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Sample records for actin retrograde flow

  1. Model for how retrograde actin flow regulates adhesion traction stresses.

    PubMed

    Li, Ying; Bhimalapuram, Prabhakar; Dinner, Aaron R

    2010-05-19

    Cells from animals adhere to and exert mechanical forces on their surroundings. Cells must control these forces for many biological processes, and dysfunction can lead to pathologies. How the actions of molecules within a cell are coordinated to regulate the adhesive interaction with the extracellular matrix remains poorly understood. It has been observed that cytoplasmic proteins that link integrin cell-surface receptors with the actin cytoskeleton flow with varying rates from the leading edge toward the center of a cell. Here, we explore theoretically how measurable subcellular traction stresses depend on the local speed of retrograde actin flow. In the model, forces result from the stretching of molecular complexes in response to the drag from the flow; because these complexes break with extension-dependent kinetics, the flow results in a decrease in their number when sufficiently large. Competition between these two effects naturally gives rise to a clutch-like behavior and a nonmonotonic trend in the measured stresses, consistent with recent data for epithelial cells. We use this basic framework to evaluate slip and catch bond mechanisms for integrins; better fits of experimental data are obtained with a catch bond representation. Extension of the model to one comprising multiple molecular interfaces shifts the peak stress to higher speeds. Connections to other models and cell movement are discussed. PMID:21386439

  2. A new link between the retrograde actin flow and focal adhesions.

    PubMed

    Yamashiro, Sawako; Watanabe, Naoki

    2014-11-01

    The retrograde actin flow, continuous centripetal movement of the cell peripheral actin networks, is widely observed in adherent cells. The retrograde flow is believed to facilitate cell migration when linked to cell adhesion molecules. In this review, we summarize our current knowledge regarding the functional relationship between the retrograde actin flow and focal adhesions (FAs). We also introduce our recent study in which single-molecule speckle (SiMS) microscopy dissected the complex interactions between FAs and the local actin flow. FAs do not simply impede the actin flow, but actively attract and remodel the local actin network. Our findings provide a new insight into the mechanisms for protrusion and traction force generation at the cell leading edge. Furthermore, we discuss possible roles of the actin flow-FA interaction based on the accumulated knowledge and our SiMS study. PMID:25190817

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

    PubMed

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

    2012-06-25

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

  4. F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation

    PubMed Central

    Babich, Alexander; Li, Shuixing; O'Connor, Roddy S.; Milone, Michael C.; Freedman, Bruce D.

    2012-01-01

    Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca2+ signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca2+ signaling. PMID:22665519

  5. Comparative Dynamics of Retrograde Actin Flow and Focal Adhesions: Formation of Nascent Adhesions Triggers Transition from Fast to Slow Flow

    PubMed Central

    Alexandrova, Antonina Y.; Arnold, Katya; Schaub, Sébastien; Vasiliev, Jury M.; Meister, Jean-Jacques; Bershadsky, Alexander D.; Verkhovsky, Alexander B.

    2008-01-01

    Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base. PMID:18800171

  6. Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow.

    PubMed

    Alexandrova, Antonina Y; Arnold, Katya; Schaub, Sébastien; Vasiliev, Jury M; Meister, Jean-Jacques; Bershadsky, Alexander D; Verkhovsky, Alexander B

    2008-01-01

    Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base. PMID:18800171

  7. New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales.

    PubMed

    Yamashiro, Sawako; Mizuno, Hiroaki; Smith, Matthew B; Ryan, Gillian L; Kiuchi, Tai; Vavylonis, Dimitrios; Watanabe, Naoki

    2014-04-01

    Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein-actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8-8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network. PMID:24501425

  8. Causes of retrograde flow in fish keratocytes.

    PubMed

    Fuhs, Thomas; Goegler, Michael; Brunner, Claudia A; Wolgemuth, Charles W; Kaes, Josef A

    2014-01-01

    Confronting motile cells with obstacles doubling as force sensors we tested the limits of the driving actin and myosin machinery. We could directly measure the force necessary to stop actin polymerization as well as the force present in the retrograde actin flow. Combined with detailed measurements of the retrograde flow velocity and specific manipulation of actin and myosin we found that actin polymerization and myosin contractility are not enough to explain the cells behavior. We show that ever-present depolymerization forces, a direct entropic consequence of actin filament recycling, are sufficient to fill this gap, even under heavy loads. PMID:24127260

  9. Retrograde Flow and Myosin II Activity within the Leading Cell Edge Deliver F-Actin to the Lamella to Seed the Formation of Graded Polarity Actomyosin II Filament Bundles in Migrating Fibroblasts

    PubMed Central

    Anderson, Tom W.; Vaughan, Andrew N.

    2008-01-01

    In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles. PMID:18799629

  10. Coupling of Retrograde Flow to Force Production During Malaria Parasite Migration.

    PubMed

    Quadt, Katharina A; Streichfuss, Martin; Moreau, Catherine A; Spatz, Joachim P; Frischknecht, Friedrich

    2016-02-23

    Migration of malaria parasites is powered by a myosin motor that moves actin filaments, which in turn link to adhesive proteins spanning the plasma membrane. The retrograde flow of these adhesins appears to be coupled to forward locomotion. However, the contact dynamics between the parasite and the substrate as well as the generation of forces are complex and their relation to retrograde flow is unclear. Using optical tweezers we found retrograde flow rates up to 15 μm/s contrasting with parasite average speeds of 1-2 μm/s. We found that a surface protein, TLP, functions in reducing retrograde flow for the buildup of adhesive force and that actin dynamics appear optimized for the generation of force but not for maximizing the speed of retrograde flow. These data uncover that TLP acts by modulating actin dynamics or actin filament organization and couples retrograde flow to force production in malaria parasites. PMID:26792112

  11. Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions

    PubMed Central

    Schelhaas, Mario; Ewers, Helge; Rajamäki, Minna-Liisa; Day, Patricia M.; Schiller, John T.; Helenius, Ari

    2008-01-01

    The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing. PMID:18773072

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

    PubMed Central

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

    1992-01-01

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

  13. Forces from the rear: deformed microtubules in neuronal growth cones influence retrograde flow and advancement

    NASA Astrophysics Data System (ADS)

    Rauch, Philipp; Heine, Paul; Goettgens, Barbara; Käs, Josef A.

    2013-01-01

    The directed motility of growth cones at the tip of neuronal processes is a key function in neuronal path-finding and relies on a complex system of interacting cytoskeletal components. Despite intensive research in this field, many aspects of the mechanical roles of actin structures and, in particular, of microtubules throughout this process remain unclear. Mostly, force generation is ascribed to actin-myosin-based structures such as filopodia bundles and the dynamic polymer gel within the lamellipodium. Our analysis of microtubule buckling and deformation in motile growth cones reveals that extending microtubule filaments contribute significantly to the overall protrusion force. In this study, we establish a relationship of the local variations in stored bending energy and deformation characteristics to growth cone morphology and retrograde actin flow. This implies the relevance of microtubule pushing and deformation for general neurite advancement as well as steering processes.

  14. Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions

    PubMed Central

    Nichol, Robert H.; Hagen, Kate M.; Lumbard, Derek C.; Dent, Erik W.

    2016-01-01

    Growth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or “clutch” myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show in Xenopus spinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-d-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuated in vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues. SIGNIFICANCE STATEMENT Here, we correlate point contact adhesions directly with clutching of filamentous actin retrograde flow (RF), which our findings strongly suggest guides developing axons. Acute assembly of new point contact

  15. Hemodynamic changes and retrograde flow in LVAD failure.

    PubMed

    Giridharan, Guruprasad A; Koenig, Steven C; Soucy, Kevin G; Choi, Young; Pirbodaghi, Tohid; Bartoli, Carlo R; Monreal, Gretel; Sobieski, Michael A; Schumer, Erin; Cheng, Allen; Slaughter, Mark S

    2015-01-01

    In the event of left ventricular assist device (LVAD) failure, we hypothesized that rotary blood pumps will experience significant retrograde flow and induce adverse physiologic responses. Catastrophic LVAD failure was investigated in computer simulation with pulsatile, axial, and centrifugal LVAD, mock flow loop with pulsatile (PVAD) and centrifugal (ROTAFLOW), and healthy and chronic ischemic heart failure bovine models with pulsatile (PVAD), axial (HeartMate II), and centrifugal (HVAD) pumps. Simulated conditions were LVAD "off" with outflow graft clamped (baseline), LVAD "off" with outflow graft unclamped (LVAD failure), and LVAD "on" (5 L/min). Hemodynamics (aortic and ventricular blood pressures, LVAD flow, and left ventricular volume), echocardiography (cardiac volumes), and end-organ perfusion (regional blood flow microspheres) were measured and analyzed. Retrograde flow was observed with axial and centrifugal rotary pumps during LVAD failure in computer simulation (axial = -3.4 L/min, centrifugal = -2.8 L/min), mock circulation (pulsatile = -0.1 L/min, centrifugal = -2.7 L/min), healthy (pulsatile = -1.2 ± 0.3 L/min, axial = -2.2 ± 0.2 L/min, centrifugal = -1.9 ± 0.3 L/min), and ischemic heart failure (centrifugal = 2.2 ± 0.7 L/min) bovine models for all test conditions (p < 0.05). Differences between axial and centrifugal LVAD were statistically indiscernible. Retrograde flow increased ventricular end-systolic and end-diastolic volumes and workload, and decreased myocardial and end-organ perfusion during LVAD failure compared with baseline, LVAD support, and pulsatile LVAD failure. PMID:25635935

  16. Measuring Actin Flow in 3D Cell Protrusions

    PubMed Central

    Chiu, Chi-Li; Digman, Michelle A.; Gratton, Enrico

    2013-01-01

    Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of

  17. Retrograde Transvenous Ethanol Embolization of High-flow Peripheral Arteriovenous Malformations

    SciTech Connect

    Linden, Edwin van der; Baalen, Jary M. van; Pattynama, Peter M. T.

    2012-08-15

    Purpose: To report the clinical efficiency and complications in patients treated with retrograde transvenous ethanol embolization of high-flow peripheral arteriovenous malformations (AVMs). Retrograde transvenous ethanol embolization of high-flow AVMs is a technique that can be used to treat AVMs with a dominant outflow vein whenever conventional interventional procedures have proved insufficient. Methods: This is a retrospective study of the clinical effectiveness and complications of retrograde embolization in five patients who had previously undergone multiple arterial embolization procedures without clinical success. Results: Clinical outcomes were good in all patients but were achieved at the cost of serious, although transient, complications in three patients. Conclusion: Retrograde transvenous ethanol embolization is a highly effective therapy for high-flow AVMs. However, because of the high complication rate, it should be reserved as a last resort, to be used after conventional treatment options have failed.

  18. Control of respiration-driven retrograde flow in the subdiaphragmatic venous return of the Fontan circulation

    PubMed Central

    Vukicevic, M; Conover, T; Jaeggli, M; Zhou, J; Pennati, G; Hsia, TY; Figliola, RS

    2014-01-01

    Respiration influences the subdiaphragmatic venous return in the total cavopulmonary connection (TCPC) of the Fontan circulation whereby both the inferior vena cava (IVC) and hepatic vein flows can experience retrograde motion. Controlling retrograde flows could improve patient outcomes. Using a patient-specific model within a Fontan mock circulatory system with respiration, we inserted a valve into the IVC to examine its effects on local hemodynamics while varying retrograde volumes by changing vascular impedances. A bovine valved conduit reduced IVC retrograde flow to within 3% of antegrade flow in all cases. The valve closed only under conditions supporting retrograde flow and its effects on local hemodynamics increased with larger retrograde volume. Liver and TCPC pressures improved only while the valve leaflets were closed while cycle-averaged pressures improved only slightly (italic>1 mm Hg). Increased pulmonary vascular resistance raised mean circulation pressures but the valve functioned and cardiac output improved and stabilized. Power loss across the TCPC improved by 12–15% (pbold>0.05) with a valve. The effectiveness of valve therapy is dependent on patient vascular impedance. PMID:24814833

  19. Control of respiration-driven retrograde flow in the subdiaphragmatic venous return of the Fontan circulation.

    PubMed

    Vukicevic, Marija; Conover, Timothy; Jaeggli, Michael; Zhou, Jian; Pennati, Giancarlo; Hsia, Tain-Yen; Figliola, Richard S

    2014-01-01

    Respiration influences the subdiaphragmatic venous return in the total cavopulmonary connection (TCPC) of the Fontan circulation whereby both the inferior vena cava (IVC) and hepatic vein flows can experience retrograde motion. Controlling retrograde flows could improve patient outcomes. Using a patient-specific model within a Fontan mock circulatory system with respiration, we inserted a valve into the IVC to examine its effects on local hemodynamics while varying retrograde volumes by changing vascular impedances. A bovine valved conduit reduced IVC retrograde flow to within 3% of antegrade flow in all cases. The valve closed only under conditions supporting retrograde flow and its effects on local hemodynamics increased with larger retrograde volume. Liver and TCPC pressures improved only when the valve leaflets were closed whereas cycle-averaged pressures improved only slightly (<1 mm Hg). Increased pulmonary vascular resistance raised mean circulation pressures, but the valve functioned and cardiac output improved and stabilized. Power loss across the TCPC improved by 12%-15% (p < 0.05) with a valve. The effectiveness of valve therapy is dependent on patient vascular impedance. PMID:24814833

  20. Exercise intensity modulates brachial artery retrograde blood flow and shear rate during leg cycling in hypoxia.

    PubMed

    Iwamoto, Erika; Katayama, Keisho; Ishida, Koji

    2015-06-01

    The purpose of this study was to elucidate the effect of exercise intensity on retrograde blood flow and shear rate (SR) in an inactive limb during exercise under normoxic and hypoxic conditions. The subjects performed two maximal exercise tests on a semi-recumbent cycle ergometer to estimate peak oxygen uptake (V˙O2peak) while breathing normoxic (inspired oxygen fraction [FIO2 = 0.21]) and hypoxic (FIO2 = 0.12 or 0.13) gas mixtures. Subjects then performed four exercise bouts at the same relative intensities (30 and 60% V˙O2peak) for 30 min under normoxic or hypoxic conditions. Brachial artery diameter and blood velocity were simultaneously recorded, using Doppler ultrasonography. Retrograde SR was enhanced with increasing exercise intensity under both conditions at 10 min of exercise. Thereafter, retrograde blood flow and SR in normoxia returned to pre-exercise levels, with no significant differences between the two exercise intensities. In contrast, retrograde blood flow and SR in hypoxia remained significantly elevated above baseline and was significantly greater at 60% than at 30% V˙O2peak. We conclude that differences in exercise intensity affect brachial artery retrograde blood flow and SR during prolonged exercise under hypoxic conditions. PMID:26038470

  1. Exercise intensity modulates brachial artery retrograde blood flow and shear rate during leg cycling in hypoxia

    PubMed Central

    Iwamoto, Erika; Katayama, Keisho; Ishida, Koji

    2015-01-01

    The purpose of this study was to elucidate the effect of exercise intensity on retrograde blood flow and shear rate (SR) in an inactive limb during exercise under normoxic and hypoxic conditions. The subjects performed two maximal exercise tests on a semi-recumbent cycle ergometer to estimate peak oxygen uptake (O2peak) while breathing normoxic (inspired oxygen fraction [FIO2 = 0.21]) and hypoxic (FIO2 = 0.12 or 0.13) gas mixtures. Subjects then performed four exercise bouts at the same relative intensities (30 and 60% O2peak) for 30 min under normoxic or hypoxic conditions. Brachial artery diameter and blood velocity were simultaneously recorded, using Doppler ultrasonography. Retrograde SR was enhanced with increasing exercise intensity under both conditions at 10 min of exercise. Thereafter, retrograde blood flow and SR in normoxia returned to pre-exercise levels, with no significant differences between the two exercise intensities. In contrast, retrograde blood flow and SR in hypoxia remained significantly elevated above baseline and was significantly greater at 60% than at 30% O2peak. We conclude that differences in exercise intensity affect brachial artery retrograde blood flow and SR during prolonged exercise under hypoxic conditions. PMID:26038470

  2. Effect of high-speed jet on flow behavior, retrogradation, and molecular weight of rice starch.

    PubMed

    Fu, Zhen; Luo, Shun-Jing; BeMiller, James N; Liu, Wei; Liu, Cheng-Mei

    2015-11-20

    Effects of high-speed jet (HSJ) treatment on flow behavior, retrogradation, and degradation of the molecular structure of indica rice starch were investigated. Decreasing with the number of HSJ treatment passes were the turbidity of pastes (degree of retrogradation), the enthalpy of melting of retrograded rice starch, weight-average molecular weights and weight-average root-mean square radii of gyration of the starch polysaccharides, and the amylopectin peak areas of SEC profiles. The areas of lower-molecular-weight polymers increased. The chain-length distribution was not significantly changed. Pastes of all starch samples exhibited pseudoplastic, shear-thinning behavior. HSJ treatment increased the flow behavior index and decreased the consistency coefficient and viscosity. The data suggested that degradation of amylopectin was mainly involved and that breakdown preferentially occurred in chains between clusters. PMID:26344255

  3. Brachial artery retrograde flow increases with age: relationship to physical function

    PubMed Central

    Credeur, Daniel P.; Dobrosielski, Devon A.; Arce-Esquivel, Arturo A.; Welsch, Michael A.

    2010-01-01

    The purpose of this study was to examine the flow velocity pattern of the brachial artery and to determine its relationship to measures of physical function. Subjects from the Louisiana Healthy Aging Study (n = 95; age = 84 ± 10 years) were evaluated. Brachial artery flow velocities and dimensions were measured using high-resolution ultrasonography. The continuous scale of physical function and performance test (CS-PFP10) was used to assess physical function. This test is based on the performance of 11 activities of daily living. Total CS-PFP10 score was 39.51 ± 21.21 U. Mean antegrade and retrograde velocities at rest were 14.2 ± 4.7 and 3.6 ± 2.2 cm/s, respectively. Ante-/retrograde ratio was 5.5 ± 4.6. Brachial artery diameter was 4.3 ± 0.7 mm. Pulse pressure and vascular conductance were 66 ± 18 mmHg, and 0.9 ± 0.5 ml/min/mmHg, respectively. Vascular conductance (r = −0.34), ante-/retrograde ratio (r = −0.42) and CS-PFP10 (r = −0.65) were inversely and retrograde velocity (r = 0.40) and pulse pressure (r = 0.36), were directly associated with age. Retrograde velocity was inversely related to vascular conductance (r = −0.27) and CS-PFP10 total score (r = −0.45). A MANOVA revealed that those with the higher CS-PFP10 scores had a lower retrograde velocity (P = 0.0001), but this association was, in part, age-dependent. Among nonagenarians (n = 52), those in the lower tertiles of the CS-PFP10 scores had significantly higher retrograde velocities compared to those in the higher tertiles (P = 0.035). These data indicate an increase in brachial retrograde velocity with age. These hemodynamic changes are related to a decline in physical function. PMID:19565260

  4. Phase-contrast magnetic resonance imaging reveals net retrograde aqueductal flow in idiopathic normal pressure hydrocephalus.

    PubMed

    Ringstad, Geir; Emblem, Kyrre Eeg; Eide, Per Kristian

    2016-06-01

    OBJECT The objective of this study was to assess the net aqueductal stroke volume (ASV) and CSF aqueductal flow rate derived from phase-contrast MRI (PC-MRI) in patients with probable idiopathic normal pressure hydrocephalus (iNPH) before and after ventriculoperitoneal shunt surgery, and to compare observations with intracranial pressure (ICP) scores. METHODS PC-MRI at the level of the sylvian aqueduct was undertaken in patients undergoing assessment for probable iNPH. Aqueductal flow in the craniocaudal direction was defined as positive, or antegrade flow, and net ASV was calculated by subtracting retrograde from antegrade aqueductal flow. Aqueductal flow rate per minute was calculated by multiplying net ASV by heart rate. During the same hospital admission, clinical examination was performed using NPH score and overnight continuous ICP monitoring. Twelve patients were followed prospectively 12 months after shunt placement with clinical assessment and a second PC-MRI. The study also included 2 healthy controls. RESULTS Among 21 patients examined for iNPH, 17 (81%) received a shunt (shunt group), and 4 were treated conservatively (conservative group). Among the patients with shunts, a clinical improvement was observed in 16 (94%) of the 17. Net ASV was negative in 16 (76%) of 21 patients before shunt placement and in 5 (42%) of 12 patients after shunt placement, and increased from a median of -5 μl (range -175 to 27 μl) to a median of 1 μl (range -61 to 30 μl; p = 0.04). Among the 12 patients with PC-MRI after shunt placement, 11 were shunt responders, and in 9 of these 11 either a reduced magnitude of retrograde aqueductal flow, or a complete reversal from retrograde to antegrade flow, occurred. Net ASV was significantly lower in the shunt group than in the conservative group (p = 0.01). The aqueductal flow rate increased from -0.56 ml/min (range -12.78 to 0.58 ml/min) to 0.06 ml/min (range -4.51 to 1.93 ml/min; p = 0.04) after shunt placement. CONCLUSIONS In

  5. Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells.

    PubMed

    Melchior, Benoît; Frangos, John A

    2012-08-15

    Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow

  6. Bilateral vertebral artery occlusion with retrograde basilary flow in three cases of giant cell arteritis

    PubMed Central

    Boettinger, Markus Robert; Sebastian, Schreglmann Robert; Gamulescu, Maria-Andreea Robert; Grauer, Oliver; Ritzka, Markus; Schuierer, Gerhard Robert; Bogdahn, Ulrich Robert; Steinbrecher, Andreas; Schlachetzki, Felix

    2009-01-01

    Vertebrobasilar ischaemia is a rare life-threatening complication in giant cell arteritis (GCA). We report three patients with bilateral vertebral artery occlusion. Neurovascular imaging, including CT-angiography, MR-angiography and colour-coded duplex sonography revealed flow reversal in the basilar artery as well as inflammation of the vertebral vessel wall. The first patient died from massive brainstem infarction, the other two patients survived the initial inflammatory phase of GCA. No stroke recurrence at 12 months’ follow-up on warfarin and steroid treatment was observed. Bilateral distal vertebral artery occlusion and retrograde basilar artery flow persisted. Outcome in these patients is dependant on potent immunosuppression, concurrent atherosclerotic steno-occlusive disease and presence and/or rapid development of sufficient collateral pathways into the vertebrobasilar circulation. The identification of patients with high risk of ischaemia due to compromised vertebrobasilar flow may be important to select adjunct treatment to immunosuppression, such as anticoagulation in GCA. PMID:21691390

  7. Salvage Free Anterolateral Thigh Composite Flap Transfer Based on the Musculocutaneous Perforator Retrograde Blood Flow Principle.

    PubMed

    Zhao, Jingyi; Chan, Fuan Chiang; Yang, Xiaonan; Zong, Xianlei; Sun, Hengyun; Qi, Zuoliang; Jin, Xiaolei

    2016-03-01

    An anterolateral thigh myo-adipofascial flap was used in the definitive management of a patient presented with chronic infective process associated with a large fronto-nasal defect. Unfortunately, the risk of free flap transfer failure emerged when intraoperative dissection showed absence of a reliable ipsilateral superficial temporal artery as the recipient artery. This rare incident happened at the stage whereby the anterolateral thigh flap was nearly completely raised with a distal perforator in situ. In this article, the authors presented an innovative strategy to salvage the flap by transforming the flap into a modified composite flap based on the retrograde blood flow principle. To the best of our knowledge, this is the first report of using such a technique in reconstructive microsurgery. This successful salvage strategy has clinical application and could potentially minimize free flap transfer failure. PMID:26854778

  8. Integrating bio-prosthetic valves in the Fontan operation - Novel treatment to control retrograde flow in caval veins

    NASA Astrophysics Data System (ADS)

    Vukicevic, Marija; Conover, Timothy; Zhou, Jian; Hsia, Tain-Yen; Figliola, Richard

    2012-11-01

    For a child born with only one functional heart ventricle, the sequence of palliative surgeries typically culminates in the Fontan operation. This procedure is usually successful initially, but leads to later complications, for reasons not fully understood. Examples are respiratory-dependent retrograde flows in the caval and hepatic veins, and increased pulmonary vascular resistance (PVR), hypothesized to be responsible for elevated pressure in the liver and disease of the liver and intestines. Here we study the parameters responsible for retrograde flows in the inferior vena cava (IVC) and hepatic vein (HV), and investigate two novel interventions to control retrograde flow: implanting either a Medtronic Contegra valved conduit or an Edwards lifescience pericardial aortic valve in the IVC or HV. We performed the experiments in a multi-scale, patient specific mock circuit, with normal and elevated PVR, towards the optimization of the Fontan circulation. The results show that both valves can significantly reduce retrograde flows in the veins, suggesting potential advantages in the treatment of the patients with congenital heart diseases. Fondation Leducq

  9. Myosin Vs organize actin cables in fission yeast

    PubMed Central

    Lo Presti, Libera; Chang, Fred; Martin, Sophie G.

    2012-01-01

    Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV∆ defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7–Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces. PMID:23051734

  10. Myosin Vs organize actin cables in fission yeast.

    PubMed

    Lo Presti, Libera; Chang, Fred; Martin, Sophie G

    2012-12-01

    Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7-Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces. PMID:23051734

  11. Actin flows in cell migration: from locomotion and polarity to trajectories.

    PubMed

    Callan-Jones, Andrew C; Voituriez, Raphaël

    2016-02-01

    Eukaryotic cell movement is characterized by very diverse migration modes. Recent studies show that cells can adapt to environmental cues, such as adhesion and geometric confinement, thereby readily switching their mode of migration. Among this diversity of motile behavior, actin flows have emerged as a highly conserved feature of both mesenchymal and amoeboid migration, and have also been identified as key regulators of cell polarity. This suggests that the various observed migration modes are continuous variations of elementary locomotion mechanisms, based on a very robust physical property of the actin/myosin system - its ability to sustain flows at the cell scale. This central role of actin/myosin flows is shown to affect the large scale properties of cell trajectories. PMID:26827283

  12. Polymer dynamics and fluid flow in actin-based cell motility

    NASA Astrophysics Data System (ADS)

    Theriot, Julie

    2005-03-01

    In living cells, nonequilibrium protein polymerization reactions are frequently used to convert chemical energy into mechanical energy and thereby generate useful force for cellular movements. We have examined the polymer and fluid dynamics in two biological cases where the assembly of branched actin filament networks generates force: the intracellular movement of the bacterial pathogen Listeria monocytogenes, and the extension of the leading edge of skin epithelial cells during wound-healing. In both cases, net actin filament assembly occurs at the front of the network structure and net disassembly occurs at the rear. Actin protein subunits and other network components must be recycled through the fluid phase to the front of the polymerizing network in order for forward movement to continue at steady state. For actin-based movement of Listeria monocytogenes, we have found that actin recycling is not rate-limiting; instead, the speed of movement is governed by the cooperative dissociation of groups of noncovalent protein-protein bonds attaching the filamentous network to the bacterial surface. In contrast, rapid actin-based extension at the leading edge of moving epithelial cells is associated with unusual perturbations in intracellular fluid flow.

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

    PubMed Central

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

    2015-01-01

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

  14. Recovery, Visualization, and Analysis of Actin and Tubulin Polymer Flow in Live Cells: A Fluorescent Speckle Microscopy Study

    PubMed Central

    Vallotton, P.; Ponti, A.; Waterman-Storer, C. M.; Salmon, E. D.; Danuser, G.

    2003-01-01

    Fluorescent speckle microscopy (FSM) is becoming the technique of choice for analyzing in vivo the dynamics of polymer assemblies, such as the cytoskeleton. The massive amount of data produced by this method calls for computational approaches to recover the quantities of interest; namely, the polymerization and depolymerization activities and the motions undergone by the cytoskeleton over time. Attempts toward this goal have been hampered by the limited signal-to-noise ratio of typical FSM data, by the constant appearance and disappearance of speckles due to polymer turnover, and by the presence of flow singularities characteristic of many cytoskeletal polymer assemblies. To deal with these problems, we present a particle-based method for tracking fluorescent speckles in time-lapse FSM image series, based on ideas from operational research and graph theory. Our software delivers the displacements of thousands of speckles between consecutive frames, taking into account that speckles may appear and disappear. In this article we exploit this information to recover the speckle flow field. First, the software is tested on synthetic data to validate our methods. We then apply it to mapping filamentous actin retrograde flow at the front edge of migrating newt lung epithelial cells. Our results confirm findings from previously published kymograph analyses and manual tracking of such FSM data and illustrate the power of automated tracking for generating complete and quantitative flow measurements. Third, we analyze microtubule poleward flux in mitotic metaphase spindles assembled in Xenopus egg extracts, bringing new insight into the dynamics of microtubule assemblies in this system. PMID:12885672

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  16. Excitable actin dynamics in lamellipodial protrusion and retraction.

    PubMed

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

    2012-04-01

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

  17. Retrograde ejaculation

    MedlinePlus

    Retrograde ejaculation occurs when semen enters the bladder instead of going out through the urethra during ejaculation. ... bladder (bladder neck) does not close. This causes semen to go backwards into the bladder rather than ...

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  1. Simultaneous tracking of 3D actin and microtubule strains in individual MLO-Y4 osteocytes under oscillatory flow.

    PubMed

    Baik, Andrew D; Qiu, Jun; Hillman, Elizabeth M C; Dong, Cheng; Guo, X Edward

    2013-02-22

    Osteocytes in vivo experience complex fluid shear flow patterns to activate mechanotransduction pathways. The actin and microtubule (MT) cytoskeletons have been shown to play an important role in the osteocyte's biochemical response to fluid shear loading. The dynamic nature of physiologically relevant fluid flow profiles (i.e., 1Hz oscillatory flow) impedes the ability to image and study both actin and MT cytoskeletons simultaneously in the same cell with high spatiotemporal resolution. To overcome these limitations, a multi-channel quasi-3D microscopy technique was developed to track the actin and MT networks simultaneously under steady and oscillatory flow. Cells displayed high intercellular variability and intracellular cytoskeletal variability in strain profiles. Shear Exz was the predominant strain in both steady and oscillatory flows in the form of viscoelastic creep and elastic oscillations, respectively. Dramatic differences were seen in oscillatory flow, however. The actin strains displayed an oscillatory strain profile more often than the MT networks in all the strains tested and had a higher peak-to-trough strain magnitude. Taken together, the actin networks are the more responsive cytoskeletal networks in osteocytes under oscillatory flow and may play a bigger role in mechanotransduction pathway activation and regulation. PMID:23352617

  2. [Antegrade flow in the aorta ascendens despite aortic atresia: 2 case reports with retrograde coronary perfusion through coronary fistulas and sinusoids].

    PubMed

    Jux, C; Kaulitz, R; von Wasielewski, R; Peuster, M; Fink, C; Paul, T; Hausdorf, G

    2000-06-01

    In aortic atresia, coronary perfusion normally occurs through retrograde blood flow in the ascending aorta. We report on two patients with antegrade flow in the ascending aorta despite aortic atresia. In one patient with hypoplastic left heart syndrome (aortic atresia, severe mitral stenosis), an intact interatrial septum/premature closure of the foramen ovale was found. While no other way of left atrial or ventricular decompression was found, echocardiography, angiography and the post-mortem examination showed left ventricular to coronary sinusoids as the sole pathway for systemic oxygenation. In a second patient with complex congenital heart disease, including aortic atresia, antegrade flow in the ascending aorta was through a left coronary fistula with shunt flow originating from the pulmonary trunk. This report describes systemic perfusion depending on retrograde coronary flow due to coronary-cameral (sinusoids) and coronary arterio-venous fistulas leading to the phenomenon of antegrade blood flow in the ascending aorta despite aortic atresia. PMID:10929434

  3. Preserved Renal Function in Kidney Transplantation over a Thrombosed Aortobifemoral Bypass Graft: The Role of Retrograde Flow and Early Thrombolysis

    PubMed Central

    Jiménez-Alvaro, Sara; Fernández-Rodríguez, Ana; Rivera-Gorrín, Maite; Sánchez, Juan; Chinchilla, Antonio; Marcén, Roberto

    2016-01-01

    Aortobifemoral bypass (ABFB) thrombosis is not uncommon, and when the artery of a renal graft is implanted on a bypass the risk of graft loss is high. We report the case of a 48-year-old woman with a previous history of ABFB under antiplatelet therapy and a kidney allograft implanted on the vascular prosthesis, who presented with acute limb ischemia and severe renal impairment. Imaging techniques revealed a complete thrombosis of the proximal left arm of the ABFB. However, a faint retrograde flow over the graft was observed thanks to the recanalization of distal left bypass by collateral native arteries. This unusual situation not previously reported in a kidney transplant setting, together with an early diagnosis, allowed graft survival until an early local thrombolysis resolved the problem. Two years later, renal function remains normal. PMID:27579209

  4. Preserved Renal Function in Kidney Transplantation over a Thrombosed Aortobifemoral Bypass Graft: The Role of Retrograde Flow and Early Thrombolysis.

    PubMed

    Pampa-Saico, Saúl; Jiménez-Alvaro, Sara; Caravaca-Fontán, Fernando; Fernández-Rodríguez, Ana; Rivera-Gorrín, Maite; Sánchez, Juan; Chinchilla, Antonio; Marcén, Roberto

    2016-01-01

    Aortobifemoral bypass (ABFB) thrombosis is not uncommon, and when the artery of a renal graft is implanted on a bypass the risk of graft loss is high. We report the case of a 48-year-old woman with a previous history of ABFB under antiplatelet therapy and a kidney allograft implanted on the vascular prosthesis, who presented with acute limb ischemia and severe renal impairment. Imaging techniques revealed a complete thrombosis of the proximal left arm of the ABFB. However, a faint retrograde flow over the graft was observed thanks to the recanalization of distal left bypass by collateral native arteries. This unusual situation not previously reported in a kidney transplant setting, together with an early diagnosis, allowed graft survival until an early local thrombolysis resolved the problem. Two years later, renal function remains normal. PMID:27579209

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

    NASA Astrophysics Data System (ADS)

    Burroughs, N. J.; Marenduzzo, D.

    2007-06-01

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

  6. Retrograde ejaculation

    MedlinePlus

    ... problem. Alternative Names Ejaculation retrograde; Dry climax Images Male reproductive system References Bhasin S, Basson R. Sexual dysfunction in men and women. In: Kronenberg HM, Melmed S, Polonsky KS, Larsen PR, eds. Williams ... management of male infertility. In: Wein AJ, ed. Campbell-Walsh Urology . ...

  7. Relationship between retrograde coronary blood flow and the extent of no-reflow and infarct size in a porcine ischemia-reperfusion model.

    PubMed

    Stavrakis, Stavros; Terrovitis, John; Tsolakis, Elias; Drakos, Stavros; Dalianis, Argirios; Bonios, Michael; Koudoumas, Dimitrios; Malliaras, Konstantinos; Nanas, John

    2011-02-01

    Recanalization of an infarct-related artery does not predictably reflect tissue reperfusion. We examined the relationship between coronary blood flow (CBF) pattern during reperfusion and infarcted (IA) and no-reflow (NR) area in a porcine ischemia-reperfusion model. The mid-left anterior descending artery of 18 pigs was occluded for 1 h and reperfused for 2 h. CBF during reperfusion was measured with a transit-time ultrasound flowmeter, while systemic arterial and left atrial pressures were monitored. IA and NR were measured with triphenyl tetrazolium chloride and thioflavin staining, respectively. In 13 pigs, early systolic retrograde CBF developed within the first 30 min and persisted throughout reperfusion. No retrograde CBF was observed in five pigs. Mean retrograde CBF at 2 h of reperfusion predicted a larger IA (r = 0.71; p = 0.001). Time-to-development of retrograde CBF was inversely related to IA (r = -0.55; p = 0.019) and NR (r = -0.62; p = 0.006). A larger IA (OR 1.12, 95% CI 1.01-1.24, p = 0.037) and NR (OR 1.09, 95% CI 1.01-1.18, p = 0.037) predicted the presence of retrograde CBF. Retrograde CBF during recanalization of the infarct-related artery predicts IA and NR and might be used as an index of successful reperfusion at the tissue level. PMID:21153063

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

    PubMed Central

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

    2013-01-01

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

  9. Whole Cell Model of Actin Diffusion and Reaction based on Single Molecule Speckle Microscopy Measurements

    NASA Astrophysics Data System (ADS)

    McMillen, Laura; Vavylonis, Dimitrios; Vavylonis Group Team

    It is debated whether transport of actin across the cell by diffusion alone is sufficiently fast to account for the rapid reorganization of actin filaments at the leading edge of motile cells. In order to investigate this question, we created a 3D model of the whole cell that includes reaction and diffusion of actin using a particle Monte Carlo method. For the lamellipodium of the simulated cell we use the model by Smith et al. Biophys. J 104:247 (2013), which includes two diffuse pools of actin, one which is slowly diffusing and the other which diffuses more quickly, as well as a pool of filamentous actin undergoing retrograde flow towards the cell center. We adjusted this model to fit a circular geometry around the whole cell. We also consider actin in the cell center which is either diffusing or in stationary filamentous form, representing cortical actin or actin in stress fibers. The local rates of polymerization and the lifetime distributions of polymerized actin were estimated from single molecule speckle microscopy experiments by the group of N. Watanabe. With this model we are able to simulate prior experiments that monitored the redistribution of actin after photoactivation or fluorescence recovery after photobleaching in various parts of the cell. We find that transport by diffusion is sufficient to fit these data, without the need for an active transport mechanism, however significant concentration gradients may develop at steady state.

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

    PubMed

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

    2009-11-13

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

  11. 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.

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

    PubMed Central

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

    2013-01-01

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

  13. Actinic Keratosis

    MedlinePlus

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

  14. Membranous IVC Obstruction Presenting with Antegrade/Retrograde Respiratory Flow in the Intrahepatic Segment in Doppler Imaging and Prostatic and Urethral Congestion

    PubMed Central

    Sood, Dinesh; Mistry, Kewal A.; Chadha, Veenal; Sharma, Sarthak; Morey, Parikshit D.; Suthar, Pokhraj P.; Patel, Dhruv G.

    2015-01-01

    Summary Background Obstruction of the inferior vena cava (IVC) is infrequent, membranous obstruction of the IVC (MOIVC) being one of its rare causes. Early diagnosis is important, as it can lead to hepatic congestion, cirrhosis and Budd-Chiari syndrome (BCS) and can predispose to development of hepatocellular carcinoma (HCC) in severe cases. Case Report We report a case of membranous IVC obstruction at the junction of hepatic and suprahepatic segments in a young male with extensive collateralization and venous aneurysms. Unique findings involved antegrade and retrograde flow during respiration in the upper part of intrahepatic IVC proximal to a large collateral vein as well as prostatic and urethral congestion leading to intermittent urinary hesitancy, which have not yet been described in such cases. Conclusions MOIVC is a rare cause of IVC obstruction with typical radiological features. Early diagnosis and management is required due to risk of cirrhosis and HCC. Antegrade and retrograde flow may be seen in incomplete MOIVC above the level of a large collateral vein and it may lead to prostatic and urethral congestion. PMID:26191114

  15. Ureteral retrograde brush biopsy

    MedlinePlus

    Biopsy - brush - urinary tract; Retrograde ureteral brush biopsy cytology; Cytology - ureteral retrograde brush biopsy ... to be biopsied is rubbed with the brush. Biopsy forceps may be used instead to collect a ...

  16. Retrograde rotation of the large-scale flow in turbulent rotating Rayleigh-Benard convection with high Rossby number

    NASA Astrophysics Data System (ADS)

    Zhong, Jin-Qiang; Li, Hui-Min; Wang, Xue-Ying

    2015-11-01

    We present measurements of the azimuthal orientation θ (t) of the large-scale circulation (LSC) for turbulent Rayleigh-Bénard convection in the presence of week rotations Ω . Linear retrograde rotations of the LSC circulating plane are observed over the entire Rossby-number range (1 <= Ro <= 300) studied. When the Ro increases, the ratio of the retrograde rotation rate, γ = - < . θ > / Ω remains nearly a constant 0.12 in the range of (1 <= Ro <= 80) and starts to increases when Ro > 80 . When Ro ~= 300 , γ approaches a value of 0.36 close to the prediction from previous theoretical models. In a background of linear rotations, erratic changes in θ (t) accompanied by decreasing in the LSC amplitude δ are observed. These small- δ events give rise to the increasing γ with very high Ro numbers (80 <= Ro <= 300). In this range, the diffusivity of θ is proportional to δ-2 . Moreover, the occurrence frequency of the small- δ events, and their average duration are independent on Ro. We propose a model to include additional viscous damping for the LSC azimuthal motion due to turbulent viscosity and provide theoretical interpretations of the experimental results. Work supported by NSFC Grant No. 11202151.

  17. Actinic keratosis

    MedlinePlus

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

  18. Retrograde peri-implantitis.

    PubMed

    Mohamed, Jumshad B; Shivakumar, B; Sudarsan, Sabitha; Arun, K V; Kumar, T S S

    2010-01-01

    Retrograde peri-implantitis constitutes an important cause for implant failure. Retrograde peri-implantitis may sometimes prove difficult to identify and hence institution of early treatment may not be possible. This paper presents a report of four cases of (the implant placed developing to) retrograde peri-implantitis. Three of these implants were successfully restored to their fully functional state while one was lost due to extensive damage. The paper highlights the importance of recognizing the etiopathogenic mechanisms, preoperative assessment, and a strong postoperative maintenance protocol to avoid retrograde peri-implant inflammation. PMID:20922082

  19. Coupling actin flow, adhesion, and morphology in a computational cell motility model

    NASA Astrophysics Data System (ADS)

    Levine, Herbert

    2014-03-01

    Eukaryotic cells crawl by means of the coordinated spatiotemporal dynamics of an active polymer gel, consisting of actin, myosin and regulators thereof. Motility is necessarily coupled to shape, as the force generating mechanisms such as polymerization-based protrusions interact with the elasticity of the cell membrane and thereby determine the cell morphology. We have introduced a ``phase-field'' model of crawling cells, utilizing a mathematical approach originally developed for morphology problems arising in the field of liquid-solid phase transitions. Our model can be used to explain the pattern of traction forces applied to the substrate as well as some recent observations concerning oscillatory instabilities of cells moving on one-dimensional fiber tracks.

  20. Effectiveness and safety of the Proxis system in demonstrating retrograde coronary blood flow during proximal occlusion and in capturing embolic material.

    PubMed

    Sievert, Horst; Wahr, Dennis W; Schuler, Gerhard; Schofer, Joachim J; Sutsch, Gabor; Pavliska, Jim; Skowasch, Marijke

    2004-11-01

    The Feasibility And Safety Trial for its embolic protection device during transluminal intervention in coronary vessels: a European Registry (FASTER) was designed to demonstrate that (1) the Proxis embolic protection system can control anterograde flow and reverse blood flow in native coronary arteries and saphenous vein grafts; and (2) this system can capture embolic debris. Percutaneous coronary intervention on stenotic coronary lesions revolutionized treatment of coronary disease, but is associated with the risk of major adverse cardiac events. This prospective, nonrandomized, multicenter clinical feasibility and safety study enrolled 40 patients with 51 lesions at 4 centers who underwent treatment of stenotic lesions with proximal emboli protection (Proxis system). Proxis was successfully used 95% of the time, and embolic debris was qualitatively identified in all cases. Major adverse cardiac events occurred in 2 patients (5.0%): 1 late in-stent thrombosis resulting in death and 1 non-Q-wave infarction when a lesion was crossed before deployment of the Proxis system. Mean vessel occlusion time was 4.3 +/- 2.4 minutes. Native flow reversal was sufficient in 31 patients, with a mean aspirate volume of 11.8 +/- 6.5 ml. When the infusion catheter was used to augment reflow, mean occlusion time was 4.6 minutes. In conclusion, this trial is the first to demonstrate that retrograde blood flow can be achieved during proximal occlusion and that the Proxis system can be used safely during intervention of saphenous vein grafts and native coronary arterial lesions to capture embolic material. PMID:15518607

  1. Actinic keratosis

    MedlinePlus

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

  2. Actinic Cheilitis

    MedlinePlus

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

  3. Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones.

    PubMed

    Munnamalai, Vidhya; Weaver, Cory J; Weisheit, Corinne E; Venkatraman, Prahatha; Agim, Zeynep Sena; Quinn, Mark T; Suter, Daniel M

    2014-08-01

    NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones. PMID:24702317

  4. Actinic reticuloid

    SciTech Connect

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

    1982-09-01

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

  5. Emerging Trends in Retrograde Signaling.

    PubMed

    Suvarna, Yashasvi; Maity, Nivedita; Shivamurthy, M C

    2016-05-01

    Retrograde signaling is defined as the signaling events leading from the plastids to the nucleus in plants and across the chemical synapse, from the postsynaptic neuron to the presynaptic neuron in animals. The discovery of various retrograde messengers has opened many avenues and clouds of thoughts as to the role of retrograde signaling. They have been implicated particularly in long-term potentiation (LTP) and synaptic plasticity. But the basic assumptions about retrograde signaling have not been studied upon for many years. This review focuses on established facts and hypothesis put forward in retrograde signaling. PMID:26081150

  6. Quantifying actin wave modulation on periodic topography

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  7. Association between tensin 1 and p130Cas at focal adhesions links actin inward flux to cell migration

    PubMed Central

    Zhao, Zhihai; Tan, Song Hui; Machiyama, Hiroaki; Kawauchi, Keiko; Araki, Keigo; Hirata, Hiroaki; Sawada, Yasuhiro

    2016-01-01

    ABSTRACT Cell migration is a highly dynamic process that plays pivotal roles in both physiological and pathological processes. We have previously reported that p130Cas supports cell migration through the binding to Src as well as phosphorylation-dependent association with actin retrograde flow at focal adhesions. However, it remains elusive how phosphorylated Cas interacts with actin cytoskeletons. We observe that the actin-binding protein, tensin 1, co-localizes with Cas, but not with its phosphorylation-defective mutant, at focal adhesions in leading regions of migrating cells. While a truncation mutant of tensin 1 that lacks the phosphotyrosine-binding PTB and SH2 domains (tensin 1-SH2PTB) poorly co-localizes or co-immunoprecitates with Cas, bacterially expressed recombinant tensin 1-SH2PTB protein binds to Cas in vitro in a Cas phosphorylation-dependent manner. Furthermore, exogenous expression of tensin 1-SH2PTB, which is devoid of the actin-interacting motifs, interferes with the Cas-driven cell migration, slows down the inward flux of Cas molecules, and impedes the displacement of Cas molecules from focal adhesions. Taken together, our results show that tensin 1 links inwardly moving actin cytoskeletons to phosphorylated Cas at focal adhesions, thereby driving cell migration. PMID:27029899

  8. Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones

    PubMed Central

    Munnamalai, Vidhya; Weaver, Cory J.; Weisheit, Corinne E.; Venkatraman, Prahatha; Agim, Zeynep Sena; Quinn, Mark T.; Suter, Daniel M.

    2014-01-01

    NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NOX2-type NADPH oxidase complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91phox localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40phox. p40phox itself exhibited co-localization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91phox and p40phox with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in co-localization of p40phox with NOX2/gp91phox at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. PMID:24702317

  9. Synaptopodin-2 induces assembly of peripheral actin bundles and immature focal adhesions to promote lamellipodia formation and prostate cancer cell migration.

    PubMed

    Kai, FuiBoon; Fawcett, James P; Duncan, Roy

    2015-05-10

    Synaptopodin-2 (Synpo2), an actin-binding protein and invasive cancer biomarker, induces formation of complex stress fiber networks in the cell body and promotes PC3 prostate cancer cell migration in response to serum stimulation. The role of these actin networks in enhanced cancer cell migration is unknown. Using time-course analysis and live cell imaging of mock- and Synpo2-transduced PC3 cells, we now show that Synpo2 induces assembly of actin fibers near the cell periphery and Arp2/3-dependent lamellipodia formation. Lamellipodia formed in a non-directional manner or repeatedly changed direction, explaining the enhanced chemokinetic activity of PC3 cells in response to serum stimulation. Myosin contraction promotes retrograde flow of the Synpo2-associated actin filaments at the leading edge and their merger with actin networks in the cell body. Enhanced PC3 cell migration correlates with Synpo2-induced formation of lamellipodia and immature focal adhesions (FAs), but is not dependent on myosin contraction or FA maturation. The previously reported correlation between Synpo2-induced stress fiber assembly and enhanced PC3 cell migration therefore reflects the role of Synpo2 as a newly identified regulator of actin bundle formation and nascent FA assembly near the leading cell edge. PMID:25883213

  10. Actinic Prurigo.

    PubMed

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

    2015-01-01

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

  11. [Actinic Keratosis].

    PubMed

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

    2015-07-01

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

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

    PubMed

    Yamashiro, Sawako; Mizuno, Hiroaki; Watanabe, Naoki

    2015-01-01

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

  13. Helical buckling of actin inside filopodia generates traction

    PubMed Central

    Leijnse, Natascha; Oddershede, Lene B.; Bendix, Poul M.

    2015-01-01

    Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell–cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle. PMID:25535347

  14. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    PubMed

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

    2002-03-01

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

  16. Transcatheter closure of ruptured sinus Valsalva aneurysm with retrograde approach.

    PubMed

    Narin, Nazmi; Ozyurt, Abdullah; Baykan, Ali; Uzüm, Kazım

    2014-04-01

    A three-year-old girl with multiple heart malformations admitted to the pediatric cardiology unit because of excessive sweating and fatigue. Abnormal color Doppler flow was detected into the right atrium from the dilated coronary sinus on the echocardiographic examination, and ruptured sinus Valsalva aneurysm (SVA) was diagnosed. Although in most such cases, an antegrade transcatheter approach has been used, a retrograde approach can be used as a cost-effective treatment modality in those cases with selective high-risk surgery. In this report, we present a patient with ruptured SVA, which was closed via Amplatzer vascular plug-4 by retrograde approach. PMID:24769826

  17. Retrograde Fluxes of Focal Adhesion Proteins in Response to Cell Migration and Mechanical Signals

    PubMed Central

    Guo, Wei-hui

    2007-01-01

    Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell. PMID:17804814

  18. Actin in Herpesvirus Infection

    PubMed Central

    Roberts, Kari L.; Baines, Joel D.

    2011-01-01

    Actin is important for a variety of cellular processes, including uptake of extracellular material and intracellular transport. Several emerging lines of evidence indicate that herpesviruses exploit actin and actin-associated myosin motors for viral entry, intranuclear transport of capsids, and virion egress. The goal of this review is to explore these processes and to highlight potential future directions for this area of research. PMID:21994736

  19. Actin from Saccharomyces cerevisiae.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

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

  20. Actin Rings of Power.

    PubMed

    Schwayer, Cornelia; Sikora, Mateusz; Slováková, Jana; Kardos, Roland; Heisenberg, Carl-Philipp

    2016-06-20

    Circular or ring-like actin structures play important roles in various developmental and physiological processes. Commonly, these rings are composed of actin filaments and myosin motors (actomyosin) that, upon activation, trigger ring constriction. Actomyosin ring constriction, in turn, has been implicated in key cellular processes ranging from cytokinesis to wound closure. Non-constricting actin ring-like structures also form at cell-cell contacts, where they exert a stabilizing function. Here, we review recent studies on the formation and function of actin ring-like structures in various morphogenetic processes, shedding light on how those different rings have been adapted to fulfill their specific roles. PMID:27326928

  1. Retrograde signaling: Organelles go networking.

    PubMed

    Kleine, Tatjana; Leister, Dario

    2016-08-01

    The term retrograde signaling refers to the fact that chloroplasts and mitochondria utilize specific signaling molecules to convey information on their developmental and physiological states to the nucleus and modulate the expression of nuclear genes accordingly. Signals emanating from plastids have been associated with two main networks: 'Biogenic control' is active during early stages of chloroplast development, while 'operational' control functions in response to environmental fluctuations. Early work focused on the former and its major players, the GUN proteins. However, our view of retrograde signaling has since been extended and revised. Elements of several 'operational' signaling circuits have come to light, including metabolites, signaling cascades in the cytosol and transcription factors. Here, we review recent advances in the identification and characterization of retrograde signaling components. We place particular emphasis on the strategies employed to define signaling components, spanning the entire spectrum of genetic screens, metabolite profiling and bioinformatics. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26997501

  2. Direct dynamin–actin interactions regulate the actin cytoskeleton

    PubMed Central

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

    2010-01-01

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

  3. Actin Mechanics and Fragmentation*

    PubMed Central

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

    2015-01-01

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

  4. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

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

    2016-01-01

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

  5. Actin Automata with Memory

    NASA Astrophysics Data System (ADS)

    Alonso-Sanz, Ramón; Adamatzky, Andy

    Actin is a globular protein which forms long polar filaments in eukaryotic. The actin filaments play the roles of cytoskeleton, motility units, information processing and learning. We model actin filament as a double chain of finite state machines, nodes, which take states “0” and “1”. The states are abstractions of absence and presence of a subthreshold charge on actin units corresponding to the nodes. All nodes update their state in parallel to discrete time. A node updates its current state depending on states of two closest neighbors in the node chain and two closest neighbors in the complementary chain. Previous models of actin automata consider momentary state transitions of nodes. We enrich the actin automata model by assuming that states of nodes depend not only on the current states of neighboring node but also on their past states. Thus, we assess the effect of memory of past states on the dynamics of acting automata. We demonstrate in computational experiments that memory slows down propagation of perturbations, decrease entropy of space-time patterns generated, transforms traveling localizations to stationary oscillators, and stationary oscillations to still patterns.

  6. The Molecular Evolution of Actin

    PubMed Central

    Hightower, Robin C.; Meagher, Richard B.

    1986-01-01

    We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3–7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated <3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11–15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8–10%, whereas these members within the soybean actin gene family ranged from 6–9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence

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

    PubMed Central

    Buttery, Shawnna M.; Yoshida, Satoshi

    2007-01-01

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

  8. Interactions among endoplasmic reticulum, microtubules, and retrograde movements of the cell surface.

    PubMed

    Terasaki, M; Reese, T S

    1994-01-01

    Relationships among the endoplasmic reticulum (ER), microtubules, and bead movements on the cell surface were investigated in the thin peripheral region of A6 cells, a frog kidney cell line. ER tubules were often aligned with microtubules, as shown by double-labeling with DiOC6(3) and anti-tubulin in fixed cells. In living cells stained with DiOC6(3) and observed in time lapse, there were frequent extensions, but few retractions, of ER tubules. In addition, there was a steady retrograde (towards the cell center) movement of all of the ER at approximately 0.3 microns/min. Since microtubules are often aligned with the ER, microtubules must also be moving retrogradely. By simultaneous imaging, it was found that the ER moves retrogradely at the same rate as aminated latex beads on the cell surface. This indicates that the mechanisms for ER and bead movement are closely related. Cytochalasin B stopped bead and ER movement in most of the cells, providing evidence that actin is involved in both retrograde movements. The ER retracted towards the cell center in nocodazole while both ER and microtubules retracted in taxol. Time lapse observations showed that for both drugs, the retraction of the ER is the result of retrograde movement in the absence of new ER extensions. Presumably, ER extensions do not occur in nocodazole because of the absence of microtubules, and do not occur in taxol because taxol-stabilized microtubules move retrogradely and there is no polymerization of new microtubule tracks for ER elongation. PMID:7859292

  9. Photosynthetic light reactions: integral to chloroplast retrograde signalling.

    PubMed

    Gollan, Peter J; Tikkanen, Mikko; Aro, Eva-Mari

    2015-10-01

    Chloroplast retrograde signalling is ultimately dependent on the function of the photosynthetic light reactions and not only guides the acclimation of the photosynthetic apparatus to changing environmental and metabolic cues, but has a much wider influence on the growth and development of plants. New information generated during the past few years about regulation of photosynthetic light reactions and identification of the underlying regulatory proteins has paved the way towards better understanding of the signalling molecules produced in chloroplasts upon changes in the environment. Likewise, the availability of various mutants lacking regulatory functions has made it possible to address the role of excitation energy distribution and electron flow in the thylakoid membrane in inducing the retrograde signals from chloroplasts to the nucleus. Such signalling molecules also induce and interact with hormonal signalling cascades to provide comprehensive information from chloroplasts to the nucleus. PMID:26318477

  10. Intranuclear Actin Regulates Osteogenesis

    PubMed Central

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

    2016-01-01

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

  11. Myosins, Actin and Autophagy.

    PubMed

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

    2016-08-01

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

  12. Ionic wave propagation along actin filaments.

    PubMed

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

    2004-04-01

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

  13. Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles

    PubMed Central

    Bittins, Claudia Margarethe; Eichler, Tilo Wolf; Hammer, John A.; Gerdes, Hans-Hermann

    2013-01-01

    Axonal transport of peptide and hormone-containing large dense core vesicles (LDCVs) is known to be a microtubule-dependent process. Here, we suggest a role for the actin-based motor protein myosin Va specifically in retrograde axonal transport of LDCVs. Using live-cell imaging of transfected hippocampal neurons grown in culture, we measured the speed, transport direction, and the number of LDCVs that were labeled with ectopically expressed neuropeptide Y fused to EGFP. Upon expression of a dominant-negative tail construct of myosin Va, a general reduction of movement in both dendrites and axons was observed. In axons, it was particularly interesting that the retrograde speed of LDCVs was significantly impaired, although anterograde transport remained unchanged. Moreover, particles labeled with the dominant-negative construct often moved in the retrograde direction but rarely in the anterograde direction. We suggest a model where myosin Va acts as an actin-dependent vesicle motor that facilitates retrograde axonal transport. PMID:19787448

  14. Concentration profiles of actin-binding molecules in lamellipodia

    NASA Astrophysics Data System (ADS)

    Falcke, Martin

    2016-04-01

    Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

  15. Myosin Va bound to phagosomes binds to F-actin and delays microtubule-dependent motility.

    PubMed

    Al-Haddad, A; Shonn, M A; Redlich, B; Blocker, A; Burkhardt, J K; Yu, H; Hammer, J A; Weiss, D G; Steffen, W; Griffiths, G; Kuznetsov, S A

    2001-09-01

    We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome-F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an "antagonistic/cooperative mechanism" to explain the saltatory phagosome movement toward the cell center in normal macrophages. PMID:11553713

  16. Linker of nucleoskeleton and cytoskeleton (LINC) complex-mediated actin-dependent nuclear positioning orients centrosomes in migrating myoblasts

    PubMed Central

    Chang, Wakam; Antoku, Susumu; Östlund, Cecilia; Worman, Howard J; Gundersen, Gregg G

    2015-01-01

    Myoblast migration is essential for muscle development and repair; however, the factors that contribute to the polarity of migrating myoblasts are relatively unknown. We find that randomly migrating C2C12 myoblasts orient their centrosomes in the direction of migration. Using wounded monolayers, we further show that centrosome orientation is stimulated by the serum factor lysophosphatidic acid (LPA) and involves the rearward movement of the nucleus while the centrosome is maintained at the cell centroid. The rate of nuclear movement correlated with that of actin retrograde flow and both cytochalasin D and blebbistatin prevented nuclear movement and centrosome orientation. Actin-dependent rearward nuclear movement in fibroblasts is mediated by assembly of nuclear membrane nesprin-2G and SUN2 LINC complexes into transmembrane actin-associated nuclear (TAN) lines anchored by A-type lamins and emerin. In C2C12 myoblasts, depletion of nesprin-2G, SUN2 or lamin A/C prevented nuclear movement and endogenous nesprin-2G and a chimeric GFP-mini-nesprin-2G formed TAN lines during nuclear movement. Depleting nesprin-2G strongly interfered with directed cell migration and reduced the efficiency of myoblast fusion into multinucleated myotubes. Our results show that nuclear movement contributes to centrosome orientation and polarity for efficient migration and fusion of myoblasts. Given that mutations in the genes encoding A-type lamins, nesprin-2 and SUN2 cause Emery-Dreifuss muscular dystrophy and related myopathies, our results have implications for understanding the mechanism of disease pathogenesis. PMID:25587885

  17. Retrogradation enthalpy does not always reflect the retrogradation behavior of gelatinized starch

    PubMed Central

    Wang, Shujun; Li, Caili; Zhang, Xiu; Copeland, Les; Wang, Shuo

    2016-01-01

    Starch retrogradation is a term used to define the process in which gelatinized starch undergoes a disorder-to-order transition. A thorough understanding of starch retrogradation behavior plays an important role in maintaining the quality of starchy foods during storage. By means of DSC, we have demonstrated for the first time that at low water contents, the enthalpy change of retrograded starch is higher than that of native starch. In terms of FTIR and Raman spectroscopic results, we showed that the molecular order of reheated retrograded starch samples is lower than that of DSC gelatinized starch. These findings have led us to conclude that enthalpy change of retrograded starch at low water contents involves the melting of recrystallized starch during storage and residual starch crystallites after DSC gelatinization, and that the endothermic transition of retrograded starch gels at low water contents does not fully represent the retrogradation behavior of starch. Very low or high water contents do not favor the occurrence of starch retrogradation. PMID:26860788

  18. A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes.

    PubMed

    Hiroyasu, Sho; Colburn, Zachary T; Jones, Jonathan C R

    2016-06-01

    During wound healing of the skin, keratinocytes disassemble hemidesmosomes and reorganize their actin cytoskeletons in order to exert traction forces on and move directionally over the dermis. Nonetheless, the transmembrane hemidesmosome component collagen XVII (ColXVII) is found in actin-rich lamella, situated behind the lamellipodium. A set of actin bundles, along which ColXVII colocalizes with actinin4, is present at each lamella. Knockdown of either ColXVII or actinin4 not only inhibits directed migration of keratinocytes but also relieves constraints on actin bundle retrograde movement at the site of lamella, such that actin bundle movement is enhanced more than 5-fold. Moreover, whereas control keratinocytes move in a stepwise fashion over a substrate by generating alternating traction forces, of up to 1.4 kPa, at each flank of the lamellipodium, ColXVII knockdown keratinocytes fail to do so. In summary, our data indicate that ColXVII-actinin4 complexes at the lamella of a moving keratinocyte regulate actin dynamics, thereby determining the direction of cell movement.-Hiroyasu, S., Colburn, Z. T., Jones, J. C. R. A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes. PMID:26936359

  19. Symmetry breaking in reconstituted actin cortices.

    PubMed

    Abu Shah, Enas; Keren, Kinneret

    2014-01-01

    The actin cortex plays a pivotal role in cell division, in generating and maintaining cell polarity and in motility. In all these contexts, the cortical network has to break symmetry to generate polar cytoskeletal dynamics. Despite extensive research, the mechanisms responsible for regulating cortical dynamics in vivo and inducing symmetry breaking are still unclear. Here we introduce a reconstituted system that self-organizes into dynamic actin cortices at the inner interface of water-in-oil emulsions. This artificial system undergoes spontaneous symmetry breaking, driven by myosin-induced cortical actin flows, which appears remarkably similar to the initial polarization of the embryo in many species. Our in vitro model system recapitulates the rich dynamics of actin cortices in vivo, revealing the basic biophysical and biochemical requirements for cortex formation and symmetry breaking. Moreover, this synthetic system paves the way for further exploration of artificial cells towards the realization of minimal model systems that can move and divide.DOI: http://dx.doi.org/10.7554/eLife.01433.001. PMID:24843007

  20. Symmetry breaking in reconstituted actin cortices

    PubMed Central

    Abu Shah, Enas; Keren, Kinneret

    2014-01-01

    The actin cortex plays a pivotal role in cell division, in generating and maintaining cell polarity and in motility. In all these contexts, the cortical network has to break symmetry to generate polar cytoskeletal dynamics. Despite extensive research, the mechanisms responsible for regulating cortical dynamics in vivo and inducing symmetry breaking are still unclear. Here we introduce a reconstituted system that self-organizes into dynamic actin cortices at the inner interface of water-in-oil emulsions. This artificial system undergoes spontaneous symmetry breaking, driven by myosin-induced cortical actin flows, which appears remarkably similar to the initial polarization of the embryo in many species. Our in vitro model system recapitulates the rich dynamics of actin cortices in vivo, revealing the basic biophysical and biochemical requirements for cortex formation and symmetry breaking. Moreover, this synthetic system paves the way for further exploration of artificial cells towards the realization of minimal model systems that can move and divide. DOI: http://dx.doi.org/10.7554/eLife.01433.001 PMID:24843007

  1. Modeling cytoskeletal flow over adhesion sites: competition between stochastic bond dynamics and intracellular relaxation.

    PubMed

    Sabass, Benedikt; Schwarz, Ulrich S

    2010-05-19

    In migrating cells, retrograde flow of the actin cytoskeleton is related to traction at adhesion sites located at the base of the lamellipodium. The coupling between the moving cytoskeleton and the stationary adhesions is mediated by the continuous association and dissociation of molecular bonds. We introduce a simple model for the competition between the stochastic dynamics of elastic bonds at the moving interface and relaxation within the moving actin cytoskeleton represented by an internal viscous friction coefficient. Using exact stochastic simulations and an analytical mean field theory, we show that the stochastic bond dynamics lead to biphasic friction laws as observed experimentally. At low internal dissipation, stochastic bond dynamics lead to a regime of irregular stick-and-slip motion. High internal dissipation effectively suppresses cooperative effects among bonds and hence stabilizes the adhesion. PMID:21386438

  2. Process for forming retrograde profiles in silicon

    DOEpatents

    Weiner, Kurt H.; Sigmon, Thomas W.

    1996-01-01

    A process for forming retrograde and oscillatory profiles in crystalline and polycrystalline silicon. The process consisting of introducing an n- or p-type dopant into the silicon, or using prior doped silicon, then exposing the silicon to multiple pulses of a high-intensity laser or other appropriate energy source that melts the silicon for short time duration. Depending on the number of laser pulses directed at the silicon, retrograde profiles with peak/surface dopant concentrations which vary from 1-1e4 are produced. The laser treatment can be performed in air or in vacuum, with the silicon at room temperature or heated to a selected temperature.

  3. Process for forming retrograde profiles in silicon

    DOEpatents

    Weiner, K.H.; Sigmon, T.W.

    1996-10-15

    A process is disclosed for forming retrograde and oscillatory profiles in crystalline and polycrystalline silicon. The process consisting of introducing an n- or p-type dopant into the silicon, or using prior doped silicon, then exposing the silicon to multiple pulses of a high-intensity laser or other appropriate energy source that melts the silicon for short time duration. Depending on the number of laser pulses directed at the silicon, retrograde profiles with peak/surface dopant concentrations which vary are produced. The laser treatment can be performed in air or in vacuum, with the silicon at room temperature or heated to a selected temperature.

  4. External security stitch for retrograde cardioplegia cannula.

    PubMed

    Gabbieri, Davide; Pedulli, Marco; Bianchi, Tiziano; Ghidoni, Italo

    2009-01-01

    Retrograde cardioplegia catheter displacement represents a troublesome complication, frequently forcing the surgeon to interrupt the operative procedure and cannulate newly the coronary sinus. However, this maneuver is time consuming, often implies the loss of surgical exposure, and exposes again the coronary sinus to the risk of iatrogenic injuries. We describe the use of an external security stitch through the muscular right atrial wall to avoid the displacement of a retrograde cardioplegia catheter and analyze the anatomic conditions which predispose to this complication. PMID:19583611

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

    PubMed

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

    2012-10-01

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

  6. Bidirectional actin transport is influenced by microtubule and actin stability.

    PubMed

    Chetta, Joshua; Love, James M; Bober, Brian G; Shah, Sameer B

    2015-11-01

    Local and long-distance transport of cytoskeletal proteins is vital to neuronal maintenance and growth. Though recent progress has provided insight into the movement of microtubules and neurofilaments, mechanisms underlying the movement of actin remain elusive, in large part due to rapid transitions between its filament states and its diverse cellular localization and function. In this work, we integrated live imaging of rat sensory neurons, image processing, multiple regression analysis, and mathematical modeling to perform the first quantitative, high-resolution investigation of GFP-actin identity and movement in individual axons. Our data revealed that filamentous actin densities arise along the length of the axon and move short but significant distances bidirectionally, with a net anterograde bias. We directly tested the role of actin and microtubules in this movement. We also confirmed a role for actin densities in extension of axonal filopodia, and demonstrated intermittent correlation of actin and mitochondrial movement. Our results support a novel mechanism underlying slow component axonal transport, in which the stability of both microtubule and actin cytoskeletal components influence the mobility of filamentous actin. PMID:26043972

  7. Endoscopic retrograde cholangio pancreatography (ERCP) - slideshow

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Contact Us Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Endoscopic retrograde cholangio pancreatography (ERCP) - series URL of this ...

  8. Retrograde Lymphatic Spread of Esophageal Cancer

    PubMed Central

    Oshiro, Hisashi; Osaka, Yoshiaki; Tachibana, Shingo; Aoki, Takaya; Tsuchiya, Takayoshi; Nagao, Toshitaka

    2015-01-01

    Abstract The concept of the retrograde lymphatic spread of cancer cells appears to account for a subset of the essential mechanisms of cancer metastasis in various organs. However, no adequate data currently exist to illustrate the pathology of the retrograde lymphatic metastasis of cancer cells in human bodies. To shed light on this phenomenon, we report a case of a 63-year-old Japanese man who underwent an esophagectomy and lymph node dissection for early-stage esophageal cancer. The patient's clinical information was evaluated by board-certified surgeons and internists. Surgically excised materials were histopathologically evaluated by attending pathologists. Postoperative pathological examination revealed that the patient's tumor was a well-differentiated squamous cell carcinoma with negative surgical margins (T1N0M0, stage I). Apart from the primary lesion, a single lymphatic vessel invasion was found between the lamina propria and lamina muscularis of the esophagus where intralymphatic cancer cells had spread against the direction of backflow prevention valves and skipped beyond these valves without destroying them. The present case demonstrated that the retrograde lymphatic spread of cancer cells can occur in valve-equipped lymphatic vessels. Our study may not only provide a scientific basis for the concept of retrograde lymphatic metastasis but also explain a portion of the complexities associated with the lymphogenous metastasis of esophageal cancer. PMID:26166121

  9. Actin-based phagosome motility.

    PubMed

    Zhang, Fangliang; Southwick, Frederick S; Purich, Daniel L

    2002-10-01

    Despite abundant evidence of actin's involvement at the particle internalization stage of phagocytosis, little is known about whether phagosomes undergo the same type of actin-based motility as observed with endocytic vesicles or such intracellular pathogens as Listeria and Shigella. By employing video microscopy to follow the fate of latex bead-containing phagosomes within the cytoplasm of bone marrow macrophages, we have made the novel observation of actin-based phagosome motility. Immunofluorescence microscopy confirmed that phagosomes containing IgG-opsonized, bovine serum albumin (or BSA) -coated or uncoated latex beads all formed actin-rich rocket tails that persisted only during a brief, 1-2 min period of actin-based motility. Average speeds of actin-based phagosome motility were 0.13 +/- 0.06 microm/s for IgG-coated beads, 0.14 +/- 0.04 microm/s for BSA-coated beads, and 0.11+/- 0.03 microm/s for uncoated beads. Moreover, the speeds and motile-phase duration of each type of phagosome were comparable to the behavior of pinosomes [Merrifield et al., 1999: Nat. Cell Biol. 1:72-74.]. Determination of optimal conditions for observing and analyzing actin-based phagosome motility should facilitate future investigations of phagocytosis and phagosome maturation. PMID:12211106

  10. Effect of vinpocetine on retrograde axoplasmic transport.

    PubMed

    Knyihar-Csillik, Elizabeth; Vecsei, Laszlo; Mihaly, Andras; Fenyo, Robert; Farkas, Ibolya; Krisztin-Peva, Beata; Csillik, Bertalan

    2007-01-01

    Vinpocetine, a derivate of vincamine, is widely used in the clinical pharmacotherapy of cerebral circulatory diseases. Herewith we report on a novel effect of vinpocetine: inhibition of retrograde axoplasmic transport of nerve growth factor (NGF) in the peripheral nerve. Blockade of retrograde transport of NGF results in transganglionic degenerative atrophy (TDA) in the segmentally related ipsilateral superficial spinal dorsal horn, which is characterized by depletion of the marker enzymes fluoride-resistant acid phosphatase (FRAP) and thiamine monophosphatase (TMP). At the same time, pain-related neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), are depleted from lamina I-III from the segmentally related, ipsitateral Rolando substance of the spinal cord. On the basis of these experiments it is suggested that vinpocetine may result in a locally restricted decrease of nociception, that might be useful in clinical treatment of intractable pain. Pilot self-experiments support this assumption. PMID:17319607

  11. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner. PMID:24637338

  12. Bacterial actins and their diversity

    PubMed Central

    Ozyamak, Ertan; Kollman, Justin M.; Komeili, Arash

    2015-01-01

    For many years bacteria were considered rather simple organisms, but the dogmatic notion that subcellular organization is a eukaryotic trait has been overthrown for more than a decade. The discovery of homologs of the eukaryotic cytoskeletal proteins actin, tubulin, and intermediate filaments in bacteria has been instrumental in changing this view. Over the recent years we gained an incredible level of insight into the diverse family of bacterial actins and their molecular workings. Here we review the functional, biochemical and structural features of the most well-studied bacterial actins. PMID:24015924

  13. Are Wnts Retrogradely Transported to the ER?

    PubMed

    Tang, Bor Luen

    2016-11-01

    A recent report showed that Drosophila miR-307a initiates endoplasmic reticulum (ER) stress in wingless (wg)-expressing cells by suppression of the evolutionarily conserve Wnt secretion factor Wntless (Wls). Interestingly, the authors noted that wg has a putative C-terminal dilysine motif (KKVY), which is required for its apparent retrograde Golgi-to-ER transport. Wls suppression resulted in ER stress, which was phenocopied by several other manipulations that impaired wg secretion in flies, as well as Wnt5a secretion in mammalian cells. The authors surmised that their data "reveals a previously unknown Golgi-to-ER retrograde route of wg, and elucidates a correlation between Wnt secretion and ER stress." However, there are obvious caveats to this interpretation, as ER stress resulting from Wnt secretion impairment could be readily explained by its inability to leave the ER, and not resulting from Golgi-to-ER retrograde transport. J. Cell. Physiol. 231: 2315-2316, 2016. © 2016 Wiley Periodicals, Inc. PMID:26916992

  14. Manipulation of arterial stiffness, wave reflections, and retrograde shear rate in the femoral artery using lower limb external compression

    PubMed Central

    Heffernan, Kevin S; Lefferts, Wesley K; Kasprowicz, Ari G; Tarzia, Brendan J; Thijssen, Dick H; Brutsaert, Tom D

    2013-01-01

    Exposure of the arterial wall to retrograde shear acutely leads to endothelial dysfunction and chronically contributes to a proatherogenic vascular phenotype. Arterial stiffness and increased pressure from wave reflections are known arbiters of blood flow in the systemic circulation and each related to atherosclerosis. Using distal external compression of the calf to increase upstream retrograde shear in the superficial femoral artery (SFA), we examined the hypothesis that changes in retrograde shear are correlated with changes in SFA stiffness and pressure from wave reflections. For this purpose, a pneumatic cuff was applied to the calf and inflated to 0, 35, and 70 mmHg (5 min compression, randomized order, separated by 5 min) in 16 healthy young men (23 ± 1 years of age). Doppler ultrasound and wave intensity analysis was used to measure SFA retrograde shear rate, reflected pressure wave intensity (negative area [NA]), elastic modulus (Ep), and a single-point pulse wave velocity (PWV) during acute cuff inflation. Cuff inflation resulted in stepwise increases in retrograde shear rate (P < 0.05 for main effect). There were also significant cuff pressure-dependent increases in NA, Ep, and PWV across conditions (P < 0.05 for main effects). Change in NA, but not Ep or PWV, was associated with change in retrograde shear rate across conditions (P < 0.05). In conclusion, external compression of the calf increases retrograde shear, arterial stiffness, and pressure from wave reflection in the upstream SFA in a dose-dependent manner. Wave reflection intensity, but not arterial stiffness, is correlated with changes in peripheral retrograde shear with this hemodynamic manipulation. PMID:24303111

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

    PubMed Central

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

    2015-01-01

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

  16. Epidemiology of actinic keratoses.

    PubMed

    Green, Adèle C

    2015-01-01

    The epidemiology of actinic keratoses (AKs) reflects their causation by cumulative sun exposure, with the highest prevalence seen in pale-skinned people living at low latitudes and on the most sun-exposed body sites, namely the hands, forearms and face. AKs are markers of increased risk of basal cell carcinoma, squamous cell carcinoma and melanoma, especially when they are numerous and have coalesced into an area of 'field cancerisation'. The major risk factors are male sex, advanced age, sun-sensitive complexion, high lifetime sun exposure and prolonged immunosuppression. Clinical counts of AKs enable the assessment and monitoring of AK burden, but accurate counting is notoriously difficult, especially when skin is severely sun damaged. AK counting has been repeatedly shown to be unreliable, even among expert dermatologists. Notwithstanding these challenges, qualitative assessment of the natural history of AKs shows a high turnover, with new lesions developing and with other lesions regressing. A very small proportion of AKs undergo malignant transformation, but the precise rate of transformation is unknown due to the inaccuracies in monitoring AK lesions over time. Primary prevention of AKs is achieved by limiting intense sun exposure through sun-protective behaviour, including seeking deep shade, wearing sun-protective clothing and applying sunscreen regularly to exposed skin, from an early age. PMID:25561199

  17. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

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

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

  18. A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

    PubMed Central

    Bai, Zhiyong; Grant, Barth D.

    2015-01-01

    Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling. PMID:25775511

  19. Mitochondrial emitted electromagnetic signals mediate retrograde signaling.

    PubMed

    Bagkos, Georgios; Koufopoulos, Kostas; Piperi, Christina

    2015-12-01

    Recent evidence shows that mitochondria regulate nuclear transcriptional activity both in normal and cell stress conditions, known as retrograde signaling. Under normal mitochondrial function, retrograde signaling is associated with mitochondrial biogenesis, normal cell phenotype and metabolic profile. In contrast, mitochondrial dysfunction leads to abnormal (oncogenic) cell phenotype and altered bio-energetic profile (nucleus reprogramming). Despite intense research efforts, a concrete mechanism through which mitochondria determine the group of genes expressed by the nucleus is still missing. The present paper proposes a novel hypothesis regarding retrograde signaling. More specifically, it reveals the mitochondrial membrane potential (MMP) and the accompanied strong electromagnetic field (EF) as key regulatory factors of nuclear activity. Mitochondrial emitted EFs extend in long distance and affect the function of nuclear membrane receptors. Depending on their frequencies, EFs can directly activate or deactivate different groups of nuclear receptors and so determine nuclear gene expression. One of the key features of the above hypothesis is that nuclear membrane receptors, besides their own endogenous or chemical ligands (hormones, lipids, etc.), can also be activated by electromagnetic signals. Moreover, normal MMP values (about -140 mV) are associated with the production of high ATP quantities and small levels of reactive oxygen species (ROS) while the hyperpolarization observed in all cancer cell types leads to a dramatic fall in ATP production and an analogous increase in ROS. The diminished ATP and increased ROS production negatively affect the function of all cellular systems including nucleus. Restoration of mitochondrial function, which is characterized by the fluctuation of MMP and EF values within a certain (normal) range, is proposed as a necessary condition for normal nuclear function and cancer therapy. PMID:26474928

  20. Mitochondrial Retrograde Signaling: Triggers, Pathways, and Outcomes

    PubMed Central

    da Cunha, Fernanda Marques; Torelli, Nicole Quesada; Kowaltowski, Alicia J.

    2015-01-01

    Mitochondria are essential organelles for eukaryotic homeostasis. Although these organelles possess their own DNA, the vast majority (>99%) of mitochondrial proteins are encoded in the nucleus. This situation makes systems that allow the communication between mitochondria and the nucleus a requirement not only to coordinate mitochondrial protein synthesis during biogenesis but also to communicate eventual mitochondrial malfunctions, triggering compensatory responses in the nucleus. Mitochondria-to-nucleus retrograde signaling has been described in various organisms, albeit with differences in effector pathways, molecules, and outcomes, as discussed in this review. PMID:26583058

  1. Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell.

    PubMed

    Guevorkian, Karine; Manzi, John; Pontani, Léa-Lætitia; Brochard-Wyart, Françoise; Sykes, Cécile

    2015-12-15

    Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics. PMID:26682806

  2. Retrograde intrarenal surgery in pediatric patients.

    PubMed

    Resorlu, Berkan; Sancak, Eyup Burak; Resorlu, Mustafa; Gulpinar, Murat Tolga; Adam, Gurhan; Akbas, Alpaslan; Ozdemir, Huseyin

    2014-11-01

    Urinary tract stone disease is seen at a level of 1%-2% in childhood (< 18 years). In recent years, however, there has been a marked increased in pediatric stone disease, particularly in adolescence. A carbohydrate- and salt-heavy diet and a more sedentary lifestyle are implicated in this increase. Although stone disease is rare in childhood, its presence is frequently associated with metabolic or anatomical disorders or infectious conditions, for which reason there is a high possibility of post-therapeutic recurrence. Factors such as a high possibility of recurrence and increasing incidence further enhance the importance of minimally invasive therapeutic options in children, with their expectations of a long life. In children in whom active stone removal is decided on, the way to achieve the highest level of success with the least morbidity is to select the most appropriate treatment modality. Thanks to today's advanced technology, renal stones that were once treated only by surgery can now be treated with minimally invasive techniques, from invasion of the urinary system in an antegrade (percutaneous nephrolithotomy) or retrograde (retrograde intrarenal surgery) manner or shock wave lithotripsy to laparoscopic stone surgery. This compilation study examined studies involving the RIRS procedure, the latest minimally invasive technique, in children and compared the results of those studies with those from other techniques. PMID:25374812

  3. Terminal retrograde turn of rolling rings

    NASA Astrophysics Data System (ADS)

    Jalali, Mir Abbas; Sarebangholi, Milad S.; Alam, Mohammad-Reza

    2015-09-01

    We report an unexpected reverse spiral turn in the final stage of the motion of rolling rings. It is well known that spinning disks rotate in the same direction of their initial spin until they stop. While a spinning ring starts its motion with a kinematics similar to disks, i.e., moving along a cycloidal path prograde with the direction of its rigid body rotation, the mean trajectory of its center of mass later develops an inflection point so that the ring makes a spiral turn and revolves in a retrograde direction around a new center. Using high speed imaging and numerical simulations of models featuring a rolling rigid body, we show that the hollow geometry of a ring tunes the rotational air drag resistance so that the frictional force at the contact point with the ground changes its direction at the inflection point and puts the ring on a retrograde spiral trajectory. Our findings have potential applications in designing topologically new surface-effect flying objects capable of performing complex reorientation and translational maneuvers.

  4. Terminal retrograde turn of rolling rings.

    PubMed

    Jalali, Mir Abbas; Sarebangholi, Milad S; Alam, Mohammad-Reza

    2015-09-01

    We report an unexpected reverse spiral turn in the final stage of the motion of rolling rings. It is well known that spinning disks rotate in the same direction of their initial spin until they stop. While a spinning ring starts its motion with a kinematics similar to disks, i.e., moving along a cycloidal path prograde with the direction of its rigid body rotation, the mean trajectory of its center of mass later develops an inflection point so that the ring makes a spiral turn and revolves in a retrograde direction around a new center. Using high speed imaging and numerical simulations of models featuring a rolling rigid body, we show that the hollow geometry of a ring tunes the rotational air drag resistance so that the frictional force at the contact point with the ground changes its direction at the inflection point and puts the ring on a retrograde spiral trajectory. Our findings have potential applications in designing topologically new surface-effect flying objects capable of performing complex reorientation and translational maneuvers. PMID:26465546

  5. Subtrochanteric fractures after retrograde femoral nailing.

    PubMed

    Mounasamy, Varatharaj; Mallu, Sathya; Khanna, Vishesh; Sambandam, Senthil

    2015-10-18

    Secondary fractures around femoral nails placed for the management of hip fractures are well known. We report, two cases of a fracture of the femur at the interlocking screw site in the subtrochanteric area after retrograde femoral nailing of a femoral shaft fracture. Only a few reports in the existing literature have described these fractures. Two young men after sustaining a fall presented to us with pain, swelling and deformity in the upper thigh region. On enquiring, examining and radiographing them, peri-implant fractures of subtrochanteric nature through the distal interlocking screws were revealed in both patients who also had histories of previous falls for which retrograde intramedullary nailing was performed for their respective femora. Both patients were managed with similar surgical routines including removal of the existing hardware, open reduction and ace cephallomedullary antegrade nailing. The second case did show evidence of delayed healing and was additionally stabilized with cerclage wires. Both patients had uneventful postoperative outcomes and union was evident at the end of 6 mo postoperatively with a good range of motion at the hip and knee. Our report suggests that though seldom reported, peri-implant fractures around the subtrochanteric region can occur and pose a challenge to the treating orthopaedic surgeon. We suggest these be managed, after initial stabilization and resuscitation, by implant removal, open reduction and interlocking intramedullary antegrade nailing. Good results and progression to union can be expected in these patients by adhering to basic principles of osteosynthesis. PMID:26495251

  6. Dual Balloon-Occluded Retrograde Transvenous Obliteration of Gastric Varix Draining into the Left Adrenal Vein and Left Inferior Phrenic Vein

    SciTech Connect

    Nishida, Norifumi Ninoi, Teruhisa; Kitayama, Toshiaki; Yamamoto, Akira; Sakai, Yukimasa; Sato, Kimihiko; Hamuro, Masao; Nakamura, Kenji; Inoue, Yuichi; Yamada, Ryusaku

    2004-09-15

    A 66-year-old woman with a gastric varix, draining into a dilated left adrenal vein and a left inferior phrenic vein, was treated with dual balloon-occluded retrograde transvenous obliteration (B-RTO). Under balloon occlusion of the left adrenal vein and the left inferior phrenic vein, retrograde injection of a sclerosant (5% ethanolamine oleate) into the gastric varix was performed. Two weeks later, disappearance of flow in the gastric varix was confirmed on endoscopic ultrasound examination.

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  8. Bacterial Actins? An Evolutionary Perspective

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

    According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

  9. Actin engine in immunological synapse.

    PubMed

    Piragyte, Indre; Jun, Chang-Duk

    2012-06-01

    T cell activation and function require physical contact with antigen presenting cells at a specialized junctional structure known as the immunological synapse. Once formed, the immunological synapse leads to sustained T cell receptor-mediated signalling and stabilized adhesion. High resolution microscopy indeed had a great impact in understanding the function and dynamic structure of immunological synapse. Trends of recent research are now moving towards understanding the mechanical part of immune system, expanding our knowledge in mechanosensitivity, force generation, and biophysics of cell-cell interaction. Actin cytoskeleton plays inevitable role in adaptive immune system, allowing it to bear dynamic and precise characteristics at the same time. The regulation of mechanical engine seems very complicated and overlapping, but it enables cells to be very sensitive to external signals such as surface rigidity. In this review, we focus on actin regulators and how immune cells regulate dynamic actin rearrangement process to drive the formation of immunological synapse. PMID:22916042

  10. Formation of actin networks in microfluidic concentration gradients

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  11. Redistribution of actin during assembly and reassembly of the contractile ring in grasshopper spermatocytes.

    PubMed

    Alsop, G Bradley; Chen, Wei; Foss, Margit; Tseng, Kuo-Fu; Zhang, Dahong

    2009-01-01

    Cytokinesis in animal cells requires the assembly of an actomyosin contractile ring to cleave the cell. The ring is highly dynamic; it assembles and disassembles during each cell cleavage, resulting in the recurrent redistribution of actin. To investigate this process in grasshopper spermatocytes, we mechanically manipulated the spindle to induce actin redistribution into ectopic contractile rings, around reassembled lateral spindles. To enhance visualization of actin, we folded the spindle at its equator to convert the remnants of the partially assembled ring into a concentrated source of actin. Filaments from the disintegrating ring aligned along reorganizing spindle microtubules, suggesting that their incorporation into the new ring was mediated by microtubules. We tracked incorporation by speckling actin filaments with Qdots and/or labeling them with Alexa 488-phalloidin. The pattern of movement implied that actin was transported along spindle microtubules, before entering the ring. By double-labeling dividing cells, we imaged actin filaments moving along microtubules near the contractile ring. Together, our findings indicate that in one mechanism of actin redistribution, actin filaments are transported along spindle microtubule tracks in a plus-end-directed fashion. After reaching the spindle midzone, the filaments could be transported laterally to the ring. Notably, actin filaments undergo a dramatic trajectory change as they enter the ring, implying the existence of a pulling force. Two other mechanisms of actin redistribution, cortical flow and de novo assembly, are also present in grasshopper, suggesting that actin converges at the nascent contractile ring from diffuse sources within the cytoplasm and cortex, mediated by spindle microtubules. PMID:19287500

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

    PubMed

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

    2016-02-15

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

  13. Phytochrome and retrograde signalling pathways coverage to antogonistically regulate a light-induced transcription network

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plastid-to-nucleus retrograde signals emitted by dysfunctional chloroplasts impact photomorphogenic development, but the molecular link between retrograde and photosensory-receptor signaling has remained undefined. Here, we show that the phytochrome (phy) and retrograde signaling pathways converge a...

  14. Using Kinesthetic Activities to Teach Ptolemaic and Copernican Retrograde Motion

    ERIC Educational Resources Information Center

    Richards, Ted

    2012-01-01

    This paper describes a method for teaching planetary retrograde motion, and the Ptolemaic and Copernican accounts of retrograde motion, by means of a series kinesthetic learning activities (KLAs). In the KLAs described, the students literally walk through the motions of the planets in both systems. A retrospective statistical analysis shows that…

  15. Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton.

    PubMed

    Schiffhauer, Eric S; Luo, Tianzhi; Mohan, Krithika; Srivastava, Vasudha; Qian, Xuyu; Griffis, Eric R; Iglesias, Pablo A; Robinson, Douglas N

    2016-06-01

    To change shape, divide, form junctions, and migrate, cells reorganize their cytoskeletons in response to changing mechanical environments [1-4]. Actin cytoskeletal elements, including myosin II motors and actin crosslinkers, structurally remodel and activate signaling pathways in response to imposed stresses [5-9]. Recent studies demonstrate the importance of force-dependent structural rearrangement of α-catenin in adherens junctions [10] and vinculin's molecular clutch mechanism in focal adhesions [11]. However, the complete landscape of cytoskeletal mechanoresponsive proteins and the mechanisms by which these elements sense and respond to force remain to be elucidated. To find mechanosensitive elements in mammalian cells, we examined protein relocalization in response to controlled external stresses applied to individual cells. Here, we show that non-muscle myosin II, α-actinin, and filamin accumulate to mechanically stressed regions in cells from diverse lineages. Using reaction-diffusion models for force-sensitive binding, we successfully predicted which mammalian α-actinin and filamin paralogs would be mechanoaccumulative. Furthermore, a "Goldilocks zone" must exist for each protein where the actin-binding affinity must be optimal for accumulation. In addition, we leveraged genetic mutants to gain a molecular understanding of the mechanisms of α-actinin and filamin catch-bonding behavior. Two distinct modes of mechanoaccumulation can be observed: a fast, diffusion-based accumulation and a slower, myosin II-dependent cortical flow phase that acts on proteins with specific binding lifetimes. Finally, we uncovered cell-type- and cell-cycle-stage-specific control of the mechanosensation of myosin IIB, but not myosin IIA or IIC. Overall, these mechanoaccumulative mechanisms drive the cell's response to physical perturbation during proper tissue development and disease. PMID:27185555

  16. Association of actin with alpha crystallins

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Boyle, D.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The alpha crystallins are cytosolic proteins that co-localize and co-purify with actin-containing microfilaments. Affinity column chromatography employing both covalently-coupled actin or alpha crystallin was used to demonstrate specific and saturable binding of actin with alpha crystallin. This conclusion was confirmed by direct visualization of alpha aggregates bound to actin polymerized in vitro. The significance of this interaction in relation to the functional properties of these two polypeptides will be discussed.

  17. Distant retrograde orbits for the Moon's exploration

    NASA Astrophysics Data System (ADS)

    Sidorenko, Vladislav

    We discuss the properties of the distant retrograde orbits (which are called quasi-satellite orbits also) around Moon. For the first time the distant retrograde orbits were described by J.Jackson in studies on restricted three body problem at the beginning of 20th century [1]. In the synodic (rotating) reference frame distant retrograde orbit looks like an ellipse whose center is slowly drifting in the vicinity of minor primary body while in the inertial reference frame the third body is orbiting the major primary body. Although being away the Hill sphere the third body permanently stays close enough to the minor primary. Due to this reason the distant retrograde orbits are called “quasi-satellite” orbits (QS-orbits) too. Several asteroids in solar system are in a QS-orbit with respect to one of the planet. As an example we can mention the asteroid 2002VE68 which circumnavigates Venus [2]. Attention of specialists in space flight mechanics was attracted to QS-orbits after the publications of NASA technical reports devoted to periodic moon orbits [3,4]. Moving in QS-orbit the SC remains permanently (or at least for long enough time) in the vicinity of small celestial body even in the case when the Hill sphere lies beneath the surface of the body. The properties of the QS-orbit can be studied using the averaging of the motion equations [5,6,7]. From the theoretical point of view it is a specific case of 1:1 mean motion resonance. The integrals of the averaged equations become the parameters defining the secular evolution of the QS-orbit. If the trajectory is robust enough to small perturbations in the simplified problem (i.e., restricted three body problem) it may correspond to long-term stability of the real-world orbit. Our investigations demonstrate that under the proper choice of the initial conditions the QS-orbits don’t escape from Moon or don’t impact Moon for long enough time. These orbits can be recommended as a convenient technique for the large

  18. Asteroids in Retrograde Orbits: Interesting Cases

    NASA Astrophysics Data System (ADS)

    Kankiewicz, Paweł; Włodarczyk, Ireneusz

    2014-12-01

    We present the most interesting examples of the orbital evolution of asteroids in retrograde orbits (i > 90°). First, we used the latest observational data to determine nominal and averaged orbital elements of these objects. Next, the equations of motion of these asteroids were integrated backward 1 My, taking into account the propagation of observational errors. We used so-called 'cloning' procedure to reproduce the reliability of initial data. We obtained some possible scenarios of the orbit inversion in the past, what is often caused by the long-term influence of outer planets. For two most interesting cases (Apollo and Amor type) we did additional calculations: 100 My in the future. Additionally, we investigated the potential influence of Yarkovski/YORP effects on the long-time orbital evolution.

  19. Retrograde Epidural Catheter Relieves Intractable Sacral Pain.

    PubMed

    Gupta, Ruchir; Shodhan, Shivam; Hosny, Amr

    2016-01-01

    Pain caused by tumor infiltration of the sacral area remains a major clinical challenge. Patients with poor pain control despite comprehensive medical management may be treated with neuraxial techniques such as continuous epidural or spinal anesthetic. We report a case in which a patient with metastatic breast cancer experienced inadequate pain relief after multiple intravenous pain management regimens as well as intrathecal (IT) drug delivery. The concentration of local anesthetics delivered via the IT catheter was limited due to the patient's baseline motor weakness which would be exacerbated with higher concentrations of local anesthetics. Thus, a decision was made to insert an epidural catheter via a retrograde technique to provide the patient with a "band of anesthesia" which would provide profound sensory blockade without concomitant motor weakness. Pain refractory to other modalities of pain control was successfully treated with the epidural technique. PMID:27162431

  20. Retrograde Epidural Catheter Relieves Intractable Sacral Pain

    PubMed Central

    Gupta, Ruchir; Shodhan, Shivam; Hosny, Amr

    2016-01-01

    Pain caused by tumor infiltration of the sacral area remains a major clinical challenge. Patients with poor pain control despite comprehensive medical management may be treated with neuraxial techniques such as continuous epidural or spinal anesthetic. We report a case in which a patient with metastatic breast cancer experienced inadequate pain relief after multiple intravenous pain management regimens as well as intrathecal (IT) drug delivery. The concentration of local anesthetics delivered via the IT catheter was limited due to the patient's baseline motor weakness which would be exacerbated with higher concentrations of local anesthetics. Thus, a decision was made to insert an epidural catheter via a retrograde technique to provide the patient with a “band of anesthesia” which would provide profound sensory blockade without concomitant motor weakness. Pain refractory to other modalities of pain control was successfully treated with the epidural technique. PMID:27162431

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2013-10-01

    A number of studies in the last decade have irrevocably promoted actin into a fully fledged member of the nuclear compartment, where it, among other crucial tasks, facilitates transcription and chromatin remodeling. Changes in nuclear actin levels have been linked to different cellular processes: decreased nuclear actin to quiescence and increased nuclear actin to differentiation. Importin 9 and exportin 6 transport factors are responsible for the continuous nucleocytoplasmic shuttling of actin, but the mechanisms, which result in modulated actin levels, have not been characterized. We find that in cells growing under normal growth conditions, the levels of nuclear actin vary considerably from cell to cell. To understand the basis for this, we have extensively quantified several cellular parameters while at the same time recording the import and export rates of green fluorescent protein (GFP)-tagged actin. Surprisingly, our dataset shows that the ratio of nuclear to cytoplasmic fluorescence intensity, but not nuclear shape, size, cytoplasm size, or their ratio, correlates negatively with both import and export rate of actin. This suggests that high-nuclear actin content is maintained by both diminished import and export. The high nuclear actin containing cells still show high mobility of actin, but it is not export competent, suggesting increased binding of actin to nuclear complexes. Creation of such export incompetent actin pool would ensure enough actin is retained in the nucleus and make it available for the various nuclear functions described for actin. PMID:23749625

  3. Actin dynamics: from nanoscale to microscale.

    PubMed

    Carlsson, Anders E

    2010-01-01

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

  4. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

    PubMed Central

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2016-01-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis. PMID:25664724

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

    PubMed Central

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

    2011-01-01

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

  6. Biomechanical performance of retrograde nail for supracondylar fractures stabilization.

    PubMed

    Chantarapanich, Nattapon; Sitthiseripratip, Kriskrai; Mahaisavariya, Banchong; Siribodhi, Pongwit

    2016-06-01

    The study compared the biomechanical performance of retrograde nail used to stabilize supracondylar fracture (three different levels) by means of finite element analysis. Three different nail lengths (200, 260, and 300 mm) of stainless steel and titanium nails were under consideration. Intact femur model was reconstructed from Digital Imaging and Communications in Medicine images of Thai cadaveric femur scanned by computed tomography spiral scanner, whereas geometry of retrograde nail was reconstructed with the data obtained from three-dimensional laser scanner. The retrograde nail was virtually attached to the femur before nodes and elements were generated for finite element model. The finite element models were analyzed in two stages, the early stage of fracture healing and the stage after fracture healing. The finding indicated that purchasing proximal locking screw in the bowing region of the femur may be at risk due to the high stresses at the implant and bone. There were no differences in stress level, elastic strain at a fracture gap, and bone stress between stainless steel and titanium implant. Since the intramedullary canal requires reaming to accommodate the retrograde nail, the length of retrograde nail should be as long as necessary. However, in case that the retrograde nail can be accommodated into the intramedullary canal without reaming, the longer retrograde nail can be used. PMID:27032932

  7. PRODUCTION OF NEAR-EARTH ASTEROIDS ON RETROGRADE ORBITS

    SciTech Connect

    Greenstreet, S.; Gladman, B.; Ngo, H.; Granvik, M.; Larson, S.

    2012-04-20

    While computing an improved near-Earth object (NEO) steady-state orbital distribution model, we discovered in the numerical integrations the unexpected production of retrograde orbits for asteroids that had originally exited from the accepted main-belt source regions. Our model indicates that {approx}0.1% (a factor of two uncertainty) of the steady-state NEO population (perihelion q < 1.3 AU) is on retrograde orbits. These rare outcomes typically happen when asteroid orbits flip to a retrograde configuration while in the 3:1 mean-motion resonance with Jupiter and then live for {approx}0.001 to 100 Myr. The model predicts, given the estimated near-Earth asteroid (NEA) population, that a few retrograde 0.1-1 km NEAs should exist. Currently, there are two known MPC NEOs with asteroidal designations on retrograde orbits which we therefore claim could be escaped asteroids instead of devolatilized comets. This retrograde NEA population may also answer a long-standing question in the meteoritical literature regarding the origin of high-strength, high-velocity meteoroids on retrograde orbits.

  8. 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.

  9. Using Kinesthetic Activities to Teach Ptolemaic and Copernican Retrograde Motion

    NASA Astrophysics Data System (ADS)

    Richards, Ted

    2012-06-01

    This paper describes a method for teaching planetary retrograde motion, and the Ptolemaic and Copernican accounts of retrograde motion, by means of a series kinesthetic learning activities (KLAs). In the KLAs described, the students literally walk through the motions of the planets in both systems. A retrospective statistical analysis shows that students who participated in these activities performed better on examination questions pertaining to retrograde motion than students who did not. Potential explanations for this result, including the breaking of classroom routine, the effect of body movement on conceptual memory, and egocentric spatial proprioception, are considered.

  10. Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion.

    PubMed

    Bell, Robert M; Mocanu, Mihaela M; Yellon, Derek M

    2011-06-01

    In the late 19th century, a number of investigators were working on perfecting isolated heart model, but it was Oscar Langendorff who, in 1895, pioneered the isolated perfused mammalian heart. Since that time, the Langendorff preparation has evolved and provided a wealth of data underpinning our understanding of the fundamental physiology of the heart: its contractile function, coronary blood flow regulation and cardiac metabolism. In more recent times, the procedure has been used to probe pathophysiology of ischaemia/reperfusion and disease states, and with the dawn of molecular biology and genetic manipulation, the Langendorff perfused heart has remained a stalwart tool in the study of the impact upon the physiology of the heart by pharmacological inhibitors and targeted deletion or up-regulation of genes and their impact upon intracellular signalling and adaption to clinically relevant stressful stimuli. We present here the basic structure of the Langendorff system and the fundamental experimental rules which warrant a viable heart preparation. In addition, we discuss the use of the isolated retrograde perfused heart in the model of ischaemia-reperfusion injury ex-vivo, and its applicability to other areas of study. The Langendorff perfusion apparatus is highly adaptable and this is reflected not only in the procedure's longevity but also in the number of different applications to which it has been turned. PMID:21385587

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

    PubMed Central

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

    2012-01-01

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

  12. Retrograde crossing for chronic total occlusion lesions: the Japanese way.

    PubMed

    Takano, Masamichi; Mizuno, Kyoichi

    2008-01-01

    Percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) lesions remains one of the major challenges in the field of interventional cardiology. Crossing guidewires through the CTO lesions has been conventionally performed from the proximal arteries to the lesions as an antegrade approach. To date, a retrograde approach, to penetrate PCI devices including guidewires and balloons into the distal end of CTO lesions via collateral vessels or coronary artery bypass grafts, has been attempted in order to achieve procedural success. With introduction of the retrograde approach for treatments of CTO lesions, several kinds of devices, techniques, and strategies have been developed. Although the techniques and strategies for the retrograde approach have not been worldwide accepted to interventional cardiologists, we introduce a way to obtain recanalization of the CTO lesions using the retrograde approach in this article. PMID:19276488

  13. The 'SAFARI' Technique Using Retrograde Access Via Peroneal Artery Access

    SciTech Connect

    Zhuang, Kun Da; Tan, Seck Guan; Tay, Kiang Hiong

    2012-08-15

    The 'SAFARI' technique or subintimal arterial flossing with antegrade-retrograde intervention is a method for recanalisation of chronic total occlusions (CTOs) when subintimal angioplasty fails. Retrograde access is usually obtained via the popliteal, distal anterior tibial artery (ATA)/dorsalis pedis (DP), or distal posterior tibial artery (PTA). Distal access via the peroneal artery has not been described and has a risk of continued bleeding, leading to compartment syndrome due to its deep location. We describe our experience in two patients with retrograde access via the peroneal artery and the use of balloon-assisted hemostasis for these retrograde punctures. This approach may potentially give more options for endovascular interventions in lower limb CTOs.

  14. Fundamental studies of retrograde reactions in direct liquefaction

    SciTech Connect

    Serio, M.A.; Solomon, P.R.; Bassilakis, R.; Kroo, E.

    1989-01-01

    Most of the proposed processing schemes for improving liquefaction yields involve favoring bond-breaking and radical stabilization reactions over the retrograde reactions. The retrograde reactions are often encountered before liquefaction temperatures are reached. The objective of this program is to elucidate and model the retrograde reaction chemistry in direct coal liquefaction through the application of experimental techniques and theoretical models which have been successfully employed at Advanced Fuel Research (AFR) and SRI International (a subcontractor) to understand and predict coal reaction behavior. The study of retrograde reactions is being done using an integrated approach using extensive characterization of the liquefaction chemistry of three kinds of systems: (1) model polymers; (2) coal; and (3) modified coals.

  15. Retrograde Melting and Internal Liquid Gettering in Silicon

    SciTech Connect

    Hudelson, Steve; Newman, Bonna K.; Bernardis, Sarah; Fenning, David P.; Bertoni, Mariana I.; Marcus, Matthew A.; Fakra, Sirine C.; Lai, Barry; Buonassisi, Tonio

    2011-07-01

    Retrograde melting (melting upon cooling) is observed in silicon doped with 3d transition metals, via synchrotron-based temperature-dependent X-ray microprobe measurements. Liquid metal-silicon droplets formed via retrograde melting act as efficient sinks for metal impurities dissolved within the silicon matrix. Cooling results in decomposition of the homogeneous liquid phase into solid multiple-metal alloy precipitates. These phenomena represent a novel pathway for engineering impurities in semiconductor-based systems.

  16. Retrograde intrarenal surgery in cross-fused ectopic kidney.

    PubMed

    Resorlu, Mustafa; Kabar, Mucahit; Resorlu, Berkan; Doluoglu, Omer Gokhan; Kilinc, Muhammet Fatih; Karakan, Tolga

    2015-02-01

    Cross-fused renal ectopia is a rare congenital anomaly in which both kidneys are fused and located on the same side. We report a case of right-to-left cross-fused renal ectopia and nephrolithiasis, in whom retrograde intrarenal surgery was used to treat the stone disease. To our knowledge, this is the first case of retrograde intrarenal surgery of a crossed-fused ectopic kidney. PMID:25481231

  17. The Design of MACs (Minimal Actin Cortices)

    PubMed Central

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

    2013-01-01

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

  18. Mesoscopic model of actin-based propulsion.

    PubMed

    Zhu, Jie; Mogilner, Alex

    2012-01-01

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

  19. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

  20. The interaction of vinculin with actin.

    PubMed

    Golji, Javad; Mofrad, Mohammad R K

    2013-04-01

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

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

    NASA Astrophysics Data System (ADS)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

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

  2. Elasticity, adhesion and actin based propulsion

    NASA Astrophysics Data System (ADS)

    Gopinathan, Ajay

    2006-03-01

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

  3. Endoscopic retrograde cholangiopancreatography during pregnancy without radiation

    PubMed Central

    Akcakaya, Adem; Ozkan, Orhan Veli; Okan, Ismail; Kocaman, Orhan; Sahin, Mustafa

    2009-01-01

    AIM: To present our experience with pregnant patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) without using radiation, and to evaluate the acceptability of this alternative therapeutic pathway for ERCP during pregnancy. METHODS: Between 2000 and 2008, six pregnant women underwent seven ERCP procedures. ERCP was performed under mild sedoanalgesia induced with pethidine HCl and midazolam. The bile duct was cannulated with a guidewire through the papilla. A catheter was slid over the guidewire and bile aspiration and/or visualization of the bile oozing around the guidewire was used to confirm correct cannulation. Following sphincterotomy, the bile duct was cleared by balloon sweeping. When indicated, stents were placed. Confirmation of successful biliary cannulation and stone extraction was made by laboratory, radiological and clinical improvement. Neither fluoroscopy nor spot radiography was used during the procedure. RESULTS: The mean age of the patients was 28 years (range, 21-33 years). The mean gestational age for the fetus was 23 wk (range, 14-34 wk). Five patients underwent ERCP because of choledocholithiasis and/or choledocholithiasis-induced acute cholangitis. In one case, a stone was extracted after precut papillotomy with a needle-knife, since the stone was impacted. One patient had ERCP because of persistent biliary fistula after hepatic hydatid disease surgery. Following sphincterotomy, scoleces were removed from the common bile duct. Two weeks later, because of the absence of fistula closure, repeat ERCP was performed and a stent was placed. The fistula was closed after stent placement. Neither post-ERCP complications nor premature birth or abortion was seen. CONCLUSION: Non-radiation ERCP in experienced hands can be performed during pregnancy. Stent placement should be considered in cases for which complete common bile duct clearance is dubious because of a lack of visualization of the biliary tree. PMID:19653343

  4. Functional interdependence between septin and actin cytoskeleton

    PubMed Central

    Schmidt, Katja; Nichols, Benjamin J

    2004-01-01

    Background Septin2 is a member of a highly conserved GTPase family found in fungi and animals. Septins have been implicated in a diversity of cellular processes including cytokinesis, formation of diffusion barriers and vesicle trafficking. Septin2 partially co-localises with actin bundles in mammalian interphase cells and Septin2-filamentmorphology depends upon an intact actin cytoskeleton. How this interaction is regulated is not known. Moreover, evidence that Septin2 is remodelled or redistributed in response to other changes in actin organisation is lacking. Results Septin2 filaments are associated with actin fibres, but Septin2 is not associated with actin at the leading edge of moving cells or in ruffles where actin is highly dynamic. Rather, Septin2 is spatially segregated from these active areas and forms O- and C-shaped structures, similar to those previously observed after latrunculin treatment. FRAP experiments showed that all assemblies formed by Septin2 are highly dynamic with a constant exchange of Septin2 in and out of these structures, and that this property is independent of actin. A combination of RNAi experiments and expression of truncated forms of Septin2 showed that Septin2 plays a significant role in stabilising or maintaining actin bundles. Conclusion We show that Septin2 can form dynamic structures with differing morphologies in living cells, and that these morphologies are dependent on the functional state of the actin cytoskeleton. Our data provide a link between the different morphological states of Septin2 and functions of Septin2 in actin-dynamics, and are consistent with the model proposed by Kinoshita and colleagues, that Septin2 filaments play a role in stabilisation of actin stress fibres thus preventing actin turnover. PMID:15541171

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

    PubMed Central

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

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

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

    PubMed

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

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

  7. Calcium control of Saccharomyces cerevisiae actin assembly.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

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

  8. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

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

    2016-03-01

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

  9. Pushing with actin: from cells to pathogens.

    PubMed

    Small, J Victor

    2015-02-01

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

  10. Dynamic actin gene family evolution in primates.

    PubMed

    Zhu, Liucun; Zhang, Ying; Hu, Yijun; Wen, Tieqiao; Wang, Qiang

    2013-01-01

    Actin is one of the most highly conserved proteins and plays crucial roles in many vital cellular functions. In most eukaryotes, it is encoded by a multigene family. Although the actin gene family has been studied a lot, few investigators focus on the comparison of actin gene family in relative species. Here, the purpose of our study is to systematically investigate characteristics and evolutionary pattern of actin gene family in primates. We identified 233 actin genes in human, chimpanzee, gorilla, orangutan, gibbon, rhesus monkey, and marmoset genomes. Phylogenetic analysis showed that actin genes in the seven species could be divided into two major types of clades: orthologous group versus complex group. Codon usages and gene expression patterns of actin gene copies were highly consistent among the groups because of basic functions needed by the organisms, but much diverged within species due to functional diversification. Besides, many great potential pseudogenes were found with incomplete open reading frames due to frameshifts or early stop codons. These results implied that actin gene family in primates went through "birth and death" model of evolution process. Under this model, actin genes experienced strong negative selection and increased the functional complexity by reproducing themselves. PMID:23841080

  11. Stochastic model of profilin-actin polymerization

    NASA Astrophysics Data System (ADS)

    Horan, Brandon; Vavylonis, Dimitrios

    A driving factor in cell motility and other processes that involve changes of cell shape is the rapid polymerization of actin subunits into long filaments. This process is regulated by profilin, a protein which binds to actin subunits and regulates elongation of actin filaments. Whether profilin stimulates polymerization by coupling to hydrolysis of ATP-bound actin is debated. Previous studies have proposed indirect coupling to ATP hydrolysis using rate equations, but did not include the effects of fluctuations that are important near the critical concentration. We developed stochastic simulations using the Gillespie algorithm to study single filament elongation at the barbed end in the presence of profilin. We used recently measured rate constants and estimated the rate of profilin binding to the barbed end such that detailed balance is satisfied. Fast phosphate release at the tip of the filament was accounted for. The elongation rate and length diffusivity as functions of profilin and actin concentration were calculated and used to extract the critical concentrations of free actin and of total actin. We show under what conditions profilin leads to an increase in the critical concentration of total actin but a decrease in the critical concentration of free actin.

  12. The Retrograde Transvenous Push-Through Method: A Novel Treatment of Peripheral Arteriovenous Malformations with Dominant Venous Outflow

    SciTech Connect

    Wohlgemuth, Walter A. Müller-Wille, René Teusch, Veronika I.; Dudeck, Oliver; Cahill, Anne M.; Alomari, Ahmad I.; Uller, Wibke

    2015-06-15

    PurposeTo evaluate the efficacy and safety of a novel retrograde transvenous embolization technique of peripheral arteriovenous malformations (AVMs) using Onyx.Materials and MethodsWe conducted a retrospective analysis of all patients who underwent transvenous retrograde Onyx embolization of peripheral AVMs with dominant venous outflow over a 29-month period. The embolization is aimed at retrograde filling of the nidus after building a solid plug in the dominant venous outflow (push-through). Classification, clinical signs, technical aspects, clinical and technical success rates, and complications were recorded. Short-term outcome was assessed.Results11 Symptomatic patients (8 female; mean age 31.4 years) were treated at our Vascular Anomalies Center with this method between January 2012 and May 2014. The AVMs were located on the upper extremity (n = 3), pelvis (n = 2), buttock (n = 2), and lower extremity (n = 4). Retrograde embolization was successfully carried out after preparatory transarterial-flow reduction in eight cases (73 %) and venous-flow reduction with Amplatzer Vascular Plugs in four cases (36 %). Complete devascularization (n = 10; 91 %) or 95 % devascularization (n = 1; 9 %) led to complete resolution (n = 8; 73 %) or improvement of clinical symptoms (n = 3; 27 %). One minor complication occurred (pain and swelling). During a mean follow-up time of 8 months, one clinically asymptomatic recurrence of AVM was detected.ConclusionInitial results suggest that retrograde transvenous Onyx embolization of peripheral AVMs with dominant venous outflow is a safe and effective novel technique with a low complication rate.

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

    PubMed

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

    2016-04-01

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

  14. Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes.

    PubMed

    Hannezo, Edouard; Dong, Bo; Recho, Pierre; Joanny, Jean-François; Hayashi, Shigeo

    2015-07-14

    An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting. PMID:26077909

  15. The Interplay between Viscoelastic and Thermodynamic Properties Determines the Birefringence of F-Actin Gels

    PubMed Central

    Helfer, Emmanuèle; Panine, Pierre; Carlier, Marie-France; Davidson, Patrick

    2005-01-01

    F-actin gels of increasing concentrations (25–300 μM) display in vitro a progressive onset of birefringence due to orientational ordering of actin filaments. At F-actin concentrations <100 μM, this birefringence can be erased and restored at will by sonication and gentle flow, respectively. Hence, the orientational ordering does not result from a thermodynamic transition to a nematic phase but instead is due to mechanical stresses stored in the gels. In contrast, at F-actin concentrations ≥100 μM, gels display spontaneous birefringence recovery, at rest, which is the sign of true nematic ordering, in good agreement with statistical physics models of the isotropic/nematic transition. Well-aligned samples of F-actin gels could be produced and their small-angle x-ray scattering patterns are quite anisotropic. These patterns show no sign of filament positional short-range order and could be modeled by averaging the form factor with the Maier-Saupe nematic distribution function. The derived nematic order parameter S of the gels ranged from S = 0.7 at 300 μM to S = 0.4 at 25 μM. Both birefringence and small-angle x-ray scattering data indicate that, even in absence of cross-linking proteins, spontaneous cooperative alignment of actin filaments may arise in motile regions of living cells where F-actin concentrations can reach values of a few 100 μM. PMID:15863487

  16. LeftyA decreases Actin Polymerization and Stiffness in Human Endometrial Cancer Cells

    PubMed Central

    Salker, Madhuri S.; Schierbaum, Nicolas; Alowayed, Nour; Singh, Yogesh; Mack, Andreas F.; Stournaras, Christos; Schäffer, Tilman E.; Lang, Florian

    2016-01-01

    LeftyA, a cytokine regulating stemness and embryonic differentiation, down-regulates cell proliferation and migration. Cell proliferation and motility require actin reorganization, which is under control of ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explored whether LeftyA modifies actin cytoskeleton, shape and stiffness of Ishikawa cells, a well differentiated endometrial carcinoma cell line. The effect of LeftyA on globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry. Rac1 and PAK1 transcript levels were measured by qRT-PCR as well as active Rac1 and PAK1 by immunoblotting. Cell stiffness (quantified by the elastic modulus), cell surface area and cell volume were studied by atomic force microscopy (AFM). As a result, 2 hours treatment with LeftyA (25 ng/ml) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin, and decreased cell stiffness, surface area and volume. The effect of LeftyA on actin polymerization was mimicked by pharmacological inhibition of Rac1 and PAK1. In the presence of the Rac1 or PAK1 inhibitor LeftyA did not lead to significant further actin depolymerization. In conclusion, LeftyA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization, cell softening and cell shrinkage. PMID:27404958

  17. Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes

    PubMed Central

    Hannezo, Edouard; Dong, Bo; Recho, Pierre; Joanny, Jean-François; Hayashi, Shigeo

    2015-01-01

    An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting. PMID:26077909

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

    PubMed Central

    Deshpande, Siddharth; Pfohl, Thomas

    2015-01-01

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

  19. Cellular chirality arising from the self-organization of the actin cytoskeleton.

    PubMed

    Tee, Yee Han; Shemesh, Tom; Thiagarajan, Visalatchi; Hariadi, Rizal Fajar; Anderson, Karen L; Page, Christopher; Volkmann, Niels; Hanein, Dorit; Sivaramakrishnan, Sivaraj; Kozlov, Michael M; Bershadsky, Alexander D

    2015-04-01

    Cellular mechanisms underlying the development of left-right asymmetry in tissues and embryos remain obscure. Here, the development of a chiral pattern of actomyosin was revealed by studying actin cytoskeleton self-organization in cells with isotropic circular shape. A radially symmetrical system of actin bundles consisting of α-actinin-enriched radial fibres (RFs) and myosin-IIA-enriched transverse fibres (TFs) evolved spontaneously into the chiral system as a result of the unidirectional tilting of all RFs, which was accompanied by a tangential shift in the retrograde movement of TFs. We showed that myosin-IIA-dependent contractile stresses within TFs drive their movement along RFs, which grow centripetally in a formin-dependent fashion. The handedness of the chiral pattern was shown to be regulated by α-actinin-1. Computational modelling demonstrated that the dynamics of the RF-TF system can explain the pattern transition from radial to chiral. Thus, actin cytoskeleton self-organization provides built-in machinery that potentially allows cells to develop left-right asymmetry. PMID:25799062

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

    PubMed Central

    2010-01-01

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

  1. Force of an actin spring

    NASA Astrophysics Data System (ADS)

    Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul

    2003-03-01

    The acrosomal process of the horseshoe crab sperm is a novel mechanochemical molecular spring that converts its elastic stain energy to mechanical work upon the chemical activation by Ca2+. Twisted and bent, the initial state of the acrosomal bundle features a high degree of complexity in its structure and the energy is believed to be stored in the highly strained actin filaments as an elastic potential energy. When activated, the bundle relaxes from the coil of the highly twisted and bent filaments to its straight conformation at a mean velocity of 15um/s. The mean extension velocity increases dramatically from 3um/s to 27um/s when temperature of the medium is changed from 9.6C to 32C (respective viscosities of 1.25-0.75cp), yet it exhibits a very weak dependence on changes in the medium viscosity (1cp-33cp). These experiments suggest that the uncoiling of the actin spring should be limited not by the viscosity of the medium but by the unlatching events of involved proteins at a molecular level. Unlike the viscosity-limited processes, where force is directly related to the rate of the reaction, a direct measurement is required to obtain the spring force of the acrosomal process. The extending acrosomal bundle is forced to push against a barrier and its elastic buckling response is analyzed to measure the force generated during the uncoiling.

  2. Dynamics of active actin networks

    NASA Astrophysics Data System (ADS)

    Koehler, Simone

    2014-03-01

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

  3. Actin-Regulator Feedback Interactions during Endocytosis.

    PubMed

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

    2016-03-29

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

  4. Effect of alpha-actinin on actin structure. Actin ATPase activity.

    PubMed

    Singh, I; Goll, D E; Robson, R M

    1981-08-28

    Alpha-Actinin increases the ATPase activity of actin by up to 84%, depending un pH, divalent cations present and the added Mg2+: ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of alpha-actinin to increase actin ATP activity. Increasing amounts of added alpha-actinin up to 1 mos alpha-actinin to 49 mol actin cause in increasing increment in actin ATPase activity, but adding alpha-actinin beyond 1 mol alpha-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol Pi/mg actin per h (4.3 mol Pi/mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added mg2+ to approx. 12.5 nmol Pi/mg actin per h (0.52 mol Pi/mol actin per h) at high pH (8.5) or at low levels (0.5-1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg2+ ATp uncomplexed with Mg2+ inhibits the ability of alpha-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg2+-modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that alpha-actinin can alter the structure of actin monomers. PMID:6456018

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

    DOEpatents

    Sederoff, Heike; Huber, Steven C; Larabell, Carolyn A

    2011-10-18

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

  6. Extracellular signaling cues for nuclear actin polymerization.

    PubMed

    Plessner, Matthias; Grosse, Robert

    2015-01-01

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

  7. Profilin connects actin assembly with microtubule dynamics.

    PubMed

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

    2016-08-01

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

  8. Actin cytoskeleton redox proteome oxidation by cadmium

    PubMed Central

    Go, Young-Mi; Orr, Michael

    2013-01-01

    Epidemiological studies associate environmental cadmium (Cd) exposure with the risk of lung diseases. Although mechanisms are not fully elucidated, several studies demonstrate Cd effects on actin and actin-associated proteins. In a recent study of Cd at concentrations similar to environmental exposures, we found that redox-dependent inflammatory signaling by NF-κB was sensitive to the actin-disrupting agent, cytochalasin D. The goal of the present study was to use mass spectrometry-based redox proteomics to investigate Cd effects on the actin cytoskeleton proteome and related functional pathways in lung cells at low environmental concentrations. The results showed that Cd under conditions that did not alter total protein thiols or glutathione redox state caused significant oxidation of peptidyl Cys of proteins regulating actin cytoskeleton. Immunofluorescence microscopy of lung fibroblasts and pulmonary artery endothelial cells showed that low-dose Cd exposure stimulated filamentous actin formation and nuclear localization of destrin, an actin-depolymerizing factor. Taken together, the results show that redox states of peptidyl Cys in proteins associated with actin cytoskeleton pathways are selectively oxidized in lung by Cd at levels thought to occur from environmental exposure. PMID:24077948

  9. Actin motility: formin a SCAry tail.

    PubMed

    Alberts, Art; Way, Michael

    2011-01-11

    A new biochemical analysis has revealed that the Rickettsia bacterial protein Sca2--recently shown to be essential for virulence and actin-dependent motility--assembles actin filaments using a mechanism that functionally resembles the processive elongation tactics used by formins. PMID:21215933

  10. Effect of ATP on actin filament stiffness.

    PubMed

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

    1990-09-01

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

  11. Myelination: actin disassembly leads the way

    PubMed Central

    Samanta, Jayshree; Salzer, James L.

    2016-01-01

    The mechanisms that drive the spiral wrapping of the myelin sheath around axons are poorly understood. Two papers in this issue of Developmental Cell demonstrate that actin disassembly, rather than actin assembly, predominates during oligodendrocyte maturation and is critical for the genesis of the central myelin sheath. PMID:26218317

  12. Colchicine activates actin polymerization by microtubule depolymerization.

    PubMed

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

    1997-06-30

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

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

    PubMed

    Lappalainen, Pekka

    2016-08-15

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

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

    PubMed Central

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

    2016-01-01

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

  15. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. PMID:24630119

  16. Study of the prograde and retrograde Chandler excitation

    NASA Astrophysics Data System (ADS)

    Zotov, , L.; Bizouard, , C.

    2014-12-01

    Observed motion of the Earth's rotation axis consists of components at both positive and negative frequencies. New generalized equations of Bizouard, which takes into account triaxiality of the Earth and asymmetry of the ocean tide, show that retrograde and prograde excitations are coupled. In this work using designed narrow-band filter and inversion we reconstruct geodetic excitation at the prograde and retrograde Chandler frequencies. Then we compare it with geophysical excitation, filtered out from the series of the oceanic angular momentum (OAM) and atmospheric angular momentum (AAM) for 1960-2000 yrs. Their sum coincides well with geodetic excitation only in the prograde Chandler band. The retrograde excitation coincides worse, probably in result of amplification of observational noises.

  17. Fundamental studies of retrograde reactions in direct liquefaction

    SciTech Connect

    Serio, M.A.; Solomon, P.R.; Kroo, E.; Charpenay, S.; Bassilakis, R.

    1991-12-17

    The overall objective of the program was to improve the understanding of retrograde reactions and their dependencies on coal rank and structure, and/or coal modifications and reaction conditions. Because retrograde reactions are competitive with bond breaking reactions, an understanding of both is required to shift the competition in favor of the latter. Related objectives were to clarify the conflicting observations reported in literature on such major topics as the role of oxygen groups in retrograde reactions and to provide a bridge from very fundamental studies on pure compounds to phenomenological studies on actual coal. This information was integrated into the FG-DVC model, which was improved and extended to the liquefaction context.

  18. Large retrograde Centaurs: visitors from the Oort cloud?

    NASA Astrophysics Data System (ADS)

    de la Fuente Marcos, C.; de la Fuente Marcos, R.

    2014-08-01

    Among all the asteroid dynamical groups, Centaurs have the highest fraction of objects moving in retrograde orbits. The distribution in absolute magnitude, H, of known retrograde Centaurs with semi-major axes in the range 6-34 AU exhibits a remarkable trend: 10 % have H<10 mag, the rest have H>12 mag. The largest objects, namely (342842) 2008 YB3, 2011 MM4 and 2013 LU28, move in almost polar, very eccentric paths; their nodal points are currently located near perihelion and aphelion. In the group of retrograde Centaurs, they are obvious outliers both in terms of dynamics and size. Here, we show that these objects are also trapped in retrograde resonances that make them unstable. Asteroid 2013 LU28, the largest, is a candidate transient co-orbital to Uranus and it may be a recent visitor from the trans-Neptunian region. Asteroids 342842 and 2011 MM4 are temporarily submitted to various high-order retrograde resonances with the Jovian planets but 342842 may be ejected towards the trans-Neptunian region within the next few hundred kyr. Asteroid 2011 MM4 is far more stable. Our analysis shows that the large retrograde Centaurs form an heterogeneous group that may include objects from various sources. Asteroid 2011 MM4 could be a visitor from the Oort cloud but an origin in a relatively stable closer reservoir cannot be ruled out. Minor bodies like 2011 MM4 may represent the remnants of the primordial planetesimals and signal the size threshold for catastrophic collisions in the early Solar System.

  19. Dynein is the motor for retrograde axonal transport of organelles

    SciTech Connect

    Schnapp, B.J.; Reese, T.S.

    1989-03-01

    Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport.

  20. Actin dynamics shape microglia effector functions.

    PubMed

    Uhlemann, Ria; Gertz, Karen; Boehmerle, Wolfgang; Schwarz, Tobias; Nolte, Christiane; Freyer, Dorette; Kettenmann, Helmut; Endres, Matthias; Kronenberg, Golo

    2016-06-01

    Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion. PMID:25989853

  1. Probing actin incorporation into myofibrils using Asp11 and His73 actin mutants.

    PubMed

    Xia, D; Peng, B; Sesok, D A; Peng, I

    1993-01-01

    We used a cell free system Bouché et al.: J. Cell Biol. 107:587-596, 1988] to study the incorporation of actin into myofibrils. We used alpha-skeletal muscle actin and actins with substitutions of either His73 [Solomon and Rubenstein: J. Biol.Chem. 262:11382, 1987], or Asp11 [Solomon et al.: J. Biol. Chem. 263:19662, 1988]. Actins were translated in reticulocyte lysate and incubated with myofibrils. The incorporated wild type actin could be cross-linked into dimers using N,N'-1,4-phenylenebismaleimide (PBM), indicating that the incorporated actin is actually inserted into the thin filaments of the myofibril. The His73 mutants incorporated to the same extent as wild type actin and was also cross-linked with PBM. Although some of the Asp11 mutants co-assembled with carrier actin, only 1-3% of the Asp11 mutant actins incorporated after 2 min and did not increase after 2 hr. Roughly 17% of wild type actin incorporated after 2 min and 31% after 2 hr. ATP increased the release of wild type actin from myofibrils, but did not increase the release of Asp11 mutants. We suggest that (1) the incorporation of wild type and His73 mutant actins was due to a physiological process whereas association of Asp11 mutants with myofibrils was non-specific, (2) the incorporation of wild type actin involved a rapid initial phase, followed by a slower phase, and (3) since some of the Asp11 mutants can co-assemble with wild type actin, the ability to self-assemble was not sufficient for incorporation into myofibrils. Thus, incorporation probably includes interaction between actin and a thin filament associated protein. We also showed that incorporation occurred at actin concentrations which would cause disassembly of F-actin. Since the myofibrils did not show large scale disassembly but incorporated actin, filament stability and monomer incorporation are likely to be mediated by actin associated proteins of the myofibril. PMID:8287497

  2. Dynamics of an actin spring

    NASA Astrophysics Data System (ADS)

    Riera, Christophe; Mahadevan, L.; Shin, Jennifer; Matsudaira, Paul

    2003-03-01

    The acrosome of the sperm of the horseshoe crab (Limulus Polyphemus) is an unusual actin based system that shows a spectacular dynamical transition in the presence of Ca++ that is present in abundance in the neighborhood of the egg. During this process, the bundle, which is initially bent and twisted uncoils and becomes straight in a matter of a few seconds. Based on microstructural data, we propose a model for the dynamics of uncoiling that is best represented by a triple-well potential corresponding to the different structural arrangements of the supertwisted filaments. Each of the false, true and coiled states corresponds to a local minimum of the energy, with the true state being the one with the lowest energy. Using an evolution equation derived by balancing torques, we investigate the nucleation and propagation of the phase transition and compare the results with those of experiments. Our model quantifies the hypothesis that the acrosomal bundle behaves like a mechano-chemical spring.

  3. Orbital Evolution and Impact Hazard of Asteroids on Retrograde Orbits

    NASA Astrophysics Data System (ADS)

    Kankiewicz, P.; Włodarczyk, I.

    2014-07-01

    We present the past evolutional scenarios of known group of asteroids in retrograde orbits. Applying the latest observational data, we determined their nominal and averaged orbital elements. Next, we studied the behaviour of their orbital motion 1~My in the past (100~My in the future for two NEAs) taking into account the limitations of observational errors. It has been shown that the influence of outer planets perturbations in many cases can import small bodies on high inclination or retrograde orbits into the inner Solar System.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-02-01

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

  6. Nuclear actin and myosins in adenovirus infection.

    PubMed

    Fuchsova, Beata; Serebryannyy, Leonid A; de Lanerolle, Primal

    2015-11-01

    Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus. PMID:26226218

  7. Erbium laser resurfacing for actinic cheilitis.

    PubMed

    Cohen, Joel L

    2013-11-01

    Actinic cheilitis is a precancerous condition characterized by grayish-whitish area(s) of discoloration on the mucosal lip, often blunting the demarcation between mucosa and cutaneous lip. Actinic cheilitis is considered to be an early part of the spectrum of squamous cell carcinoma. Squamous cell carcinoma specifically of the lip has a high rate of recurrence and metastasis through the oral cavity leading to a poor overall survival. Risk factors for the development of actinic cheilitis include chronic solar irradiation, increasing age, male gender, light skin complexion, immunosuppression, and possibly tobacco and alcohol consumption. Treatment options include topical pharmacotherapy (eg, fluorouracil, imiquimod) or procedural interventions (eg, cryotherapy, electrosurgery, surgical vermillionectomy, laser resurfacing), each with their known advantages and disadvantages. There is little consensus as to which treatment options offer the most clinical utility given the paucity of comparative clinical data. In my practice, laser resurfacing has become an important tool for the treatment of actinic cheilitis owing to its ease of use and overall safety, tolerability, and cosmetic acceptability. Herein the use of erbium laser resurfacing is described for three actinic cheilitis presentations for which I find it particularly useful: clinically prominent actinic cheilitis, biopsy-proven actinic cheilitis, and treatment of the entire lip following complete tumor excision of squamous cell carcinoma. All patients were treated with a 2940-nm erbium laser (Sciton Profile Contour Tunable Resurfacing Laser [TRL], Sciton, Inc., Palo Alto, CA). PMID:24196339

  8. 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

  9. Actinic Granuloma with Focal Segmental Glomerulosclerosis

    PubMed Central

    Phasukthaworn, Ruedee; Chanprapaph, Kumutnart; Vachiramon, Vasanop

    2016-01-01

    Actinic granuloma is an uncommon granulomatous disease, characterized by annular erythematous plaque with central clearing predominately located on sun-damaged skin. The pathogenesis is not well understood, ultraviolet radiation is recognized as precipitating factor. We report a case of a 52-year-old woman who presented with asymptomatic annular erythematous plaques on the forehead and both cheeks persisting for 2 years. The clinical presentation and histopathologic findings support the diagnosis of actinic granuloma. During that period of time, she also developed focal segmental glomerulosclerosis. The association between actinic granuloma and focal segmental glomerulosclerosis needs to be clarified by further studies. PMID:27293392

  10. Dynamic reorganization of the actin cytoskeleton

    PubMed Central

    Gressin, Laurène; Théry, Manuel; Blanchoin, Laurent

    2015-01-01

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

  11. Binding of actin to lens alpha crystallins

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Actin has been coupled to a cyanogen bromide-activated Sepharose 4B column, then tested for binding to alpha, beta, and gamma crystallin preparations from the bovine lens. Alpha, but not beta or gamma, crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.

  12. Chloroplast Retrograde Regulation of Heat Stress Responses in Plants.

    PubMed

    Sun, Ai-Zhen; Guo, Fang-Qing

    2016-01-01

    It is well known that intracellular signaling from chloroplast to nucleus plays a vital role in stress responses to survive environmental perturbations. The chloroplasts were proposed as sensors to heat stress since components of the photosynthetic apparatus housed in the chloroplast are the major targets of thermal damage in plants. Thus, communicating subcellular perturbations to the nucleus is critical during exposure to extreme environmental conditions such as heat stress. By coordinating expression of stress specific nuclear genes essential for adaptive responses to hostile environment, plants optimize different cell functions and activate acclimation responses through retrograde signaling pathways. The efficient communication between plastids and the nucleus is highly required for such diverse metabolic and biosynthetic functions during adaptation processes to environmental stresses. In recent years, several putative retrograde signals released from plastids that regulate nuclear genes have been identified and signaling pathways have been proposed. In this review, we provide an update on retrograde signals derived from tetrapyrroles, carotenoids, reactive oxygen species (ROS) and organellar gene expression (OGE) in the context of heat stress responses and address their roles in retrograde regulation of heat-responsive gene expression, systemic acquired acclimation, and cellular coordination in plants. PMID:27066042

  13. A systematic review of in vitro retrograde obturation materials.

    PubMed

    Theodosopoulou, Joanna N; Niederman, Richard

    2005-05-01

    The purpose of this review was two-fold: (a) to determine which retrograde obturation material(s) best prevents dye/ink penetration in vitro; and (b) to determine whether in vitro results agree with in vivo results. A MEDLINE search was conducted to identify in vitro studies published between January 1966 and October, week 4, 2003, conducted on human teeth, and published in English, German, or French language, testing the resistance to retrograde penetration of retrograde filling materials. The MEDLINE search identified 278 published articles. Of those, 115 studies examined the resistance to penetration of various retrograde filling materials, in vitro. Thirty-four studies met all the inclusion and validity criteria. The results indicate that, beyond 10 days in vitro, the most effective retrofilling materials, when measured by dye/ink penetration are: composites>glass ionomer cement>amalgam>orthograde gutta-percha>EBA. The results of these in vitro studies are not congruent with in vivo study results, suggesting a need to re-evaluate the clinical validity and importance of in vitro studies. PMID:15851926

  14. Water dynamics and retrogradation of ultrahigh pressurized wheat starch.

    PubMed

    Doona, Christopher J; Feeherry, Florence E; Baik, Moo-Yeol

    2006-09-01

    The water dynamics and retrogradation kinetics behavior of gelatinized wheat starch by either ultrahigh pressure (UHP) processing or heat are investigated. Wheat starch completely gelatinized in the condition of 90, 000 psi at 25 degrees C for 30 min (pressurized gel) or 100 degrees C for 30 min (heated gel). The physical properties of the wheat starches were characterized in terms of proton relaxation times (T2 times) measured using time-domain nuclear magnetic resonance spectroscopy and evaluated using commercially available continuous distribution modeling software. Different T2 distributions in both micro- and millisecond ranges between pressurized and heated wheat starch gels suggest distinctively different water dynamics between pressurized and heated wheat starch gels. Smaller water self-diffusion coefficients were observed for pressurized wheat starch gels and are indicative of more restricted translational proton mobility than is observed with heated wheat starch gels. The physical characteristics associated with changes taking place during retrogradation were evaluated using melting curves obtained with differential scanning calorimetry. Less retrogradation was observed in pressurized wheat starch, and it may be related to a smaller quantity of freezable water in pressurized wheat starch. Starches comprise a major constituent of many foods proposed for commercial potential using UHP, and the present results furnish insight into the effect of UHP on starch gelatinization and the mechanism of retrogradation during storage. PMID:16939331

  15. The kinematics of turnaround and retrograde axonal transport.

    PubMed

    Snyder, R E

    1986-11-01

    Rapid axonal transport of a pulse of 35S-methionine-labelled material was studied in vitro in the sensory neurons of amphibian sciatic nerve using a position-sensitive detector. For 10 nerves studied at 23.0 +/- 0.2 degrees C it was found that a pulse moved in the anterograde direction characterized by front edge, peak, and trailing edge transport rates of (mm/d) 180.8 +/- 2.2 (+/- SEM), 176.6 +/- 2.3, and 153.7 +/- 3.0, respectively. Following its arrival at a distal ligature, a smaller pulse was observed to move in the retrograde direction characterized by front edge and peak transport rates of 158.0 +/- 7.3 and 110.3 +/- 3.5, respectively, indicating that retrograde transport proceeds at a rate of 0.88 +/- 0.04 that of anterograde. The retrograde pulse was observed to disperse at a rate greater than the anterograde. Reversal of radiolabel at the distal ligature began 1.49 +/- 0.15 h following arrival of the first radiolabel. Considerable variation was seen between preparations in the way radiolabel accumulated in the end (ligature) regions of the nerve. Although a retrograde pulse was seen in all preparations, in 7 of 10 preparations there was no evidence of this pulse accumulating within less than 2-3 mm of a proximal ligature; however, accumulation was observed within less than 5 mm in all preparations. PMID:2432169

  16. Chloroplast Retrograde Regulation of Heat Stress Responses in Plants

    PubMed Central

    Sun, Ai-Zhen; Guo, Fang-Qing

    2016-01-01

    It is well known that intracellular signaling from chloroplast to nucleus plays a vital role in stress responses to survive environmental perturbations. The chloroplasts were proposed as sensors to heat stress since components of the photosynthetic apparatus housed in the chloroplast are the major targets of thermal damage in plants. Thus, communicating subcellular perturbations to the nucleus is critical during exposure to extreme environmental conditions such as heat stress. By coordinating expression of stress specific nuclear genes essential for adaptive responses to hostile environment, plants optimize different cell functions and activate acclimation responses through retrograde signaling pathways. The efficient communication between plastids and the nucleus is highly required for such diverse metabolic and biosynthetic functions during adaptation processes to environmental stresses. In recent years, several putative retrograde signals released from plastids that regulate nuclear genes have been identified and signaling pathways have been proposed. In this review, we provide an update on retrograde signals derived from tetrapyrroles, carotenoids, reactive oxygen species (ROS) and organellar gene expression (OGE) in the context of heat stress responses and address their roles in retrograde regulation of heat-responsive gene expression, systemic acquired acclimation, and cellular coordination in plants. PMID:27066042

  17. Actin Depletion Initiates Events Leading to Granule Secretion at the Immunological Synapse

    PubMed Central

    Ritter, Alex T.; Asano, Yukako; Stinchcombe, Jane C.; Dieckmann, N.M.G.; Chen, Bi-Chang; Gawden-Bone, C.; van Engelenburg, Schuyler; Legant, Wesley; Gao, Liang; Davidson, Michael W.; Betzig, Eric; Lippincott-Schwartz, Jennifer; Griffiths, Gillian M.

    2015-01-01

    Summary Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion. PMID:25992860

  18. Retroperitoneal hematoma: an unexpected complication during intervention on an occluded superficial femoral artery via a retrograde popliteal artery approach.

    PubMed

    Akkus, Nuri I; Beedupalli, Jagan; Varma, Jai

    2013-01-01

    Peripheral arterial disease involvement of the superficial femoral artery (SFA) is common. Different endovascular techniques are used successfully for revascularization of this artery. A retrograde approach to chronic total occlusion (CTO) of the SFA through the ipsilateral popliteal artery has been used occasionally if an antegrade approach is not feasible or has failed. Some of the known complications encountered during this approach are arteriovenous fistula formation at the access site, occlusion of the popliteal artery if closure devices are used, and bleeding. There are no reports of perforation or bleeding of the SFA or the external iliac artery (EIA) during a popliteal approach, probably due to lack of flow in the occluded segment of the SFA. We report a case in which a retroperitoneal hematoma occurred due to retrograde blood flow through the established true channel in the proximal SFA and subsequently to the dissection plane with a wire tip perforation in the EIA, which was treated by stopping retrograde filling with prolonged balloon inflation in the distal SFA before the CTO. PMID:23890758

  19. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  20. Retrograde fluids in granulites: Stable isotope evidence of fluid migration

    SciTech Connect

    Morrison, J. ); Valley, J.W. )

    1991-07-01

    Widespread retrograde alteration assemblages document the migration of mixed H{sub 2}O-CO{sub 2} fluids into granulite facies rocks in the Adirondack Mountains. Fluid migration is manifest by (1) veins and patchy intergrowths of chlorite {plus minus} sericite {plus minus} calcite, (2) small veins of calcite, many only identifiable by cathodoluminescence, and (3) high-density, CO{sub 2}-rich or mixed H{sub 2}O-CO{sub 2} fluid inclusions. The distinct and varied textural occurrences of the alteration minerals indicate that fluid-rock ratios were low and variable on a local scale. Stable isotope ratios of C, O, and S have been determined in retrograde minerals from samples of the Marcy anorthosite massif and adjacent granitic gneisses (charnockites). Retrograde calcite in the anorthosite has a relatively small range in both {delta}{sup 18}O{sub SMOW} and {delta}{sup 13}C{sub PDB} (8.6 to 14.9% and {minus}4.1 to 0.4%, respectively), probably indicating that the hydrothermal fluids that precipitated the calcite had exchanged with a variety of crustal lithologies including marbles and orthogneisses, and that calcite was precipitated over a relatively narrow temperature interval. Values of {delta}{sup 34}S{sub CDT} that range from 2.8 to 8.3% within the anorthosite can also be interpreted to reflect exchange between orthogneisses and metasediments. The recognition of retrograde fluid migration is particularly significant in granulite facies terranes because the controversy surrounding the origin of granulites arises in part from differing interpretations of fluid inclusion data, specifically, the timing of entrapment of high-density, CO{sub 2}-rich inclusions. Results indicate that retrograde fluid migration, which in some samples may leave only cryptic petrographic evidence, is a process capable of producing high-density, CO{sub 2}-rich fluid inclusions.

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

    PubMed

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

    2015-07-23

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

  2. Nuclear actin levels as an important transcriptional switch

    PubMed Central

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

    2012-01-01

    Nuclear actin levels have recently been linked to different cellular fates, suggesting that actin could act as a switch between altered transcriptional states. Here we discuss our latest results on the mechanisms by which nuclear actin levels are regulated and their implications to the functional significance of nuclear actin. PMID:22771994

  3. Genetics Home Reference: actin-accumulation myopathy

    MedlinePlus

    ... 7(3):160-8. Citation on PubMed Laing NG, Dye DE, Wallgren-Pettersson C, Richard G, Monnier ... Vigneron J, Wallgren-Pettersson C, Beggs AH, Laing NG. Mutations in the skeletal muscle alpha-actin gene ...

  4. Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea).

    PubMed

    Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José

    2013-08-01

    Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980

  5. Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

    PubMed Central

    Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José

    2013-01-01

    Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980

  6. [Actin in the wound healing process].

    PubMed

    Nowak, Dorota; Popow-Woźniak, Agnieszka; Raźnikiewicz, Linda; Malicka-Błaszkiewicz, Maria

    2009-01-01

    Wound healing is an important biological process of crucial value for organisms survival and retention of its proper functions. The recognition of molecular mechanisms of these phenomenon is still under investigation. The transition of mesenchymal fibroblasts to myofibroblasts is a key point in wound healing. The contraction ability of myofibroblast enables the shrinkage of a wound and closes its edges. Alpha smooth muscle actin (alpha-SMA), one of six actin isoforms, is a marker of compeletely differentiated myofibroblast. The regulation of differentiation process depends on many growth factors (especially TGF beta 1), the level of active thymosin beta 4, extracellular matrix proteins--including fibronectin, and also on specificity of microenvironment. Thymosin beta 4 is responsible for maintenance of pool of monomeric actin and actin filaments depolymerization. It can also act as a transcription factor, migration stimulator and immunomodulator, so this protein deserves for more attention in wound healing research field. PMID:19824469

  7. 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.

  8. Structural dynamics of an actin spring.

    PubMed

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

    2011-02-16

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

  9. Actinic review of EUV masks

    NASA Astrophysics Data System (ADS)

    Feldmann, Heiko; Ruoff, Johannes; Harnisch, Wolfgang; Kaiser, Winfried

    2010-04-01

    Management of mask defects is a major challenge for the introduction of EUV for HVM production. Once a defect has been detected, its printing impact needs to be predicted. Potentially the defect requires some repair, the success of which needs to be proven. This defect review has to be done with an actinic inspection system that matches the imaging conditions of an EUV scanner. During recent years, several concepts for such an aerial image metrology system (AIMS™) have been proposed. However, until now no commercial solution exists for EUV. Today, advances in EUV optics technology allow envisioning a solution that has been discarded before as unrealistic. We present this concept and its technical cornerstones.While the power requirement for the EUV source is less demanding than for HVM lithography tools, radiance, floor space, and stability are the main criteria for source selection. The requirement to emulate several generations of EUV scanners demands a large flexibility for the ilumination and imaging systems. New critical specifications to the EUV mirrors in the projection microscope can be satisfied using our expertise from lithographic mirrors. In summary, an EUV AIMS™ meeting production requirements seems to be feasible.

  10. The actin cytoskeleton in presynaptic assembly.

    PubMed

    Nelson, Jessica C; Stavoe, Andrea K H; Colón-Ramos, Daniel A

    2013-01-01

    Dramatic morphogenetic processes underpin nearly every step of nervous system development, from initial neuronal migration and axon guidance to synaptogenesis. Underlying this morphogenesis are dynamic rearrangements of cytoskeletal architecture. Here we discuss the roles of the actin cytoskeleton in the development of presynaptic terminals, from the elaboration of terminal arbors to the recruitment of presynaptic vesicles and active zone components. The studies discussed here underscore the importance of actin regulation at every step in neuronal circuit assembly. PMID:23628914

  11. Mechanism of Actin Filament Bundling by Fascin

    SciTech Connect

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

    2013-03-07

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

  12. Actin filament curvature biases branching direction

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  13. The Bacterial Actin-Like Cytoskeleton

    PubMed Central

    Carballido-López, Rut

    2006-01-01

    Recent advances have shown conclusively that bacterial cells possess distant but true homologues of actin (MreB, ParM, and the recently uncovered MamK protein). Despite weak amino acid sequence similarity, MreB and ParM exhibit high structural homology to actin. Just like F-actin in eukaryotes, MreB and ParM assemble into highly dynamic filamentous structures in vivo and in vitro. MreB-like proteins are essential for cell viability and have been implicated in major cellular processes, including cell morphogenesis, chromosome segregation, and cell polarity. ParM (a plasmid-encoded actin homologue) is responsible for driving plasmid-DNA partitioning. The dynamic prokaryotic actin-like cytoskeleton is thought to serve as a central organizer for the targeting and accurate positioning of proteins and nucleoprotein complexes, thereby (and by analogy to the eukaryotic cytoskeleton) spatially and temporally controlling macromolecular trafficking in bacterial cells. In this paper, the general properties and known functions of the actin orthologues in bacteria are reviewed. PMID:17158703

  14. Osmotically induced cell volume changes alter anterograde and retrograde transport, Golgi structure, and COPI dissociation.

    PubMed

    Lee, T H; Linstedt, A D

    1999-05-01

    Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment (ERGIC)-to-Golgi trafficking steps were blocked, but retrograde transport was active, and it mediated ERGIC and Golgi collapse into the ER. Extensive tubulation and relatively rapid Golgi resident redistribution were observed under hypo-osmotic conditions, whereas a slower redistribution of the same markers, without apparent tubulation, was observed under hyperosmotic conditions. The osmotic stress response correlated with the perturbation of COPI function, because both hypo- and hyperosmotic conditions slowed brefeldin A-induced dissociation of betaCOP from Golgi membranes. Remarkably, Golgi residents reemerged after several hours of sustained incubation in hypotonic or hypertonic medium. Reemergence was independent of new protein synthesis but required PKC, an activity known to mediate cell volume recovery. Taken together these results indicate the existence of a coupling between cell volume and constitutive traffic that impacts organelle structure through independent effects on anterograde and retrograde flow and that involves, in part, modulation of COPI function. PMID:10233155

  15. Properties of retrograded and acetylated starch produced via starch extrusion or starch hydrolysis with pullulanase.

    PubMed

    Kapelko, M; Zięba, T; Gryszkin, A; Styczyńska, M; Wilczak, A

    2013-09-12

    The aim of the present study was to determine the impact of serial modifications of starch, including firstly starch extrusion or hydrolysis with pullulanase, followed by retrogradation (through freezing and defrosting of pastes) and acetylation (under industrial conditions), on its susceptibility to amylolysis. The method of production had a significant effect on properties of the resultant preparations, whilst the direction and extent of changes depended on the type of modification applied. In the produced starch esters, the degree of substitution, expressed by the per cent of acetylation, ranged from 3.1 to 4.4 g/100 g. The acetylation had a significant impact on contents of elements determined with the atomic emission spectrometry, as it contributed to an increased Na content and decreased contents of Ca and K. The DSC thermal characteristics enabled concluding that the modifications caused an increase in temperatures and a decrease in heat of transition (or its lack). The acetylation of retrograded starch preparations increased their solubility in water and water absorbability. The modifications were found to exert various effects on the rheological properties of pastes determined based on the Brabender's pasting characteristics and flow curves determined with the use of an oscillatory-rotating viscosimeter. All starch acetates produced were characterized by ca. 40% resistance to amylolysis. PMID:23911484

  16. Osmotically Induced Cell Volume Changes Alter Anterograde and Retrograde Transport, Golgi Structure, and COPI Dissociation

    PubMed Central

    Lee, Tina H.; Linstedt, Adam D.

    1999-01-01

    Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment (ERGIC)-to-Golgi trafficking steps were blocked, but retrograde transport was active, and it mediated ERGIC and Golgi collapse into the ER. Extensive tubulation and relatively rapid Golgi resident redistribution were observed under hypo-osmotic conditions, whereas a slower redistribution of the same markers, without apparent tubulation, was observed under hyperosmotic conditions. The osmotic stress response correlated with the perturbation of COPI function, because both hypo- and hyperosmotic conditions slowed brefeldin A-induced dissociation of βCOP from Golgi membranes. Remarkably, Golgi residents reemerged after several hours of sustained incubation in hypotonic or hypertonic medium. Reemergence was independent of new protein synthesis but required PKC, an activity known to mediate cell volume recovery. Taken together these results indicate the existence of a coupling between cell volume and constitutive traffic that impacts organelle structure through independent effects on anterograde and retrograde flow and that involves, in part, modulation of COPI function. PMID:10233155

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

    SciTech Connect

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

    2013-11-20

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

  18. Nuclear and cytoplasmic actin in dinoflagellates.

    PubMed

    Soyer-Gobillard, M O; Ausseil, J; Géraud, M L

    1996-01-01

    Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the

  19. Effects of chitin nano-whiskers on the gelatinization and retrogradation of maize and potato starches.

    PubMed

    Ji, Na; Liu, Chengzhen; Zhang, Shuangling; Yu, Jing; Xiong, Liu; Sun, Qingjie

    2017-01-01

    Starch is very prone to retrogradation after gelatinization. Inhibition of starch retrogradation has been an important factor in improving the quality of food. For the first time, we investigated the effect of nano-materials, represented by chitin nano-whiskers (CNWs), on the short- and long-term retrogradation of maize and potato starches. Rapid Visco-Analyser results showed that the addition of CNWs significantly decreased the setback values of maize and potato starches, which suggested that CNWs could retard the short-term retrogradation of starch. Differential scanning calorimetry and X-ray diffraction results showed that the percentage of retrogradation of maize and potato starches significantly decreased (P<0.05), suggesting the inhibition of long-term retrogradation. The CNWs could be used as a new inhibitor of starch retrogradation to develop starch-based food with longer shelf life. PMID:27507508

  20. Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein

    PubMed Central

    Umeki, Nobuhisa; Hirose, Keiko; Uyeda, Taro Q. P.

    2016-01-01

    To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive. PMID:26842224

  1. New technique for retrograde cerebral perfusion during arch aneurysm repair.

    PubMed

    Bartoccioni, S; Lanzillo, G; deJong, A A; Fiaschini, P; Martinelli, G; Fedeli, C; Di Lazarro, D; Mercati, U

    1995-09-01

    Many techniques are used to reduce brain damage during surgery for dissecting aneurysms of the ascending aorta and arch. Recently, new techniques of protection were proposed, consistent with hypothermic circulatory arrest in association with retrograde cerebral perfusion via superior vena cava. We propose a simple, time-saving method, which does not require any manipulation of the heart. We use a multilumen cannula for cardioplegia (D 860-DIDECO FUNDARO') with pressure transducer. This cannula is inserted in superior vena cava by means of a simple purse-string, and linked to the arterial line with a "Y" derivation, allowing retrograde perfusion of the brain and monitoring the perfusion pressure at every moment. The superior vena cava placed downstream from the cannula is closed by a small vascular clamp, to avoid blood reflux in the right atrium. This method is time- and money-saving, is readily available, and can be prepared whenever necessary, also in the middle of the surgical procedure. PMID:7488786

  2. Learning the Languages of the Chloroplast: Retrograde Signaling and Beyond.

    PubMed

    Chan, Kai Xun; Phua, Su Yin; Crisp, Peter; McQuinn, Ryan; Pogson, Barry J

    2016-04-29

    The chloroplast can act as an environmental sensor, communicating with the cell during biogenesis and operation to change the expression of thousands of proteins. This process, termed retrograde signaling, regulates expression in response to developmental cues and stresses that affect photosynthesis and yield. Recent advances have identified many signals and pathways-including carotenoid derivatives, isoprenes, phosphoadenosines, tetrapyrroles, and heme, together with reactive oxygen species and proteins-that build a communication network to regulate gene expression, RNA turnover, and splicing. However, retrograde signaling pathways have been viewed largely as a means of bilateral communication between organelles and nuclei, ignoring their potential to interact with hormone signaling and the cell as a whole to regulate plant form and function. Here, we discuss new findings on the processes by which organelle communication is initiated, transmitted, and perceived, not only to regulate chloroplastic processes but also to intersect with cellular signaling and alter physiological responses. PMID:26735063

  3. Ureteroscopy assisted retrograde nephrostomy for complete staghorn renal calculi.

    PubMed

    Kawahara, Takashi; Ito, Hiroki; Terao, Hideyuki; Ogawa, Takehiko; Uemura, Hiroji; Kubota, Yoshinobu; Matsuzaki, Junichi

    2012-09-01

    Complete staghorn calculi are typically managed with percutaneous nephrolithotomy (PCNL). However, dilating nephrostomy and inserting a nephro access sheath can be difficult to perform without hydronephrosis. We reported the procedure of ureteroscopy-assisted retrograde nephrostomy (UARN) during PCNL. UARN is effective without dilating the renal collecting system in cases of complete staghorn calculi. A 63-year old female with a left complete staghorn renal calculus was referred to our hospital. Under general and epidural anesthesia, the patient was placed in a modified-Valdivia position. A flexible ureteroscope was inserted and a Lawson retrograde nephrostomy puncture wire was advanced into the flexible ureteroscope. The puncture wire was forwarded along the route from the renal pelvis to the exit skin. Calculus fragmentation was done using a pneumatic lithotripter and the Ho: YAG laser. UARN during PCNL was effective for the treatment of a complete staghorn calculus. PMID:24917723

  4. Ureteroscopy-Assisted Retrograde Nephrostomy (UARN) after Anatrophic Nephrolithotomy

    PubMed Central

    Kawahara, Takashi; Ito, Hiroki; Terao, Hideyuki; Kato, Yoshitake; Ogawa, Takehiko; Uemura, Hiroji; Kubota, Yoshinobu; Matsuzaki, Junichi

    2012-01-01

    Introduction. Open surgical anatrophic nephrolithotomy (ANL) had been the standard treatment for large renal calculi prior to the development of endoscopic devices and endoscopic techniques. A previous report described the efficacy of ureteroscopy-assisted retrograde nephrostomy (UARN) and presented a case of renal calculi successfully treated with UARN during percutaneous nephrolithotomy (PCNL) in a patient after ANL. Case Presentation. A 61-year-old male with left renal calculi was referred for further treatment. The patient was placed under general and epidural anesthesia, in a Galdakao-modified Valdivia position. A flexible ureteroscope (URS) was inserted, and a Lawson retrograde nephrostomy puncture wire was advanced into the flexible URS. The puncture wire then followed the route from the renal pelvis to the exit skin. Calculus fragmentation was undertaken using a pneumatic lithotripter. Conclusions. UARN for PCNL was therefore found to be a safe, effective, and appropriate treatment for a patient presenting with renal calculi after undergoing ANL. PMID:22924043

  5. Subversion of Retrograde Trafficking by Translocated Pathogen Effectors.

    PubMed

    Personnic, Nicolas; Bärlocher, Kevin; Finsel, Ivo; Hilbi, Hubert

    2016-06-01

    Intracellular bacterial pathogens subvert the endocytic bactericidal pathway to form specific replication-permissive compartments termed pathogen vacuoles or inclusions. To this end, the pathogens employ type III or type IV secretion systems, which translocate dozens, if not hundreds, of different effector proteins into their host cells, where they manipulate vesicle trafficking and signaling pathways in favor of the intruders. While the distinct cocktail of effectors defines the specific processes by which a pathogen vacuole is formed, the different pathogens commonly target certain vesicle trafficking routes, including the endocytic or secretory pathway. Recently, the retrograde transport pathway from endosomal compartments to the trans-Golgi network emerged as an important route affecting pathogen vacuole formation. Here, we review current insight into the host cell's retrograde trafficking pathway and how vacuolar pathogens of the genera Legionella, Coxiella, Salmonella, Chlamydia, and Simkania employ mechanistically distinct strategies to subvert this pathway, thus promoting intracellular survival and replication. PMID:26924068

  6. Sealing ability of five different retrograde filling materials.

    PubMed

    Gerhards, F; Wagner, W

    1996-09-01

    The sealing ability of Amalgam, Harvard-Cement, Diaket, gold-leaf, and Ketac-Endo as retrofilling materials was investigated. Paper cones were fixed with Harvard-Cement in the instrumented roots of 100 extracted human incisors. Apicectomy was performed and a 2-mm-deep retrograde cavity was prepared. Teeth were assigned to five groups (n = 20); each group received a different filling material. Surfaces of the roots were isolated with nail polish. Teeth, were stored in 1% methylene blue dye for 72 h. Roots were sectioned, and the depth of dye penetration was evaluated through a stereomicroscope. Retrofills with Ketac-Endo showed significantly less leakage compared with amalgam. There was no significant difference between the amalgam and Diaket groups. The sealing ability of Harvard-Cement and gold foil was lower than amalgam. It was concluded that retrograde fillings with Ketac-Endo or Diaket can be considered as alternatives for amalgam. PMID:9198426

  7. The Retrograde Ulnar Dorsal Flap: Surgical Technique and Experience as Island Flap in Coverage of Hand Defects.

    PubMed

    Vergara-Amador, Enrique

    2015-09-01

    Flaps from the forearm are often used to reconstruct soft-tissue defects in the hand. The retrograde ulnar dorsal flap has the advantage that it does not sacrifice a major vascular axis. The anatomic bases of this flap are the proximal and distal branch of the ulnar dorsal artery. The distal branch is partially accompanied with the dorsal branch of the ulnar nerve, and arrives under the abductor digiti quinti muscle making anastomoses with the deep branch of the ulnar artery. The proximal branch reaching the proximal third of the forearm, and anastomose with perforating branches of the ulnar artery. I used this island flap in 12 patients with coverage defects on the hand. The biggest flap was 13×6 cm. Only 1 flap had partial necrosis which did not lead to problems. The retrograde ulnar dorsal flap is a flap designed with reverse flow from the distal branch of the ulnar dorsal artery, and which does not sacrifice the ulnar artery. The donor defect on the forearm ulnar side had a greater esthetic acceptance. Knowing other distal anastomoses, described by other authors later, dorsal at the base of the fourth interdigital space grant greater security to the retrograde ulnar dorsal flap. It is worth highlighting the importance of preserving the adipofascial tissue around the pedicle. Experience with this flap permits us to state that it is a safe and reproducible flap to cover any defect on the dorsal of the hand as well as the first web space. PMID:26079665

  8. Retrograde approach for closure of ruptured sinus of Valsalva.

    PubMed

    Jayaranganath, M; Subramanian, Anand; Manjunath, Cholenahally Nanjappa

    2010-07-01

    Though ruptured sinuses of Valsalva have been traditionally managed surgically, they are amenable to transcatheter closure. Various devices have been used for closure of these defects. We describe a novel technique of closure of a ruptured right sinus of Valsalva into the right ventricular outflow tract. A muscular ventricular septal defect occluder was deployed retrogradely, without resorting to the usual antegrade technique involving formation of an arteriovenous loop. PMID:20603510

  9. Formation Flying in Earth, Libration, and Distant Retrograde Orbits

    NASA Technical Reports Server (NTRS)

    Folta, David C.

    2004-01-01

    This slide presentation examines the current and future state of formation flying, LEO formations, control strategies for flight in the vicinity of the libration points, and distant retrograde orbit formations. This discussion of LEO formations includes background on perturbation theory/accelerations and LEO formation flying. The discussion of strategies for formation flight in the vicinity of the libration points includes libration missions and natural and controlled libration orbit formations. A reference list is included.

  10. Transit Timing Variations for Inclined and Retrograde Exoplanetary Systems

    NASA Astrophysics Data System (ADS)

    Payne, Matthew J.; Ford, Eric B.; Veras, Dimitri

    2010-03-01

    We perform numerical calculations of the expected transit timing variations (TTVs) induced on a hot-Jupiter by an Earth-mass perturber. Motivated by the recent discoveries of retrograde transiting planets, we concentrate on an investigation of the effect of varying relative planetary inclinations, up to and including completely retrograde systems. We find that planets in low-order (e.g., 2:1) mean-motion resonances (MMRs) retain approximately constant TTV amplitudes for 0° < i < 170°, only reducing in amplitude for i>170°. Systems in higher order MMRs (e.g., 5:1) increase in TTV amplitude as inclinations increase toward 45°, becoming approximately constant for 45° < i < 135°, and then declining for i>135°. Planets away from resonance slowly decrease in TTV amplitude as inclinations increase from 0° to 180°, whereas planets adjacent to resonances can exhibit a huge range of variability in TTV amplitude as a function of both eccentricity and inclination. For highly retrograde systems (135° < i <= 180°), TTV signals will be undetectable across almost the entirety of parameter space, with the exceptions occurring when the perturber has high eccentricity or is very close to an MMR. This high inclination decrease in TTV amplitude (on and away from resonance) is important for the analysis of the known retrograde and multi-planet transiting systems, as inclination effects need to be considered if TTVs are to be used to exclude the presence of any putative planetary companions: absence of evidence is not evidence of absence.

  11. Activity-Dependent Regulation of Synapses by Retrograde Messengers

    PubMed Central

    Regehr, Wade G.; Carey, Megan R.; Best, Aaron R.

    2011-01-01

    Summary Throughout the brain postsynaptic neurons release substances from their cell bodies and dendrites that regulate the strength of the synapses they receive. Diverse chemical messengers have been implicated in retrograde signaling from postsynaptic neurons to presynaptic boutons. Here we provide an overview of the signaling systems that lead to rapid changes in synaptic strength. We consider the capabilities, specializations and physiological roles of each type of signaling system. PMID:19640475

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

    NASA Astrophysics Data System (ADS)

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

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

  13. Huge biloma after endoscopic retrograde cholangiopancreatography and endoscopic biliary sphincterotomy

    PubMed Central

    Alkhateeb, Harith M.; Aljanabi, Thaer J.; Al-azzawi, Khairallh H.; Alkarboly, Taha A.

    2015-01-01

    Background Biliary leak can occur as a complication of biliary surgery, endoscopic retrograde cholangiopancreatography manipulations and endoscopic biliary sphincterotomy. Consequently, bile may collect in the abdominal cavity, a condition called biloma. Rarely, it may reach a massive size. Case presentation A 72-year-old man presented with gastric upset with gradual abdominal distension reaching a large size due to intra-abdominal bile collection (biloma) after endoscopic retrograde cholangiopancreatography plus endoscopic biliary sphincterotomy and stenting for post laparoscopic cholecystectomy common bile duct stricture. This huge biloma was treated by percutaneous insertion of a tube drain for a few days, evacuating the collection successfully without recurrence. Discussion This patient might sustain injury to the common bile duct either by the guide wire or stent, or the injury occurred at the angle between the common bile duct and duodenum during sphincterotomy of the ampulla. Although any of these rents may lead to a bile leak, causing a huge biloma, they could be successfully treated by percutaneous drainage. Conclusions (1) Following endoscopic retrograde cholangiopancreatography, a patient’s complaints should not be ignored. (2) A massive biloma can occur due to such procedures. (3) Conservative treatment with minimal invasive technique can prove to be effective. PMID:26402876

  14. Osteochondritis Dissecans Involving the Trochlear Groove Treated With Retrograde Drilling

    PubMed Central

    Kaji, Yoshio; Nakamura, Osamu; Yamaguchi, Konosuke; Yamamoto, Tetsuji

    2015-01-01

    Abstract Osteochondritis dissecans (OCD) occurs frequently in the humeral capitellum of the upper extremity, whereas OCD involving the trochlear groove (trochlear groove OCD) is rarely reported. A standard treatment for trochlear groove OCD has therefore not been determined, although several methods have been tried. The case of a 14-year-old male gymnast with bilateral trochlear groove OCD is presented. Retrograde drilling from the lateral condyle of the humerus was applied for the OCD lesion of the left elbow, since it was larger in size than that in the right elbow and was symptomatic. Conversely, since the right lesion was small and asymptomatic, it was managed conservatively. After treatment, consolidation of the OCD lesions was observed in both elbows. However, the time to healing was shorter in the left elbow treated surgically than in the right elbow managed conservatively. In conclusion, retrograde drilling is a very simple and minimally invasive treatment. This case suggests that retrograde drilling for trochlear groove OCD may be a useful procedure that may accelerate the healing process for OCD lesions. PMID:26356703

  15. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  17. Model of cellular mechanotransduction via actin stress fibers.

    PubMed

    Gouget, Cecile L M; Hwang, Yongyun; Barakat, Abdul I

    2016-04-01

    Mechanical stresses due to blood flow regulate vascular endothelial cell structure and function and play a key role in arterial physiology and pathology. In particular, the development of atherosclerosis has been shown to correlate with regions of disturbed blood flow where endothelial cells are round and have a randomly organized cytoskeleton. Thus, deciphering the relation between the mechanical environment, cell structure, and cell function is a key step toward understanding the early development of atherosclerosis. Recent experiments have demonstrated very rapid ([Formula: see text]100 ms) and long-distance ([Formula: see text]10 [Formula: see text]m) cellular mechanotransduction in which prestressed actin stress fibers play a critical role. Here, we develop a model of mechanical signal transmission within a cell by describing strains in a network of prestressed viscoelastic stress fibers following the application of a force to the cell surface. We find force transmission dynamics that are consistent with experimental results. We also show that the extent of stress fiber alignment and the direction of the applied force relative to this alignment are key determinants of the efficiency of mechanical signal transmission. These results are consistent with the link observed experimentally between cytoskeletal organization, mechanical stress, and cellular responsiveness to stress. Based on these results, we suggest that mechanical strain of actin stress fibers under force constitutes a key link in the mechanotransduction chain. PMID:26081725

  18. Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

    SciTech Connect

    Hirano, Hidemi; Matsuura, Yoshiyuki

    2011-10-22

    Highlights: {yields} MAL has a bipartite NLS that binds to Imp{alpha} in an extended conformation. {yields} Mutational analyses verified the functional significance of MAL-Imp{alpha} interactions. {yields} Induced folding and NLS-masking by G-actins inhibit nuclear import of MAL. -- Abstract: The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin {alpha}/{beta} heterodimer, and that G-actin competes with importin {alpha}/{beta} for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal 'RPEL' domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-{alpha}, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-{alpha}- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-{alpha} recognition.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  1. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    PubMed Central

    Paves, Heiti; Truve, Erkki

    2004-01-01

    Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area. PMID:15102327

  2. Actin-dependent mechanisms in AMPA receptor trafficking

    PubMed Central

    Hanley, Jonathan G.

    2014-01-01

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

  3. Crystal structure of a nuclear actin ternary complex.

    PubMed

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

    2016-08-01

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

  4. Supercoiling of f-actin filaments.

    PubMed

    Lednev, V V; Popp, D

    1990-05-01

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

  5. Impact of Carbon Nanomaterials on Actin Polymerization.

    PubMed

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

    2016-03-01

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

  6. Retrogradation behaviour of high-amylose rice starch prepared by improved extrusion cooking technology.

    PubMed

    Zhang, Yanjun; Liu, Wei; Liu, Chengmei; Luo, Shunjing; Li, Ti; Liu, Yunfei; Wu, Di; Zuo, Yanna

    2014-09-01

    Native rice starch (NRS, amylose/28.9%) was gelatinized by improved extrusion cooking technology (IECT) and retrograded (RRS) after low temperature storage (4 °C). The retrogradation behaviour of RRS was changed to low retrogradation percentage and low retrogradation rate. The retrogradation resulted in a high compact morphology. The melt enthalpy change and percentage of retrogradation of RRS was 3.68 J/g and 37.7%, respectively, compared to those of NRS (9.75 J/g, 100%). The retrogradation percentage for RRS was low during storage as shown as a low retrogradation rate (0.21 d(-1)) and a high Avrami exponent (0.89). The pattern of rice starch changed from A-type to amorphous and B-type. Both the relative crystallinity of RRS (12.7%) by the X-ray diffractograms and the ratio of the band height (0.63) in the FTIR spectra were low. The analysis of retrogradation structure and short-range molecular order further confirmed the retrogradation behaviour of rice starch after IECT treatment. PMID:24731339

  7. Structural Transitions of F-Actin:Espin Bundles

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin; Bartles, James; Wong, Gerard

    2006-03-01

    Espin is an actin bundling protein involved in the formation of the parallel bundles of filamentous actin in hair cell stereocilia. Mutations in espin are implicated in deafness phenotypes in mice and humans. We present measurements of the F-actin structures induced by wild type and by mutated espin obtained via small angle x-ray scattering and fluorescence microscopy. We found that wild type espin induced a paracrystalline hexagonal array of twisted F-actin, whereas the mutated espin only condensed the F-actin into a nematic-like phase. The possibility of coexisting nematic and bundled actin in mixtures containing both mutant and wild type espins was also investigated.

  8. Actin Filament Segmentation Using Dynamic Programming

    PubMed Central

    Li, Hongsheng; Shen, Tian; Huang, Xiaolei

    2011-01-01

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

  9. Spontaneous actin dynamics in contractile rings

    NASA Astrophysics Data System (ADS)

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

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

  10. Actin polymerization stabilizes α4β1 integrin anchors that mediate monocyte adhesion

    PubMed Central

    Becker, Henry; Hyduk, Sharon J.; Wong, Janice C.; Digby, Genevieve; Arora, Pamma D.; Cano, Adrianet Puig; Hartwig, John; McCulloch, Christopher A.

    2012-01-01

    Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces. PMID:22472442

  11. The actin binding protein adseverin regulates osteoclastogenesis.

    PubMed

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  12. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    PubMed Central

    Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  13. F-actin Severing Facilitates Distinct Mechanisms of Stress Relaxation in the Actin Cytoskeleton

    NASA Astrophysics Data System (ADS)

    Kim, Taeyoon; Jung, Wonyeong; Murrell, Michael

    Rheological behaviors of actin cytoskeleton play an important role in physiological processes including cell migration and division. The actin cytoskeleton shows a wide variety of viscoelastic responses to external mechanical cues, such as strain-stiffening and stress relaxation. It has been hypothesized that the stress relaxation originates mainly from transient nature of cross-linkers that connect pairs of F-actins. By contrast, potential impacts of rich F-actin dynamics to the stress relaxation have been neglected in most previous studies. Here, using a computational model, we demonstrated that severing of F-actins induced by buckling during strain-stiffening can facilitate a very distinct mode of stress relaxation in the actin cytoskeleton from that induced by the transient cross-linkers. We also explored conditions where the severing-induced stress relaxation becomes prominent. This finding provides a more complete understanding of rheological behaviors of the actin cytoskeleton. We gratefully acknowledge the support of the National Science Foundation (1434013-CMMI and 1434095-CMMI).

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    1998-06-01

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

  16. Bacterial lipopolysaccharide induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in pulmonary vascular endothelial cells: concurrent F-actin depolymerization and new actin synthesis.

    PubMed

    Goldblum, S E; Ding, X; Brann, T W; Campbell-Washington, J

    1993-10-01

    Bacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to Escherichia coli 0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: 1) transendothelial 14C-albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the DNAse 1 inhibition assay. LPS induced increments in 14C-albumin flux (P < 0.001) and intercellular gap formation at > or = 2-6 h. During this same time period the endothelial F-actin pool was not significantly changed compared to simultaneous media controls. Mean (+/- SE) G-actin (micrograms/mg total protein) was significantly (P < 0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F-actin stabilization with phallicidin protected against the LPS-induced increments in G-actin (P = 0.040) as well as changes in barrier function (P < 0.0001). Prior protein synthesis inhibition unmasked an LPS-induced decrement in F-actin (P = 0.0044), blunted the G-actin increment (P = 0.010), and increased LPS-induced changes in endothelial barrier function (P < 0.0001). Therefore, LPS induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P < 0.0001) and new actin synthesis (P = 0.0063) which may be a compensatory endothelial cell response to LPS-induced F-actin depolymerization. PMID:8408232

  17. CNS myelin wrapping is driven by actin disassembly.

    PubMed

    Zuchero, J Bradley; Fu, Meng-Meng; Sloan, Steven A; Ibrahim, Adiljan; Olson, Andrew; Zaremba, Anita; Dugas, Jason C; Wienbar, Sophia; Caprariello, Andrew V; Kantor, Christopher; Leonoudakis, Dmitri; Leonoudakus, Dmitri; Lariosa-Willingham, Karen; Kronenberg, Golo; Gertz, Karen; Soderling, Scott H; Miller, Robert H; Barres, Ben A

    2015-07-27

    Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility. PMID:26166300

  18. The Interaction of Caldesmon with the COOH Terminus of Actin*

    PubMed Central

    Crosbie, Rachelle; Adams, Susan; Chalovich, Joseph M.; Reisler, Emil

    2005-01-01

    Caldesmon interacts with the NH2-terminal region of actin. It is now shown in airfuge centrifugation experiments that modification of the penultimate cysteine residue of actin significantly weakens its binding to caldesmon both in the presence and absence of tropomyosin. Furthermore, as revealed by fluorescence measurements, caldesmon increases the exposure of the COOH-terminal region of actin to the solvent. This effect of caldesmon, like its inhibitory effect on actomyosin ATPase activity, is enhanced in the presence of tropomyosin. Proteolytic removal of the last three COOH-terminal residues of actin, containing the modified cysteine residue, restores the normal binding between caldesmon and actin. These results establish a correlation between the binding of caldesmon to actin and the conformation of the COOH-terminal region of actin and suggest an indirect rather than direct interaction between caldesmon and this part of actin. PMID:1939062

  19. Actin of Beta vulgaris seedlings under the clinorotation

    NASA Astrophysics Data System (ADS)

    Kozeko, L. Ye.

    We study the influence of altered gravity on actin expression in roots of Beta vulguris seedlings grown on the horizontal clinostat (2 rpm) from seed germination for three days. It is shown that the total actin quantity was not influenced. Three actin isoforms are revealed; a relative protein quantity of these isoforms was similar both in clinorotated seedlings and in ones grown in norm. This point to stable expression of actin under the altered gravity conditions.

  20. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders E.

    2010-06-01

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

  1. Curvature and torsion in growing actin networks

    NASA Astrophysics Data System (ADS)

    Shaevitz, Joshua W.; Fletcher, Daniel A.

    2008-06-01

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

  2. Actin-aggregating cucurbitacins from Physocarpus capitatus.

    PubMed

    Maloney, Katherine N; Fujita, Masaki; Eggert, Ulrike S; Schroeder, Frank C; Field, Christine M; Mitchison, Timothy J; Clardy, Jon

    2008-11-01

    Bioassay-guided fractionation of Physocarpus capitatus yielded two new cucurbitacins (3 and 4) along with the known cucurbitacin F (1) and dihydrocucurbitacin F (2). Preliminary mechanism of action studies indicate that the cucurbitacins cause actin aggregates and inhibit cell division. PMID:18959442

  3. Approximations of distant retrograde orbits for mission design

    NASA Technical Reports Server (NTRS)

    Hirani, Anil N.; Russell, Ryan P.

    2006-01-01

    Distant retrograde orbits (DROs) are stable periodic orbit solutions of the equations of motion in the circular restricted three body problem. Since no closed form expressions for DROs are known, we present methods for approximating a family of planar DROs for an arbitrary, fixed mass ratio. Furthermore we give methods for computing the first and second derivatives of the position and velocity with respect to the variables that parameterize the family. The approximation and derivative methods described allow a mission designer to target specific DROs or a range of DROs with no regard to phasing in contrast to the more limited case of targeting a six-state only.

  4. A chloroplast retrograde signal regulates nuclear alternative splicing

    PubMed Central

    Petrillo, Ezequiel; Herz, Micaela A. Godoy; Fuchs, Armin; Reifer, Dominik; Fuller, John; Yanovsky, Marcelo J.; Simpson, Craig; Brown, John W. S.; Barta, Andrea; Kalyna, Maria; Kornblihtt, Alberto R.

    2015-01-01

    Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions. PMID:24763593

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

    PubMed

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

    2016-05-27

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

  6. Transformation of actin-encapsulating liposomes induced by cytochalasin D.

    PubMed Central

    Miyata, H; Kinosita, K

    1994-01-01

    Liposomes encapsulating actin filaments were prepared by swelling at 0 degrees C lipid film consisting of a mixture of dimyristoyl phosphatidylcholine and cardiolipin (equal amounts by weight) in 100 microM rabbit skeletal muscle actin and 0.5 mM CaCl2 followed by polymerization of actin at 30 degrees C. Liposomes initially assumed either disk or dumbbell shape, but when cytochalasin D was added to the medium surrounding the liposomes, they were found to become spindle shaped. Liposomes containing bovine serum albumin that were given cytochalasin D and actin-containing liposomes that were given dimethylformamide, the solvent for cytochalasin D, did not transform. These results indicated actin-cytochalasin interaction is involved in the transformation process. Falling-ball viscometry and sedimentation analysis of actin solution indicated that cytochalasin cleaved actin filaments and caused depolymerization. The observation of polarized fluorescence of encapsulated actin labeled with acrylodan indicated that the actin filaments in the transformed liposomes aligned along the long axis of the liposomes. Because the actin filaments in the disk- or dumbbell-shaped liposomes formed bundles running along the liposome contour, the transformation was likely to be accompanied by the change in the actin filament arrangement in the liposomes, which was induced by actin-cytochalasin interaction. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:7948706

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

    PubMed

    Brugerolle, G; Bricheux, G; Coffe, G

    1996-01-01

    The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads. PMID:9175265

  8. In situ localization of F-actin microfilaments in the vasculature of the porcine retina.

    PubMed

    Strauss, B I; Langille, B L; Gotlieb, A I

    1987-10-01

    The organization of F-actin microfilaments in the vascular endothelium of the porcine retina was studied in situ using rhodamine phalloidin labelling and fluorescence microscopy. A comparison was made between arterial and venous endothelial-cell microfilament distribution. The arterial cells in straight segments, bifurcations and branch points were elongated with their long axis in the direction of flow. Venous endothelial cells, on the other hand, were ellipsoid to rhomboid in shape throughout. F-actin was localized at the periphery of both arterial and venous endothelial cells. Prominent central microfilament bundles, similar to in vitro stress fibres, were oriented parallel to the long axis of arterial cells but were rarely present in venous cells. Only occasional venous endothelial cells contained short central actin filaments which were mainly in the venules. Central microfilaments were not identified in pre-capillary, capillary, or post-capillary endothelial cells. Incubation of the retinal organ cultures for 24 hr resulted in loss of the central microfilaments while peripheral staining persisted. Short-term incubation of the retinas in organ culture with low-dose cytochalasin B resulted in disruption of the central microfilaments while the peripheral actin microfilaments remained intact. The central microfilament bundles may reflect an adaptive response to arterial blood flow and may indeed be a sensitive dynamic system reflecting the influence of environmental factors on endothelial cells. PMID:3428383

  9. Tailor-Made Ezrin Actin Binding Domain to Probe Its Interaction with Actin In-Vitro

    PubMed Central

    Shrivastava, Rohini; Köster, Darius; Kalme, Sheetal; Mayor, Satyajit; Neerathilingam, Muniasamy

    2015-01-01

    Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well. PMID:25860910

  10. FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongation

    SciTech Connect

    Thompson, Morgan E; Heimsath, Ernest G; Gauvin, Timothy J; Higgs, Henry N; Kull, F Jon

    2012-12-09

    Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.

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

    PubMed Central

    Hansen, Scott D.

    2010-01-01

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

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

    PubMed Central

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

    1988-01-01

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

  13. ATTENUATION OF REFLECTED WAVES IN MAN DURING RETROGRADE PROPAGATION FROM FEMORAL ARTERY TO PROXIMAL AORTA

    PubMed Central

    Baksi, A John; Davies, Justin E; Hadjiloizou, Nearchos; Baruah, Resham; Unsworth, Beth; Foale, Rodney A; Korolkova, Olga; Siggers, Jennifer H; Francis, Darrel P; Mayet, Jamil; Parker, Kim H; Hughes, Alun D

    2015-01-01

    Background Wave reflection may be an important influence on blood pressure, but the extent to which reflections undergo attenuation during retrograde propagation has not been studied. We quantified retrograde transmission of a reflected wave created by occlusion of the left femoral artery in man. Methods 20 subjects (age 31-83 y; 14 male) underwent invasive measurement of pressure and flow velocity with a sensor-tipped intra-arterial wire at multiple locations distal to the proximal aorta before, during and following occlusion of the left femoral artery by thigh cuff inflation. A numerical model of the circulation was also used to predict reflected wave transmission. Wave reflection was measured as the ratio of backward to forward wave energy (WRI) and the ratio of peak backward to forward pressure (Pb/Pf). Results Cuff inflation caused a marked reflection which was largest 5-10cm from the cuff (change (Δ) in WRI = 0.50 (95% CI 0.38, 0.62); p<0.001, ΔPb/Pf = 0.23 (0.18 - 0.29); p<0.001). The magnitude of the cuff-induced reflection decreased progressively at more proximal locations and was barely discernible at sites >40cm from the cuff including in the proximal aorta. Numerical modelling gave similar predictions to those observed experimentally. Conclusions Reflections due to femoral artery occlusion are markedly attenuated by the time they reach the proximal aorta. This is due to impedance mismatches of bifurcations traversed in the backward direction. This degree of attenuation is inconsistent with the idea of a large discrete reflected wave arising from the lower limb and propagating back into the aorta. PMID:26436672

  14. Structure of a Bud6/Actin Complex Reveals a Novel WH2-like Actin Monomer Recruitment Motif.

    PubMed

    Park, Eunyoung; Graziano, Brian R; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L; Eck, Michael J

    2015-08-01

    In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a "core" domain that binds to the formin and a "flank" domain that binds monomeric actin. Here, we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6(flank) interact with actin; one binds in a groove at the barbed end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation. PMID:26118535

  15. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

    2001-01-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

  16. Exploring the Stability Limits of Actin and Its Suprastructures

    PubMed Central

    Rosin, Christopher; Erlkamp, Mirko; Ecken, Julian von der; Raunser, Stefan; Winter, Roland

    2014-01-01

    Actin is the main component of the microfilament system in eukaryotic cells and can be found in distinct morphological states. Global (G)-actin is able to assemble into highly organized, supramolecular cellular structures known as filamentous (F)-actin and bundled (B)-actin. To evaluate the structure and stability of G-, F-, and B-actin over a wide range of temperatures and pressures, we used Fourier transform infrared spectroscopy in combination with differential scanning and pressure perturbation calorimetry, small-angle x-ray scattering, laser confocal scanning microscopy, and transmission electron microscopy. Our analysis was designed to provide new (to our knowledge) insights into the stabilizing forces of actin self-assembly and to reveal the stability of the actin polymorphs, including in conditions encountered in extreme environments. In addition, we sought to explain the limited pressure stability of actin self-assembly observed in vivo. G-actin is not only the least temperature-stable but also the least pressure-stable actin species. Under abyssal conditions, where temperatures as low as 1–4°C and pressures up to 1 kbar are reached, G-actin is hardly stable. However, the supramolecular assemblies of actin are stable enough to withstand the extreme conditions usually encountered on Earth. Beyond ∼3–4 kbar, filamentous structures disassemble, and beyond ∼4 kbar, complete dissociation of F-actin structures is observed. Between ∼1 and 2 kbar, some disordering of actin assemblies commences, in agreement with in vivo observations. The limited pressure stability of the monomeric building block seems to be responsible for the suppression of actin assembly in the kbar pressure range. PMID:25517163

  17. Retrograde intrarenal surgery in Nepal: an early experience.

    PubMed

    Gyawali, P R; Luitel, B R; Luitel, B R

    2013-06-01

    With the advancement in technology and miniaturization of instruments, retrograde intrarenal surgery (RIRS) with flexible ureteroscope is gaining popularity. Flexible ureteroscope is introduced into renal collecting system through the urethra. Holmium YAG (Ho-YAG) Laser fiber of different sizes is introduced through the ureteroscope and renal stones are pulverized. Removal of renal stones less than 15 mm in size with RIRS has sharply reduced post operative morbidity. This is a Prospective study comprised of 58 RIRS performed from January 2013 to July 2013. Preoperative investigations like full blood count (FBC), renal function test (RFT), serological investigations, urine culture, intravenous urogram or CT-urogram, chest ray and electrocardiogram were done in all patients. Renal stones less than 15 mm in size were included for RIRS. Intra renal stones were treated with 7.5 Fr flexible ureteroscope (Flex - X2) using Holmium-YAG laser. Mean stone burden in our study was 10.5 +/- 3.3 mm. Out of 32 male patients, 29 (90.6%) required pre-stenting before RIRS and all 58 patients with successful dusting of stones in single sitting were discharged on next day. Retrograde Intrarenal Surgery is advanced and successful technique and a viable alternative to Extracorporeal Shock Wave Lithotripsy (ESWL) and Percutaneous Nephrolithotomy (PCNL) in the treatment of selected intrarenal stones with minimum morbidity. PMID:24696935

  18. Retrograde closed orbits in a rotating triaxial potential

    NASA Astrophysics Data System (ADS)

    Heisler, J.; Merritt, D.; Schwarzschild, M.

    1982-07-01

    Four closed periodic orbit sequences are determined numerically, and their stability is investigated by the standard Floquet method, for the case of a specific, triaxial rotating potential. The sequences comprise (1) stable anomalous orbits that are tipped to the long axis which they circle, so that they also circle the short rotation axis, (2) unstable, anomalous orbits circling the intermediate axis, otherwise behaving like (1), (3) stable, normal retrograde orbits lying in the equatorial plane, which become unstable against perpendicular perturbations in Binney's instability strip, and (4) Z-axis orbits lying on the rotation axis, which, although stable in their inner section, become unstable to perturbations parallel to the intermediate axis farther out, and to the long axis farther out still. The entire set contains one composite sequence which is stable over the entire energy range, consisting of the outer section of the normal retrograde orbits, the sequence of the anomalous orbits, and the inner section of the Z-axis orbits. It is suggested that the composite sequence may be relevant to the dynamics of gas masses captured by rotating triaxial galaxies.

  19. Quantitative radioactive analysis of microleakage of four different retrograde fillings.

    PubMed

    Danin, J; Linder, L; Sund, M L; Strömberg, T; Torstenson, B; Zetterqvist, L

    1992-07-01

    Sealing properties of four different retrograde filling materials were investigated in vitro. Radioactive isotopes were applied in the root canal, and leakage into an extraradicular fluid was measured at regular intervals. The method permitted repeated observation of the specimens over prolonged periods of time. Forty single-rooted human teeth were biomechanically instrumented and obturated using calcium-hydroxide paste. Following obturation, an apicectomy was performed and retrograde cavities were filled with four different materials: group 1, non gamma 2 amalgam (Amalcap); group 2, glass ionomer cement (Ketac Silver); group 3, calcium-hydroxide-based root canal sealer (Sealapex); group 4, composite resin (Palfique Light-S). After removal of the calcium hydroxide, the teeth were immersed in a fluid. An isotope solution was then placed in the root canals. Samples were taken from the fluid at 0, 3, 7, 28, 56, 105, 210, 285 and 376 days to determine the radioactivity. It was found that Sealapex and Palfique Light-S showed significantly less leakage than amalgam and glass ionomer cement, which had the highest apical leakage. PMID:1399068

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

    PubMed Central

    Curtis, Adam; Aitchison, Gregor; Tsapikouni, Theodora

    2006-01-01

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

  1. The retrograde limb of internal mammary vessels as reliable recipient vessels in DIEP flap breast reconstruction: a clinical and radiological study.

    PubMed

    Salgarello, Marzia; Visconti, Giuseppe; Barone-Adesi, Liliana; Cina, Alessandro

    2015-04-01

    For many microsurgeons, antegrade internal mammary vessels (AIMVs) represent the recipients of choice in autogenous breast reconstruction. For the past few years, the retrograde internal mammary vessels (RIMVs) have been demonstrated to be a further reliable option when needed, according to many papers focusing more on the vein than on the artery. Besides the clinical evidence, the hemodynamic features of the retrograde system have been very seldom analyzed.In this article, we report our clinical experience with deep inferior epigastric perforator (DIEP) flaps anastomosed to RIMVs, along with a perioperative radiological follow-up to study RIMVs' hemodynamics and to further support the reliability of the retrograde system with particular focus on the retrograde internal mammary artery.Prospective, preoperative, and postoperative (3 days, 21 days, and 3 months, respectively) color Doppler sonographies of the internal mammary artery (IMA) and DIEPs have been performed to collect the velocity of flow (v) and resistive index (RI) data. Twenty-two patients agreed to undergo this protocol, of which 10 unipedicle flaps were anastomosed to AIMVs ("control" group), 10 bipedicle DIEPs to both AIMVs and RIMVs ("study" group), and 2 DIEPs anastomosed to retrograde internal mammary artery and antegrade internal mammary vein (not statistically analyzed for their paucity). Student t test was performed to compare the "control" and "study" groups.All the flaps survived, and no re-exploration was needed. Internal mammary artery and perforators v showed similar but speculate trend, whereas IMA and perforators RI looked stable during that time. Significant differences have been found in the "study" group for IMA v at 3-day period, for perforator v at 21- and 90-day periods, and for perforator RI at 90-day period, without any clinical implication for flap viability.Retrograde internal mammary vessels can be considered reliable vessels for both arterial flap input and venous flap

  2. Geometrical and Mechanical Properties Control Actin Filament Organization

    PubMed Central

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

    2015-01-01

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

  3. Cytoplasmic Actin: Purification and Single Molecule Assembly Assays

    PubMed Central

    Hansen, Scott D.; Zuchero, J. Bradley; Mullins, R. Dyche

    2014-01-01

    The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy. PMID:23868587

  4. A complex gene superfamily encodes actin in petunia.

    PubMed Central

    Baird, W V; Meagher, R B

    1987-01-01

    We have shown by several independent criteria that actin is encoded by a very large and complex superfamily of genes in Petunia. Several cDNA and genomic probes encoding actins from diverse organisms (Dictyostelium, Drosophila, chicken and soybean) hybridize to hundreds of restriction fragments in the petunia genome. Actin-hybridizing sequences were isolated from a petunia genomic library at a rate of at least 200 per genome equivalent. Twenty randomly selected actin-hybridizing clones were characterized in more detail. DNA sequence data from four representative and highly divergent clones, PAc2, PAc3, PAc4 and PAc7, demonstrate that these actin-like sequences are related to functional actin genes. Intron positions typical of other known plant actin genes are conserved in these clones. Four of six clones analyzed (PAc1, PAc2, PAc3, PAc4) hybridize to leaf mRNA of the same size (1.7 kb) as that reported for other plant actin mRNAs and to a slightly smaller mRNA species (1.5 kb). Five distinct subfamilies of actin-related genes were characterized which varied in size from a few members to several dozen members. It is clear from our data that other actin gene subfamilies must also exist within the genome. Possible mechanisms of actin gene amplification and genome turnover are discussed. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3428258

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

    PubMed

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

    2015-05-01

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

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

    PubMed

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

    2013-01-01

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

  7. Bundling actin filaments from membranes: some novel players

    PubMed Central

    Thomas, Clément

    2012-01-01

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

  8. Tropomyosin diffusion over actin subunits facilitates thin filament assembly

    PubMed Central

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

    2016-01-01

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

  9. The Structural Basis of Actin Organization by Vinculin and Metavinculin.

    PubMed

    Kim, Laura Y; Thompson, Peter M; Lee, Hyunna T; Pershad, Mihir; Campbell, Sharon L; Alushin, Gregory M

    2016-01-16

    Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. Here we present an 8.5-Å-resolution cryo-electron microscopy reconstruction and pseudo-atomic model of the vinculin tail (Vt) domain bound to F-actin. Upon actin engagement, the N-terminal "strap" and helix 1 are displaced from the Vt helical bundle to mediate actin bundling. We find that an analogous conformational change also occurs in the H1' helix of the tail domain of metavinculin (MVt) upon actin binding, a muscle-specific splice isoform that suppresses actin bundling by Vt. These data support a model in which metavinculin tunes the actin bundling activity of vinculin in a tissue-specific manner, providing a mechanistic framework for understanding metavinculin mutations associated with hereditary cardiomyopathies. PMID:26493222

  10. The Potential Roles of Actin in The Nucleus

    PubMed Central

    Falahzadeh, Khadijeh; Banaei-Esfahani, Amir; Shahhoseini, Maryam

    2015-01-01

    Over the past few decades, actin’s presence in the nucleus has been demonstrated. Actin is a key protein necessary for different nuclear processes. Although actin is well known for its functional role in dynamic behavior of the cytoskeleton, emerging studies are now highlighting new roles for actin. At the present time there is no doubt about the presence of actin in the nucleus. A number of studies have uncovered the functional involvement of actin in nuclear processes. Actin as one of the nuclear components has its own structured and functional rules, such as nuclear matrix association, chromatin remodeling, transcription by RNA polymerases I, II, III and mRNA processing. In this historical review, we attempt to provide an overview of our current understanding of the functions of actin in the nucleus. PMID:25870830

  11. Regulation of cellular actin architecture by S100A10.

    PubMed

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

    2010-04-15

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

  12. Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

    PubMed Central

    Salinas, Patricia C.

    2015-01-01

    Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling. PMID:26252776

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

    NASA Astrophysics Data System (ADS)

    Blanpied, Thomas A.

    2013-03-01

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

  14. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  15. Anterograde and Retrograde Amnesia of Place Discrimination in Retrosplenial Cortex and Hippocampal Lesioned Rats

    ERIC Educational Resources Information Center

    Haijima, Asahi; Ichitani, Yukio

    2008-01-01

    Retrograde and anterograde amnesic effects of excitotoxic lesions of the rat retrosplenial cortex (RS) and hippocampus (HPC) were investigated. To test retrograde amnesia, rats were trained with two-arm place discrimination in a radial maze 4 wk and 1 d before surgery with a different arm pair, respectively. In the retention test 1 wk after…

  16. Isolation and characterization of six different chicken actin genes.

    PubMed Central

    Chang, K S; Zimmer, W E; Bergsma, D J; Dodgson, J B; Schwartz, R J

    1984-01-01

    Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene. Images PMID:6513927

  17. Actinic keratoses: past, present and future.

    PubMed

    Fenske, Neil Alan; Spencer, James; Adam, Friedman

    2010-05-01

    Actinic keratoses (AKs) are cutaneous neoplasms composed of proliferations of cytologically aberrant, epidermal keratinocytes caused by prolonged exposure to ultraviolet radiation. Combining the evidence that AKs are the second most common reason for visits to the dermatologist and it is generally believed that they should be treated, it is no surprise that the direct cost of the management of actinic keratoses in the United States (U.S.) is exceedingly high. There are currently numerous treatment modalities with more on the way as there is a demand for formulating newer, cheaper, less painful and less invasive means. The future of AK treatment involves both the continued investigation of current novel therapies, as well as the development of new treatment modalities. PMID:20518359

  18. Actin-mediated motion of meiotic chromosomes

    PubMed Central

    Koszul, R.; Kim, K. P.; Prentiss, M.; Kleckner, N.; Kameoka, S.

    2008-01-01

    Summary Chromosome movement is prominent during meiosis. Here, using a combination of in vitro and in vivo approaches, we elucidate the basis for dynamic mid-prophase chromosome movement in budding yeast. Diverse finding reveal a process in which, at the pachytene stage, individual telomere/nuclear envelope (NE) ensembles attach passively to, and then move in concert with, nucleus-hugging actin cables that are continuous with the global cytoskeletal actin network. Other chromosomes move in concert with lead chromosome(s). The same process, in modulated form, explains the zygotene "bouquet" configuration in which, immediately preceding pachytene, chromosome ends colocalize dynamically in a restricted region of the NE. Mechanical properties of the system and biological roles of mid-prophase movement for meiosis, including recombination, are discussed. PMID:18585353

  19. Regulation of actin nucleation and autophagosome formation.

    PubMed

    Coutts, Amanda S; La Thangue, Nicholas B

    2016-09-01

    Autophagy is a process of self-eating, whereby cytosolic constituents are enclosed by a double-membrane vesicle before delivery to the lysosome for degradation. This is an important process which allows for recycling of nutrients and cellular components and thus plays a critical role in normal cellular homeostasis as well as cell survival during stresses such as starvation or hypoxia. A large number of proteins regulate various stages of autophagy in a complex and still incompletely understood series of events. In this review, we will discuss recent studies which provide a growing body of evidence that actin dynamics and proteins that influence actin nucleation play an important role in the regulation of autophagosome formation and maturation. PMID:27147468

  20. Caffeine relaxes smooth muscle through actin depolymerization.

    PubMed

    Tazzeo, Tracy; Bates, Genevieve; Roman, Horia Nicolae; Lauzon, Anne-Marie; Khasnis, Mukta D; Eto, Masumi; Janssen, Luke J

    2012-08-15

    Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using β-escin and depleting the internal Ca(2+) store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca(2+), suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle. PMID:22683573

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2011-01-01

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

  8. Left Subclavian Artery Occlusion: Femoro-Axillary Artery Retrograde Bypass

    PubMed Central

    Nakashima, Masaya; Kobayashi, Hideaki; Kobayashi, Masayoshi

    2016-01-01

    The treatment tactics for subclavian artery occlusion include the more commonly used endovascular therapy rather than surgical intervention. We present a case of a 61-year-old woman with dialysis-dependent chronic renal failure who experienced left finger necrosis in the left upper extremity. To salvage the limb, we performed femoro-axillary (fem-ax) artery bypass using an autologous saphenous vein graft. However, 10 months later, she experienced coldness in the left forearm. Angiography revealed chronic total occlusion of the venous bypass. Despite emergent thrombectomy, redo fem-ax artery bypass operation was performed using a prosthetic graft. Upper limb salvage can be achieved by fem-ax artery retrograde bypass. PMID:27386454

  9. Emotion-induced retrograde amnesia and trait anxiety.

    PubMed

    Miu, Andrei C; Heilman, Renata M; Opre, Adrian; Miclea, Mircea

    2005-11-01

    Emotional arousal can both enhance and impair memory. Considering that both emotional memory and trait anxiety (TA) have been associated with adrenergic activity, the authors investigated whether there is an association between 2 opposite emotional memory biases and the TA. The authors used a procedure recently put forward by B. A. Strange, R. Hurlemann, and R. J. Dolan (2003) to elicit an emotion-induced retrograde amnesia (ERA) coupled to an emotional memory enhancement (EME). The authors contrasted the association between these emotional memory biases and the TA in several conditions involving different levels of encoding and types of recall. The results presented here indicated a significant interaction of the TA with EME and ERA and the dependency of these biases on the consciously controlled use of memory. PMID:16393044

  10. Inhibition of wheat starch retrogradation by tea derivatives.

    PubMed

    Zhang, Haihua; Sun, Binghua; Zhang, Shikang; Zhu, Yuejin; Tian, Yaoqi

    2015-12-10

    The effect of four industrial tea derivatives (tea polyphenols [TPS], tea water-soluble extracts [TSE], tea polysaccharides [TSS], and green tea powder [GTP]), on the retrogradation of wheat starch was investigated using texture profile analysis (TPA), differential scanning calorimetry (DSC), rapid viscosity analysis (RVA), and the α-amylase-iodine method. The addition of the four tea derivatives resulted in decreased hardness and increased cohesiveness of the starch gel as shown by the TPA test. The DSC data demonstrated an increase in the enthalpy change of starch gelatinization and a decrease in the enthalpy change of starch recrystallite dissociation. The RVA results indicated that the peak viscosity, representing the intermolecular forces of wheat starch, was reduced after addition of TPS, TSE, and TSS, respectively, but was increased by GTP. Furthermore, the half crystallization time in the Avrami equation almost doubled after the separate addition of the tea derivatives. PMID:26428142

  11. Retrograde nailing for distal femur fractures in the elderly

    PubMed Central

    Giddie, Jasdeep; Sawalha, Seif; Parker, Martyn

    2015-01-01

    Introduction: We report the results of treating a series of 56 fractures in 54 elderly patients with a distal femur fracture with a retrograde femoral nail. Methods: Fifty-four of the nails were inserted percutaneously with a closed reduction. After surgery all patients were allowed to weight bear as tolerated. Four fractures were supported in a temporary external splint. Results: The mean age of patients was 80.6 years (range 51–103 years), 52/54 (96%) were females. There were no cases of nail related complications and no re-operations were required. One patient was lost to follow up. The 30-day mortality was 5/54 (9.3%) and the one year mortality was 17/54 (31.5%). Conclusions: Distal femoral nail fixation provides a good method of fixation allowing immediate mobilisation for this group of patients. PMID:27163086

  12. CONFIRMATION OF A RETROGRADE ORBIT FOR EXOPLANET WASP-17b

    SciTech Connect

    Bayliss, Daniel D. R.; Sackett, Penny D.; Winn, Joshua N.; Mardling, Rosemary A.

    2010-10-20

    We present high-precision radial velocity observations of WASP-17 throughout the transit of its close-in giant planet, using the MIKE spectrograph on the 6.5 m Magellan Telescope at Las Campanas Observatory. By modeling the Rossiter-McLaughlin effect, we find the sky-projected spin-orbit angle to be {lambda} = 167.4 {+-} 11.2 deg. This independently confirms the previous finding that WASP-17b is on a retrograde orbit, suggesting it underwent migration via a mechanism other than just the gravitational interaction between the planet and the disk. Interestingly, our result for {lambda} differs by 45 {+-} 13 deg from the previously announced value, and we also find that the spectroscopic transit occurs 15 {+-} 5 minutes earlier than expected, based on the published ephemeris. The discrepancy in the ephemeris highlights the need for contemporaneous spectroscopic and photometric transit observations whenever possible.

  13. Gelsolin mediates calcium-dependent disassembly of Listeria actin tails

    PubMed Central

    Larson, Laura; Arnaudeau, Serge; Gibson, Bruce; Li, Wei; Krause, Ryoko; Hao, Binghua; Bamburg, James R.; Lew, Daniel P.; Demaurex, Nicolas; Southwick, Frederick

    2005-01-01

    The role of intracellular Ca2+ in the regulation of actin filament assembly and disassembly has not been clearly defined. We show that reduction of intracellular free Ca2+ concentration ([Ca2+]i) to <40 nM in Listeria monocytogenes-infected, EGFP–actin-transfected Madin–Darby canine kidney cells results in a 3-fold lengthening of actin filament tails. This increase in tail length is the consequence of marked slowing of the actin filament disassembly rate, without a significant change in assembly rate. The Ca2+-sensitive actin-severing protein gelsolin concentrates in the Listeria rocket tails at normal resting [Ca2+]i and disassociates from the tails when [Ca2+]i is lowered. Reduction in [Ca2+]i also blocks the severing activity of gelsolin, but not actin-depolymerizing factor (ADF)/cofilin microinjected into Listeria-infected cells. In Xenopus extracts, Listeria tail lengths are also calcium-sensitive, markedly shortening on addition of calcium. Immunodepletion of gelsolin, but not Xenopus ADF/cofilin, eliminates calcium-sensitive actin-filament shortening. Listeria tail length is also calcium-insensitive in gelsolin-null mouse embryo fibroblasts. We conclude that gelsolin is the primary Ca2+-sensitive actin filament recycling protein in the cell and is capable of enhancing Listeria actin tail disassembly at normal resting [Ca2+]i (145 nM). These experiments illustrate the unique and complementary functions of gelsolin and ADF/cofilin in the recycling of actin filaments. PMID:15671163

  14. The neuronal and actin commitment: Why do neurons need rings?

    PubMed

    Leite, Sérgio Carvalho; Sousa, Mónica Mendes

    2016-09-01

    The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc. PMID:26784007

  15. Sequence and comparative genomic analysis of actin-related proteins.

    PubMed

    Muller, Jean; Oma, Yukako; Vallar, Laurent; Friederich, Evelyne; Poch, Olivier; Winsor, Barbara

    2005-12-01

    Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4. PMID:16195354

  16. Actin is required for IFT regulation in Chlamydomonas reinhardtii

    PubMed Central

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

    2014-01-01

    Summary Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Since actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here, we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation, and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor suggesting actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length. PMID:25155506

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

    PubMed

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

    2014-09-01

    Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length. PMID:25155506

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

    PubMed

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

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition. PMID:26240174

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

    PubMed Central

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

    2006-01-01

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

  20. Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.

    PubMed

    Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo

    2014-10-15

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase. PMID:25131196

  1. Dissociation of F-actin induced by hydrostatic pressure.

    PubMed

    Garcia, C R; Amaral Júnior, J A; Abrahamsohn, P; Verjovski-Almeida, S

    1992-11-01

    F-actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N-(1-pyrenyl)iodoacetamide-labeled actin were monitored; electron microscopy of samples fixed under pressure; size-exclusion HPLC of pressurized actin. The effect of pressure upon F-actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 +/- 14 ml/mol for Mg-F-actin, 79 +/- 12 ml/mol for Ca-F-actin and 328 +/- 63 ml/mol for K-F-actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F-actin filament there is a predominant intersubunit interaction interface, most likely the head-to-tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 micrograms to 200 micrograms/ml Mg-F-actin did not cause a change in the pressure required for half-maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg-F-actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits. PMID:1425683

  2. Comparison of antegrade and retrograde laparoscopic radical prostatectomy techniques.

    PubMed

    Tugcu, Volkan; Sahin, Selcuk; Resorlu, Berkan; Yigitbasi, Ismail; Yavuzsan, Abdullah H; Tasci, Ali I

    2016-08-01

    We evaluated the effect of antegrade and retrograde approaches on functional recovery and surgical outcomes of extraperitoneal laparoscopic radical prostatectomy (LRP). We analyzed 135 patients who underwent extraperitoneal LRP, with the retrograde technique performed on 42 (31%; Group 1) and the antegrade technique on 93 (69%; Group 2). Both groups were statistically similar with respect to age, clinical stage, preoperative prostate-specific antigen (PSA) and American Society of Anesthesiologists (ASA) scores, prostate volume, and previous surgical history. Mean operative time was significantly longer in Group 1 (244±18.3 vs. 203.3±18.4 min, p<0.001), whereas mean anastomosis times for both groups were similar (35.8±7.2 vs. 34.7±5.8 min, p=0.155). Estimated blood loss and transfusion rates were significantly lower in Group 2. A significant difference was observed for both hospitalization (6.79±3.3 vs. 5.46±3.08 days, respectively; p=0.026) and catheterization times (12.24±2.1 vs. 11±1.08 days, respectively; p=0.001) for Group 2. The total complication rate was 47.6% in Group 1, and 11.8% in Group 2 (p<0.01). Rates of positive surgical margins were 14.2% and 15% for Groups 1 and 2, respectively. At the 12-month interval from operation, similar recoveries in urinary continence were obtained for both groups (81% in Group 1; 91% in Group 2). Upon comparison of the two LRP techniques, we found that both were effective; however, the latter resulted in lower minor complication rate, lower blood loss, shorter operation time, and shorter length of hospital stay. PMID:27523453

  3. Primary Retrograde Tibiotalocalcaneal Nailing For Fragility Ankle Fractures

    PubMed Central

    Taylor, Benjamin C.; Hansen, Dane C.; Harrison, Ryan; Lucas, Douglas E; Degenova, Daniel

    2016-01-01

    Background Ankle fragility fractures are difficult to treat due to poor bone quality and soft tissues as well as the near ubiquitous presence of comorbidities including diabetes mellitus and peripheral neuropathy. Conventional open reduction and internal fixation in this population has been shown to lead to a significant rate of complications. Given the high rate of complications with contemporary fixation methods, the present study aims to critically evaluate the use of acute hindfoot nailing as a percutaneous fixation technique for high-risk ankle fragility fractures. Methods In this study, we retrospectively evaluated 31 patients treated with primary retrograde tibiotalocalcaneal nail without joint preparation for a mean of 13.6 months postoperatively from an urban Level I trauma center during the years 2006-2012. Results Overall, there were two superficial infections (6.5%) and three deep infections (9.7%) in the series. There were 28 (90.3%) patients that went on to radiographic union at a mean of 22.2 weeks with maintenance of foot and ankle alignment. There were three cases of asymptomatic screw breakage observed at a mean of 18.3 months postoperatively, which were all treated conservatively.. Conclusions This study shows that retrograde hindfoot nailing is an acceptable treatment option for treatment of ankle fragility fractures. Hindfoot nailing allows early weightbearing, limited soft tissue injury, and a relatively low rate of complications, all of which are advantages to conventional open reduction internal fixation techniques. Given these findings, larger prospective randomized trials comparing this treatment with conventional open reduction internal fixation techniques are warranted. PMID:27528840

  4. Fibroblast-mediated contraction in actinically exposed and actinically protected aging skin

    SciTech Connect

    Marks, M.W.; Morykwas, M.J.; Wheatley, M.J. )

    1990-08-01

    The changes in skin morphology over time are a consequence of both chronologic aging and the accumulation of environmental exposure. Through observation, we know that actinic radiation intensifies the apparent aging of skin. We have investigated the effects of aging and actinic radiation on the ability of fibroblasts to contract collagen-fibroblast lattices. Preauricular and postauricular skin samples were obtained from eight patients aged 49 to 74 undergoing rhytidectomy. The samples were kept separate, and the fibroblasts were grown in culture. Lattices constructed with preauricular fibroblasts consistently contracted more than lattices containing postauricular fibroblasts. The difference in amount of contraction in 7 days between sites was greatest for the younger patients and decreased linearly as donor age increased (r = -0.96). This difference may be due to preauricular fibroblasts losing their ability to contract a lattice as aging skin is exposed to more actinic radiation.

  5. Structure of the F-actin-tropomyosin complex.

    PubMed

    von der Ecken, Julian; Müller, Mirco; Lehman, William; Manstein, Dietmar J; Penczek, Pawel A; Raunser, Stefan

    2015-03-01

    Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted

  6. Actin depolymerisation and crosslinking join forces with myosin II to contract actin coats on fused secretory vesicles.

    PubMed

    Miklavc, Pika; Ehinger, Konstantin; Sultan, Ayesha; Felder, Tatiana; Paul, Patrick; Gottschalk, Kay-Eberhard; Frick, Manfred

    2015-03-15

    In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and α-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by α-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion. PMID:25637593

  7. Cortical actin regulation modulates vascular contractility and compliance in veins

    PubMed Central

    Saphirstein, Robert J; Gao, Yuan Z; Lin, Qian Qian; Morgan, Kathleen G

    2015-01-01

    Abstract The literature on arterial mechanics is extensive, but far less is known about mechanisms controlling mechanical properties of veins. We use here a multi-scale approach to identify subcellular sources of venous stiffness. Portal vein tissue displays a severalfold decrease in passive stiffness compared to aortic tissues. The α-adrenergic agonist phenylephrine (PE) increased tissue stress and stiffness, both attenuated by cytochalasin D (CytoD) and PP2, inhibitors of actin polymerization and Src activity, respectively. We quantify, for the first time, cortical cellular stiffness in freshly isolated contractile vascular smooth muscle cells using magnetic microneedle technology. Cortical stiffness is significantly increased by PE and CytoD inhibits this increase but, surprisingly, PP2 does not. No detectable change in focal adhesion size, measured by immunofluorescence of FAK and zyxin, accompanies the PE-induced changes in cortical stiffness. Probing with phospho-specific antibodies confirmed activation of FAK/Src and ERK pathways and caldesmon phosphorylation. Thus, venous tissue stiffness is regulated both at the level of the smooth muscle cell cortex, via cortical actin polymerization, and by downstream smooth muscle effectors of Src/ERK signalling pathways. These findings identify novel potential molecular targets for the modulation of venous capacitance and venous return in health and disease. Key points Most cardiovascular research focuses on arterial mechanisms of disease, largely ignoring venous mechanisms. Here we examine ex vivo venous stiffness, spanning tissue to molecular levels, using biomechanics and magnetic microneedle technology, and show for the first time that venous stiffness is regulated by a molecular actin switch within the vascular smooth muscle cell in the wall of the vein. This switch connects the contractile apparatus within the cell to adhesion structures and facilitates stiffening of the vessel wall, regulating blood flow return

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

    PubMed

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

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

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

    PubMed Central

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

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

  10. Selective biliary cannulation techniques for endoscopic retrograde cholangiopancreatography procedures and prevention of post- endoscopic retrograde cholangiopancreatography pancreatitis.

    PubMed

    Mukai, Shuntaro; Itoi, Takao

    2016-06-01

    Numerous endoscopic retrograde cholangiopancreatography (ERCP) techniques have been reported to achieve selective biliary cannulation success. For standard biliary cannulation procedures, the wire-guided cannulation technique has been reported to reduce the rate of post-ERCP pancreatitis (PEP) and increase the biliary cannulation success rate, although conflicting reports exist. The pancreatic or double-guidewire technique and several precut techniques have been reported as useful techniques in difficult biliary cannulation cases. Although ERCP is a useful endoscopic procedure, the risk of adverse events, particularly post-ERCP pancreatitis, is inevitable. Previous studies and analyses have revealed the risk factors for PEP. The efficacy of prophylactic pancreatic duct stent placement and the administration of rectal nonsteroidal anti-inflammatory drugs for preventing PEP has also been reported. Herein, we reviewed reports in the literature regarding the current status of selective biliary cannulation techniques and PEP prevention. PMID:26782710

  11. Transfer of a Redox-Signal through the Cytosol by Redox-Dependent Microcompartmentation of Glycolytic Enzymes at Mitochondria and Actin Cytoskeleton

    PubMed Central

    Wojtera-Kwiczor, Joanna; Groß, Felicitas; Leffers, Hans-Martin; Kang, Minhee; Schneider, Markus; Scheibe, Renate

    2013-01-01

    The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12, GapC) plays an important role in glycolysis by providing the cell with ATP and NADH. Interestingly, despite its glycolytic function in the cytosol, GAPDH was reported to possess additional non-glycolytic activities, correlating with its nuclear, or cytoskeletal localization in animal cells. In transiently transformed mesophyll protoplasts from Arabidopsis thaliana colocalization and interaction of the glycolytic enzymes with the mitochondria and with the actin cytoskeleton was visualized by confocal laser scanning microscopy (cLSM) using fluorescent protein fusions and by bimolecular fluorescence complementation, respectively. Yeast two-hybrid screens, dot-blot overlay assays, and co-sedimentation assays were used to identify potential protein–protein interactions between two cytosolic GAPDH isoforms (GapC1, At3g04120; GapC2, At1g13440) from A. thaliana with the neighboring glycolytic enzyme, fructose 1,6-bisphosphate aldolase (FBA6, At2g36460), the mitochondrial porin (VDAC3; At5g15090), and actin in vitro. From these experiments, a mitochondrial association is suggested for both glycolytic enzymes, GAPDH and aldolase, which appear to bind to the outer mitochondrial membrane, in a redox-dependent manner. In addition, both glycolytic enzymes were found to bind to F-actin in co-sedimentation assays, and lead to bundling of purified rabbit actin, as visualized by cLSM. Actin-binding and bundling occurred reversibly under oxidizing conditions. We speculate that such dynamic formation of microcompartments is part of a redox-dependent retrograde signal transduction network for adaptation upon oxidative stress. PMID:23316205

  12. Spatial control of actin polymerization during neutrophil chemotaxis

    PubMed Central

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

    2010-01-01

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

  13. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  14. Effect of pullulan on the short-term and long-term retrogradation of rice starch.

    PubMed

    Chen, Long; Ren, Fei; Zhang, Zipei; Tong, Qunyi; Rashed, Marwan M A

    2015-01-22

    The effect of pullulan (PUL) on the retrogradation of rice starch (RS) was investigated by means of rapid visco-analyzer (RVA), rotational rheometer, differential scanning calorimetry (DSC), and X-ray diffraction (XRD). RVA results showed that addition of pullulan significantly decreased the breakdown and setback values, which meant that the short-term retrogradation of RS was inhibited. The dynamic time sweep of samples also proved the retarding effect of pullulan on the retrogradation of RS. DSC curves showed clearly that pullulan significantly reduced the retrogradation enthalpy of amylopectin, and the kinetics of retrogradation was analyzed using the Avrami model. XRD results showed that recrystallinity of RS was reduced from 11.565% to 8.841% with the addition of pullulan and this was in line with the DSC results. It could be concluded that the addition of pullulan apparently influenced not only the short-term retrogradation of amylose, but also the long-term retrogradation of amylopectin. PMID:25439913

  15. Identification of the main retrogradation-related properties of rice starch.

    PubMed

    Lian, Xijun; Kang, Haiqi; Sun, Haibo; Liu, Lizeng; Li, Lin

    2015-02-11

    The retrogradation of rice in shelf life is the biggest barrier to the industrial production of traditional foods using rice as material. Many rice breeders have tried their best to screen low-retrogradation rice cultivars without a specific indicator. To identify the main retrogradation-related properties of rice, the starch, amylose, and amylopectin from 16 rice cultivars were extracted from rice powder and their physicochemical properties, such as visible absorbance, infrared, average molecule weight (amylopectin), chain-length distribution (amylopectin), X-ray diffraction, and differential scanning calorimetry, were determined. The correlation between starch retrogradation rates and those physicochemical properties was investigated. The results show that a significant positive correlation (R(2) = 0.85; r = 0.926; p < 0.01) exists only between proportions of the chains [degree of polymerization (DP) > 10] in amylopectin and the retrogradation rates of different rice starches. The findings in the paper offer a shortcut for rice breeders to screen cultivars with a low retrogradation rate. Because the genes related to the branching enzyme control the DP of amylopectin, they can be exploited as molecular markers to screen low-retrogradation rice cultivars. PMID:25615262

  16. Electrophoresis and orientation of F-actin in agarose gels.

    PubMed Central

    Borejdo, J; Ortega, H

    1989-01-01

    F-Actin was electrophoresed on agarose gels. In the presence of 2 mM MgCl2 and above pH 8.5 F-actin entered 1% agarose; when the electric field was 2.1 V/cm and the pH was 8.8, F-actin migrated through a gel as a single band at a rate of 2.5 mm/h. Labeling of actin with fluorophores did not affect its rate of migration, but an increase in ionic strength slowed it down. After the electrophoresis actin was able to bind phalloidin and heavy meromyosin (HMM) and it activated Mg2+-dependent ATPase activity of HMM. The mobility of F-actin increased with the rise in pH. Acto-S-1 complex was also able to migrate in agarose at basic pH, but at a lower rate than F-actin alone. The orientation of fluorescein labeled F-actin and of fluorescein labeled S-1 which formed rigor bonds with F-actin was measured during the electrophoresis by the fluorescence detected linear dichroism method. The former showed little orientation, probably because the dye was mobile on the surface of actin, but we were able to measure the orientation of the absorption dipole of the dye bound to S-1 which was attached to F-actin, and found that it assumed an orientation largely parallel to the direction of the electric field. These results show that actin can migrate in agarose gels in the F form and that it is oriented during the electrophoresis. Images FIGURE 1 FIGURE 3 FIGURE 4 PMID:2528384

  17. Measuring F-actin properties in dendritic spines

    PubMed Central

    Koskinen, Mikko; Hotulainen, Pirta

    2014-01-01

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

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

    PubMed

    Schroeter, Mechthild; Chalovich, Joseph M

    2004-11-01

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

  19. RAB-6.1 and RAB-6.2 Promote Retrograde Transport in C. elegans

    PubMed Central

    Zhang, Donglei; Dubey, Jyoti; Koushika, Sandhya P.; Rongo, Christopher

    2016-01-01

    Retrograde transport is a critical mechanism for recycling certain membrane cargo. Following endocytosis from the plasma membrane, retrograde cargo is moved from early endosomes to Golgi followed by transport (recycling) back to the plasma membrane. The complete molecular and cellular mechanisms of retrograde transport remain unclear. The small GTPase RAB-6.2 mediates the retrograde recycling of the AMPA-type glutamate receptor (AMPAR) subunit GLR-1 in C. elegans neurons. Here we show that RAB-6.2 and a close paralog, RAB-6.1, together regulate retrograde transport in both neurons and non-neuronal tissue. Mutants for rab-6.1 or rab-6.2 fail to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. Loss of both rab-6.1 and rab-6.2 results in an additive effect on GLR-1 retrograde recycling, indicating that these two C. elegans Rab6 isoforms have overlapping functions. MIG-14 (Wntless) protein, which undergoes retrograde recycling, undergoes a similar degradation in intestinal epithelia in both rab-6.1 and rab-6.2 mutants, suggesting a broader role for these proteins in retrograde transport. Surprisingly, MIG-14 is localized to separate, spatially segregated endosomal compartments in rab-6.1 mutants compared to rab-6.2 mutants. Our results indicate that RAB-6.1 and RAB-6.2 have partially redundant functions in overall retrograde transport, but also have their own unique cellular- and subcellular functions. PMID:26891225

  20. A Robust Actin Filaments Image Analysis Framework.

    PubMed

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

    2016-08-01

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

  1. A Robust Actin Filaments Image Analysis Framework

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-04-01

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

  3. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  4. Structure and Mechanics of Actin Cortex Contained in Vesicles

    NASA Astrophysics Data System (ADS)

    Limozin, Laurent; Roth, Alexander; Sackmann, Erich

    2003-03-01

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

  5. Force Generation by Endocytic Actin Patches in Budding Yeast

    PubMed Central

    Carlsson, Anders E.; Bayly, Philip V.

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2008-08-15

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

  8. VASP Governs Actin Dynamics by Modulating Filament Anchoring

    PubMed Central

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

    2007-01-01

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

  9. Solubilization of native actin monomers from human erythrocyte membranes.

    PubMed

    Tilley, L; Dwyer, M; Ralston, G B

    1986-01-01

    Up to 50% of the actin in erythrocyte membranes can be solubilized at low ionic strength in a form capable of inhibiting DNAse I, in the presence of 0.4 mM ATP and 0.05 mM calcium. In the absence of calcium and ATP, actin is released but is apparently rapidly denatured. Solubilization of G-actin increases with temperature up to 37 degrees C. At higher temperatures, actin is released rapidly but quickly loses its ability to inhibit DNAse I. PMID:3789986

  10. Electrostatics control actin filament nucleation and elongation kinetics.

    PubMed

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

    2013-04-26

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

  11. Retrograde mechanochemical ablation of the small saphenous vein for the treatment of a venous ulcer.

    PubMed

    Moore, Hayley M; Lane, Tristan R A; Franklin, Ian J; Davies, Alun H

    2014-10-01

    We present the first case of retrograde ablation of the small saphenous vein to treat active venous ulceration. A 73-year-old gentleman with complicated varicose veins of the left leg and a non-healing venous ulcer despite previous successful endovenous treatment to his left great saphenous vein underwent mechanochemical ablation of his small saphenous vein with the ClariVein® system, under local anaesthetic, using a retrograde cannulation technique. Post-operatively the patient had improved symptomatically and the ulcer size had reduced. This report highlights that patients with small saphenous vein incompetence and active ulceration can be treated successfully with retrograde mechanochemical ablation. PMID:24347131

  12. TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport.

    PubMed

    Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R; Mans, Dorus A; Huber, Celine; Boldt, Karsten; Patel, Mitali; van Reeuwijk, Jeroen; Plaza, Jean-Marc; van Beersum, Sylvia E C; Yap, Zhi Min; Letteboer, Stef J F; Taylor, S Paige; Herridge, Warren; Johnson, Colin A; Scambler, Peter J; Ueffing, Marius; Kayserili, Hulya; Krakow, Deborah; King, Stephen M; Beales, Philip L; Al-Gazali, Lihadh; Wicking, Carol; Cormier-Daire, Valerie; Roepman, Ronald; Mitchison, Hannah M; Witman, George B

    2015-01-01

    The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions. PMID:26044572

  13. TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport

    PubMed Central

    Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R.; Mans, Dorus A.; Huber, Celine; Boldt, Karsten; Patel, Mitali; van Reeuwijk, Jeroen; Plaza, Jean-Marc; van Beersum, Sylvia E. C.; Yap, Zhi Min; Letteboer, Stef J. F.; Taylor, S. Paige; Herridge, Warren; Johnson, Colin A.; Scambler, Peter J.; Ueffing, Marius; Kayserili, Hulya; Krakow, Deborah; King, Stephen M.; Beales, Philip L.; Al-Gazali, Lihadh; Wicking, Carol; Cormier-Daire, Valerie; Roepman, Ronald; Mitchison, Hannah M.; Witman, George B.; Al-Turki, Saeed; Anderson, Carl; Anney, Richard; Antony, Dinu; Asimit, Jennifer; Ayub, Mohammad; Barrett, Jeff; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Blackwood, Douglas; Bobrow, Martin; Bochukova, Elena; Bolton, Patrick; Boustred, Chris; Breen, Gerome; Brion, Marie-Jo; Brown, Andrew; Calissano, Mattia; Carss, Keren; Chatterjee, Krishna; Chen, Lu; Cirak, Sebhattin; Clapham, Peter; Clement, Gail; Coates, Guy; Collier, David; Cosgrove, Catherine; Cox, Tony; Craddock, Nick; Crooks, Lucy; Curran, Sarah; Daly, Allan; Danecek, Petr; Smith, George Davey; Day-Williams, Aaron; Day, Ian; Durbin, Richard; Edkins, Sarah; Ellis, Peter; Evans, David; Farooqi, I. Sadaf; Fatemifar, Ghazaleh; Fitzpatrick, David; Flicek, Paul; Floyd, Jamie; Foley, A. Reghan; Franklin, Chris; Futema, Marta; Gallagher, Louise; Gaunt, Tom; Geschwind, Daniel; Greenwood, Celia; Grozeva, Detelina; Guo, Xiaosen; Gurling, Hugh; Hart, Deborah; Hendricks, Audrey; Holmans, Peter; Huang, Jie; Humphries, Steve E.; Hurles, Matt; Hysi, Pirro; Jackson, David; Jamshidi, Yalda; Jewell, David; Chris, Joyce; Kaye, Jane; Keane, Thomas; Kemp, John; Kennedy, Karen; Kent, Alastair; Kolb-Kokocinski, Anja; Lachance, Genevieve; Langford, Cordelia; Lee, Irene; Li, Rui; Li, Yingrui; Ryan, Liu; Lönnqvist, Jouko; Lopes, Margarida; MacArthur, Daniel G.; Massimo, Mangino; Marchini, Jonathan; Maslen, John; McCarthy, Shane; McGuffin, Peter; McIntosh, Andrew; McKechanie, Andrew; McQuillin, Andrew; Memari, Yasin; Metrustry, Sarah; Min, Josine; Moayyeri, Alireza; Morris, James; Muddyman, Dawn; Muntoni, Francesco; Northstone, Kate; O'Donovan, Michael; O'Rahilly, Stephen; Onoufriadis, Alexandros; Oualkacha, Karim; Owen, Michael; Palotie, Aarno; Panoutsopoulou, Kalliope; Parker, Victoria; Parr, Jeremy; Paternoster, Lavinia; Paunio, Tiina; Payne, Felicity; Perry, John; Pietilainen, Olli; Plagnol, Vincent; Quail, Michael A.; Quaye, Lydia; Raymond, Lucy; Rehnström, Karola; Brent Richards, J.; Ring, Sue; Ritchie, Graham R S; Savage, David B.; Schoenmakers, Nadia; Semple, Robert K.; Serra, Eva; Shihab, Hashem; Shin, So-Youn; Skuse, David; Small, Kerrin; Smee, Carol; Soler, Artigas María; Soranzo, Nicole; Southam, Lorraine; Spector, Tim; St Pourcain, Beate; St. Clair, David; Stalker, Jim; Surdulescu, Gabriela; Suvisaari, Jaana; Tachmazidou, Ioanna; Tian, Jing; Timpson, Nic; Tobin, Martin; Valdes, Ana; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Wain, Louise; Walter, Klaudia; Wang, Jun; Ward, Kirsten; Wheeler, Ellie; Whittall, Ros; Williams, Hywel; Williamson, Kathy; Wilson, Scott G.; Wong, Kim; Whyte, Tamieka; ChangJiang, Xu; Zeggini, Eleftheria; Zhang, Feng; Zheng, Hou-Feng

    2015-01-01

    The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions. PMID:26044572

  14. Retrograde Proximal Anterior Tibial Artery Access for Treating Femoropopliteal Segment Occlusion: A Novel Approach.

    PubMed

    Affonso, Breno Boueri; Golghetto Domingos, Fernanda Uchiyama; da Motta Leal Filho, Joaquim Maurício; Maciel, Macello José Sampaio; Cavalcante, Rafael Noronha; Bortolini, Edgar; Carnevale, Francisco Cesar

    2016-05-01

    Some challenges have been detected when there are long and complex lesions of femoropopliteal arterial occlusive disease, even with descriptions of the retrograde pedal approaches. The aim of this article is to describe the retrograde proximal anterior tibial artery access for treatment of femoropopliteal segment occlusion when antegrade recanalization failed (retrograde recanalization and rearranging the system into an antegrade position). Technical and clinical success was achieved in 100% of 4 cases, with an improvement of at least 2 Rutherford classes. Minor complication, small hematoma in an anterior compartment of the limb, occurred in 1 patient. No sign of compartmental syndrome was observed. PMID:26902943

  15. Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

    PubMed

    Walther, Christa G; Whitfield, Robert; James, David C

    2016-04-01

    The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions. PMID:26679704

  16. Circulating Complexes of the Vitamin D Binding Protein with G-Actin Induce Lung Inflammation by Targeting Endothelial Cells

    PubMed Central

    Ge, Lingyin; Trujillo, Glenda; Miller, Edmund J.; Kew, Richard R.

    2013-01-01

    This study investigated the actin scavenger function of the vitamin D binding protein (DBP) in vivo using DBP null (−/−) mice. Intravenous injection of G-actin into wild-type (DBP+/+) and DBP−/− mice showed that contrary to expectations, DBP+/+ mice developed more severe acute lung inflammation. Inflammation was restricted to the lung and pathological changes were clearly evident at 1.5 and 4 hours post-injection but were largely resolved by 24 hours. Histology of DBP+/+ lungs revealed noticeably more vascular leakage, hemorrhage and thickening of the alveolar wall. Flow cytometry analysis of whole lung homogenates showed significantly increased neutrophil infiltration into DBP+/+ mouse lungs at 1.5 and 4 hours. Increased amounts of protein and leukocytes were also noted in bronchoalveolar lavage fluid from DBP+/+ mice 4 hours after actin injection. In vitro, purified DBP-actin complexes did not activate complement or neutrophils but induced injury and death of cultured human lung microvascular endothelial cells (HLMVEC) and human umbilical vein endothelial cells (HUVEC). Cells treated with DBP-actin showed a significant reduction in viability at 4 hours, this effect was reversible if cells were cultured in fresh media for another 24 hours. However, a 24-hour treatment with DBP-actin complexes showed a significant increase in cell death (95% for HLMVEC, 45% for HUVEC). The mechanism of endothelial cell death was via both caspase-3 dependent (HUVEC) and independent (HLMVEC) pathways. These results demonstrate that elevated levels and/or prolonged exposure to DBP-actin complexes may induce endothelial cell injury and death, particularly in the lung microvasculature. PMID:24268110

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

    PubMed Central

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

    2014-01-01

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

  18. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

    PubMed Central

    Bonello, Teresa T.; Janco, Miro; Hook, Jeff; Byun, Alex; Appaduray, Mark; Dedova, Irina; Hitchcock-DeGregori, Sarah; Hardeman, Edna C.; Stehn, Justine R.; Böcking, Till; Gunning, Peter W.

    2016-01-01

    The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development. PMID:26804624

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2006-12-18

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

  1. Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms*

    PubMed Central

    Yamashiro, Sawako; Gokhin, David S.; Sui, Zhenhua; Bergeron, Sarah E.; Rubenstein, Peter A.; Fowler, Velia M.

    2014-01-01

    Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ∼5-fold more weakly than Tmod1 to α/βTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (αsk-actin) filaments coated with α/βTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated αsk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH2 terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet β/cytoplasmic γ (γcyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate β/γcyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic β- and γcyto-actin (but not αsk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-αsk-actin or TM-β/γcyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester β- and γcyto-actin (but not αsk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells. PMID:24644292

  2. Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas.

    PubMed

    Onishi, Masayuki; Pringle, John R; Cross, Frederick R

    2016-03-01

    Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses. PMID:26715672

  3. Isolation of Functional Golgi-derived Vesicles with a Possible Role in Retrograde Transport

    PubMed Central

    Love, Harold D.; Lin, Chung-Chih; Short, Craig S.; Ostermann, Joachim

    1998-01-01

    Secretory proteins enter the Golgi apparatus when transport vesicles fuse with the cis-side and exit in transport vesicles budding from the trans-side. Resident Golgi enzymes that have been transported in the cis-to-trans direction with the secretory flow must be recycled constantly by retrograde transport in the opposite direction. In this study, we describe the functional characterization of Golgi-derived transport vesicles that were isolated from tissue culture cells. We found that under the steady-state conditions of a living cell, a fraction of resident Golgi enzymes was found in vesicles that could be separated from cisternal membranes. These vesicles appeared to be depleted of secretory cargo. They were capable of binding to and fusion with isolated Golgi membranes, and after fusion their enzymatic contents most efficiently processed cargo that had just entered the Golgi apparatus. Those results indicate a possible role for these structures in recycling of Golgi enzymes in the Golgi stack. PMID:9456315

  4. Fluoroscopic analysis of lumbar epidural contrast spread after retrograde interlaminar ventral epidural injection (RIVEI)

    PubMed Central

    Jeong, Ji Seon; Woo, Jung Pil; Shim, Jae Hang

    2013-01-01

    Background Retrograde interlaminar ventral epidural injection (RIVEI) may hypothetically be more effective if the catheter is placed at the ventrocaudal aspect of the exiting nerve. We tested that hypothesis by measuring ventral and dorsal epidural contrast flow during RIVEI. Methods To perform RIVEI, a 17 G Tuohy needle was inserted to access the epidural space. A 19 G epidural catheter was inserted and advanced through the needle, passing in a caudal direction to the lower aspect of the contralateral pedicle. Fluoroscopic images were recorded at 1.5 ml increments of contrast. Based on the images of contrast dispersal, the extent of contrast spreading was assessed in 82 patients. Results All 82 patients (100%) injected with 3.0 ml contrast medium demonstrated ventral epidural spreading. Mean spreading level from the catheter tip was 2.21 ± 0.93 with 3.0 ml of contrast. Spreading to the superior aspect of the supra-adjacent intervertebral disc was observed in 67/82 (81.7%) of RIVEIs with 3.0 ml of contrast injected into the ventral epidural space. We found that 3.0 ml of contrast reached the inferior aspect of the infra-adjacent intervertebral disc in 95.1% (78/82) of RIVEIs performed. Conclusions Our findings imply that a one-level RIVEI may be sufficient in situations where a two-level injection would currently be used. PMID:24363846

  5. Actin Foci Adhesion of D. discoideum

    NASA Astrophysics Data System (ADS)

    Flanders, Bret; Paneru, Govind

    2014-03-01

    Amoeboid migration is a fast (10 μm min-1) integrin-independent mode of migration that is important with D. discoideum, leukocytes, and breast cancer cells. It is poorly understood, but depends on the establishment of adhesive contacts to the substrate where the cell transmits traction forces. In pre-aggregative D. discoideum, a model system for learning about amoeboid migration, these adhesive contacts are discrete complexes that are known as actin-foci. They have an area of ~ 0.5 μm2 and a lifetime of ~ 20 s. This talk will present measurements of the adhesive character of actin foci that have been obtained using a submicron force transducer that was designed for this purpose. Results on the rupture stresses and lifetimes of individual acting foci under nano-newton level forces will be described in the context of a general theory for cellular adhesion. This theory depends on, essentially, three cellular properties: the membrane-medium surface tension, the number density of adhesion receptors in the membrane, and the receptor-substrate potential energy surface. Therefore, the use of the transducer to determine the surface tension will be presented, as well.

  6. Encoding Mechano-Memories in Actin Networks

    NASA Astrophysics Data System (ADS)

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

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

  7. Force Transmission in the Actin Cytoskeleton

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret

    2012-02-01

    The ability of cells to sense and generate mechanical forces is essential to numerous aspects of their physiology, including adhesion, migration, division and differentiation. To a large degree, cellular tension is regulated by the transmission of myosin II-generated forces through the filamentous actin (F-actin) cytoskeleton. While transmission of myosin-generated stresses from the molecular to cellular length scale is well understood in the context of highly organized sarcomeres found in striated muscle, non-muscle and smooth muscle cells contain a wide variety of bundles and networks lacking sarcomeric organization. I will describe the in vitro and in vivo approaches we use to study force transmission in such disordered actomyosin assemblies. Our in vivo results are showing that highly organized stress fibers contribute surprisingly little to the overall extent of cellular tension as compared to disordered actomyosin meshworks. Our in vitro results are demonstrating the mechanisms of symmetry breaking in disordered actomyosin bundles that facilitate the formation of contractile bundles with well-defined ``contractile elements.'' These results provide insight into the self-organization of actomyosin cytoskeleton in non-muscle cells that regulate and maintain cellular tension.

  8. Actin and Endocytosis in Budding Yeast

    PubMed Central

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  9. How cofilin severs an actin filament.

    PubMed

    De La Cruz, Enrique M

    2009-05-15

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

  10. Actin binding proteins, spermatid transport and spermiation*

    PubMed Central

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  11. Drebrin inhibits cofilin-induced severing of F-actin.

    PubMed

    Grintsevich, Elena E; Reisler, Emil

    2014-08-01

    Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer's disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor's binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments. PMID:25047716

  12. Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ.

    PubMed

    Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; Zischka, H; Mueller, R; Vollmar, A M

    2014-01-01

    Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as

  13. Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ

    PubMed Central

    Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; Zischka, H; Mueller, R; Vollmar, A M

    2014-01-01

    Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as

  14. Mechanics of composite actin networks: in vitro and cellular perspectives

    NASA Astrophysics Data System (ADS)

    Upadhyaya, Arpita

    2014-03-01

    Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-12-01

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

  18. Retrograde Approach Using Surgical Cutdown Technique for Limb Salvage in a Case of Critical Limb Ischemia With Severely Calcified Tibial Occlusive Disease.

    PubMed

    Shiraki, Tatsuya; Iida, Osamu; Suemitsu, Kotaro; Tsuji, Yoriko; Uematsu, Masaaki

    2016-05-01

    We here report a successful angioplasty for tibial artery occlusion using direct tibial puncture and subsequent retrograde approach under surgical cutdown technique. An 82-year-old man with ulcer/gangrene in first and second digits was referred to our hospital for endovascular therapy (EVT) of lower extremity ischemia. Diagnostic angiogram revealed anterior tibial artery (ATA) occlusion with severe calcification. Subintimal angioplasty was attempted using a 0.014-inch hydrophilic guidewire but was unsuccessful. A retrograde approach was subsequently attempted for ATA recanalization. However, because of severe calcification of dorsal pedis artery (DPA), percutaneous distal puncture was also unsuccessful. Direct puncture under surgical cutdown technique for DPA was subsequently performed and was successful. A 0.014-inch hydrophilic wire was advanced in retrograde fashion across the ATA occlusion and was used to access the microcatheter positioned at the proximal ATA via antegrade approach. Angioplasty of the ATA occlusion was performed using a 2.5-/3.0-mm tapered balloon. Completion angiogram revealed restoration of flow without dissection. Skin perfusion pressure was dramatically improved. Complete wound healing was achieved 5 months after EVT. PMID:27207678

  19. Yeast as a tool to study mitochondrial retrograde pathway en route to cell stress response.

    PubMed

    Ždralević, Maša; Guaragnella, Nicoletta; Giannattasio, Sergio

    2015-01-01

    Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions by changes in nuclear gene expression. The best comprehension of components and regulation of retrograde signaling have been obtained in Saccharomyces cerevisiae, where retrograde target gene expression is regulated by RTG genes. In this chapter, we describe the methods to measure mitochondrial retrograde pathway activation in yeast cells by monitoring the mRNA levels of RTG target genes, such as those encoding for peroxisomal citrate synthase, aconitase, and NAD(+)-specific isocitrate dehydrogenase subunit 1, as well as the phosphorylation status of Rtg1/3p transcriptional factor which controls RTG target gene transcription. PMID:25634284

  20. Pancreatitis with an unusual fatal complication following endoscopic retrograde cholangiopancreaticography: a case report

    PubMed Central

    Kanen, Boris; Loffeld, Ruud JLF

    2008-01-01

    Introduction Endoscopic retrograde cholangiopancreaticography has been the treatment of choice for stones in the common bile duct. Although the procedure is usually safe, procedure-related complications do occur. Case presentation A case of pancreatitis following endoscopic retrograde cholangiopancreaticography is described in a 55-year-old woman. After an uneventful recovery the patient's condition deteriorated rapidly 16 days after the endoscopic retrograde cholangiopancreaticography, and the patient died within 1 hour. Post-mortem examination revealed massive intrapulmonary fat embolism. The complications of endoscopic retrograde cholangiopancreaticography and pancreatitis are described. Conclusion Fat embolism can occur after the remission of pancreatitis and pancreatic necrosis may be overlooked on contrast-enhanced computed tomography scanning. PMID:18577211

  1. Reducing retrogradation and lipid oxidation of normal and glutinous rice flours by adding mango peel powder.

    PubMed

    Siriamornpun, Sirithon; Tangkhawanit, Ekkarat; Kaewseejan, Niwat

    2016-06-15

    Green and ripe mango peel powders (MPP) were added to normal rice flour (NRF) and glutinous rice flour (GRF) at three levels (400, 800 and 1200 ppm) and their effects on physicochemical properties and lipid oxidation inhibition were investigated. Overall, MPP increased the breakdown viscosity and reduced the final viscosity in rice flours when compared to the control. Decreasing in retrogradation was observed in both NRF and GRF with MPP added of all levels. MPP addition also significantly inhibited the lipid oxidation of all flours during storage (30 days). Retrogradation values were strongly negatively correlated with total phenolic and flavonoid contents, but not with fiber content. The hydrogen bonds and hydrophilic interactions between phenolic compounds with amylopectin molecule may be involved the decrease of starch retrogradation, especially GRF. We suggest that the addition of MPP not only reduced the retrogradation but also inhibited the lipid oxidation of rice flour. PMID:26868561

  2. PULMONARY IMPEDANCE IN DOGS MEASURED BY FORCED RANDOM NOISE WITH A RETROGRADE CATHETER

    EPA Science Inventory

    Retrograde catheter and forced random noise techniques were combined to study the distribution of resistance and compliance in dogs following the inhalation of aerosols containing 2.5 and 5.0 mg/ml of histamine.

  3. Vesicourethral fistula after retrograde primary endoscopic realignment in posterior urethral injury.

    PubMed

    Arora, Rajat; John, Nirmal Thampi; Kumar, Santosh

    2015-01-01

    A 22-year-old male patient presented with iatrogenic vesicourethral fistula after immediate retrograde endoscopic realignment of urethra after a posterior urethral injury associated with pelvic fracture. PMID:25440761

  4. NGF-stimulated retrograde transport of trkA in the mammalian nervous system.

    PubMed

    Ehlers, M D; Kaplan, D R; Price, D L; Koliatsos, V E

    1995-07-01

    The present study was designed to clarify the in vivo function of trkA as an NGF receptor in mammalian neurons. Using the rat sciatic nerve as a model system, we examined whether trkA is retrogradely transported and whether transport is influenced by physiological manipulations. Following nerve ligation, trkA protein accumulates distal to the ligation site as shown by Western blot analysis. The distally accumulating trkA species were tyrosine phosphorylated. The trkA retrograde transport and phosphorylation were enhanced by injecting an excess of NGF in the footpad and were abolished by blocking endogenous NGF with specific antibodies. These results provide evidence that, upon NGF binding, trkA is internalized and retrogradely transported in a phosphorylated state, possibly together with the neurotrophin. Furthermore, our results suggest that trkA is a primary retrograde NGF signal in mammalian neurons in vivo. PMID:7540615

  5. Fundamental studies of retrograde reactions in direct liquefaction. Topical report, September 30, 1988--September 30, 1989

    SciTech Connect

    Serio, M.A.; Solomon, P.R.; Bassilakis, R.; Kroo, E.

    1989-12-31

    Most of the proposed processing schemes for improving liquefaction yields involve favoring bond-breaking and radical stabilization reactions over the retrograde reactions. The retrograde reactions are often encountered before liquefaction temperatures are reached. The objective of this program is to elucidate and model the retrograde reaction chemistry in direct coal liquefaction through the application of experimental techniques and theoretical models which have been successfully employed at Advanced Fuel Research (AFR) and SRI International (a subcontractor) to understand and predict coal reaction behavior. The study of retrograde reactions is being done using an integrated approach using extensive characterization of the liquefaction chemistry of three kinds of systems: (1) model polymers; (2) coal; and (3) modified coals.

  6. Retrograde signalling at the synapse: a role for Wnt proteins.

    PubMed

    Salinas, P C

    2005-12-01

    The formation of functional synapses requires a proper dialogue between incoming axons and their future synaptic targets. As axons approach their target, they are instructed to slow down and remodel to form proper presynaptic terminals. Although significant progress has been made in the identification of the mechanisms that control axon guidance, little is known about the mechanisms that regulate the conversion of actively growing axon into a presynaptic terminal. We found that Wnt secreted proteins are retrograde signals that regulate the terminal arborization of axons and synaptic differentiation. Wnts released from postsynaptic neurons induce extensive remodelling on incoming axons. This remodelling is manifested by a decrease in axon extension with a concomitant increase in growth-cone size. This morphological change is correlated with changes in the dynamics and organization of microtubules. Studies of a vertebrate synapse and the Drosophila neuromuscular junction suggest that a conserved Wnt signalling pathway modulates presynaptic microtubules as axons remodel during synapse formation. In this paper I discuss the role of the Wnt-Dvl (Dishevelled protein)-GSK-3beta (glycogen synthase kinase-3beta) signalling pathway in axon remodelling during synapse formation in the central nervous system. PMID:16246102

  7. Endoscopic retrograde cholangiopancreatography in periampullary diverticulum: The challenge of cannulation.

    PubMed

    Altonbary, Ahmed Youssef; Bahgat, Monir Hussein

    2016-03-25

    Periampullary diverticulum (PAD) is duodenal outpunching defined as herniation of the mucosa or submucosa that occurs via a defect in the muscle layer within an area of 2 to 3 cm around the papilla. Although PAD is usually asymptomatic and discovered incidentally during endoscopic retrograde cholangiopancreatography (ERCP), it is associated with different pathological conditions such as common bile duct obstruction, pancreatitis, perforation, bleeding, and rarely carcinoma. ERCP has a low rate of success in patients with PAD, suggesting that this condition may complicate the technical application of the ERCP procedure. Moreover, cannulation of PAD can be challenging, time consuming, and require the higher level of skill of more experienced endoscopists. A large portion of the failures of cannulation in patients with PAD can be attributed to inability of the endoscopist to detect the papilla. In cases where the papilla is identified but does not point in a suitable direction for cannulation, different techniques have been described. Endoscopists must be aware of papilla identification in the presence of PAD and of different cannulation techniques, including their technical feasibility and safety, to allow for an informed decision and ensure the best outcome. Herein, we review the literature on this practical topic and propose an algorithm to increase the success rate of biliary cannulation. PMID:27014423

  8. Early Results of Retrograde Transpopliteal Angioplasty of Iliofemoral Lesions

    SciTech Connect

    Saha, Saumitra; Gibson, Matthew; Magee, Timothy R.; Galland, Robert B.; Torrie, E. Peter H.

    2001-12-15

    Purpose: To assess whether the retrograde transpopliteal approach is a safe, practical and effective alternative to femoral puncture for percutaneous transluminal angioplasty (PTA).Methods: Forty PTAs in 38 patients were evaluated. Intentional subintimal recanalization was performed in 13 limbs. Ultrasound evaluation of the popliteal fossa was carried out 30 min and 24 hr post procedurally in the first 10 patients to exclude local complications. All patients had a follow-up of at least 6 weeks.Results: The indication for PTA was critical ischemia in seven limbs and disabling claudication in the remainder.Stenoses (single or multiple) were present in 24 and occlusion in 15.The superficial femoral artery (SFA) was the commonest segment affected(36) followed by common femoral artery (CFA) in four and iliac artery in four. Technical success was achieved in 38 of 39 limbs where angioplasty was carried out. In one limb no lesion was found.Immediate complications were distal embolization in two and thrombosis in one. None of these required immediate surgery. There were no puncture site hematomas or popliteal arteriovenous fistulae.Symptomatic patency at 6 weeks was 85%. Further reconstructive surgery was required in three limbs and amputation in two.Conclusion: The transpopliteal approach has a high technical success rate and a low complication rate with a potential to develop into an outpatient procedure. It should be considered for flush SFA occulsions or iliac disease with tandem CFA/SFA disease where the contralateral femoral approach is often technically difficult.

  9. Advances in endoscopic retrograde cholangiopancreatography for the treatment of cholangiocarcinoma

    PubMed Central

    Uppal, Dushant S; Wang, Andrew Y

    2015-01-01

    Cholangiocarcinoma (CCA) is a malignancy of the bile ducts that carries high morbidity and mortality. Patients with CCA typically present with obstructive jaundice, and associated complications of CCA include cholangitis and biliary sepsis. Endoscopic retrograde cholangiopancreatography (ERCP) is a valuable treatment modality for patients with CCA, as it enables internal drainage of blocked bile ducts and hepatic segments by using plastic or metal stents. While there remains debate as to if bilateral (or multi-segmental) hepatic drainage is required and/or superior to unilateral drainage, the underlying tenant of draining any persistently opacified bile ducts is paramount to good ERCP practice and good clinical outcomes. Endoscopic therapy for malignant biliary strictures from CCA has advanced to include ablative therapies via ERCP-directed photodynamic therapy (PDT) or radiofrequency ablation (RFA). While ERCP techniques cannot cure CCA, advancements in the field of ERCP have enabled us to improve upon the quality of life of patients with inoperable and incurable disease. ERCP-directed PDT has been used in lieu of brachytherapy to provide neoadjuvant local tumor control in patients with CCA who are awaiting liver transplantation. Lastly, mounting evidence suggests that palliative ERCP-directed PDT, and probably ERCP-directed RFA as well, offer a survival advantage to patients with this difficult-to-treat malignancy. PMID:26140095

  10. Endoscopic retrograde cholangiopancreatography in periampullary diverticulum: The challenge of cannulation

    PubMed Central

    Altonbary, Ahmed Youssef; Bahgat, Monir Hussein

    2016-01-01

    Periampullary diverticulum (PAD) is duodenal outpunching defined as herniation of the mucosa or submucosa that occurs via a defect in the muscle layer within an area of 2 to 3 cm around the papilla. Although PAD is usually asymptomatic and discovered incidentally during endoscopic retrograde cholangiopancreatography (ERCP), it is associated with different pathological conditions such as common bile duct obstruction, pancreatitis, perforation, bleeding, and rarely carcinoma. ERCP has a low rate of success in patients with PAD, suggesting that this condition may complicate the technical application of the ERCP procedure. Moreover, cannulation of PAD can be challenging, time consuming, and require the higher level of skill of more experienced endoscopists. A large portion of the failures of cannulation in patients with PAD can be attributed to inability of the endoscopist to detect the papilla. In cases where the papilla is identified but does not point in a suitable direction for cannulation, different techniques have been described. Endoscopists must be aware of papilla identification in the presence of PAD and of different cannulation techniques, including their technical feasibility and safety, to allow for an informed decision and ensure the best outcome. Herein, we review the literature on this practical topic and propose an algorithm to increase the success rate of biliary cannulation. PMID:27014423

  11. Radiation dose to patients during endoscopic retrograde cholangiopancreatography

    PubMed Central

    Boix, Jaume; Lorenzo-Zúñiga, Vicente

    2011-01-01

    Endoscopic retrograde cholangiopancreatography (ERCP) is an important tool for the diagnosis and treatment of the hepatobiliary system. The use of fluoroscopy to aid ERCP places both the patient and the endoscopy staff at risk of radiation-induced injury. Radiation dose to patients during ERCP depends on many factors, and the endoscopist cannot control some variables, such as patient size, procedure type, or fluoroscopic equipment used. Previous reports have demonstrated a linear relationship between radiation dose and fluoroscopy duration. When fluoroscopy is used to assist ERCP, the shortest fluoroscopy time possible is recommended. Pulsed fluoroscopy and monitoring the length of fluoroscopy have been suggested for an overall reduction in both radiation exposure and fluoroscopy times. Fluoroscopy time is shorter when ERCP is performed by an endoscopist who has many years experience of performing ERCP and carried out a large number of ERCPs in the preceding year. In general, radiation exposure is greater during therapeutic ERCP than during diagnostic ERCP. Factors associated with prolonged fluoroscopy have been delineated recently, but these have not been validated. PMID:21860683

  12. Endoscopic prevention of post-endoscopic retrograde cholangiopancreatography pancreatitis

    PubMed Central

    Lee, Tae Hoon; Park, Do Hyun

    2014-01-01

    Post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) is not an uncommon adverse event but may be an avoidable complication. Although pancreatitis of severe grade is reported in 0.1%-0.5% of ERCP patients, a serious clinical course may be lethal. For prevention of severe PEP, patient risk stratification, appropriate selection of patients using noninvasive diagnostic imaging methods such as magnetic resonance cholangiopancreatography or endoscopic ultrasonography (EUS), and avoidance of unnecessary invasive procedures, are important measures to be taken before any procedure. Pharmacological prevention is also commonly attempted but is usually ineffective. No ideal agent has not yet been found and the available data conflict. Currently, rectal non-steroidal anti-inflammatory drugs are used to prevent PEP in high-risk patients, but additional studies using larger numbers of subjects are necessary to confirm any prophylactic effect. In this review, we focus on endoscopic procedures seeking to prevent or decrease the severity of PEP. Among various cannulation methods, wire-guided cannulation, precut fistulotomy, and transpancreatic septostomy are reviewed. Prophylactic pancreatic stent placement, which is the best-known prophylactic method, is reviewed with reference to the ideal stent type, adequate duration of stent placement, and stent-related complications. Finally, we comment on other treatment alternatives, and make the point that further advances in EUS-guided techniques may afford useful PEP prophylaxis. PMID:25469026

  13. Retrograde amnesia in praying mantis after two successive learning processes.

    PubMed

    Zabala, N A; Maldonado, H

    1990-01-01

    Learning and memory consolidation was investigated in the mantis Stagmatoptera biocellata, by two successive trainings: a) the attack (A) avoidance training in which animals were not allowed to catch a mobile star, followed by the deimatic reaction (DR) avoidance training of not displaying this defensive response, or b) DR-avoidance training followed by A-avoidance training. The results showed that the presence and the 1 of A-avoidance learning and DR-avoidance learning did not affect the learning of the other training. Total retention of A-avoidance memory was demonstrated when A-avoidance training was given first, whereas DR-avoidance memory was significantly lost (716%) when the DR-avoidance training was given first. The retroactive forgetting of DR-avoidance memory is considered as retrograde amnesia rather than retroactive memory inhibition because it depends on the activity during A-avoidance training (number of attacks) and not the quantity of A-avoidance learning. The magnitude of the amnesic effect due to this distracting activity in the A-avoidance training appears to be remarkably similar to that reported in a previous work using nitrogen-induced anoxia as the amnesic factor. PMID:2152377

  14. Endoscopic retrograde cholangiopancreatography in ruptured liver hydatid cyst.

    PubMed

    Borahma, Mohamed; Afifi, Rajaa; Benelbarhdadi, Imane; Ajana, Fatima Zahra; Essamri, Wafaa; Essaid, Abdellah

    2015-07-01

    One of the most common and serious complications of hepatic hydatid cyst disease is communication between the cyst and the biliary tree. Surgical management of biliary fistula is associated with high morbidity and mortality. We retrospectively reviewed the effectiveness of endoscopic treatment of ruptured hydatid cyst into intrahepatic bile ducts. Diagnosis of intrabiliary rupture of hydatid cyst was mostly suspected by acute cholangitis, jaundice, pain, and/or persistent external biliary fistula after surgery. The diagnosis was confirmed by radiology and endoscopic retrograde cholangiopancreatography (ERCP) findings. We retrospectively reviewed clinical, laboratory, imagery, and ERCP findings for all patients. The therapeutic methods performed were endoscopic sphincterotomy, extraction by balloon or Dormia basket, stenting, or nasobiliary drainage. Sixteen patients with ruptured hepatic hydatid cyst into bile ducts were seen in 9 years. Nine of 16 patients had a surgical history of hepatic hydatid cyst and three patients had a percutanous treatment history. We carried out ERCP with sphincterotomy and extraction of hydatid materials (extraction balloon n = 11; Dormia basket n = 5) or biliary drainage (nasobiliary drainage n = 1; biliary stenting n = 1). The fistula healed in 80 % of patients with a median time of 6 weeks [range, 1-12] after endoscopic treatment. ERCP was an effective method of treatment for hepatic hydatid cyst with biliary fistula. PMID:26345677

  15. Acute pulmonary embolism during an endoscopic retrograde cholangiopancreatography.

    PubMed

    Painter, Nate P; Kumar, Priya A; Arora, Harendra

    2014-01-01

    A 76-year-old female patient presented for an endoscopic retrograde cholangiopancreatography (ERCP) for the removal of a biliary stent and lithotripsy. During the procedure, an acute drop in the end-tidal CO 2 , followed by cardiovascular collapse prompted the initiation of the advanced cardiac life support protocol. Transesophageal echocardiography (TEE) demonstrated direct evidence of pulmonary embolism. The patient was promptly treated with thrombolytic therapy and subsequently discharged home on oral warfarin therapy, with no noted sequelae. Although, there have been case reports of air embolism during an ERCP presenting with cardiovascular collapse, to the best of our knowledge, there are no reported cases of acute pulmonary embolus during this procedure. While the availability of TEE in the operating suites is quite common, quick access and interpretation capabilities in remote locations may not be as common. With the expansion of anesthesia services outside of the operating rooms, it may be prudent to develop rapid response systems that incorporate resources such as TEE and trained personnel to deal with such emergent situations. PMID:24732617

  16. Retrograde root filling with dentin-bonded modified resin composite.

    PubMed

    Rud, J; Rud, V; Munksgaard, E C

    1996-09-01

    A retrograde root-end cover with a special resin composite (Retroplast) combined with the dentin bonding agent Gluma (Bayer AG) has been used since 1984 by the authors. Its content of silver, added to promote radiopacity, has been found to lower the working time and storage stability of the composite and might cause discolorations. Since 1990, silver has therefore been replaced with ytterbium trifluoride, which eliminates these side effects. The purpose of this study was to compare the clinical results obtained with these two resin composites and to evaluate the healing results after several years in function. Apical fillings (351) with the modified Retroplast showed the following radiographic healing pattern 1 yr after surgery: 80% complete healing, 2% scar tissue, 12% uncertain healing, and 6% failure. No significant difference in this healing pattern was found, compared with that obtained with the silver-containing Retroplast. Cases with ytterbium trifluoride classified as scar tissue and uncertain healing at 1 yr when examined at 2 to 4 yr postoperatively showed 89% complete healing. 0% scar tissue, 1% uncertain healing, and 9% failure. This result is significantly different from that obtained 1 yr after surgery. Based on calculations, it was predicted that with time 90% will become complete healing, whereas 10% will become failure. PMID:9198430

  17. Posterior tibial artery access using transradial techniques: retrograde approach to inaccessible lower extremity lesions.

    PubMed

    Londoño, Juan Carlos; Singh, Vikas; Martinez, Claudia A

    2012-06-01

    Percutaneous intervention of chronic limb ischemia is often limited by vascular access especially in patients with previous surgical interventions. This warrants development of alternative endovascular techniques, particularly for patients in whom traditional ipsilateral antegrade or contralateral retrograde access has failed or is not possible. We describe a novel approach to the posterior tibial artery using retrograde access with transradial techniques including closure devices in two patients with inaccessible antegrade access. PMID:21432983

  18. Emergent retrograde tracheal intubation in a 3-year-old with stevens-johnsons syndrome.

    PubMed

    He, Michael

    2014-01-01

    A 3-year-old girl suffering from Stevens-Johnsons Syndrome with severe sloughing of the oropharyngeal mucosa was brought to the operating room for an emergent tracheostomy after multiple failed attempts to intubate the trachea in the pediatric intensive care unit. However, a retrograde tracheal intubation was successfully performed in the operating room to secure her airway, after which a tracheostomy was performed. Retrograde intubation can be a quick and effective method for securing the difficult airway. PMID:25612259

  19. Retrograde solubility of formamidinium and methylammonium lead halide perovskites enabling rapid single crystal growth.

    PubMed

    Saidaminov, Makhsud I; Abdelhady, Ahmed L; Maculan, Giacomo; Bakr, Osman M

    2015-12-28

    Here we show the retrograde solubility of various hybrid perovskites through the correct choice of solvent(s) and report their solubility curves. Retrograde solubility enables to develop inverse temperature crystallization of FAPbX3 (FA = HC(NH2)2(+), X = Br(-)/I(-)). FAPbI3 crystals exhibit a 1.4 eV bandgap--considerably narrower than their polycrystalline counterparts. PMID:26511771

  20. Surgical Repair of Retrograde Type A Aortic Dissection after Thoracic Endovascular Aortic Repair

    PubMed Central

    Kim, Chang-Young; Kim, Yeon Soo; Ryoo, Ji Yoon

    2014-01-01

    It is expected that the stent graft will become an alternative method for treating aortic diseases or reducing the extent of surgery; therefore, thoracic endovascular aortic repair has widened its indications. However, it can have rare but serious complications such as paraplegia and retrograde type A aortic dissection. Here, we report a surgical repair of retrograde type A aortic dissection that was performed after thoracic endovascular aortic repair. PMID:24570865

  1. Local translation and retrograde axonal transport of CREB regulates IL-6-induced nociceptive plasticity

    PubMed Central

    2014-01-01

    Transcriptional regulation of genes by cyclic AMP response element binding protein (CREB) is essential for the maintenance of long-term memory. Moreover, retrograde axonal trafficking of CREB in response to nerve growth factor (NGF) is critical for the survival of developing primary sensory neurons. We have previously demonstrated that hindpaw injection of interleukin-6 (IL-6) induces mechanical hypersensitivity and hyperalgesic priming that is prevented by the local injection of protein synthesis inhibitors. However, proteins that are locally synthesized that might lead to this effect have not been identified. We hypothesized that retrograde axonal trafficking of nascently synthesized CREB might link local, activity-dependent translation to nociceptive plasticity. To test this hypothesis, we determined if IL-6 enhances the expression of CREB and if it subsequently undergoes retrograde axonal transport. IL-6 treatment of sensory neurons in vitro caused an increase in CREB protein and in vivo treatment evoked an increase in CREB in the sciatic nerve consistent with retrograde transport. Importantly, co-injection of IL-6 with the methionine analogue azido-homoalanine (AHA), to assess nascently synthesized proteins, revealed an increase in CREB containing AHA in the sciatic nerve 2 hrs post injection, indicating retrograde transport of nascently synthesized CREB. Behaviorally, blockade of retrograde transport by disruption of microtubules or inhibition of dynein or intrathecal injection of cAMP response element (CRE) consensus sequence DNA oligonucleotides, which act as decoys for CREB DNA binding, prevented the development of IL-6-induced mechanical hypersensitivity and hyperalgesic priming. Consistent with previous studies in inflammatory models, intraplantar IL-6 enhanced the expression of BDNF in dorsal root ganglion (DRG). This effect was blocked by inhibition of retrograde axonal transport and by intrathecal CRE oligonucleotides. Collectively, these findings

  2. Successful Balloon-Occluded Retrograde Transvenous Obliteration for Gastric Varix Mainly Draining into the Pericardiophrenic Vein

    SciTech Connect

    Kageyama, Ken; Nishida, N. Matsui, H.; Yamamoto, A.; Nakamura, K.; Miki, Y.

    2012-02-15

    Two cases of gastric varices were treated by balloon-occluded retrograde transvenous obliteration via the pericardiophrenic vein at our hospital, and both were successful. One case developed left hydrothorax. Gastric varices did not bled and esophageal varices were not aggravated in both cases for 24-30 months thereafter. These outcomes indicate the feasibility of balloon-occluded retrograde transvenous obliteration via the pericardiophrenic vein.

  3. Yeast studies reveal moonlighting functions of the ancient actin cytoskeleton.

    PubMed

    Sattlegger, Evelyn; Chernova, Tatiana A; Gogoi, Neeku M; Pillai, Indu V; Chernoff, Yury O; Munn, Alan L

    2014-08-01

    Classic functions of the actin cytoskeleton include control of cell size and shape and the internal organization of cells. These functions are manifest in cellular processes of fundamental importance throughout biology such as the generation of cell polarity, cell migration, cell adhesion, and cell division. However, studies in the unicellular model eukaryote Saccharomyces cerevisiae (Baker's yeast) are giving insights into other functions in which the actin cytoskeleton plays a critical role. These include endocytosis, control of protein translation, and determination of protein 3-dimensional shape (especially conversion of normal cellular proteins into prions). Here, we present a concise overview of these new "moonlighting" roles for the actin cytoskeleton and how some of these roles might lie at the heart of important molecular switches. This is an exciting time for researchers interested in the actin cytoskeleton. We show here how studies of actin are leading us into many new and exciting realms at the interface of genetics, biochemistry, and cell biology. While many of the pioneering studies have been conducted using yeast, the conservation of the actin cytoskeleton and its component proteins throughout eukaryotes suggests that these new roles for the actin cytoskeleton may not be restricted to yeast cells but rather may reflect new roles for the actin cytoskeleton of all eukaryotes. PMID:25138357

  4. Yeast studies reveal moonlighting functions of the ancient actin cytoskeleton

    PubMed Central

    Sattlegger, Evelyn; Chernova, Tatiana A.; Gogoi, Neeku M.; Pillai, Indu V.; Chernoff, Yury O.; Munn, Alan L.

    2014-01-01

    Classic functions of the actin cytoskeleton include control of cell size and shape and the internal organisation of cells. These functions are manifest in cellular processes of fundamental importance throughout biology such as the generation of cell polarity, cell migration, cell adhesion and cell division. However, studies in the unicellular model eukaryote Saccharomyces cerevisiae (Baker's yeast) are giving insights into other functions in which the actin cytoskeleton plays a critical role. These include endocytosis, control of protein translation and determination of protein 3-dimensional shape (especially conversion of normal cellular proteins into prions). Here we present a concise overview of these new "moonlighting" roles for the actin cytoskeleton and how some of these roles might lie at the heart of important molecular switches. This is an exciting time for researchers interested in the actin cytoskeleton. We show here how studies of actin are leading us into many new and exciting realms at the interface of genetics, biochemistry and cell biology. While many of the pioneering studies have been conducted using yeast, the conservation of the actin cytoskeleton and its component proteins throughout eukaryotes suggests that these new roles for the actin cytoskeleton may not be restricted to yeast cells but rather may reflect new roles for the actin cytoskeleton of all eukaryotes. PMID:25138357

  5. Deafness and espin-actin self-organization in stereocilia

    NASA Astrophysics Data System (ADS)

    Wong, Gerard C. L.

    2009-03-01

    Espins are F-actin-bundling proteins associated with large parallel actin bundles found in hair cell stereocilia in the ear, as well as brush border microvilli and Sertoli cell junctions. We examine actin bundle structures formed by different wild-type espin isoforms, fragments, and naturally-occurring human espin mutants linked to deafness and/or vestibular dysfunction. The espin-actin bundle structure consisted of a hexagonal arrangement of parallel actin filaments in a non-native twist state. We delineate the structural consequences caused by mutations in espin's actin-bundling module. For espin mutation with a severely damaged actin-bundling module, which are implicated in deafness in mice and humans, oriented nematic-like actin filament structures, which strongly impinges on bundle mechanical stiffness. Finally, we examine what makes espin different, via a comparative study of bundles formed by espin and those formed by fascin, a prototypical bundling protein found in functionally different regions of the cell, such as filopodia.

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

    PubMed

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

    2015-02-27

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

  7. Actin dynamics and the evolution of the memory trace.

    PubMed

    Rudy, Jerry W

    2015-09-24

    The goal of this essay is to link the regulation of actin dynamics to the idea that the synaptic changes that support long-term potentiation and memory evolve in temporally overlapping stages-generation, stabilization, and consolidation. Different cellular/molecular processes operate at each stage to change the spine cytoarchitecture and, in doing so, alter its function. Calcium-dependent processes that degrade the actin cytoskeleton network promote a rapid insertion of AMPA receptors into the post synaptic density, which increases a spine's capacity to express a potentiated response to glutamate. Other post-translation events then begin to stabilize and expand the actin cytoskeleton by increasing the filament actin content of the spine and reorganizing it to be resistant to depolymerizing events. Disrupting actin polymerization during this stabilization period is a terminal event-the actin cytoskeleton shrinks and potentiated synapses de-potentiate and memories are lost. Late-arriving, new proteins may consolidate changes in the actin cytoskeleton. However, to do so requires a stabilized actin cytoskeleton. The now enlarged spine has properties that enable it to capture other newly transcribed mRNAs or their protein products and thus enable the synaptic changes that support LTP and memory to be consolidated and maintained. This article is part of a Special Issue entitled SI: Brain and Memory. PMID:25498985

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

    PubMed Central

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

    2014-01-01

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

  9. Actin-based spindle positioning: new insights from female gametes.

    PubMed

    Almonacid, Maria; Terret, Marie-Émilie; Verlhac, Marie-Hélène

    2014-02-01

    Asymmetric divisions are essential in metazoan development, where they promote the emergence of cell lineages. The mitotic spindle has astral microtubules that contact the cortex, which act as a sensor of cell geometry and as an integrator to orient cell division. Recent advances in live imaging revealed novel pools and roles of F-actin in somatic cells and in oocytes. In somatic cells, cytoplasmic F-actin is involved in spindle architecture and positioning. In starfish and mouse oocytes, newly discovered meshes of F-actin control chromosome gathering and spindle positioning. Because oocytes lack centrosomes and astral microtubules, F-actin networks are key players in the positioning of spindles by transmitting forces over long distances. Oocytes also achieve highly asymmetric divisions, and thus are excellent models to study the roles of these newly discovered F-actin networks in spindle positioning. Moreover, recent studies in mammalian oocytes provide a further understanding of the organisation of F-actin networks and their biophysical properties. In this Commentary, we present examples of the role of F-actin in spindle positioning and asymmetric divisions, with an emphasis on the most up-to-date studies from mammalian oocytes. We also address specific technical issues in the field, namely live imaging of F-actin networks and stress the need for interdisciplinary approaches. PMID:24413163

  10. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins

    PubMed Central

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R.

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  11. Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex

    PubMed Central

    Heinemann, Fabian; Vogel, Sven K.; Schwille, Petra

    2013-01-01

    Diffusion of lipids and proteins within the cell membrane is essential for numerous membrane-dependent processes including signaling and molecular interactions. It is assumed that the membrane-associated cytoskeleton modulates lateral diffusion. Here, we use a minimal actin cortex to directly study proposed effects of an actin meshwork on the diffusion in a well-defined system. The lateral diffusion of a lipid and a protein probe at varying densities of membrane-bound actin was characterized by fluorescence correlation spectroscopy (FCS). A clear correlation of actin density and reduction in mobility was observed for both the lipid and the protein probe. At high actin densities, the effect on the protein probe was ∼3.5-fold stronger compared to the lipid. Moreover, addition of myosin filaments, which contract the actin mesh, allowed switching between fast and slow diffusion in the minimal system. Spot variation FCS was in accordance with a model of fast microscopic diffusion and slower macroscopic diffusion. Complementing Monte Carlo simulations support the analysis of the experimental FCS data. Our results suggest a stronger interaction of the actin mesh with the larger protein probe compared to the lipid. This might point toward a mechanism where cortical actin controls membrane diffusion in a strong size-dependent manner. PMID:23561523

  12. Building an artificial actin cortex on microscopic pillar arrays.

    PubMed

    Ayadi, R; Roos, W H

    2015-01-01

    Eukaryotic cells obtain their morphology and mechanical strength from the cytoskeleton and in particular from the cross-linked actin network that branches throughout the whole cell. This actin cortex lies like a quasi-two-dimensional (2D) biopolymer network just below the cell membrane, to which it is attached. In the quest for building an artificial cell, one needs to make a biomimetic model of the actin cortex and combine this in a bottom-up approach with other "synthetic" components. Here, we describe a reconstitution method for such an artificial actin cortex, which is freely suspended on top of a regular array of pillars. By this immobilization method, the actin network is only attached to a surface at discrete points and can fluctuate freely in between. By discussing the method to make the micropillars and the way to reconstitute a quasi-2D actin network on top, we show how one can study an isolated, reconstituted part of a cell. This allows the study of fundamental interaction mechanisms of actin networks, providing handles to design a functional actin cortex in an artificial cell. PMID:25997345

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

    PubMed Central

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

    2008-01-01

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

  14. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53

    PubMed Central

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  15. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53.

    PubMed

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1-393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using "hot-spot" p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  16. Retrogradation of Waxy Rice Starch Gel in the Vicinity of the Glass Transition Temperature

    PubMed Central

    Charoenrein, Sanguansri; Udomrati, Sunsanee

    2013-01-01

    The retrogradation rate of waxy rice starch gel was investigated during storage at temperatures in the vicinity of the glass transition temperature of a maximally concentrated system (Tg′), as it was hypothesized that such temperatures might cause different effects on retrogradation. The Tg′ value of fully gelatinized waxy rice starch gel with 50% water content and the enthalpy of melting retrograded amylopectin in the gels were investigated using differential scanning calorimetry. Starch gels were frozen to −30°C and stored at 4, 0, −3, −5, and −8°C for 5 days. The results indicated that the Tg′ value of gelatinized starch gel annealed at −7°C for 15 min was −3.5°C. Waxy rice starch gels retrograded significantly when stored at 4°C with a decrease in the enthalpy of melting retrograded starch in samples stored for 5 days at −3, −5, and −8°C, respectively, perhaps due to the more rigid glass matrix and less molecular mobility facilitating starch chain recrystallization at temperatures below Tg′. This suggests that retardation of retrogradation of waxy rice starch gel can be achieved at temperature below Tg′. PMID:26904602

  17. Focal retrograde amnesia: voxel-based morphometry findings in a case without MRI lesions.

    PubMed

    Sehm, Bernhard; Frisch, Stefan; Thöne-Otto, Angelika; Horstmann, Annette; Villringer, Arno; Obrig, Hellmuth

    2011-01-01

    Focal retrograde amnesia (FRA) is a rare neurocognitive disorder presenting with an isolated loss of retrograde memory. In the absence of detectable brain lesions, a differentiation of FRA from psychogenic causes is difficult. Here we report a case study of persisting FRA after an epileptic seizure. A thorough neuropsychological assessment confirmed severe retrograde memory deficits while anterograde memory abilities were completely normal. Neurological and psychiatric examination were unremarkable and high-resolution MRI showed no neuroradiologically apparent lesion. However, voxel-based morphometry (VBM)-comparing the MRI to an education-, age-and sex-matched control group (n = 20) disclosed distinct gray matter decreases in left temporopolar cortex and a region between right posterior parahippocampal and lingual cortex. Although the results of VBM-based comparisons between a single case and a healthy control group are generally susceptible to differences unrelated to the specific symptoms of the case, we believe that our data suggest a causal role of the cortical areas detected since the retrograde memory deficit is the preeminent neuropsychological difference between patient and controls. This was paralleled by grey matter differences in central nodes of the retrograde memory network. We therefore suggest that these subtle alterations represent structural correlates of the focal retrograde amnesia in our patient. Beyond the implications for the diagnosis and etiology of FRA, our results advocate the use of VBM in conditions that do not show abnormalities in clinical radiological assessment, but show distinct neuropsychological deficits. PMID:22028902

  18. Retrograde reactions in coal processing: The behavior of ether and sulfide model compounds

    SciTech Connect

    Buchanan, A.C. III; Britt, P.F.; Skeen, J.T.

    1997-04-01

    Retrograde reactions that produce more refractory molecular structures are undesirable in coal liquefaction. The authors previously found that restricted mass transport, induced by immobilization on a silica support, promotes retrograde reactions for 1,2-diphenylethane (C{sub 6}H{sub 5}CH{sub 2}CH{sub 2}C{sub 6}H{sub 5}) by both skeletal rearrangement and ring growth (cyclization-dehydrogenation) pathways involving free-radical intermediates. They are now examining the influence of heteroatoms on the retrograde pathways for the corresponding surface-immobilized ether (C{sub 6}H{sub 5}OCH{sub 2}C{sub 6}H{sub 5}) and sulfide (C{sub 6}H{sub 5}SCH{sub 2}C{sub 6}H{sub 5}) model compounds at 275--350 C. Cyclization-dehydrogenation pathways are not detected for either model compound. However, retrograde skeletal rearrangements involving 1,2-phenyl shifts in C{sub 6}H{sub 5}XCH{center_dot}C{sub 6}H{sub 5} (X = O,S) are found to be significant under restricted diffusion, and for X = O, radical coupling at ring carbons to form benzylphenols is also observed as a major pathway. For surface-immobilized benzyl phenyl ether, the two retrograde processes account for ca. 50% of the thermolysis products, and also generate reactive hydroxyl and keto functionalities that can be involved in additional retrograde reactions.

  19. Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

    PubMed Central

    Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

    2015-01-01

    The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. PMID:26157166

  20. 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

  1. Actin network disassembly powers dissemination of Listeria monocytogenes.

    PubMed

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

    2014-01-01

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

  2. Actin-associated Proteins in the Pathogenesis of Podocyte Injury

    PubMed Central

    He, Fang-Fang; Chen, Shan; Su, Hua; Meng, Xian-Fang; Zhang, Chun

    2013-01-01

    Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies. PMID:24396279

  3. Actinic prurigo of the lip: Two case reports.

    PubMed

    Miranda, Ana Mo; Ferrari, Thiago M; Werneck, Juliana T; Junior, Arley Silva; Cunha, Karin S; Dias, Eliane P

    2014-08-16

    Actinic prurigo is a photodermatosis that can affect the skin, conjunctiva and lips. It is caused by an abnormal reaction to sunlight and is more common in high-altitude living people, mainly in indigenous descendants. The diagnosis of actinic prurigo can be challenging, mainly when lip lesions are the only manifestation, which is not a common clinical presentation. The aim of this article is to report two cases of actinic prurigo showing only lip lesions. The patients were Afro-American and were unaware of possible Indian ancestry. Clinical exam, photographs, videoroscopy examination and biopsy were performed, and the diagnosis of actinic prurigo was established. Topical corticosteroid and lip balm with ultraviolet protection were prescribed with excellent results. The relevance of this report is to show that although some patients may not demonstrate the classical clinical presentation of actinic prurigo, the associated clinical and histological exams are determinants for the correct diagnosis and successful treatment of this disease. PMID:25133153

  4. Actinic prurigo of the lip: Two case reports

    PubMed Central

    Miranda, Ana MO; Ferrari, Thiago M; Werneck, Juliana T; Junior, Arley Silva; Cunha, Karin S; Dias, Eliane P

    2014-01-01

    Actinic prurigo is a photodermatosis that can affect the skin, conjunctiva and lips. It is caused by an abnormal reaction to sunlight and is more common in high-altitude living people, mainly in indigenous descendants. The diagnosis of actinic prurigo can be challenging, mainly when lip lesions are the only manifestation, which is not a common clinical presentation. The aim of this article is to report two cases of actinic prurigo showing only lip lesions. The patients were Afro-American and were unaware of possible Indian ancestry. Clinical exam, photographs, videoroscopy examination and biopsy were performed, and the diagnosis of actinic prurigo was established. Topical corticosteroid and lip balm with ultraviolet protection were prescribed with excellent results. The relevance of this report is to show that although some patients may not demonstrate the classical clinical presentation of actinic prurigo, the associated clinical and histological exams are determinants for the correct diagnosis and successful treatment of this disease. PMID:25133153

  5. 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

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

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

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

  7. IFT88 influences chondrocyte actin organization and biomechanics

    PubMed Central

    Wang, Z.; Wann, A.K.T.; Thompson, C.L.; Hassen, A.; Wang, W.; Knight, M.M.

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. Results IFT88orpk cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88orpk cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88orpk cells. Following membrane blebbing, IFT88orpk cells exhibited slower reformation of the actin cortex. IFT88orpk cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. Conclusions This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. PMID:26493329

  8. A dynamic formin-dependent deep F-actin network in axons

    PubMed Central

    Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe

    2015-01-01

    Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902

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

    PubMed

    DiNubile, M J; Huang, S

    1997-01-01

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

  10. Endoscopic retrograde cholangiopancreatography-related perforations: Diagnosis and management

    PubMed Central

    Vezakis, Antonios; Fragulidis, Georgios; Polydorou, Andreas

    2015-01-01

    Endoscopic retrograde cholangiopancreatography (ERCP) has become an important therapeutic modality for biliary and pancreatic disorders. Perforation is one of the most feared complications of ERCP and endoscopic sphincterotomy. A MEDLINE search was performed from 2000-2014 using the keywords “perforation”, “ERCP” and “endoscopic sphincterotomy”. All articles including more than nine cases were reviewed. The incidence of ERCP-related perforations was low (0.39%, 95%CI: 0.34-0.69) with an associated mortality of 7.8% (95%CI: 3.80-13.07). Endoscopic sphincterotomy was responsible for 41% of perforations, insertion and manipulations of the endoscope for 26%, guidewires for 15%, dilation of strictures for 3%, other instruments for 4%, stent insertion or migration for 2% and in 7% of cases the etiology was unknown. The diagnosis was made during ERCP in 73% of cases. The mechanism, site and extent of injury, suggested by clinical and radiographic findings, should guide towards operative or non-operative management. In type I perforations early surgical repair is indicated, unless endoscopic closure can be achieved. Patients with type II perforations should be treated initially non-operatively. Non-operative treatment includes biliary stenting, fasting, intravenous fluid resuscitation, nasogastric drainage, broad spectrum antibiotics, percutaneous drainage of fluid collections. Non-operative treatment was successful in 79% of patients with type II injuries, with an overall mortality of 9.4%. Non-operative treatment was sufficient in all patients with type III injuries. Surgical technique depends on timing, site and size of defect and clinical condition of the patient. In conclusion, diagnosis is based on clinical suspicion and clinical and radiographic findings. Whilst surgery is usually indicated in patients with type I injuries, patients with type II or III injuries should be treated initially non-operatively. A minority of them will finally require surgical

  11. Properties of Cores Formed by Retrograde Minor Mergers.

    NASA Astrophysics Data System (ADS)

    Bak, J.

    1999-09-01

    In the last 10 years over a dozen elliptical galaxies have been observed to posses a core which rotates counter to the rest of the galaxy. In one formation scenario, dynamical friction causes a compact companion to spiral into the center of a much larger elliptical galaxy on a retrograde orbit relative to the larger galaxy's rotation. If the core of the smaller galaxy is not tidally disrupted it may carry some of it's orbital angular momentum to the center. I present results from N-body simulations, which cover the parameter space over which satellite accretion is most likely to form counter rotating cores. The kinematic parts of the results are analyzed using the penalized likelihood method of Merritt to calculate 2D line-of-sight velocity fields, including third and fourth order Gauss-Hermite terms. By combining this method with IRAF, the photometric aspects of the results are analyzed and compared with observations. The results indicate that dissipationless satellite accretion can only form counter rotating cores when the larger galaxy's intrinsic angular momentum is almost perfectly antiparallel to the orbital angular momentum of the satellite. In most other cases a kinematically distinct core is formed. I present statistical properties of the cores, which include the deviations from pure isophote ellipses as well as deviations of the line-of-sight velocity profiles from a pure Gaussian form. To test the robustness of the results, some of the simulations are redone with a minor amount of dissipation added to the satellite. These simulations indicate that including small amounts of gas does not significantly effect the conclusions. I would like to thank the Student Stipend Committee for making this presentation possible.

  12. Triggering Actin Comets Versus Membrane Ruffles: Distinctive Effects of Phosphoinositides on Actin Reorganization

    PubMed Central

    Ueno, Tasuku; Falkenburger, Björn H.; Pohlmeyer, Christopher; Inoue, Takanari

    2012-01-01

    A limited set of phosphoinositide membrane lipids regulate diverse cellular functions including proliferation, differentiation, and migration. We developed two techniques based on rapamycin-induced protein dimerization to rapidly change the concentration of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. First, we increased PI(4,5)P2 synthesis from phosphatidylinositol 4-phosphate [PI(4)P] using a membrane recruitable form of PI(4)P 5-kinase, and found that COS-7, HeLa, and HEK293 cells formed bundles of motile actin filaments known as actin comets. In contrast, a second technique that increased the concentration of PI(4,5)P2 without consuming PI(4)P induced membrane ruffles. These distinct phenotypes were mediated by dynamin-mediated vesicular trafficking and mutually inhibitory crosstalk between the small guanosine triphosphatases Rac and RhoA. Our results indicate that the effect of PI(4,5)P2 on actin reorganization depends on the abundance of other phosphoinositides, such as PI(4)P. Thus, combinatorial regulation of phosphoinositide concentrations may contribute to the diversity of phosphoinositide functions. PMID:22169478

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

    PubMed Central

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

    2016-01-01

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

  14. Endothelin-1 impairs retrograde axonal transport and leads to axonal injury in rat optic nerve.

    PubMed

    Taniguchi, Takazumi; Shimazawa, Masamitsu; Sasaoka, Masaaki; Shimazaki, Atsushi; Hara, Hideaki

    2006-05-01

    The purpose of this study was to examine the effects of endothelin-1 (ET-1) on retrograde axonal transport in the rat optic nerve. Vehicle or ET-1 (0.2, 1, or 5 pmol/eye) were injected into the vitreous body in Sprague-Dawley rats. Retinal vessels were observed, using a fundus camera, before, and at 10 min, 3 days and 7 days after a single intravitreous injection. Two days after the injection, a neuronal tracer, fluoro gold, was administered via the superior colliculi to retrogradely label active retinal ganglion cells (RGCs). Five days after the tracer administration, retrogradely labeled RGCs were evaluated in the flat-mounted retina, and cross sections from each optic nerve were graded for injury by four independent, masked observers. ET-1 at 5 pmol/eye caused a significant constriction of retinal vessels (versus the vehicle-treated group) at 10 min after the injection. Intravitreous injection of ET-1 caused a dose-related decrease in the number of retrogradely labeled RGCs. Injection of 5 pmol/eye ET-1 led to a statistically significant decrease in the number of retrogradely labeled RGCs (versus the vehicle-treated group). ET-1 at 1 and 5 pmol/eye caused histological optic nerve damage (evaluated using a graded scale). The histological optic nerve damage correlated with the number of retrogradely labeled RGCs. In conclusion, a single intravitreous injection of ET-1 impaired retrograde axonal transport in the rat optic nerve and this impairment correlated with the histological optic nerve damage. PMID:16719791

  15. Combination treatment of transjugular retrograde obliteration and endoscopic embolization for portosystemic encephalopathy with esophageal varices.

    PubMed

    Chikamori, Fumio; Kuniyoshi, Nobutoshi; Shibuya, Susumu; Takase, Yasuhiro

    2004-01-01

    The treatment of chronic portosystemic encephalopathy with esophageal varices has not yet been established. We were able to control a case of chronic portosystemic encephalopathy with esophageal varices using a combination treatment of transjugular retrograde obliteration and endoscopic embolization. A 57-year-old man came to our hospital in a confused, apathetic and tremulous state. The grade of encephalopathy was II. The plasma ammonia level was abnormally elevated to 119 microg/dL, and the ICGR15 was 59%. Endoscopic examination revealed nodular esophageal varices with cherry-red spots. There were no gastric varices. Ultrasonography and CT revealed liver cirrhosis with a splenorenal shunt. We first applied endoscopic embolization for the esophageal varices before transjugular retrograde obliteration. We injected 5% ethanolamine oleate with iopamidol retrogradely into the esophageal varices and their associated blood routes under fluoroscopy and obliterated the palisade vein, the cardiac venous plexus and left gastric vein. Transjugular retrograde obliteration was performed 14 days after endoscopic embolization. Retrograde shunt venography visualized the splenorenal shunt and communicating route to the retroperitoneal vein. There was no communicating route to the azygos vein. After obliteration of the communicating route to the retroperitoneal vein with absolute ethanol, 5% ethanolamine oleate with iopamidol was injected into the splenorenal shunt as far as the root of the posterior gastric vein. After transjugular retrograde obliteration, the encephalopathy improved to grade 0 even without the administration of lactulose and branched-chain amino acid. The plasma ammonia level and ICGR15 were reduced to 62 microg/dL and 26%. We conclude that combination treatment of transjugular retrograde obliteration and endoscopic embolization is a rational, effective and safe treatment for chronic portosystemic encephalopathy complicated with esophageal varices. PMID:15362757

  16. On remembering and forgetting our autobiographical pasts: retrograde amnesia and Andrew Mayes's contribution to neuropsychological method.

    PubMed

    Kopelman, M D; Bright, P

    2012-11-01

    Andrew Mayes's contribution to the neuropsychology of memory has consisted in steadily teasing out the nature of the memory deficit in the amnesic syndrome. This has been done with careful attention to matters of method at all stages. This particularly applies to his investigations of forgetting rates in amnesia and to his studies of retrograde amnesia. Following a brief outline of his work, the main current theories of retrograde amnesia are considered: consolidation theory, episodic-to-semantic shift theory, and multiple trace theory. Findings across the main studies in Alzheimer dementia are reviewed to illustrate what appears to be consistently found, and what is much more inconsistent. A number of problems and issues in current theories are then highlighted--including the nature of the temporal gradient, correlations with the extent of temporal lobe damage, what we would expect 'normal' remote memory curves to look like, how they would appear in focal retrograde amnesia, and whether we can pinpoint retrograde amnesia to hippocampal/medial temporal damage on the basis of existing studies. A recent study of retrograde amnesia is re-analysed to demonstrate temporal gradients on recollected episodic memories in hippocampal/medial temporal patients. It is concluded that there are two requirements for better understanding of the nature of retrograde amnesia: (i) a tighter, Mayesian attention to method in terms of both the neuropsychology and neuroimaging in investigations of retrograde amnesia; and (ii) acknowledging that there may be multiple factors underlying a temporal gradient, and that episodic and semantic memory show important interdependencies at both encoding and retrieval. Such factors may be critical to understanding what is remembered and what is forgotten from our autobiographical pasts. PMID:22884958

  17. The ADF/cofilin family: actin-remodeling proteins

    PubMed Central

    Maciver, Sutherland K; Hussey, Patrick J

    2002-01-01

    The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms. PMID:12049672

  18. The centrosome is an actin-organizing center

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed Central

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

    1987-01-01

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

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

    NASA Astrophysics Data System (ADS)

    John, Karin; Peyla, Philippe; Misbah, Chaouqi

    2007-03-01

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

  2. Self-organizing actin waves as planar phagocytic cup structures

    PubMed Central

    Ecke, Mary; Schroth-Diez, Britta; Gerwig, Silke; Engel, Ulrike; Maddera, Lucinda; Clarke, Margaret

    2009-01-01

    Actin waves that travel on the planar membrane of a substrate-attached cell underscore the capability of the actin system to assemble into dynamic structures by the recruitment of proteins from the cytoplasm. The waves have no fixed shape, can reverse their direction of propagation and can fuse or divide. Actin waves separate two phases of the plasma membrane that are distinguished by their lipid composition. The area circumscribed by a wave resembles in its phosphoinositide content the interior of a phagocytic cup, leading us to explore the possibility that actin waves are in-plane phagocytic structures generated without the localized stimulus of an attached particle. Consistent with this view, wave-forming cells were found to exhibit a high propensity for taking up particles. Cells fed rod-shaped particles produced elongated phagocytic cups that displayed a zonal pattern that reflected in detail the actin and lipid pattern of free-running actin waves. Neutrophils and macrophages are known to spread on surfaces decorated with immune complexes, a process that has been interpreted as “frustrated” phagocytosis. We suggest that actin waves enable a phagocyte to scan a surface for particles that might be engulfed. PMID:19855162

  3. Actin Dynamics in Growth Cone Motility and Navigation

    PubMed Central

    Gomez, Timothy M.; Letourneau, Paul C.

    2014-01-01

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

  4. Nuclear Actin Extends, with No Contraction in Sight

    PubMed Central

    Pederson, Thoru; Aebi, Ueli

    2005-01-01

    Within the past two years, actin has been implicated in eukaryotic gene transcription by all three classes of RNA polymerase. Moreover, within just the past year, actin has been identified as a constituent of filaments attached to the nuclear pore complexes and extending into the nucleus. This review summarizes these and other very recent advances in the nuclear actin field and emphasizes the key present issues. On the one hand, we are confronted with a body of evidence for a role of actin in gene transcription but with no known structural basis; on the other hand, there is now evidence for polymeric actin—not likely in the classical F-actin conformation—in the nuclear periphery with no known function. In addition, numerous proteins that interact with either G- or F-actin are increasingly being detected in the nucleus, suggesting that both monomeric and oligomeric or polymeric forms of actin are at play and raising the possibility that the equilibrium between them, perhaps differentially regulated at various intranuclear sites, may be a major determinant of nuclear function. PMID:16148048

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

    PubMed

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

    2016-05-10

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Mizuno, Hiroaki; Watanabe, Naoki

    2012-01-01

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

  8. Gcn1 and Actin Binding to Yih1

    PubMed Central

    Sattlegger, Evelyn; Barbosa, João A. R. G.; Moraes, Maria Carolina S.; Martins, Rafael M.; Hinnebusch, Alan G.; Castilho, Beatriz A.

    2011-01-01

    Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1–132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68–258, encompassing part of the RWD and the C-terminal “ancient domain”; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68–258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation. PMID:21239490

  9. Role of actin in auxin transport and transduction of gravity

    NASA Astrophysics Data System (ADS)

    Hu, S.; Basu, S.; Brady, S.; Muday, G.

    Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

  10. Pathogenic microbes manipulate cofilin activity to subvert actin cytoskeleton.

    PubMed

    Zheng, Kai; Kitazato, Kaio; Wang, Yifei; He, Zhendan

    2016-09-01

    Actin-depolymerizing factor (ADF)/cofilin proteins are key players in controlling the temporal and spatial extent of actin dynamics, which is crucial for mediating host-pathogen interactions. Pathogenic microbes have evolved molecular mechanisms to manipulate cofilin activity to subvert the actin cytoskeletal system in host cells, promoting their internalization into the target cells, modifying the replication niche and facilitating their intracellular and intercellular dissemination. The study of how these pathogens exploit cofilin pathways is crucial for understanding infectious disease and providing potential targets for drug therapies. PMID:25853495

  11. A Nucleator Arms Race: Cellular Control of Actin Assembly

    PubMed Central

    Campellone, Kenneth G.; Welch, Matthew D.

    2010-01-01

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

  12. Actin purification from a gel of rat brain extracts.

    PubMed

    Levilliers, N; Peron-Renner, M; Coffe, G; Pudles, J

    1984-01-01

    Actin, 99% pure, has been recovered from rat brain with a high yield (greater than 15 mg/100 g brain). We have shown that: 1. a low ionic strength extract from rat brain tissue is capable of giving rise to a gel; 2. actin is the main gel component and its proportion is one order of magnitude higher than in the original extract; 3. actin can be isolated from this extract by a three-step procedure involving gelation, dissociation of the gel in 0.6 M KCl, followed by one or two depolymerization-polymerization cycles. PMID:6529588

  13. Interactions Between DNA and Actin in Model Cystic Fibrosis Sputum

    NASA Astrophysics Data System (ADS)

    Kyung, Hee; Sanders, Lori; Angelini, Thomas; Butler, John; Wong, Gerard

    2003-03-01

    Cystic fibrosis sputum is a complex fluid which has a high concentration of DNA and F-actin, two anionic biological polyelectrolytes. In this work, we study the interactions between DNA and actin in an aqueous environment over a wide range of polyelectrolyte lengths and salt levels, using synchrotron Small Angle X-ray Scattering(SAXS) and confocal microscopy. Perliminary results indicate the existence of a compressed phase of nematic F-actin in the presence of DNA. This work was supported by NSF DMR-0071761, the Beckman Young Investigator Program, and the Cystic Fibrosis Foundation.

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

    PubMed

    Hagan, Iain M

    2016-01-01

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

  15. Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

    PubMed Central

    Lin, Wei-Chi; Wang, Liang-Chen; Pang, Te-Ling; Chen, Mei-Yu

    2015-01-01

    Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG− cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG− cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG− cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion. PMID:25609090

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

    PubMed Central

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

    2014-01-01

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

  17. Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum.

    PubMed

    Lin, Wei-Chi; Wang, Liang-Chen; Pang, Te-Ling; Chen, Mei-Yu

    2015-03-15

    Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971-amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG(-) cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401-600 and aa 501-550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501-550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG(-) cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG(-) cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion. PMID:25609090

  18. Retrograde binaries of massive black holes in circumbinary accretion discs

    NASA Astrophysics Data System (ADS)

    Amaro-Seoane, Pau; Maureira-Fredes, Cristián; Dotti, Massimo; Colpi, Monica

    2016-06-01

    accretion only explores the late evolution stages of the binary in an otherwise unperturbed retrograde disc to illustrate how eccentricity evolves with time in relation to the shape of the underlying surface density distribution.

  19. Kinematical evolution of tidally limited star clusters: the role of retrograde stellar orbits

    NASA Astrophysics Data System (ADS)

    Tiongco, Maria A.; Vesperini, Enrico; Varri, Anna Lisa

    2016-09-01

    The presence of an external tidal field often induces significant dynamical evolutionary effects on the internal kinematics of star clusters. Previous studies investigating the restricted three-body problem with applications to star cluster dynamics have shown that unbound stars on retrograde orbits (with respect to the direction of the cluster's orbit) are more stable against escape than prograde orbits, and predicted that a star cluster might acquire retrograde rotation through preferential escape of stars on prograde orbits. In this study, we present evidence of this prediction, but we also illustrate that there are additional effects that cannot be accounted for by the preferential escape of prograde orbits alone. Specifically, in the early evolution, initially underfilling models increase their fraction of retrograde stars without losing significant mass, and acquire a retrograde angular velocity. We attribute this effect to the development of preferentially eccentric/radial orbits in the outer regions of star clusters as they are expanding into their tidal limitation. We explore the implications of the evolution of the fraction of prograde and retrograde stars for the evolution of the cluster internal rotation, and its dependence on the initial structural properties. Although all the systems studied here evolve towards an approximately solid-body internal rotation with angular velocity equal to about half of the angular velocity of the cluster orbital motion around the host galaxy, the evolutionary history of the radial profile of the cluster internal angular velocity depends on the cluster initial structure.

  20. Profound loss of general knowledge in retrograde amnesia: evidence from an amnesic artist

    PubMed Central

    Gregory, Emma; McCloskey, Michael; Landau, Barbara

    2014-01-01

    Studies of retrograde amnesia have focused on autobiographical memory, with fewer studies examining how non-autobiographical memory is affected. Those that have done so have focused primarily on memory for famous people and public events—relatively limited aspects of memory that are tied to learning during specific times of life and do not deeply tap into the rich and extensive knowledge structures that are developed over a lifetime. To assess whether retrograde amnesia can also cause impairments to other forms of general world knowledge, we explored losses across a broad range of knowledge domains in a newly-identified amnesic. LSJ is a professional artist, amateur musician and history buff with extensive bilateral medial temporal and left anterior temporal damage. We examined LSJ's knowledge across a range of everyday domains (e.g., sports) and domains for which she had premorbid expertise (e.g., famous paintings). Across all domains tested, LSJ showed losses of knowledge at a level of breadth and depth never before documented in retrograde amnesia. These results show that retrograde amnesia can involve broad and deep deficits across a range of general world knowledge domains. Thus, losses that have already been well-documented (famous people and public events) may severely underestimate the nature of human knowledge impairment that can occur in retrograde amnesia. PMID:24834048

  1. The effect of partial gelatinization of corn starch on its retrogradation.

    PubMed

    Fu, Zong-qiang; Wang, Li-jun; Li, Dong; Zhou, Yu-guang; Adhikari, Benu

    2013-09-12

    The objective of this work was to investigate the effect of partial gelatinization of starch on its retrogradation using differential scanning calorimetry (DSC) and X-ray diffraction (XRD) techniques. The Avrami equation was used to predict the evolution of starch retrogradation kinetics. The degree of retrogradation in starch samples partially gelatinized 64°C (S64), 68°C (S68) and 70°C (S70) and control (S25) increased with storage time. The retrogradation enthalpies of S68 and S70 were almost four times as high as that of S64. The S25 and S64 had dominant A-type crystalline pattern while S68 and S70 showed dominant B-type crystalline pattern. The growth of remainder crystals was faster in S25 and S64, while both the nucleation and growth rates of new crystals were faster in S68 and S70. The Avrami model was found to represent the retrogradation kinetics data of these partially gelatinized starch samples quite satisfactorily (R(2)>0.95). PMID:23911478

  2. Retrogradation of Maize Starch after High Hydrostatic Pressure Gelation: Effect of Amylose Content and Depressurization Rate

    PubMed Central

    Yang, Zhi; Swedlund, Peter; Gu, Qinfen; Hemar, Yacine; Chaieb, Sahraoui

    2016-01-01

    High hydrostatic pressure (HHP) has been employed to gelatinize or physically modify starch dispersions. In this study, waxy maize starch, normal maize starch, and two high amylose content starch were processed by a HHP of the order of 600 MPa, at 25°C for 15min. The effect of HHP processing on the crystallization of maize starches with various amylose content during storage at 4°C was investigated. Crystallization kinetics of HHP treated starch gels were investigated using rheology and FTIR. The effect of crystallization on the mechanical properties of starch gel network were evaluated in terms of dynamic complex modulus (G*). The crystallization induced increase of short-range helices structures were investigated using FTIR. The pressure releasing rate does not affect the starch retrogradation behaviour. The rate and extent of retrogradation depends on the amylose content of amylose starch. The least retrogradation was observed in HHP treated waxy maize starch. The rate of retrogradation is higher for HHP treated high amylose maize starch than that of normal maize starch. A linear relationship between the extent of retrogradation (phase distribution) measured by FTIR and G* is proposed. PMID:27219066

  3. Kinematical evolution of tidally limited star clusters: the role of retrograde stellar orbits

    NASA Astrophysics Data System (ADS)

    Tiongco, Maria A.; Vesperini, Enrico; Varri, Anna Lisa

    2016-06-01

    The presence of an external tidal field often induces significant dynamical evolutionary effects on the internal kinematics of star clusters. Previous studies investigating the restricted three-body problem with applications to star cluster dynamics have shown that unbound stars on retrograde orbits (with respect to the direction of the cluster's orbit) are more stable against escape than prograde orbits, and predicted that a star cluster might acquire retrograde rotation through preferential escape of stars on prograde orbits. In this study we present evidence of this prediction, but we also illustrate that there are additional effects that cannot be accounted for by the preferential escape of prograde orbits alone. Specifically, in the early evolution, initially underfilling models increase their fraction of retrograde stars without losing significant mass, and acquire a retrograde angular velocity. We attribute this effect to the development of preferentially eccentric/radial orbits in the outer regions of star clusters as they are expanding into their tidal limitation. We explore the implications of the evolution of the fraction of prograde and retrograde stars for the evolution of the cluster internal rotation, and its dependence on the initial structural properties. Although all the systems studied here evolve towards an approximately solid-body internal rotation with angular velocity equal to about half of the angular velocity of the cluster orbital motion around the host galaxy, the evolutionary history of the radial profile of the cluster internal angular velocity depends on the cluster initial structure.

  4. A radiographic study of the effect of various retrograde fillings on periapical healing after replantation.

    PubMed

    Andreasen, J O; Pitt Ford, T R

    1994-12-01

    An effective retrograde sealing procedure places great demands upon both technique and materials. Prevention of micro-leakage, biocompatibility and stability of the material in the apical tissues are very important. To evaluate potential retrograde filling materials, a replantation model has been developed in which extracted permanent molars were replanted in monkeys after apicectomy of each root, preparation of a 2-mm deep retrograde cavity and its sealing with various dental materials. Prior to retro-filling the remaining pulp was exposed to saliva. Apicected molars which were infected and did not receive retrograde fillings served as positive controls. Periapical healing was evaluated radiographically after 8 weeks based on planimetric measurements of the size of the periapical radiolucency. The following dental materials were tested: amalgam, glass ionomer cement, calcium-hydroxide lining cement, AH 26 root canal sealer, various zinc oxide-eugenol cements, Cavit, and gutta-percha with various sealers. The materials which were associated with better apical healing than the infected controls were glass ionomer cement, Cavit, and the zinc oxide-eugenol cements. When plain zinc oxide-eugenol or IRM were combined with a gutta-percha core, healing was best and not statistically different from normal apices. It was concluded that radiographic assessment at 8 weeks of molar teeth retrograde filled prior to replantation could be a valuable method for discrimination of potentially useful materials in vivo. PMID:7867616

  5. Application of retrograde dissection method for isolation of bone marrow cells from rat femurs and tibiae.

    PubMed

    Li, C M; Fu, B M; Zhang, L C; Tang, B; Zhu, L; Zhao, Y; Zhang, J

    2016-01-01

    Currently, there is no practical and efficient method for the isolation of bone marrow cells (BMCs) from rat femurs and tibiae. Here, we attempted to develop a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae. Rat femurs and tibiae were dissected from the ankle to the hip joint; subsequently, a three-step "locate-slide-twist" procedure was performed using scissors and forceps to remove the femurs and tibiae completely, from the surrounding musculature. The bones were flushed with phosphate-buffered saline to harvest BMCs. The femurs and tibiae were dissected in 1.8 ± 0.6 min, and the BMC suspension preparation time was 13.1 ± 2.3 min. The bone marrow cavities did not incur any fractures or injuries during the isolation. Culture of harvested BMCs for 72 h led to a significant increase in cell number from 4.4 ± 0.3 x 106 to 6.9 ± 0.7 x 10(6) (P < 0.01) with no significant decrease in viability (98.1 ± 0.6% vs 96.2 ± 1.1%; P > 0.05). Microscopic examination of the isolated BMCs after the 72-h incubation period revealed the no-microbial or muscle cell contamination. Furthermore, flow cytometry revealed that cultured BMCs (72-h culture) grew well. Here, we have reported a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae by using retrograde dissection. This method can be used to harvest a large number of viable BMCs without the risk of contamination from muscle and connective tissues. PMID:27323101

  6. GBT Reveals Satellite of Milky Way in Retrograde Orbit

    NASA Astrophysics Data System (ADS)

    2003-05-01

    New observations with National Science Foundation's Robert C. Byrd Green Bank Telescope (GBT) suggest that what was once believed to be an intergalactic cloud of unknown distance and significance, is actually a previously unrecognized satellite galaxy of the Milky Way orbiting backward around the Galactic center. Path of Complex H Artist's rendition of the path of satellite galaxy Complex H (in red) in relation to the orbit of the Sun (in yellow) about the center of the Milky Way Galaxy. The outer layers of Complex H are being stripped away by its interaction with the Milky Way. The hydrogen atmosphere (in blue) is shown surrounding the visible portion (in white) of the Galaxy. CREDIT: Lockman, Smiley, Saxton; NRAO/AUI Jay Lockman of the National Radio Astronomy Observatory (NRAO) in Green Bank, West Virginia, discovered that this object, known as "Complex H," is crashing through the outermost parts of the Milky Way from an inclined, retrograde orbit. Lockman's findings will be published in the July 1 issue of the Astrophysical Journal, Letters. "Many astronomers assumed that Complex H was probably a distant neighbor of the Milky Way with some unusual velocity that defied explanation," said Lockman. "Since its motion appeared completely unrelated to Galactic rotation, astronomers simply lumped it in with other high velocity clouds that had strange and unpredictable trajectories." High velocity clouds are essentially what their name implies, fast-moving clouds of predominately neutral atomic hydrogen. They are often found at great distances from the disk of the Milky Way, and may be left over material from the formation of our Galaxy and other galaxies in our Local Group. Over time, these objects can become incorporated into larger galaxies, just as small asteroids left over from the formation of the solar system sometimes collide with the Earth. Earlier studies of Complex H were hindered because the cloud currently is passing almost exactly behind the outer disk of

  7. The Structure and Function of Bacterial Actin Homologs

    PubMed Central

    Shaevitz, Joshua W.; Gitai, Zemer

    2010-01-01

    During the past decade, the appreciation and understanding of how bacterial cells can be organized in both space and time have been revolutionized by the identification and characterization of multiple bacterial homologs of the eukaryotic actin cytoskeleton. Some of these bacterial actins, such as the plasmid-borne ParM protein, have highly specialized functions, whereas other bacterial actins, such as the chromosomally encoded MreB protein, have been implicated in a wide array of cellular activities. In this review we cover our current understanding of the structure, assembly, function, and regulation of bacterial actins. We focus on ParM as a well-understood reductionist model and on MreB as a central organizer of multiple aspects of bacterial cell biology. We also discuss the outstanding puzzles in the field and possible directions where this fast-developing area may progress in the future. PMID:20630996

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

    PubMed Central

    Ammer, Amanda Gatesman; Weed, Scott A.

    2008-01-01

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

  9. Nanosecond electric pulses trigger actin responses in plant cells

    SciTech Connect

    Berghoefer, Thomas; Eing, Christian; Flickinger, Bianca; Hohenberger, Petra; Wegner, Lars H.; Frey, Wolfgang; Nick, Peter

    2009-09-25

    We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

  10. 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.

  11. Direct interaction of beta-dystroglycan with F-actin.

    PubMed Central

    Chen, Yun-Ju; Spence, Heather J; Cameron, Jacqueline M; Jess, Thomas; Ilsley, Jane L; Winder, Steven J

    2003-01-01

    Dystroglycans are essential transmembrane adhesion receptors for laminin. Alpha-dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of beta-dystroglycan. Beta-dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin-glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of beta-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells. PMID:12892561

  12. Mechanism of Actin Network Attachment to Moving Membranes

    PubMed Central

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

    2007-01-01

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

  13. Differential requirements for actin during yeast and mammalian endocytosis.

    PubMed

    Aghamohammadzadeh, Soheil; Ayscough, Kathryn R

    2009-08-01

    Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination. PMID:19597484

  14. Stable high capacity, F-actin affinity column

    SciTech Connect

    Luna, E.J.; Wang, Y.L.; Voss, E.W. Jr.; Branton, D.; Taylor, D.L.

    1982-11-10

    A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds /sup 125/I-heavy meromyosin and /sup 125/I-tropomyosin. The associations between the F-actin-binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.