpful when optimized engineered cells are needed based on mechanical condition for functional engineered tissue and cell therapy.

  4. Vibration induced osteogenic commitment of mesenchymal stem cells is enhanced by cytoskeletal remodeling but not fluid shear.

    PubMed

    Uzer, Gunes; Pongkitwitoon, Suphannee; Ete Chan, M; Judex, Stefan

    2013-09-03

    Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell's sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04 Pa to 5 Pa. Vibrations were applied at magnitudes of 0.15 g, 1g, and 2g using frequencies of both 100 Hz and 30 Hz. After 14 d and under low fluid shear conditions associated with 100 Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30 Hz vibrations enhanced mineralization only in the 2g group. Over 3d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott-Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity.

  5. Cytoskeletal studies on Lowicryl K4M embedded and Affi-Gel 731 attached rat peritoneal mast cells.

    PubMed

    Nielsen, E H; Jahn, H

    1984-01-01

    The subplasmalemmal network in mast cells consists of irregularly arranged 6-7 nm filaments (actin) connected by thinner filaments. In places oblique filaments with crossbridges or short, perpendicular filaments (11-12 nm) connect cell and granule membrane. Filaments attaching subplasmalemmal network to cell membrane divide like a Y and attach cell membrane end-on with a conical, hook-like bending. Each granule is surrounded by a regular network of filaments.

  6. IGE RECEPTOR-MEDIATED ALTERATION OF MEMBRANE-CYTOSKELETAL INTERACTIONS REVEALED BY MASS SPECTROMETRIC ANALYSIS OF DETERGENT-RESISTANT MEMBRANES†

    PubMed Central

    Han, Xuemei; Smith, Norah L.; Sil, Dwaipayan; Holowka, David A.; McLafferty, Fred W.; Baird, Barbara A.

    2009-01-01

    We use electrospray ionization mass spectrometry to quantify >100 phospholipid (PL) components in detergent-resistant membrane (DRM) domains that are related to ordered membrane compartments commonly known as lipid rafts. We previously compared PL compositions of DRMs with plasma membrane vesicles and whole cell lipid extracts from RBL mast cells, and we made the initial observation that antigen stimulation of IgE receptors (FcεRI) causes a significant change in the PL composition of DRMs (Fridriksson, et al., Biochemistry 38: 8056, 1999). We now characterize the signaling requirements and time course for this change, which is manifested as an increase in the recovery of polyunsaturated PL in DRM, particularly in phosphatidylinositol species. We find that this change is largely independent of tyrosine phosphorylation, stimulated by engagement of FcεRI, and can be activated by Ca2+ ionophore independent of antigen stimulation. Unexpectedly, we found that inhibitors of actin polymerization (cytochalasin D, latrunculin A) cause a similar, but more rapid, change in the PL composition of DRMs in the absence of FcεRI activation, indicating that perturbations in the actin cytoskeleton affect the organization of plasma membrane domains. Consistent with this interpretation, a membrane-permeable stabilizer of F-actin, jasplakinolide, prevents antigen-stimulated changes in DRM PL composition. These results are confirmed by a detailed analysis of multiple experiments, showing that receptor and cytochalasin D-stimulated changes in DRM lipid composition follow first-order kinetics. Analysis in terms of the number of double bonds in the fatty acid chains is valid for total PL of the major head groups and for head groups individually. In this manner we show that, on average, concentrations of saturated or monounsaturated PL decrease in the DRM, whereas concentrations of PL with two or more double bonds (polyunsaturated PL) increase due to cytoskeletal perturbation. We find that

  7. Differential effects of two phospholipase D inhibitors, 1-butanol and N-acylethanolamine, on in vivo cytoskeletal organization and Arabidopsis seedling growth.

    PubMed

    Motes, Christy M; Pechter, Priit; Yoo, Cheol Min; Wang, Yuh-Shuh; Chapman, Kent D; Blancaflor, Elison B

    2005-12-01

    Plant development is regulated by numerous chemicals derived from a multitude of metabolic pathways. However, we know very little about the biological effects and functions of many of these metabolites in the cell. N-Acylethanolamines (NAEs) are a group of lipid mediators that play important roles in mammalian physiology. Despite the intriguing similarities between animals and plants in NAE metabolism and perception, not much is known about the precise function of these metabolites in plant physiology. In plants, NAEs have been shown to inhibit phospholipase Dalpha (PLDalpha) activity, interfere with abscisic acid-induced stomatal closure, and retard Arabidopsis seedling development. 1-Butanol, an antagonist of PLD-dependent phosphatidic acid production, was reported to induce defects in Arabidopsis seedling development that were somewhat similar to effects induced by elevated levels of NAE. This raised the possibility that the impact of NAE on seedling growth could be mediated in part via its influence on PLD activity. To begin to address this possibility, we conducted a detailed, comparative analysis of the effects of 1-butanol and N-lauroylethanolamine (NAE 12:0) on Arabidopsis root cell division, in vivo cytoskeletal organization, seed germination, and seedling growth. Although both NAE 12:0 and 1-butanol induced profound cytoskeletal and morphological alterations in seedlings, there were distinct differences in their overall effects. 1-Butanol induced more pronounced modifications in cytoskeletal organization, seedling growth, and cell division at concentrations severalfold higher than NAE 12:0. We propose that these compounds mediate their differential effects on cellular organization and seedling growth, in part through the differential modulation of specific PLD isoforms.

  8. Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

    PubMed Central

    Casolari, Jason M.; Thompson, Michael A.; Salzman, Julia; Champion, Lowry M.; Moerner, W. E.; Brown, Patrick O.

    2012-01-01

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches. PMID:22359641

  9. Apo and calcium-bound crystal structures of cytoskeletal protein alpha-14 giardin (annexin E1) from the intestinal protozoan parasite Giardia lamblia.

    PubMed

    Pathuri, Puja; Nguyen, Emily Tam; Ozorowski, Gabriel; Svärd, Staffan G; Luecke, Hartmut

    2009-01-30

    Alpha-14 giardin (annexin E1), a member of the alpha giardin family of annexins, has been shown to localize to the flagella of the intestinal protozoan parasite Giardia lamblia. Alpha giardins show a common ancestry with the annexins, a family of proteins most of which bind to phospholipids and cellular membranes in a Ca(2+)-dependent manner and are implicated in numerous membrane-related processes including cytoskeletal rearrangements and membrane organization. It has been proposed that alpha-14 giardin may play a significant role during the cytoskeletal rearrangement during differentiation of Giardia. To gain a better understanding of alpha-14 giardin's mode of action and its biological role, we have determined the three-dimensional structure of alpha-14 giardin and its phospholipid-binding properties. Here, we report the apo crystal structure of alpha-14 giardin determined in two different crystal forms as well as the Ca(2+)-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 A, respectively. Although the overall fold of alpha-14 giardin is similar to that of alpha-11 giardin, multiwavelength anomalous dispersion phasing was required to solve the alpha-14 giardin structure, indicating significant structural differences between these two members of the alpha giardin family. Unlike most annexin structures, which typically possess N-terminal domains, alpha-14 giardin is composed of only a core domain, followed by a C-terminal extension that may serve as a ligand for binding to cytoskeletal protein partners in Giardia. In the Ca(2+)-bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calcium-binding site not previously observed in annexin structures. This novel high-affinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipid-binding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to

  10. New insights into dynamic actin-based chloroplast photorelocation movement.

    PubMed

    Kong, Sam-Geun; Wada, Masamitsu

    2011-09-01

    Chloroplast movement is essential for plants to survive under various environmental light conditions. Phototropins-plant-specific blue-light-activated receptor kinases-mediate the response by perceiving light intensity and direction. Recently, novel chloroplast actin (cp-actin) filaments have been identified as playing a pivotal role in the directional chloroplast photorelocation movement. Encouraging progress has recently been made in this field of research through molecular genetics and cell biological analyses. This review describes factors that have been identified as being involved in chloroplast movement and their roles in the regulation of cp-actin filaments, thus providing a basis for reflection on their biochemical activities and functions.

  11. Toxaphene, but not beryllium, induces human neutrophil chemotaxis and apoptosis via reactive oxygen species (ROS): involvement of caspases and ROS in the degradation of cytoskeletal proteins.

    PubMed

    Lavastre, Valérie; Roberge, Charles J; Pelletier, Martin; Gauthier, Marc; Girard, Denis

    2002-07-01

    Chemicals of environmental concern are known to alter the immune system. Recent data indicate that some contaminants possess proinflammatory properties by activating neutrophils, an area of research that is still poorly investigated. We have previously documented that toxaphene activates human neutrophils to produce reactive oxygen species (ROS) and accelerates apoptosis by a yet unknown mechanism. In this study, we found that toxaphene induces another neutrophil function, chemotaxis. Furthermore, we found that toxaphene induces both chemotaxis and apoptosis via a ROS-dependent mechanism, since these responses were blocked by the addition of catalase to the culture. In addition, toxaphene was found to induce the degradation of the cytoskeletal proteins gelsolin, paxillin, and vimentin during apoptosis, and this was reversed by the addition of z-VAD-FMK (caspase inhibitor) or catalase, demonstrating the importance of caspases and ROS in this process. In contrast to toxaphene, we found that beryllium does not induce superoxide production, and, this correlates with its inability to induce chemotaxis and apoptosis. We conclude that toxaphene induces chemotaxis and apoptosis via ROS and that caspases and ROS are involved in the degradation of cytoskeletal proteins.

  12. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation

    PubMed Central

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  13. Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes.

    PubMed Central

    Burn, P; Kupfer, A; Singer, S J

    1988-01-01

    Members of the family of transmembrane integral membrane proteins called integrins have been implicated in forming attachments to actin microfilaments of the cytoskeleton. These attachments are thought to involve one or more intervening peripheral membrane proteins linked to integrin. To detect such possible linkages in vivo, the integrin molecules on the surfaces of intact chicken peripheral blood lymphocytes were collected into caps by cross-linking with specific antibodies, and the capped cells were examined by double immunofluorescence to determine whether particular cytoskeletal proteins were co-collected with the integrin. With resting lymphocytes, the capping of integrin did not result in any detectable redistribution of either talin, vinculin, or alpha-actinin inside the cells. However, if the capping was carried out upon the addition of phorbol 12-myristate 13-acetate (PMA) to the cells, then talin, but not vinculin or alpha-actinin, was found associated with the integrin caps. PMA is known to activate protein kinase C. These results suggest that after, but not before, PMA stimulation of intact cells, talin becomes linked either directly or indirectly with integrin, reflecting the formation of a membrane-cytoskeletal association that is metabolically regulated. Images PMID:3124107

  14. Expression of cytoskeletal and molt-related genes is temporally scheduled in the hypodermis of the crayfish Procambarus clarkii during premolt.

    PubMed

    Tom, Moshe; Manfrin, Chiara; Chung, Sook J; Sagi, Amir; Gerdol, Marco; De Moro, Gianluca; Pallavicini, Alberto; Giulianini, Piero G

    2014-12-01

    The rigid crustacean exoskeleton, the cuticle, is composed of the polysaccharide chitin, structural proteins and mineral deposits. It is periodically replaced to enable growth and its construction is an energy-demanding process. Ecdysis, the shedding event of the old cuticle, is preceded by a preparatory phase, termed premolt, in which the present cuticle is partially degraded and a new one is formed underneath it. Procambarus clarkii (Girard 1852), an astacid crustacean, was used here to comprehensively examine the changing patterns of gene expression in the hypodermis underlying the cuticle of the carapace at seven time points along ~14 premolt days. Next generation sequencing was used to construct a multi-tissue P. clarkii transcript sequence assembly for general use in a variety of transcriptomic studies. A reference transcriptome was created here in order to perform digital transcript expression analysis, determining the gene expression profiles in each of the examined premolt stages. The analysis revealed a cascade of sequential expression events of molt-related genes involved in chitin degradation, synthesis and modification, as well as synthesis of collagen and four groups of cuticular structural genes. The new description of major transcriptional events during premolt and the determination of their timing provide temporal markers for future studies of molt progress and regulation. The peaks of the expression of the molt-related genes were preceded by expression peaks of cytoskeletal genes that are hypothesized to be essential for premolt progress through regulating protein synthesis and/or transport, probably by remodeling the cytoskeletal structure.

  15. Smoking correlates with increased cytoskeletal protein-related coding region mutations in the lung and head and neck datasets of the cancer genome atlas.

    PubMed

    Yavorski, John M; Blanck, George

    2016-12-01

    Cancer from smoking tobacco is considered dependent on mutagens, but significant molecular aspects of smoking-specific, cancer development remain unknown. We defined sets of coding regions for oncoproteins, tumor suppressor proteins, and cytoskeletal-related proteins that were compared between nonsmokers and smokers, for mutation occurrences, in the lung adenocarcinoma (LUAD), head and neck squamous carcinoma (HNSC), bladder carcinoma (BLCA), and pancreatic adenocarcinoma ( PAAD) datasets from the cancer genome atlas (TCGA). We uncovered significant differences in overall mutation rates, and in mutation rates in cytoskeletal protein-related coding regions (CPCRs, including extracellular matrix protein coding regions), between nonsmokers and smokers in LUAD and HNSC (P < 0.001), raising the question of whether the CPCR mutation differences lead to different clinical courses for nonsmoker and smoker cancers. Another important question inspired by these results is, whether high smoker cancer mutation rates would facilitate genotoxicity or neoantigen-based therapies. No significant, mutation-based differences were found in the BLCA or PAAD datasets, between nonsmokers and smokers. However, a significant difference was uncovered for the average number of overall cancer mutations, in LUAD, for persons who stopped smoking more than 15 years ago, compared with more recent smokers (P < 0.032).

  16. Cytoskeletal re-arrangement in TGF-β1-induced alveolar epithelial-mesenchymal transition studied by atomic force microscopy and high-content analysis.

    PubMed

    Buckley, Stephen T; Medina, Carlos; Davies, Anthony M; Ehrhardt, Carsten

    2012-04-01

    Epithelial-mesenchymal transition (EMT) is closely implicated in the pathogenesis of idiopathic pulmonary fibrosis. Associated with this phenotypic transition is the acquisition of an elongated cell morphology and establishment of stress fibers. The extent to which these EMT-associated changes influence cellular mechanics is unclear. We assessed the biomechanical properties of alveolar epithelial cells (A549) following exposure to TGF-β1. Using atomic force microscopy, changes in cell stiffness and surface membrane features were determined. Stimulation with TGF-β1 gave rise to a significant increase in stiffness, which was augmented by a collagen I matrix. Additionally, TGF-β1-treated cells exhibited a rougher surface profile with notable protrusions. Simultaneous quantitative examination of the morphological attributes of stimulated cells using an image-based high-content analysis system revealed dramatic alterations in cell shape, F-actin content and distribution. Together, these investigations point to a strong correlation between the cytoskeletal-associated cellular architecture and the mechanical dynamics of alveolar epithelial cells undergoing EMT. From the Clinical Editor: Epithelial-mesenchymal transition is implicated in the pathogenesis of pulmonary fibrosis. Using atomic force microscopy, the authors demonstrate a strong correlation between the cytoskeletal-associated cellular architecture and the mechanical dynamics of alveolar epithelial cells undergoing mesenchymal transition.

  17. Properties of rat erythrocyte membrane cytoskeletal structures produced by digitonin extraction: digitonin-insoluble beta-adrenergic receptor, adenylate cyclase, and cholera toxin substrate.

    PubMed

    LeVine, H; Sahyoun, N E; Cuatrecasas, P

    1982-01-01

    Rat erythrocyte plasma membranes have been extracted exhaustively with digitonin at low temperature, and the residual, detergent-extracted membrane cytoskeletal material is compared to that prepared with Triton X-100 with respect to protein, glycoprotein, phospholipid, and cholesterol content. Digitonin, a weaker detergent than Triton X-100, solubilizes only 26% of the phospholipids and none of the cholesterol. SDS-polyacrylamide gel electrophoresis reveals that differences between the proteins extracted by the two detergents are primarily quantitative. In terms of functional preservation, digitonin retains in the cytoskeleton 28% of the beta-adrenergic receptor binding activity (with the balance accounted for in the supernatant), greater than 90% of the adenylate cyclase and greater than 90% of the 45,000 mol wt polypeptide cholera toxin substrate. The cytoskeletal-associated beat-adrenergic receptor retains binding properties for antagonist and agonist which are identical to those of the native membrane receptor. The digitonin-extracted cytoskeleton containing the beta-adrenergic receptor may provide a useful vehicle for the reconstitution of a hormone-sensitive adenylate cyclase.

  18. The fractal architecture of cytoplasmic organization: scaling, kinetics and emergence in metabolic networks.

    PubMed

    Aon, Miguel Antonio; O'Rourke, Brian; Cortassa, Sonia

    2004-01-01

    In this work, we highlight the links between fractals and scaling in cells and explore the kinetic consequences for biochemical reactions operating in fractal media. Based on the proposal that the cytoskeletal architecture is organized as a percolation lattice, with clusters emerging as fractal forms, the analysis of kinetics in percolation clusters is especially emphasized. A key consequence of this spatiotemporal cytoplasmic organization is that enzyme reactions following Michaelis-Menten or allosteric type kinetics exhibit higher rates in fractal media (for short times and at lower substrate concentrations) at the percolation threshold than in Euclidean media. As a result, considerably faster and higher amplification of enzymatic activity is obtained. Finally, we describe some of the properties bestowed by cytoskeletal organization and dynamics on metabolic networks.

  19. Form-Finding Model Shows How Cytoskeleton Network Stiffness Is Realized

    PubMed Central

    Gong, Jinghai; Zhang, Daxu; Tseng, Yiider; Li, Baolong; Wirtz, Denis; Schafer, Benjamin William

    2013-01-01

    In eukaryotic cells the actin-cytoskeletal network provides stiffness and the driving force that contributes to changes in cell shape and cell motility, but the elastic behavior of this network is not well understood. In this paper a two dimensional form-finding model is proposed to investigate the elasticity of the actin filament network. Utilizing an initially random array of actin filaments and actin-cross-linking proteins the form-finding model iterates until the random array is brought into a stable equilibrium configuration. With some care given to actin filament density and length, distance between host sites for cross-linkers, and overall domain size the resulting configurations from the form-finding model are found to be topologically similar to cytoskeletal networks in real cells. The resulting network may then be mechanically exercised to explore how the actin filaments deform and align under load and the sensitivity of the network’s stiffness to actin filament density, length, etc. Results of the model are consistent with the experimental literature, e.g. actin filaments tend to re-orient in the direction of stretching; and the filament relative density, filament length, and actin-cross-linking protein’s relative density, control the actin-network stiffness. The model provides a ready means of extension to more complicated domains and a three-dimensional form-finding model is under development as well as models studying the formation of actin bundles. PMID:24146992

  20. Artificial microtubule cytoskeleton construction, manipulation, and modeling via holographic trapping of network nodes

    NASA Astrophysics Data System (ADS)

    Bergman, J.; Doval, F.; Vershinin, M.

    2016-09-01

    Cytoskeletal networks are 3D arrangements of filaments whose complex spatial structure contributes significantly to their intracellular functions, e.g. biomechanics and cargo motility. Microtubule networks in cells are a particular challenge for in vitro modeling because they are sparse and possess overall structure and so cannot be approximated experimentally as a random hydrogel. We have used holographic optical trapping to precisely position and hold multiple microtubule filaments in an in vitro assay, where chemical and environmental variables can be carefully controlled. Below we describe the relevant practical details of the approach and demonstrate how our approach can scale to accommodate modeling of molecular motor transport and biomechanics experiments.

  1. The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

    SciTech Connect

    Doller, Anke; Badawi, Amel

    2015-01-01

    The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D{sub 1} encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E{sub 2} synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on

  2. Time courses of post-injury mitochondrial oxidative damage and respiratory dysfunction and neuronal cytoskeletal degradation in a rat model of focal traumatic brain injury.

    PubMed

    Hill, Rachel L; Singh, Indrapal N; Wang, Juan A; Hall, Edward D

    2017-03-23

    Traumatic brain injury (TBI) results in rapid reactive oxygen species (ROS) production and oxidative damage to essential brain cellular components leading to neuronal dysfunction and cell death. It is increasingly appreciated that a major player in TBI-induced oxidative damage is the reactive nitrogen species (RNS) peroxynitrite (PN) which is produced in large part in injured brain mitochondria. Once formed, PN decomposes into highly reactive free radicals that trigger membrane lipid peroxidation (LP) of polyunsaturated fatty acids (e.g. arachidonic acid) and protein nitration (3-nitrotyrosine, 3-NT) in mitochondria and other cellular membranes causing various functional impairments to mitochondrial oxidative phosphorylation and calcium (Ca(2+)) buffering capacity. The LP also results in the formation of neurotoxic reactive aldehyde byproducts including 4-hydroxynonenal (4-HNE) and propenal (acrolein) which exacerbates ROS/RNS production and oxidative protein damage in the injured brain. Ultimately, this results in intracellular Ca(2+) overload that activates proteolytic degradation of α-spectrin, a neuronal cytoskeletal protein. Therefore, the aim of this study was to establish the temporal evolution of mitochondrial dysfunction, oxidative damage and cytoskeletal degradation in the brain following a severe controlled cortical impact (CCI) TBI in young male adult rats. In mitochondria isolated from an 8 mm diameter cortical punch including the 5 mm wide impact site and their respiratory function studied ex vivo, we observed an initial decrease in complex I and II mitochondrial bioenergetics within 3 h (h). For complex I bioenergetics, this partially recovered by 12-16 h, whereas for complex II respiration the recovery was complete by 12 h. During the first 24 h, there was no evidence of an injury-induced increase in LP or protein nitration in mitochondrial or cellular homogenates. However, beginning at 24 h, there was a gradual secondary decline in complex

  3. Optical modulation of astrocyte network using ultrashort pulsed laser

    NASA Astrophysics Data System (ADS)

    Yoon, Jonghee; Ku, Taeyun; Chong, Kyuha; Ryu, Seung-Wook; Choi, Chulhee

    2012-03-01

    Astrocyte, the most abundant cell type in the central nervous system, has been one of major topics in neuroscience. Even though many tools have been developed for the analysis of astrocyte function, there has been no adequate tool that can modulates astrocyte network without pharmaceutical or genetic interventions. Here we found that ultrashort pulsed laser stimulation can induce label-free activation of astrocytes as well as apoptotic-like cell death in a dose-dependent manner. Upon irradiation with high intensity pulsed lasers, the irradiated cells with short exposure time showed very rapid mitochondria fragmentation, membrane blebbing and cytoskeletal retraction. We applied this technique to investigate in vivo function of astrocyte network in the CNS: in the aspect of neurovascular coupling and blood-brain barrier. We propose that this noninvasive technique can be widely applied for in vivo study of complex cellular network.

  4. Differential expression of cytoskeletal proteins in the dendrites of parvalbumin-positive interneurons versus granule cells in the adult rat dentate gyrus.

    PubMed

    de Haas Ratzliff, A; Soltesz, I

    2000-01-01

    Parvalbumin-positive interneurons and granule cells of the dentate gyrus exhibit characteristic differences in morphological, cytochemical, physiological, and pathophysiological properties. Several of these defining features, including dendritic morphology, spine density, and sensitivity to insults, are likely to be influenced by the neuronal cytoskeleton. The data in this paper demonstrate striking differences in the expression levels of all three neurofilament triplet proteins, as well as alpha-internexin and beta-tubulin III, between the parvalbumin-positive interneurons and dentate granule cells. Therefore, the molecular composition of intermediate filaments and microtubules in the dendritic domain of parvalbumin-positive dentate interneurons is distinct from the cytoskeleton of neighboring granule cells, indicating the existence of highly cell type-specific cytoskeletal architecture within the dentate gyrus.

  5. Modification of Experimental Protocols for a Space Shuttle Flight and Applications for the Analysis of Cytoskeletal Structures During Fertilization, Cell Division , and Development in Sea Urchin Embryos

    NASA Technical Reports Server (NTRS)

    Chakrabarti, Amitabha; Stoecker, Andrew; Schatten, Heide

    1995-01-01

    To explore the role of microgravity on cytoskeletal organization and skeletal calcium deposition during fertilization, cell division, and early development, the sea urchin was chosen as a model developmental system. Methods were developed to employ light, immunofluorescence, and electron microscopy on cultures being prepared for flight on the Space Shuttle. For analysis of microfilaments, microtubules, centrosomes, and calcium-requiring events, our standard laboratory protocols had to be modified substantially for experimentation on the Space Shuttle. All manipulations were carried out in a closed culture chamber containing 35 ml artificial sea water as a culture fluid. Unfertilized eggs stored for 24 hours in these chambers were fertilized with sperm diluted in sea water and fixed with concentrated fixatives for final fixation in formaldehyde, taxol, EGTA, and MgCl2(exp -6)H2O for 1 cell to 16 cell stages to preserve cytoskeletal structures for simultaneous analysis with light, immunofluorescence, and electron microscopy, and 1.5 percent glutaraldehyde and 0.4 percent formaldehyde for blastula and plueus stages. The fixed samples wre maintained in chambers without degradation for up to two weeks after which the specimens were processed and analyzed with routine methods. Since complex manipulations are not possible in the closed chambers, the fertilization coat was removed from fixation using 0.5 percent freshly prepared sodium thioglycolate solution at pH 10.0 which provided reliable immunofluorescence staining for microtubules. Sperm/egg fusion, mitosis, cytokinesis, and calcium deposition during spicule formatin in early embryogenesis were found to be without artificial alterations when compared to cells fixed fresh and processed with conventional methods.

  6. Persistent oxygen-glucose deprivation induces astrocytic death through two different pathways and calpain-mediated proteolysis of cytoskeletal proteins during astrocytic oncosis.

    PubMed

    Cao, Xu; Zhang, Ying; Zou, Liangyu; Xiao, Haibing; Chu, Yinghao; Chu, Xiaofan

    2010-07-26

    Astrocytes are thought to play a role in the maintenance of homeostasis and the provision of metabolic substrates for neurons as well as the coupling of cerebral blood flow to neuronal activity. Accordingly, astrocytic death due to various types of injury can critically influence neuronal survival. The exact pathway of cell death after brain ischemia is under debate. In the present study, we used astrocytes from rat primary culture treated with persistent oxygen-glucose-deprivation (OGD) as a model of ischemia to examine the pathway of cell death and the relevant mechanisms. We observed changes in the cellular morphology, the energy metabolism of astrocytes, and the percentage of apoptosis or oncosis of the astrocytes induced by OGD. Electron microscopy revealed the co-existence of ultrastructural features in both apoptosis and oncosis in individual cells. The cellular ATP content was gradually decreased and the percentages of apoptotic and oncotic cells were increased during OGD. After 4h of OGD, ATP depletion to less than 35% of the control was observed, and oncosis became the primary pathway for astrocytic death. Increased plasma membrane permeability due to oncosis was associated with increased calpain-mediated degradation of several cytoskeletal proteins, including paxillin, vinculin, vimentin and GFAP. Pre-treatment with the calpain inhibitor 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) could delay the OGD-induced astrocytic oncosis. These results suggest that there is a narrow range of ATP that determines astrocytic oncotic death induced by persistent OGD and that calpain-mediated hydrolysis of the cytoskeletal-associated proteins may contribute to astrocytes oncosis.

  7. Internalization of Tissue Factor-Rich Microvesicles by Platelets Occurs Independently of GPIIb-IIIa, and Involves CD36 Receptor, Serotonin Transporter and Cytoskeletal Assembly.

    PubMed

    Lopez-Vilchez, Irene; Diaz-Ricart, Maribel; Galan, Ana M; Roque, Merce; Caballo, Carolina; Molina, Patricia; White, James G; Escolar, Gines

    2016-02-01

    Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized; however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF+MVs) and that platelet-associated TF enhances thrombus formation at sites of vascular damage. Here, we investigate the mechanisms implied in the interactions of TF+MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF+MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3-kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF+MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb-IIIa (GPIIb-IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF+MVs. Ultrastructural techniques revealed that uptake of TF+MVs was efficiently prevented by anti-CD36 and SERT inhibitor, but only moderately interfered by GPIIb-IIIa blockade. We conclude that internalization of TF+MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb-IIIa), involves the scavenger receptor CD36, SERT and engages PI3-Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF+MVs with platelets and possibly downregulate their prothrombotic phenotype.

  8. EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins.

    PubMed

    Huber, Robert J; O'Day, Danton H

    2012-09-01

    DdEGFL1, a synthetic epidermal growth factor-like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32kDa) as potential components of the DdEGFL1 signaling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signaling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signaling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signaling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge.

  9. Structure, chromosome location, and expression of the human. gamma. -actin gene: Differential evolution, location, and expression of the cytoskeletal BETA- and. gamma. -actin genes

    SciTech Connect

    Erba, H.P.; Eddy, R.; Shows, T.; Kedes, L.; Gunning, P.

    1988-04-01

    The accumulation of the cytoskeletal ..beta..-and ..gamma..-actin mRNAs was determined in a variety of mouse tissues and organs. The ..beta..-iosform is always expressed in excess of the ..gamma..-isoform. However, the molar ratio of ..beta..- to ..gamma..-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. The authors conclude that, whereas the cytoskeletal ..beta..- and ..gamma..-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human ..gamma..-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike ..beta..-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the ..beta..- and ..gamma..-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human ..beta..- and ..gamma..-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the ..beta..-actin gene but are conserved between the human ..gamma..-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of ..beta..- and ..gamma..-actin or to the unique regulation and function of the ..gamma..-actin gene. Finally, the authors demonstrate that the human ..gamma..-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human ..gamma..-actin gene is appropriately regulated.

  10. The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

    PubMed Central

    Malliri, Angeliki; Symons, Marc; Hennigan, Robert F.; Hurlstone, Adam F.L.; Lamb, Richard F.; Wheeler, Tricia; Ozanne, Bradford W.

    1998-01-01

    Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion. PMID:9817764

  11. The 13-kD FK506 binding protein, FKBP13, interacts with a novel homologue of the erythrocyte membrane cytoskeletal protein 4.1.

    PubMed

    Walensky, L D; Gascard, P; Fields, M E; Blackshaw, S; Conboy, J G; Mohandas, N; Snyder, S H

    1998-04-06

    We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G-CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677- 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705-708). We report the specific association of FKBP13 with 4.1G-CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G-CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G-CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G-CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds.

  12. The 13-kD FK506 Binding Protein, FKBP13, Interacts with a Novel Homologue of the Erythrocyte Membrane Cytoskeletal Protein 4.1

    PubMed Central

    Walensky, Loren D.; Gascard, Philippe; Field, Michael E.; Blackshaw, Seth; Conboy, John G.; Mohandas, Narla; Snyder, Solomon H.

    1998-01-01

    We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G–CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677– 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705–708). We report the specific association of FKBP13 with 4.1G–CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G–CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G–CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G–CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds. PMID:9531554

  13. Anomalous motor mediated cargo transport in microtubule networks

    NASA Astrophysics Data System (ADS)

    Vandal, Steven; Macveigh-Fierro, Daniel; Shen, Zhiyuan; Lemoi, Kyle; Vidali, Luis; Ross, Jennifer; Tuzel, Erkan

    Cargo transport is an important biological mechanism by which cells locomote, self-organize, and actively transport organelles. This transport is mediated by the cytoskeletal network and molecular motors; however, it is not known how network self-organization and dynamics affect these transport processes. In order to develop a mechanistic understanding of cargo transport, we use a coarse-grained Brownian dynamics model that incorporates the dynamics of these networks, as well as experimentally determined motor properties. We will test these models with two experimental systems: (1) in vitro microtubule networks with kinesin-1 motors, and quantum dot cargos on recreated microtubule networks, and (2) an excellent model organism, the moss Physcomitrella patens, in which chloroplasts are transported via the microtubule network by means of kinesin-like proteins. Phenomenological network characterizations are made, both in vivo and in vitro, and cargo motility is characterized using Mean Squared Displacement (MSD) measurements. Our simulations shed light on the role of network density and motor properties on the observed transport behavior, and improve our understanding of cargo transport in cells.

  14. The conserved mobility of mitochondria during leaf senescence reflects differential regulation of the cytoskeletal components in Arabidopsis thaliana

    PubMed Central

    2011-01-01

    Leaf senescence is an organized process, which requires fine tuning between nuclear gene expression, activity of proteases and the maintenance of primary metabolism. Recently, we reported that leaf senescence was accompanied by an early degradation of the microtubule cytoskeleton in Arabidopsis thaliana. As the cytoskeleton is essential for cell stability, vesicle shuttling and organelle mobility, it might be asked how the regulation of these cell functions occurs with such drastic modifications of the cytoskeleton. Based on confocal laser microscopy observations and a micro-array analysis, the following addendum shows that mitochondrial mobility is conserved until the late stages of leaf senescence and provides evidences that the actin-cytoskeleton is maintained longer than the microtubule network. This conservation of actin-filaments is discussed with regards to energy metabolism as well as calcium signaling during programmed cell death. PMID:21270537

  15. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  16. Crumbs is an essential regulator of cytoskeletal dynamics and cell-cell adhesion during dorsal closure in Drosophila

    PubMed Central

    Flores-Benitez, David; Knust, Elisabeth

    2015-01-01

    The evolutionarily conserved Crumbs protein is required for epithelial polarity and morphogenesis. Here we identify a novel role of Crumbs as a negative regulator of actomyosin dynamics during dorsal closure in the Drosophila embryo. Embryos carrying a mutation in the FERM (protein 4.1/ezrin/radixin/moesin) domain-binding motif of Crumbs die due to an overactive actomyosin network associated with disrupted adherens junctions. This phenotype is restricted to the amnioserosa and does not affect other embryonic epithelia. This function of Crumbs requires DMoesin, the Rho1-GTPase, class-I p21-activated kinases and the Arp2/3 complex. Data presented here point to a critical role of Crumbs in regulating actomyosin dynamics, cell junctions and morphogenesis. DOI: http://dx.doi.org/10.7554/eLife.07398.001 PMID:26544546

  17. RNA Helicase DDX5 Regulates MicroRNA Expression and Contributes to Cytoskeletal Reorganization in Basal Breast Cancer Cells

    SciTech Connect

    Wang, Daojing; Huang, Jing; Hu, Zhi

    2011-11-15

    RNA helicase DDX5 (also p68) is involved in all aspects of RNA metabolism and serves as a transcriptional co-regulator, but its functional role in breast cancer remains elusive. Here, we report an integrative biology study of DDX5 in breast cancer, encompassing quantitative proteomics, global MicroRNA profiling, and detailed biochemical characterization of cell lines and human tissues. We showed that protein expression of DDX5 increased progressively from the luminal to basal breast cancer cell lines, and correlated positively with that of CD44 in the basal subtypes. Through immunohistochemistry analyses of tissue microarrays containing over 200 invasive human ductal carcinomas, we observed that DDX5 was upregulated in the majority of malignant tissues, and its expression correlated strongly with those of Ki67 and EGFR in the triple-negative tumors. We demonstrated that DDX5 regulated a subset of MicroRNAs including miR-21 and miR-182 in basal breast cancer cells. Knockdown of DDX5 resulted in reorganization of actin cytoskeleton and reduction of cellular proliferation. The effects were accompanied by upregulation of tumor suppressor PDCD4 (a known miR-21 target); as well as upregulation of cofilin and profilin, two key proteins involved in actin polymerization and cytoskeleton maintenance, as a consequence of miR-182 downregulation. Treatment with miR-182 inhibitors resulted in morphologic phenotypes resembling those induced by DDX5 knockdown. Using bioinformatics tools for pathway and network analyses, we confirmed that the network for regulation of actin cytoskeleton was predominantly enriched for the predicted downstream targets of miR-182. Our results reveal a new functional role of DDX5 in breast cancer via the DDX5→miR-182→actin cytoskeleton pathway, and suggest the potential clinical utility of DDX5 and its downstream MicroRNAs in the theranostics of breast cancer.

  18. 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.

  19. 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

  20. p38MAPK, Rho/ROCK and PKC pathways are involved in influenza-induced cytoskeletal rearrangement and hyperpermeability in PMVEC via phosphorylating ERM.

    PubMed

    Zhang, Chenyue; Wu, Ying; Xuan, Zinan; Zhang, Shujing; Wang, Xudan; Hao, Yu; Wu, Jun; Zhang, Shu

    2014-11-04

    Severe influenza infections are featured by acute lung injury, a syndrome of pulmonary microvascular leak. A growing number of evidences have shown that the pulmonary microvascular endothelial cells (PMVEC) are critical target of influenza virus, promoting microvascular leak. It is reported that there are multiple mechanisms by which influenza virus could elicit increased pulmonary endothelial permeability, in both direct and indirect manners. Ezrin/radixin/moesin family proteins, the linkers between plasma membrane and actin cytoskeleton, have been reported to be involved in cell adhesion, motility and may modulate endothelial permeability. Studies have also shown that ERM is phosphorylated in response to various stimuli via p38MAPK, Rho/ROCK or PKC pathways. However, it is unclear that whether influenza infection could induce ERM phosphorylation and its relocalization. In the present study, we have found that there are cytoskeletal reorganization and permeability increases in the course of influenza virus infection, accompanied by upregulated levels of p-ERM. p-ERM's aggregation along the periphery of PMVEC upon influenza virus infection was detected via confocal microscopy. Furthermore, we sought to determine the role of p38MAPK, Rho/ROCK and PKC pathways in ERM phosphorylation as well as their involvement in influenza virus-induced endothelial malfunction. The activation of p38MAPK, Rho/ROCK and PKC pathways upon influenza virus stimulation were observed, as evidenced by the evaluation of phosphorylated p38 (p-p38), phosphorylated MKK (p-MKK) in p38MAPK pathway, ROCK1 in Rho/ROCK pathway and phosphorylated PKC (p-PKC) in PKC pathway. We also showed that virus-induced ERM phosphorylation was reduced by using p38MAPK inhibitor, SB203580 (20 μM), Rho/ROCK inhibitor, Y27632 (20 μM), PKC inhibitor, LY317615 (10 μM). Additionally, influenza virus-induced F-actin reorganization and hyperpermeability were attenuated by pretreatment with SB203580, Y27632 and LY317615

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

    PubMed

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

    2009-11-09

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

  2. Ultrastructural immunolocalization of nestin in the regenerating tail of lizards shows its presence during cytoskeletal modifications in the epidermis, muscles and nerves.

    PubMed

    Alibardi, Lorenzo

    2015-04-01

    Nestin has been considered a neural stem cell marker, and represents an intermediate filament protein likely involved in restructuring the cytoskeleton in different cell types. The present ultrastructural study has immunodetected nestin especially in the wound epidermis, regenerating myotubes and in the growing nerves of the regenerating tail of lizards. In keratinocytes of the stratified wound epidermis nestin is present in the irregular electron-paler meshwork located along the cell perimeter and among keratin bundles converging into desmosomes. In the regenerating muscles nestin-immunoreactivity remains confined to some external regions along the myotubes and in the cytoplasmic ends of the myotubes not occupied by myofibrils. A diffuse nestin immunolabeling is also present among the neurofilaments of growing axons, in Schwann cells and in ependymal cells of the regenerating spinal cord of the tail. The localization of nestin in sites of cytoskeletal remodeling in keratinocytes, myotubes, ependymal cells and axons, suggests that this protein is associated to the reassembling of keratin tonofilaments in moving keratinocytes, assembling of contractile proteins in myotubes, and in the organization of neurofilaments during the growth and myelination of axons within the regenerating lizard tail.

  3. Genome-wide RNA-seq of iPSC-derived motor neurons indicates selective cytoskeletal perturbation in Brown-Vialetto disease that is partially rescued by riboflavin.

    PubMed

    Rizzo, Federica; Ramirez, Agnese; Compagnucci, Claudia; Salani, Sabrina; Melzi, Valentina; Bordoni, Andreina; Fortunato, Francesco; Niceforo, Alessia; Bresolin, Nereo; Comi, Giacomo P; Bertini, Enrico; Nizzardo, Monica; Corti, Stefania

    2017-04-06

    Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown-Vialetto-Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell-selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy-lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.

  4. Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae.

    PubMed Central

    Kohno, H; Tanaka, K; Mino, A; Umikawa, M; Imamura, H; Fujiwara, T; Fujita, Y; Hotta, K; Qadota, H; Watanabe, T; Ohya, Y; Takai, Y

    1996-01-01

    The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation. We have recently shown that Pkc1p, a yeast homolog of mammalian protein kinase C, and glucan synthase are targets of Rho1p. Using the two-hybrid screening system, we cloned a gene encoding a protein which interacted with the GTP-bound form of Rho1p. This gene was identified as BNI1, known to be implicated in cytokinesis or establishment of cell polarity in S.cerevisiae. Bni1p shares homologous domains (FH1 and FH2 domains) with proteins involved in cytokinesis or establishment of cell polarity, including formin of mouse, capu and dia of Drosophila and FigA of Aspergillus. A temperature-sensitive mutation in which the RHO1 gene was replaced by the mammalian RhoA gene showed a synthetically lethal interaction with the bni1 mutation and the RhoA bni1 mutant accumulated cells with a deficiency in cytokinesis. Furthermore, this synthetic lethality was caused by the incapability of RhoA to activate Pkc1p, but not glucan synthase. These results suggest that Rho1p regulates cytoskeletal reorganization at least through Bni1p and Pkc1p. Images PMID:8947028

  5. Study of cytoskeletal changes induced by okadaic acid in HL-7702 liver cells and development of a fluorimetric microplate assay for detecting diarrhetic shellfish poisoning.

    PubMed

    Huang, Haiyan; Huang, Aijun; Zhuang, Zhixiong; Huang, Wei; Fu, Yingbin; Peng, Chaoqiong; Liu, Jianjun

    2013-02-01

    Diarrhetic shellfish poisoning (DSP) is a gastrointestinal illness with symptoms such as diarrhea, nausea, vomiting, headache, chills and moderate to severe abdominal pain. DSP has been recognized as a worldwide public health problem, causing great concern to the shellfish industry. Accumulation of DSP in shellfish is an unpredictable phenomenon that necessitates the implementation of a widespread collection and thorough monitoring program for mollusk toxicity. Therefore, development of accurate analytical protocols for the rapid determination of toxicity levels would be necessary. In this study we investigated cytoskeletal changes induced by okadaic acid in HL-7702 Liver Cells and developed a new cytotoxicity assay for detection and quantitation of DSP. This assay is based on fluorometric of F-actin depolymerization induced by okadaic acid (OA) compounds in HL-7702 liver cell line. The measurable range of OA was 2.5 ∼ 40 nmol/L. The detection limit of the F-actin assay for OA was 2.01 μg/100 g muscles in shellfish extracts. The performance of this assay has been evaluated by comparative analysis of shellfish samples by the fluorescent assay, mouse bioassay, and ELISA assay. Comparison of the results by all three methods revealed excellent consistency, the results of fluorescent assay were in significant correlation with ELISA assay (R(2) = 0.830). Examination of F-actin assay is very convenient, rapid, and sensitive, which can be used to quantify the amount of OA in shellfish samples.

  6. Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells*

    PubMed Central

    Ruperez, Patricia; Gago-Martinez, Ana; Burlingame, A. L.; Oses-Prieto, Juan A.

    2012-01-01

    Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse. PMID:22499768

  7. Genome-wide RNA-seq of iPSC-derived motor neurons indicates selective cytoskeletal perturbation in Brown–Vialetto disease that is partially rescued by riboflavin

    PubMed Central

    Rizzo, Federica; Ramirez, Agnese; Compagnucci, Claudia; Salani, Sabrina; Melzi, Valentina; Bordoni, Andreina; Fortunato, Francesco; Niceforo, Alessia; Bresolin, Nereo; Comi, Giacomo P.; Bertini, Enrico; Nizzardo, Monica; Corti, Stefania

    2017-01-01

    Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown–Vialetto–Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell–selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy–lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies. PMID:28382968

  8. A cytoskeletal localizing domain in the cyclase-associated protein, CAP/Srv2p, regulates access to a distant SH3-binding site.

    PubMed

    Yu, J; Wang, C; Palmieri, S J; Haarer, B K; Field, J

    1999-07-09

    In the yeast, Saccharomyces cerevisiae, adenylyl cyclase consists of a 200-kDa catalytic subunit (CYR1) and a 70-kDa subunit (CAP/SRV2). CAP/Srv2p assists the small G protein Ras to activate adenylyl cyclase. CAP also regulates the cytoskeleton through an actin sequestering activity and is directed to cortical actin patches by a proline-rich SH3-binding site (P2). In this report we analyze the role of the actin cytoskeleton in Ras/cAMP signaling. Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduced adenylyl cyclase binding, and cortical actin patch localization. A third mutation, L27F, also failed to localize but showed no loss of either cAMP signaling or adenylyl cyclase binding. However, all three N-terminal mutations reduced CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away. Finally, disruption of the actin cytoskeleton with latrunculin-A did not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele. These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling.

  9. Seasonal Changes in the Immunolocalization of Cytoskeletal Proteins and Laminin in the Testis of the Black-Backed Jackal (Canis mesomelas).

    PubMed

    Madekurozwa, M-C; Booyse, D

    2017-02-01

    Manipulation of the reproductive activity of jackals is dependent on a thorough understanding of the reproductive biology of this species. This study describes seasonal morphological changes in the adult testis of the black-backed jackal in relation to the immunoexpression of the basement membrane marker, laminin and the cytoskeletal proteins, cytokeratin, smooth muscle actin and vimentin. Laminin was immunolocalized in basement membranes surrounding seminiferous tubules, as well as in basement membranes associated with Leydig, peritubular myoid and vascular smooth muscle cells. Scalloped basement membranes enclosed seminiferous tubules in regressing testes. The seminiferous epithelium and interstitial tissue in all animals studied were cytokeratin immunonegative. Smooth muscle actin was demonstrated in vascular smooth muscle cells, as well as in peritubular myoid cells encircling seminiferous tubules. Vimentin immunoreactivity was exhibited in the cytoplasm of Sertoli cells, Leydig cells, vascular endothelial cells, vascular smooth muscle cells and fibrocytes. Vimentin immunostaining in Sertoli, Leydig and peritubular myoid cells varied depending on the functional state of the testis. The results of the study have shown that dramatic seasonal histological changes occur in the testes of the jackal. In addition, the use of immunohistochemistry accentuates these morphological changes.

  10. Polycystin-1 Induces Cell Migration by Regulating Phosphatidylinositol 3-kinase-dependent Cytoskeletal Rearrangements and GSK3β-dependent Cell–Cell Mechanical Adhesion

    PubMed Central

    Boca, Manila; D'Amato, Lisa; Distefano, Gianfranco; Polishchuk, Roman S.; Germino, Gregory G.

    2007-01-01

    Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1−/− mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and β-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated β-catenin through activation of GSK3β. Expression of a nondegradable form of β-catenin in PC-1 MDCK cells restores strong cell–cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis. PMID:17671167

  11. Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells.

    PubMed

    Egbowon, Biola F; Harris, Wayne; Arnott, Gordon; Mills, Chris Lloyd; Hargreaves, Alan J

    2016-04-01

    The aims of this study were to examine the effects of CdCl2 on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1 μM CdCl2 was sub-cytotoxic at all time-points. Exposure to 1 and 12 μM CdCl2 for 4 h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12 μM CdCl2 in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl2 caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd(2+) also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1-12 μM Cd(2+) is attainable in vivo, our findings are consistent with the possibility that Cd(2+) induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure.

  12. Signaling by the engulfment receptor draper: a screen in Drosophila melanogaster implicates cytoskeletal regulators, Jun N-terminal Kinase, and Yorkie.

    PubMed

    Fullard, John F; Baker, Nicholas E

    2015-01-01

    Draper, the Drosophila melanogaster homolog of the Ced-1 protein of Caenorhabditis elegans, is a cell-surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, we screened chromosomal deficiencies to identify loci that dominantly modify the phenotype of overexpression of Draper isoform II (suppressed differentiation of the posterior crossvein in the wing). We found evidence for 43 genetic modifiers of Draper II. Twenty-four of the 37 suppressor loci and 3 of the 6 enhancer loci were identified. An additional 5 suppressors and 2 enhancers were identified among mutations in functionally related genes. These studies reveal positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott-Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in Draper signaling. Relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador/Warts/Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are also discussed.

  13. Network Science

    NASA Astrophysics Data System (ADS)

    Barabási, Albert-László

    2016-07-01

    Preface; Personal introduction; 1. Introduction; 2. Graph theory; 3. Random networks; 4. The scale-free property; 5. The Barabási-Albert model; 6. Evolving networks; 7. Degree correlation; 8. Network robustness; 9. Communities; 10. Spreading phenomena; Index.

  14. Network Cosmology

    PubMed Central

    Krioukov, Dmitri; Kitsak, Maksim; Sinkovits, Robert S.; Rideout, David; Meyer, David; Boguñá, Marián

    2012-01-01

    Prediction and control of the dynamics of complex networks is a central problem in network science. Structural and dynamical similarities of different real networks suggest that some universal laws might accurately describe the dynamics of these networks, albeit the nature and common origin of such laws remain elusive. Here we show that the causal network representing the large-scale structure of spacetime in our accelerating universe is a power-law graph with strong clustering, similar to many complex networks such as the Internet, social, or biological networks. We prove that this structural similarity is a consequence of the asymptotic equivalence between the large-scale growth dynamics of complex networks and causal networks. This equivalence suggests that unexpectedly similar laws govern the dynamics of complex networks and spacetime in the universe, with implications to network science and cosmology. PMID:23162688

  15. Network cosmology.

    PubMed

    Krioukov, Dmitri; Kitsak, Maksim; Sinkovits, Robert S; Rideout, David; Meyer, David; Boguñá, Marián

    2012-01-01

    Prediction and control of the dynamics of complex networks is a central problem in network science. Structural and dynamical similarities of different real networks suggest that some universal laws might accurately describe the dynamics of these networks, albeit the nature and common origin of such laws remain elusive. Here we show that the causal network representing the large-scale structure of spacetime in our accelerating universe is a power-law graph with strong clustering, similar to many complex networks such as the Internet, social, or biological networks. We prove that this structural similarity is a consequence of the asymptotic equivalence between the large-scale growth dynamics of complex networks and causal networks. This equivalence suggests that unexpectedly similar laws govern the dynamics of complex networks and spacetime in the universe, with implications to network science and cosmology.

  16. Network Kits.

    ERIC Educational Resources Information Center

    Falk, Howard

    1999-01-01

    Describes interconnection methods, speed, and comparative equipment costs of networking starter kits. These kits supply network-connection devices that plug into or connect to each computer that is part of a network; they may also provide interconnection cables and installation software needed to set up a network. Reviews 20 kits that use a…

  17. Network Solutions.

    ERIC Educational Resources Information Center

    Vietzke, Robert; And Others

    1996-01-01

    This special section explains the latest developments in networking technologies, profiles school districts benefiting from successful implementations, and reviews new products for building networks. Highlights include ATM (asynchronous transfer mode), cable modems, networking switches, Internet screening software, file servers, network management…

  18. Confocal microscopic observation of cytoskeletal reorganizations in cultured shark rectal gland cells following treatment with hypotonic shock and high external K+.

    PubMed

    Henson, J H; Roesener, C D; Gaetano, C J; Mendola, R J; Forrest, J N; Holy, J; Kleinzeller, A

    1997-12-01

    The dogfish shark (Squalus acanthias) rectal gland (SRG) cell has served as a model experimental system for investigating the relationship between the actin cytoskeleton and cell volume regulation. Previous reports employing conventional fluorescence microscopy of tissue slices have shown that cells exposed to high external K+ and hypotonically-induced cell swelling displayed a fading of F-actin staining intensity, particularly at the basolateral cell borders. However, spectroscopic measurement of the F-actin present in similarly treated rectal gland slices failed to demonstrate a net change in F-actin amount. In an effort to resolve the structural reorganizations of F-actin which may be occurring during high K+ and hypotonic shock treatments, we have used cultured SRG cells in conjunction with confocal microscopic immunocytochemical localization techniques to examine actin filament, microtubule, and cytokeratin filament dynamics under these two experimental conditions. The results reveal that F-actin in control cells exists in an array of parallel linear bundles (which do not appear to be stress fiber-like given their lack of staining for myosin II or alpha-actinin) that is reorganized to a punctate pattern in hypotonic shock and a dense meshwork in high K+. The linear bundle pattern of F-actin returns in cells undergoing regulatory volume decrease. Quantitative western blotting of F-actin in SRG cell detergent extracted cytoskeletons indicates no significant difference in the relative amounts of F-actin present in control, hypotonic shocked, or high K+ cells. Anti-tubulin and anti-cytokeratin labeling of the treated SRG cells suggest that these other major cytoskeletal elements are not significantly altered by the treatments. Taken together, our results reinforce the concept that there is an association between the structural organization of the actin cytoskeleton and cell volume regulation in the SRG epithelial cells.

  19. Early and Late Loss of the Cytoskeletal Scaffolding Protein, Ankyrin G Reveals its Role in Maturation and Maintenance of Nodes of Ranvier in Myelinated Axons.

    PubMed

    Saifetiarova, Julia; Taylor, Anna M; Bhat, Manzoor A

    2017-02-01

    The mechanisms that govern node of Ranvier organization, stability and long-term maintenance remain to be fully elucidated. One of the molecular components of the node is the cytoskeletal scaffolding protein, Ankyrin G (AnkG), which interacts with multiple members of the nodal complex. The role of AnkG in nodal organization and maintenance is still not clearly defined, as to whether AnkG functions as an initial nodal organizer or whether it functions as a nodal stabilizer after the nodal complex has been assembled. Using a mouse model system, we report here that perinatal and juvenile neuronal ablation of AnkG has differential consequences on nodal stability. Early loss of AnkG creates immature nodes with abnormal morphology, which undergo accelerated destabilization within a month, resulting in rapid NaV channel and βIV Spectrin loss with reduced effects on Neurofascin 186. On the other hand, late ablation of AnkG from established nodal complexes leads to slow but progressive nodal destabilization over 10 months, primarily affecting βIV Spectrin, followed by NaV channels, with modest impact on Neurofascin 186. We also show that Ankyrin R and βI Spectrin are not sufficient to prevent nodal disorganization after AnkG ablation. Additionally, nodal disorganization in both early and late AnkG mutants is accompanied by axonal pathology and neurological dysfunction. Together, our results suggest that AnkG plays an indispensable role in maturation and long-term stabilization of the newly assembled nodal complex, and that loss of AnkG after nodal stabilization does not lead to rapid nodal disassembly but loss of specific nodal components in a time-dependent manner.

  20. Organization of pp60src and selected cytoskeletal proteins within adhesion plaques and junctions of Rous sarcoma virus-transformed rat cells

    PubMed Central

    1981-01-01

    The localization of pp60src within adhesion structures of epithelioid rat kidney cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus was compared to the organization of actin, alpha-actinin, vinculin (a 130,000-dalton protein), tubulin, and the 58,000-dalton intermediate filament protein. The adhesion structures included both adhesion plaques and previously uncharacterized adhesive regions formed at cell-cell junctions. We have termed these latter structures "adhesion junctions." Both adhesion plaques and adhesion junctions were identified by interference-reflection microscopy and compared to the location of pp60src and the various cytoskeletal proteins by double fluorescence. The results demonstrated that the src gene product was found within both adhesion plaques and the adhesion junctions. In addition, actin, alpha-actinin, and vinculin were also localized within the same pp60src-containing adhesion structures. In contrast, tubulin and the 58,000-dalton intermediate filament protein were not associated with either adhesion plaques or adhesion junctions. Both adhesion plaques and adhesion junctions were isolated as substratum-bound structures and characterized by scanning electron microscopy. Immunofluorescence revealed that pp60src, actin, alpha-actinin, and vinculin were organized within specific regions of the adhesion junctions. Heavy accumulations of actin and alpha-actinin were found on both sides of the junctions with a narrow gap of unstained material at the midline, whereas pp60src stain was more intense in this central region. Antibody to vinculin stained double narrow lines defining the periphery of the junctional complexes but was excluded from the intervening region. In addition, the distribution of vinculin relative to pp60src within adhesion plaques suggested an inverse relationship between the presence of these two proteins. Overall, these results establish a close link between the src gene product and components of the

  1. Cytoskeletal dynamics in rabbit synovial fibroblasts: II. Reformation of stress fibers in cells rounded by treatment with collagenase-inducing agents.

    PubMed

    Aggeler, J

    1990-01-01

    Modulation of the synthesis and secretion of extracellular matrix proteins and matrix-degrading metalloproteases by rabbit synovial fibroblasts is an important model system for studying the control of tissue-specific gene expression. Induction of collagenase expression is correlated with changes in cell shape and actin filament distribution, but the role of the cellular cytoskeleton in the sustained synthesis and secretion of metalloproteases has not been closely examined. When cells were allowed to respread after rounding by trypsin or cytochalasin, two known metalloprotease inducers, reformation of stress fibers was observed within 2 h in the presence of serum. In the absence of serum, trypsin-treated cells did not respread substantially, even after 24 h in culture. In contrast, cytochalasin-treated cells recovered almost as rapidly in the absence as in the presence of serum, showing reformation of well-formed microfilament bundles within 30 min of drug removal, especially at the spreading cell edges. High resolution electron-microscopic views of detergent-extracted cytoskeletons confirmed the rapid rebundling of peripheral microfilaments. Acrylamide-treated cells fell between these two extremes, spreading slowly in the absence of serum, but almost as rapidly as cytochalasin-treated cells in its presence. Reestablishment of normal intermediate filament distribution generally lagged slightly behind actin for all treatments, and intermediate filaments always appeared to spread back into the cellular cytoplasm within the confines of the reforming peripheral microfilament bundles. No obvious interaction between these two cytoskeletal elements was observed after any treatment, and no specific role for intermediate filaments in modulating gene expression in these cells is suggested by these results. The serum dependence displayed after trypsin or acrylamide treatment may be due to the disturbances in fibronectin synthesis observed in these cells and is consistent with

  2. Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA.

    PubMed

    Karlsson, Oskar; Berg, Anna-Lena; Hanrieder, Jörg; Arnerup, Gunnel; Lindström, Anna-Karin; Brittebo, Eva B

    2015-03-01

    The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease, and recent studies indicate that BMAA can be misincorporated into proteins. BMAA is a developmental neurotoxicant that can induce long-term learning and memory deficits, as well as regionally restricted neuronal degeneration and mineralization in the hippocampal CA1. The aim of the study was to characterize long-term changes (2 weeks to 6 months) further in the brain of adult rats treated neonatally (postnatal days 9-10) with BMAA (460 mg/kg) using immunohistochemistry (IHC), transmission electron microscopy, and laser capture microdissection followed by LC-MS/MS for proteomic analysis. The histological examination demonstrated progressive neurodegenerative changes, astrogliosis, microglial activation, and calcification in the hippocampal CA1 3-6 months after exposure. The IHC showed an increased staining for α-synuclein and ubiquitin in the area. The ultrastructural examination revealed intracellular deposition of abundant bundles of closely packed parallel fibrils in neurons, axons, and astrocytes of the CA1. Proteomic analysis of the affected site demonstrated an enrichment of chaperones (e.g., clusterin, GRP-78), cytoskeletal and intermediate filament proteins, and proteins involved in the antioxidant defense system. Several of the most enriched proteins (plectin, glial fibrillar acidic protein, vimentin, Hsp 27, and ubiquitin) are known to form complex astrocytic inclusions, so-called Rosenthal fibers, in the neurodegenerative disorder Alexander disease. In addition, TDP-43 and the negative regulator of autophagy, GLIPR-2, were exclusively detected. The present study demonstrates that neonatal exposure to BMAA may offer a novel model for the study of hippocampal fibril formation in vivo.

  3. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    PubMed Central

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-01-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin. PMID:8552082

  4. Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.

    PubMed

    Turner, C E; Brown, M C; Perrotta, J A; Riedy, M C; Nikolopoulos, S N; McDonald, A R; Bagrodia, S; Thomas, S; Leventhal, P S

    1999-05-17

    Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

  5. Cytoskeletal protein Flightless (Flii) is elevated in chronic and acute human wounds and wound fluid: neutralizing its activity in chronic but not acute wound fluid improves cellular proliferation.

    PubMed

    Ruzehaji, Nadira; Grose, Randall; Krumbiegel, Doreen; Zola, Heddy; Dasari, Pallave; Wallace, Hilary; Stacey, Michael; Fitridge, Robert; Cowin, Allison J

    2012-01-01

    Chronic non-healing wounds form a medical need which will expand as the population ages and the obesity epidemic grows. Whilst the complex mechanisms underlying wound repair are not fully understood, remodelling of the actin cytoskeleton plays a critical role. Elevated expression of the actin cytoskeletal protein Flightless I (Flii) is known to impair wound outcomes. To determine if Flii is involved in the impaired healing observed in chronic wounds, its expression in non-healing human wounds from patients with venous leg ulcers was determined and compared to its expression in acute wounds and unwounded skin. Increased expression of Flii was observed in both chronic and acute wounds with wound fluid and plasma also containing secreted Flii protein. Inflammation is a key aspect of wound repair and fluorescence-activated cell sorting (FACS) analysis revealed Flii was located in neutrophils within the blood and that it co-localised with CD16+ neutrophils in chronic wounds. The function of secreted Flii was investigated as both chronic wound fluid and Flii have previously been shown to inhibit fibroblast proliferation. To determine if the inhibitory effect of wound fluid was due in part to the presence of Flii, wound fluids were depleted of Flii using Flii-specific neutralizing antibodies (FnAb). Flii depleted chronic wound fluid no longer inhibited fibroblast proliferation, suggesting that Flii may contribute to the inhibitory effect of chronic wound fluid on fibroblast function. Application of FnAbs to chronic wounds may therefore be a novel approach used to improve the local environment of non-healing wounds and potentially improve healing outcomes.

  6. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    PubMed

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-02-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

  7. The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is required for memory consolidation of pavlovian fear conditioning in the lateral amygdala.

    PubMed

    Ploski, Jonathan E; Pierre, Vicki J; Smucny, Jason; Park, Kevin; Monsey, Melissa S; Overeem, Kathie A; Schafe, Glenn E

    2008-11-19

    The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is an immediate early gene that has been widely implicated in hippocampal-dependent learning and memory and is believed to play an integral role in synapse-specific plasticity. Here, we examined the role of Arc/Arg3.1 in amygdala-dependent Pavlovian fear conditioning. We first examined the regulation of Arc/Arg3.1 mRNA and protein after fear conditioning and LTP-inducing stimulation of thalamic inputs to the lateral amygdala (LA). Quantitative real-time PCR, in situ hybridization, Western blotting and immunohistochemistry revealed a significant upregulation of Arc/Arg3.1 mRNA and protein in the LA relative to controls. In behavioral experiments, intra-LA infusion of an Arc/Arg3.1 antisense oligodeoxynucleotide (ODN) was observed to be anatomically restricted to the LA, taken up by LA cells, and to promote significant knockdown of Arc/Arg3.1 protein. Rats given intra-LA infusions of multiple doses of the Arc/Arg3.1 ODN showed an impairment of LTM (tested approximately 24 later), but no deficit in STM (tested 3 h later) relative to controls infused with scrambled ODN. Finally, to determine whether upregulation of Arc/Arg3.1 occurs downstream of ERK/MAPK activation, we examined Arc/Arg3.1 expression in rats given intra-LA infusion of the MEK inhibitor U0126. Relative to vehicle controls, infusion of U0126 impaired training-induced increases in Arc/Arg3.1 expression. These findings suggest that Arc/Arg3.1 expression in the amygdala is required for fear memory consolidation, and further suggest that Arc/Arg3.1 regulation in the LA is downstream of the ERK/MAPK signaling pathway.

  8. 4-(1-Ethyl-4-anisyl-imidazol-5-yl)-N-hydroxycinnamide – A new pleiotropic HDAC inhibitor targeting cancer cell signalling and cytoskeletal organisation

    SciTech Connect

    Mahal, Katharina; Kahlen, Philip; Biersack, Bernhard; Schobert, Rainer

    2015-08-15

    Histone deacetylases (HDAC) which play a crucial role in cancer cell proliferation are promising drug targets. However, HDAC inhibitors (HDACi) modelled on natural hydroxamic acids such as trichostatin A frequently lead to resistance or even an increased agressiveness of tumours. As a workaround we developed 4-(1-ethyl-4-anisyl-imidazol-5-yl)-N-hydroxycinnamide (etacrox), a hydroxamic acid that combines HDAC inhibition with synergistic effects of the 4,5-diarylimidazole residue. Etacrox proved highly cytotoxic against a panel of metastatic and resistant cancer cell lines while showing greater specificity for cancer over non-malignant cells when compared to the approved HDACi vorinostat. Like the latter, etacrox and the closely related imidazoles bimacroxam and animacroxam acted as pan-HDACi yet showed some specificity for HDAC6. Akt signalling and interference with nuclear beta-catenin localisation were elicited by etacrox at lower concentrations when compared to vorinostat. Moreover, etacrox disrupted the microtubule and focal adhesion dynamics of cancer cells and inhibited the proteolytic activity of prometastatic and proangiogenic matrix metalloproteinases. As a consequence, etacrox acted strongly antimigratory and antiinvasive against various cancer cell lines in three-dimensional transwell invasion assays and also antiangiogenic in vivo with respect to blood vessel formation in the chorioallantoic membrane assay. These pleiotropic effects and its water-solubility and tolerance by mice render etacrox a promising new HDACi candidate. - Graphical abstract: A novel histone deacetylase inhibitor with pleiotropic anticancer effects. - Highlights: • Etacrox is a new HDACi with cytotoxic, antiangiogenic and antiinvasive activity. • Etacrox causes aberrant cancer cell signalling and cytoskeletal reorganisation. • Pro-metastatic and angiogenic matrix metalloproteinases are inhibited by etacrox. • Etacrox impairs blood vessel maturation in vivo and cancer cell

  9. ENH, containing PDZ and LIM domains, heart/skeletal muscle-specific protein, associates with cytoskeletal proteins through the PDZ domain.

    PubMed

    Nakagawa, N; Hoshijima, M; Oyasu, M; Saito, N; Tanizawa, K; Kuroda, S

    2000-06-07

    The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C beta (PKCbeta) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029-31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein alpha-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and alpha-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1-3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCbeta is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCbeta to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.

  10. Micro-rheological behaviour and nonlinear rheology of networks assembled from polysaccharides from the plant cell wall

    NASA Astrophysics Data System (ADS)

    Vincent, R. R. R.; Mansel, B. W.; Kramer, A.; Kroy, K.; Williams, M. A. K.

    2013-03-01

    The same fundamental questions that have driven enquiry into cytoskeletal mechanics can be asked of the considerably less-studied, yet arguably just as important, biopolymer matrix in the plant cell wall. In this case, it is well-known that polysaccharides, rather than filamentous and tubular protein assemblies, play a major role in satisfying the mechanical requirements of a successful cell wall, but developing a clear structure-function understanding has been exacerbated by the familiar issue of biological complexity. Herein, in the spirit of the mesoscopic approaches that have proved so illuminating in the study of cytoskeletal networks, the linear microrheological and strain-stiffening responses of biopolymeric networks reconstituted from pectin, a crucial cell wall polysaccharide, are examined. These are found to be well-captured by the glassy worm-like chain (GWLC) model of self-assembled semi-flexible filaments. Strikingly, the nonlinear mechanical response of these pectin networks is found to be much more sensitive to temperature changes than their linear response, a property that is also observed in F-actin networks, and is well reproduced by the GWLC model. Additionally, microrheological measurements suggest that over long timescales (>10 s) internal stresses continue to redistribute facilitating low frequency motions of tracer particles.

  11. The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks*

    PubMed Central

    Van Itallie, Christina M.; Aponte, Angel; Tietgens, Amber Jean; Gucek, Marjan; Fredriksson, Karin; Anderson, James Melvin

    2013-01-01

    The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization. PMID:23553632

  12. Networking standards

    NASA Technical Reports Server (NTRS)

    Davies, Mark

    1991-01-01

    The enterprise network is currently a multivendor environment consisting of many defacto and proprietary standards. During the 1990s, these networks will evolve towards networks which are based on international standards in both Local Area Network (LAN) and Wide Area Network (WAN) space. Also, you can expect to see the higher level functions and applications begin the same transition. Additional information is given in viewgraph form.

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

    PubMed

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

    2006-09-21

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

  14. Semantic Networks and Social Networks

    ERIC Educational Resources Information Center

    Downes, Stephen

    2005-01-01

    Purpose: To illustrate the need for social network metadata within semantic metadata. Design/methodology/approach: Surveys properties of social networks and the semantic web, suggests that social network analysis applies to semantic content, argues that semantic content is more searchable if social network metadata is merged with semantic web…

  15. Network Basics.

    ERIC Educational Resources Information Center

    Tennant, Roy

    1992-01-01

    Explains how users can find and access information resources available on the Internet. Highlights include network information centers (NICs); lists, both formal and informal; computer networking protocols, including international standards; electronic mail; remote log-in; and file transfer. (LRW)

  16. Network neuroscience.

    PubMed

    Bassett, Danielle S; Sporns, Olaf

    2017-02-23

    Despite substantial recent progress, our understanding of the principles and mechanisms underlying complex brain function and cognition remains incomplete. Network neuroscience proposes to tackle these enduring challenges. Approaching brain structure and function from an explicitly integrative perspective, network neuroscience pursues new ways to map, record, analyze and model the elements and interactions of neurobiological systems. Two parallel trends drive the approach: the availability of new empirical tools to create comprehensive maps and record dynamic patterns among molecules, neurons, brain areas and social systems; and the theoretical framework and computational tools of modern network science. The convergence of empirical and computational advances opens new frontiers of scientific inquiry, including network dynamics, manipulation and control of brain networks, and integration of network processes across spatiotemporal domains. We review emerging trends in network neuroscience and attempt to chart a path toward a better understanding of the brain as a multiscale networked system.

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

    PubMed

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

    2015-04-01

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

  18. Network science.

    PubMed

    Barabási, Albert-László

    2013-03-28

    Professor Barabási's talk described how the tools of network science can help understand the Web's structure, development and weaknesses. The Web is an information network, in which the nodes are documents (at the time of writing over one trillion of them), connected by links. Other well-known network structures include the Internet, a physical network where the nodes are routers and the links are physical connections, and organizations, where the nodes are people and the links represent communications.

  19. JWA loss promotes cell migration and cytoskeletal rearrangement by affecting HER2 expression and identifies a high-risk subgroup of HER2-positive gastric carcinoma patients

    PubMed Central

    Qian, Jing; Zhu, Weiyou; Wang, Keming; Ma, Lin; Xu, Jin; Xu, Tongpeng; Røe, Oluf Dimitri; Li, Aiping; Zhou, Jianwei; Shu, Yongqian

    2016-01-01

    Background and Aims JWA, a microtubule-associated protein (MAP) involved in apoptosis, has been identified as a suppressor of metastasis, and it affects cell migration in melanoma and its downregulation in tumor is an idependent negative prognostic factor in resectable gastric cancer. HER2 overexpression has been observed in gastric cancer (GC) cells and implicated in the metastatic phenotype. However, the biological role of JWA in migration and its clinical value in HER2-positive GC remain elusive. Results JWA suppresses EGF-induced cell migration and actin cytoskeletal rearrangement by abrogating HER2 expression and downstream PI3K/AKT signaling in HER2-overexpressing GC cell lines. The modulation of HER2 by JWA is dependent on ERK activation and consequent PEA3 upregulation and activation. Reduced JWA expression is associated with high HER2 expression and with poor survival in patients with AGC, whereas HER2 expression alone is not associated with survival. However, concomitant low JWA and high HER2 expression is associated with unfavorable outcomes. Additionally, when patients were stratified by JWA expression, those with higher HER2 expression in the low JWA expression subgroup exhibited worse survival. Methods The impact of JWA on the EGF-induced migration of HER2-positive GC cells was studied using transwell assays and G-LISA assays. Western blotting, real-time PCR, electrophoretic mobility shift assays and luciferase assays were utilized to investigate the mechanisms by which JWA affects HER2. The association of JWA with HER2 and its clinical value were further analyzed by IHC in 128 pairs of advanced gastric cancer (AGC) and adjacent normal tissue samples. Conclusions This study characterizes a novel mechanism for regulating cell motility in HER2-overexpressing GC