Sample records for actinic flux density

  1. Calibration and evaluation of CCD spectroradiometers for ground-based and airborne measurements of spectral actinic flux densities

    NASA Astrophysics Data System (ADS)

    Bohn, Birger; Lohse, Insa

    2017-09-01

    The properties and performance of charge-coupled device (CCD) array spectroradiometers for the measurement of atmospheric spectral actinic flux densities (280-650 nm) and photolysis frequencies were investigated. These instruments are widely used in atmospheric research and are suitable for aircraft applications because of high time resolutions and high sensitivities in the UV range. The laboratory characterization included instrument-specific properties like the wavelength accuracy, dark signal, dark noise and signal-to-noise ratio (SNR). Spectral sensitivities were derived from measurements with spectral irradiance standards. The calibration procedure is described in detail, and a straightforward method to minimize the influence of stray light on spectral sensitivities is introduced. From instrument dark noise, minimum detection limits ≈ 1 × 1010 cm-2 s-1 nm-1 were derived for spectral actinic flux densities at wavelengths around 300 nm (1 s integration time). As a prerequisite for the determination of stray light under field conditions, atmospheric cutoff wavelengths were defined using radiative transfer calculations as a function of the solar zenith angle (SZA) and total ozone column (TOC). The recommended analysis of field data relies on these cutoff wavelengths and is also described in detail taking data from a research flight on HALO (High Altitude and Long Range Research Aircraft) as an example. An evaluation of field data was performed by ground-based comparisons with a double-monochromator-based, highly sensitive reference spectroradiometer. Spectral actinic flux densities were compared as well as photolysis frequencies j(NO2) and j(O1D), representing UV-A and UV-B ranges, respectively. The spectra expectedly revealed increased daytime levels of stray-light-induced signals and noise below atmospheric cutoff wavelengths. The influence of instrument noise and stray-light-induced noise was found to be insignificant for j(NO2) and rather limited for j(O1D

  2. Analysis of actinic flux profiles measured from an ozonesonde balloon

    NASA Astrophysics Data System (ADS)

    Wang, P.; Allaart, M.; Knap, W. H.; Stammes, P.

    2015-04-01

    A green light sensor has been developed at KNMI to measure actinic flux profiles using an ozonesonde balloon. In total, 63 launches with ascending and descending profiles were performed between 2006 and 2010. The measured uncalibrated actinic flux profiles are analysed using the Doubling-Adding KNMI (DAK) radiative transfer model. Values of the cloud optical thickness (COT) along the flight track were taken from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) Cloud Physical Properties (CPP) product. The impact of clouds on the actinic flux profile is evaluated on the basis of the cloud modification factor (CMF) at the cloud top and cloud base, which is the ratio between the actinic fluxes for cloudy and clear-sky scenes. The impact of clouds on the actinic flux is clearly detected: the largest enhancement occurs at the cloud top due to multiple scattering. The actinic flux decreases almost linearly from cloud top to cloud base. Above the cloud top the actinic flux also increases compared to clear-sky scenes. We find that clouds can increase the actinic flux to 2.3 times the clear-sky value at cloud top and decrease it to about 0.05 at cloud base. The relationship between CMF and COT agrees well with DAK simulations, except for a few outliers. Good agreement is found between the DAK-simulated actinic flux profiles and the observations for single-layer clouds in fully overcast scenes. The instrument is suitable for operational balloon measurements because of its simplicity and low cost. It is worth further developing the instrument and launching it together with atmospheric chemistry composition sensors.

  3. Investigation of the 3-D actinic flux field in mountainous terrain

    PubMed Central

    Wagner, J.E.; Angelini, F.; Blumthaler, M.; Fitzka, M.; Gobbi, G.P.; Kift, R.; Kreuter, A.; Rieder, H.E.; Simic, S.; Webb, A.; Weihs, P.

    2011-01-01

    During three field campaigns spectral actinic flux was measured from 290–500 nm under clear sky conditions in Alpine terrain and the associated O3- and NO2-photolysis frequencies were calculated and the measurement products were then compared with 1-D- and 3-D-model calculations. To do this 3-D-radiative transfer model was adapted for actinic flux calculations in mountainous terrain and the maps of the actinic flux field at the surface, calculated with the 3-D-radiative transfer model, are given. The differences between the 3-D- and 1-D-model results for selected days during the campaigns are shown, together with the ratios of the modeled actinic flux values to the measurements. In many cases the 1-D-model overestimates actinic flux by more than the measurement uncertainty of 10%. The results of using a 3-D-model generally show significantly lower values, and can underestimate the actinic flux by up to 30%. This case study attempts to quantify the impact of snow cover in combination with topography on spectral actinic flux. The impact of snow cover on the actinic flux was ~ 25% in narrow snow covered valleys, but for snow free areas there were no significant changes due snow cover in the surrounding area and it is found that the effect snow-cover at distances over 5 km from the point of interest was below 5%. Overall the 3-D-model can calculate actinic flux to the same accuracy as the 1-D-model for single points, but gives a much more realistic view of the surface actinic flux field in mountains as topography and obstruction of the horizon are taken into account. PMID:26412915

  4. Actinic Flux Calculations: A Model Sensitivity Study

    NASA Technical Reports Server (NTRS)

    Krotkov, Nickolay A.; Flittner, D.; Ahmad, Z.; Herman, J. R.; Einaudi, Franco (Technical Monitor)

    2000-01-01

    calculate direct and diffuse surface irradiance and actinic flux (downwelling (2p) and total (4p)) for the reference model. Sensitivity analysis has shown that the accuracy of the radiative transfer flux calculations for a unit ETS (i.e. atmospheric transmittance) together with a numerical interpolation technique for the constituents' vertical profiles is better than 1% for SZA less than 70(sub o) and wavelengths longer than 310 nm. The differences increase for shorter wavelengths and larger SZA, due to the differences in pseudo-spherical correction techniques and vertical discretetization among the codes. Our sensitivity study includes variation of ozone cross-sections, ETS spectra and the effects of wavelength shifts between vacuum and air scales. We also investigate the effects of aerosols on the spectral flux components in the UV and visible spectral regions. The "aerosol correction factors" (ACFs) were calculated at discrete wavelengths and different SZAs for each flux component (direct, diffuse, reflected) and prescribed IPMMI aerosol parameters. Finally, the sensitivity study was extended to calculation of selected photolysis rates coefficients.

  5. Intramolecular Nuclear Flux Densities

    NASA Astrophysics Data System (ADS)

    Barth, I.; Daniel, C.; Gindensperger, E.; Manz, J.; PéRez-Torres, J. F.; Schild, A.; Stemmle, C.; Sulzer, D.; Yang, Y.

    The topic of this survey article has seen a renaissance during the past couple of years. Here we present and extend the results for various phenomena which we have published from 2012-2014, with gratitude to our coauthors. The new phenomena include (a) the first reduced nuclear flux densities in vibrating diatomic molecules or ions which have been deduced from experimental pump-probe spectra; these "experimental" nuclear flux densities reveal several quantum effects including (b) the "quantum accordion", i.e., during the turn from bond stretch to bond compression, the diatomic system never stands still — instead, various parts of it with different bond lengths flow into opposite directions. (c) Wavepacket interferometry has been extended from nuclear densities to flux densities, again revealing new phenomena: For example, (d) a vibrating nuclear wave function with compact initial shape may split into two partial waves which run into opposite directions, thus causing interfering flux densities. (e) Tunneling in symmetric 1-dimensional double-well systems yields maximum values of the associated nuclear flux density just below the potential barrier; this is in marked contrast with negligible values of the nuclear density just below the barrier. (f) Nuclear flux densities of pseudorotating nuclei may induce huge magnetic fields. A common methodologic theme of all topics is the continuity equation which connects the time derivative of the nuclear density to the divergence of the flux density, subject to the proper boundary conditions. (g) Nearly identical nuclear densities with different boundary conditions may be related to entirely different flux densities, e.g., during tunneling in cyclic versus non-cyclic systems. The original continuity equation, density and flux density of all nuclei, or of all nuclear degrees of freedom, may be reduced to the corresponding quantities for just a single nucleus, or just a single degree of freedom.

  6. Solar actinic flux spectroradiometry: a technique for measuring photolysis frequencies in the atmosphere.

    PubMed

    Hofzumahaus, A; Kraus, A; Müller, M

    1999-07-20

    A spectroradiometer has been developed for direct measurement of the solar actinic UV flux (scalar intensity) and determination of photolysis frequencies in the atmosphere. The instrument is based on a scanning double monochromator with an entrance optic that exhibits an isotropic angular response over a solid angle of 2pi sr. Actinic flux spectra are measured at a resolution of 1 nm across a range of 280-420 nm, which is relevant for most tropospheric photolysis processes. The photolysis frequencies are derived from the measured radiation spectra by use of published absorption cross sections and quantum yields. The advantage of this technique compared with the traditional chemical actinometry is its versatility. It is possible to determine the photolysis frequency for any photochemical reaction of interest provided that the respective molecular photodissociation parameters are known and the absorption cross section falls within a wavelength range that is accessible by the spectroradiometer. The instrument and the calibration procedures are described in detail, and problems specific to measurement of the actinic radiation are discussed. An error analysis is presented together with a discussion of the spectral requirements of the instrument for accurate measurements of important tropospheric photolysis frequencies (J(O(1))(D), J(NO(2)), J(HCHO)). An example of measurements from previous atmospheric chemistry field campaigns are presented and discussed.

  7. Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy

    NASA Astrophysics Data System (ADS)

    Clausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M.

    2017-02-01

    Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50  ±  15 nm and 120  ±  40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

  8. Fast two-stream method for computing diurnal-mean actinic flux in vertically inhomogeneous atmospheres

    NASA Technical Reports Server (NTRS)

    Filyushkin, V. V.; Madronich, S.; Brasseur, G. P.; Petropavlovskikh, I. V.

    1994-01-01

    Based on a derivation of the two-stream daytime-mean equations of radiative flux transfer, a method for computing the daytime-mean actinic fluxes in the absorbing and scattering vertically inhomogeneous atmosphere is suggested. The method applies direct daytime integration of the particular solutions of the two-stream approximations or the source functions. It is valid for any duration of period of averaging. The merit of the method is that the multiple scattering computation is carried out only once for the whole averaging period. It can be implemented with a number of widely used two-stream approximations. The method agrees with the results obtained with 200-point multiple scattering calculations. The method was also tested in runs with a 1-km cloud layer with optical depth of 10, as well as with aerosol background. Comparison of the results obtained for a cloud subdivided into 20 layers with those obtained for a one-layer cloud with the same optical parameters showed that direct integration of particular solutions possesses an 'analytical' accuracy. In the case of the source function interpolation, the actinic fluxes calculated above the one-layer and 20-layer clouds agreed within 1%-1.5%, while below the cloud they may differ up to 5% (in the worst case). The ways of enhancing the accuracy (in a 'two-stream sense') and computational efficiency of the method are discussed.

  9. 47 CFR 25.208 - Power flux density limits.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... COMMUNICATIONS Technical Standards § 25.208 Power flux density limits. (a) In the band 3650-4200 MHz, the power flux density at the Earth's surface produced by emissions from a space station for all conditions and... and 10.7-11.7 GHz for NGSO FSS space stations, the power flux-density at the Earth's surface produced...

  10. Increased fibroblast density in actinic cheilitis: association with tryptase-positive mast cells, actinic elastosis and epithelial p53 and COX-2 expression.

    PubMed

    Rojas, Isolde G; Boza, Yadira V; Spencer, Maria Loreto; Flores, Maritza; Martínez, Alejandra

    2012-01-01

    Actinic cheilitis (AC) is characterized by epithelial and connective tissue alterations caused by ultraviolet sunlight overexposure known as photodamage. Fibroblasts have been linked to photodamage and tumor progression during skin carcinogenesis; however, their role in early lip carcinogenesis remains unknown. The aim of this study was to assess the density of fibroblasts in AC and normal lip (NL) samples and determine their association with markers of lip photodamage. Fibroblasts, mast cells, p53, COX-2, and elastin were detected in NL (n = 20) and AC (n = 28) biopsies using immunohistochemistry/histochemistry. Mast cell and fibroblast density and epithelial p53 and COX-2 expression scores were then obtained. Elastosis was scored 1-4 according to elastin fiber density and tortuosity. Fibroblasts, mast cells, p53, COX-2, and elastosis were increased in AC as compared to NL (P < 0.001). Multivariate analysis showed an association between fibroblast and mast cell density at the papillary and reticular areas of AC and NL (P < 0.05). Papillary fibroblast density was also associated with epithelial p53 and COX-2 expression (P < 0.05). Increased fibroblast density, both papillary and reticular, was found in the high elastosis group (scores 3-4) as compared to the low elastosis group (scores 1-2) (P < 0.01). Increased reticular mast cell density was detected only in the high elastosis group (P < 0.01). Fibroblasts are increased in AC, and they are associated with mast cell density, epithelial p53 and COX-2 expression, and actinic elastosis. Therefore, fibroblasts may contribute to lip photodamage and could be considered useful markers of early lip carcinogenesis. © 2011 John Wiley & Sons A/S.

  11. Sensors for Metering Heat Flux Area Density and Metrological Equipment for the Heat Flux Density Measurement

    NASA Astrophysics Data System (ADS)

    Doronin, D. O.

    2018-04-01

    The demand in measuring and studies of heat conduction of various media is very urgent now. This article considers the problem of heat conduction monitoring and measurement in various media and materials in any industries and branches of science as well as metrological support of the heat flux measurement equipment. The main study objects are both the sensors manufactured and facilities onto which these sensors will be installed: different cladding structures of the buildings, awnings, rocket fairings, boiler units, internal combustion engines. The Company develops and manufactures different types of heat flux sensors: thermocouple, thin-film, heterogeneous gradient as well as metrological equipment for the gauging calibration of the heat flux density measurement. The calibration shall be performed using both referencing method in the unit and by fixed setting of the heat flux in the unit. To manufacture heterogeneous heat flux gradient sensors (HHFGS) the Company developed and designed a number of units: diffusion welding unit, HHFGS cutting unit. Rather good quality HHFGS prototypes were obtained. At this stage the factory tests on the equipment for the heat flux density measurement equipment are planned. A high-sensitivity heat flux sensor was produced, now it is tested at the Construction Physics Research Institute (Moscow). It became possible to create thin-film heat flux sensors with the sensitivity not worse than that of the sensors manufactured by Captec Company (France). The Company has sufficient premises to supply the market with a wide range of sensors, to master new sensor manufacture technologies which will enable their application range.

  12. Measurement of Flux Density of Cas A at Low Frequencies

    NASA Astrophysics Data System (ADS)

    Patil, Ajinkya; Fisher, R.

    2012-01-01

    Cas A is used as a flux calibrator throughout the radio spectrum. Therefore it is important to know the spectral and secular variations in its flux density. Earlier observations by Scott et. al. (1969) and Baars et. al. (1972) suggested a secular decrease in flux density of Cas A at a rate of about 1% per year at all frequencies. However later observations by Erickson & Perley (1975) and Read (1977) indicated anomalously high flux from Cas A at 38 MHz. Also, these observations suggested that the original idea of faster decay of the flux density rate at low frequencies may be in error or that something more complex than simple decay is affecting the flux density at low frequencies. The source changes at 38 MHz still remains a mystery. We intend to present the results of follow up observations made from 1995 to 1998 with a three element interferometer in Green Bank operating in frequency range 30 to 120 MHz. We will discuss the problems at such low frequencies due to large beamwidth and unstable ionosphere. We will also discuss the strategies we have used so far to to find the flux density of Cas A by calculating the ratio of flux density of Cas A to that of Cyg A, assuming flux density of Cyg A to be constant. Above mentioned work was performed in summer student program sponsored by National Radio Astronomy Observatory.

  13. Correlated flux densities from VLBI observations with the DSN

    NASA Technical Reports Server (NTRS)

    Coker, R. F.

    1992-01-01

    Correlated flux densities of extragalactic radio sources in the very long baseline interferometry (VLBI) astrometric catalog are required for the VLBI tracking of Galileo, Mars Observer, and future missions. A system to produce correlated and total flux density catalogs was developed to meet these requirements. A correlated flux density catalog of 274 sources, accurate to about 20 percent, was derived from more than 5000 DSN VLBI observations at 2.3 GHz (S-band) and 8.4 GHz (X-band) using 43 VLBI radio reference frame experiments during the period 1989-1992. Various consistency checks were carried out to ensure the accuracy of the correlated flux densities. All observations were made on the California-Spain and California-Australia DSN baselines using the Mark 3 wideband data acquisition system. A total flux density catalog, accurate to about 20 percent, with data on 150 sources, was also created. Together, these catalogs can be used to predict source strengths to assist in the scheduling of VLBI tracking passes. In addition, for those sources with sufficient observations, a rough estimate of source structure parameters can be made.

  14. Meteoroid stream flux densities and the zenith exponent

    NASA Astrophysics Data System (ADS)

    Molau, Sirko; Barentsen, Geert

    2013-01-01

    The MetRec software was recently extended to measure the limiting magnitude in real-time, and to determine meteoroid stream flux densities. This paper gives a short overview of the applied algorithms. We introduce the MetRec Flux Viewer, a web tool to visualize activity profiles on- line. Starting from the Lyrids 2011, high-quality flux density profiles were derived from IMO Video Network observations for every major meteor shower. They are often in good agreement with visual data. Analyzing the 2011 Perseids, we found systematic daily variations in the flux density profile, which can be attributed to a zenith exponent gamma > 1.0. We analyzed a number of meteor showers in detail and found zenith exponent variations from shower to shower in the range between 1.55 and 2.0. The average value over all analyzed showers is gamma = 1.75. In order to determine the zenith exponent precisely, the observations must cover a large altitude range (at least 45 degrees).

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

    PubMed

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

    2013-04-16

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

  16. Rheology of Membrane-Attached Minimal Actin Cortices.

    PubMed

    Nöding, Helen; Schön, Markus; Reinermann, Corinna; Dörrer, Nils; Kürschner, Aileen; Geil, Burkhard; Mey, Ingo; Heussinger, Claus; Janshoff, Andreas; Steinem, Claudia

    2018-04-26

    The actin cortex is a thin cross-linked network attached to the plasma membrane, which is responsible for the cell's shape during migration, division, and growth. In a reductionist approach, we created a minimal actin cortex (MAC) attached to a lipid membrane to correlate the filamentous actin architecture with its viscoelastic properties. The system is composed of a supported 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayer doped with the receptor lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P 2 ) to which a constitutively active mutant of ezrin, which is a direct membrane-cytoskeleton linker, is bound. The formation of the MAC on the supported lipid bilayer is analyzed as a function of increasing PtdIns(4,5)P 2 /ezrin pinning points, revealing an increase in the intersections between actin filaments, that is, the node density of the MAC. Bead tracking microrheology on the membrane-attached actin network provides information about its viscoelastic properties. The results show that ezrin serves as a dynamic cross-linker for the actin cortex attached to the lipid bilayer and that the stiffness of the network is influenced by the pinning point density, relating the plateau storage modulus G 0 to the node density of the MAC.

  17. Spitzer Mid-to-Far-Infrared Flux Densities of Distant Galaxies

    NASA Astrophysics Data System (ADS)

    Papovich, Casey J.; Rudnick, G.; Le Floc'h, E.; van Dokkum, P. G.; Rieke, G. H.; Taylor, E. N.; Armus, L.; Gawiser, E.; Marcillac, D.; Huang, J.; Franx, M.

    2007-05-01

    We study the 24, 70, and 160 μm properties of high-redshift galaxies. Our primary interest is to improve the constraints on the total infrared (IR) luminosities, L(IR), of these galaxies. We combine Spitzer data in the southern Extended Chandra Deep Field with a Ks-band-selected galaxy sample with photometric redshifts from the Multiwavelength Survey by Yale-Chile. We used a stacking analysis to measure the average 70 and 160 μm flux densities of 1.5 < zph < 2.5 galaxies as a function of 24 μm flux density, X-ray activity, and rest-frame near-IR color. Galaxies with 1.5 < zph < 2.5 and S(24) = 54-250 μJy have L(IR) derived from their average 24-160 μm flux densities within factors of 2-3 of those derived from the 24 μm flux densities only. However, L(IR) derived from the average 24-160 μm flux densities for galaxies with S(24) > 250 μJy and 1.5 < zph < 2.5 are lower than those derived using only the 24 μm flux density by factors of 2-6. Galaxies with S(24) > 250 μJy have S(70)/S(24) flux ratios comparable to sources with X-ray detections or red rest-frame IR colors, suggesting that warm dust possibly heated by AGN produces high 24 μm emission. Based on the average 24-160 μm flux densities, 24 μm-selected galaxies at 1.5 < zph < 2.5 have an upper envelope of L(IR) < 6 × 1012 L⊙, which if attributed to star formation corresponds to < 1000 M⊙ yr-1. This envelope is similar to the maximal star formation rate observed in low redshift galaxies, suggesting that high redshift galaxies have star formation efficiencies and feedback processes comparable to lower redshift analogs. Support for this work was provided by NASA through the Spitzer Space Telescope Fellowship Program, through a contract issued by JPL, Caltech under a contract with NASA.

  18. 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

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

    PubMed Central

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

    2013-01-01

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

  20. Magnetic Flux Density of Different Types of New Generation Magnetic Attachment Systems.

    PubMed

    Akin, Hakan

    2015-07-01

    The purpose of this study was to analyze the static magnetic flux density of different types of new generation laser-welded magnetic attachments in the single position and the attractive position and to determine the effect of different corrosive environments on magnetic flux density. Magnetic flux densities of four magnetic attachment systems (Hyper slim, Hicorex slim, Dyna, and Steco) were measured with a gaussmeter. Then magnetic attachment systems were immersed in two different media, namely 1% lactic acid solution (pH 2.3), and 0.9% NaCl solution (pH 7.3). Magnetic flux densities of the attachment systems were measured with a gaussmeter after immersion to compare with measurements before immersion (α = 0.05). The data were statistically evaluated with one-way ANOVA, paired-samples t-test, and post hoc Tukey-Kramer multiple comparisons tests (α = 0.05). The highest magnetic flux density was found in Dyna magnets for both single and attractive positions. In addition, after the magnets were in the corrosive environments for 2 weeks, they had a significant decrease in magnetic flux density (p < 0.05). No significant differences were found between corrosive environments (p > 0.05). The leakage flux of all the magnetic attachments did not exceed the WHO's guideline of 40 mT. The magnets exhibited a significant decrease in magnetic flux density after aging in corrosive environments including lactic acid and NaCl. © 2014 by the American College of Prosthodontists.

  1. Fourier transform magnetic resonance current density imaging (FT-MRCDI) from one component of magnetic flux density.

    PubMed

    Ider, Yusuf Ziya; Birgul, Ozlem; Oran, Omer Faruk; Arikan, Orhan; Hamamura, Mark J; Muftuler, L Tugan

    2010-06-07

    Fourier transform (FT)-based algorithms for magnetic resonance current density imaging (MRCDI) from one component of magnetic flux density have been developed for 2D and 3D problems. For 2D problems, where current is confined to the xy-plane and z-component of the magnetic flux density is measured also on the xy-plane inside the object, an iterative FT-MRCDI algorithm is developed by which both the current distribution inside the object and the z-component of the magnetic flux density on the xy-plane outside the object are reconstructed. The method is applied to simulated as well as actual data from phantoms. The effect of measurement error on the spatial resolution of the current density reconstruction is also investigated. For 3D objects an iterative FT-based algorithm is developed whereby the projected current is reconstructed on any slice using as data the Laplacian of the z-component of magnetic flux density measured for that slice. In an injected current MRCDI scenario, the current is not divergence free on the boundary of the object. The method developed in this study also handles this situation.

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

    PubMed Central

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

    2012-01-01

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

  3. 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.

  4. Method for determining transport critical current densities and flux penetration depth in bulk superconductors

    NASA Technical Reports Server (NTRS)

    Israelsson, Ulf E. (Inventor); Strayer, Donald M. (Inventor)

    1992-01-01

    A contact-less method for determining transport critical current density and flux penetration depth in bulk superconductor material. A compressor having a hollow interior and a plunger for selectively reducing the free space area for distribution of the magnetic flux therein are formed of superconductor material. Analytical relationships, based upon the critical state model, Maxwell's equations and geometrical relationships define transport critical current density and flux penetration depth in terms of the initial trapped magnetic flux density and the ratio between initial and final magnetic flux densities whereby data may be reliably determined by means of the simple test apparatus for evaluating the current density and flux penetration depth.

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

    PubMed

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

    2016-04-01

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

  6. Planck intermediate results. LII. Planet flux densities

    NASA Astrophysics Data System (ADS)

    Planck Collaboration; Akrami, Y.; Ashdown, M.; Aumont, J.; Baccigalupi, C.; Ballardini, M.; Banday, A. J.; Barreiro, R. B.; Bartolo, N.; Basak, S.; Benabed, K.; Bernard, J.-P.; Bersanelli, M.; Bielewicz, P.; Bonavera, L.; Bond, J. R.; Borrill, J.; Bouchet, F. R.; Boulanger, F.; Bucher, M.; Burigana, C.; Butler, R. C.; Calabrese, E.; Cardoso, J.-F.; Carron, J.; Chiang, H. C.; Colombo, L. P. L.; Comis, B.; Couchot, F.; Coulais, A.; Crill, B. P.; Curto, A.; Cuttaia, F.; de Bernardis, P.; de Rosa, A.; de Zotti, G.; Delabrouille, J.; Di Valentino, E.; Dickinson, C.; Diego, J. M.; Doré, O.; Ducout, A.; Dupac, X.; Elsner, F.; Enßlin, T. A.; Eriksen, H. K.; Falgarone, E.; Fantaye, Y.; Finelli, F.; Frailis, M.; Fraisse, A. A.; Franceschi, E.; Frolov, A.; Galeotta, S.; Galli, S.; Ganga, K.; Génova-Santos, R. T.; Gerbino, M.; González-Nuevo, J.; Górski, K. M.; Gruppuso, A.; Gudmundsson, J. E.; Hansen, F. K.; Helou, G.; Henrot-Versillé, S.; Herranz, D.; Hivon, E.; Jaffe, A. H.; Jones, W. C.; Keihänen, E.; Keskitalo, R.; Kiiveri, K.; Kim, J.; Kisner, T. S.; Krachmalnicoff, N.; Kunz, M.; Kurki-Suonio, H.; Lagache, G.; Lamarre, J.-M.; Lasenby, A.; Lattanzi, M.; Lawrence, C. R.; Le Jeune, M.; Lellouch, E.; Levrier, F.; Liguori, M.; Lilje, P. B.; Lindholm, V.; López-Caniego, M.; Ma, Y.-Z.; Macías-Pérez, J. F.; Maggio, G.; Maino, D.; Mandolesi, N.; Maris, M.; Martin, P. G.; Martínez-González, E.; Matarrese, S.; Mauri, N.; McEwen, J. D.; Melchiorri, A.; Mennella, A.; Migliaccio, M.; Miville-Deschênes, M.-A.; Molinari, D.; Moneti, A.; Montier, L.; Moreno, R.; Morgante, G.; Natoli, P.; Oxborrow, C. A.; Paoletti, D.; Partridge, B.; Patanchon, G.; Patrizii, L.; Perdereau, O.; Piacentini, F.; Plaszczynski, S.; Polenta, G.; Rachen, J. P.; Racine, B.; Reinecke, M.; Remazeilles, M.; Renzi, A.; Rocha, G.; Romelli, E.; Rosset, C.; Roudier, G.; Rubiño-Martín, J. A.; Ruiz-Granados, B.; Salvati, L.; Sandri, M.; Savelainen, M.; Scott, D.; Sirri, G.; Spencer, L. D.; Suur-Uski, A.-S.; Tauber, J. A.; Tavagnacco, D.; Tenti, M.; Toffolatti, L.; Tomasi, M.; Tristram, M.; Trombetti, T.; Valiviita, J.; Van Tent, F.; Vielva, P.; Villa, F.; Wehus, I. K.; Zacchei, A.

    2017-11-01

    Measurements of flux density are described for five planets, Mars, Jupiter, Saturn, Uranus, and Neptune, across the six Planck High Frequency Instrument frequency bands (100-857 GHz) and these are then compared with models and existing data. In our analysis, we have also included estimates of the brightness of Jupiter and Saturn at the three frequencies of the Planck Low Frequency Instrument (30, 44, and 70 GHz). The results provide constraints on the intrinsic brightness and the brightness time-variability of these planets. The majority of the planet flux density estimates are limited by systematic errors, but still yield better than 1% measurements in many cases. Applying data from Planck HFI, the Wilkinson Microwave Anisotropy Probe (WMAP), and the Atacama Cosmology Telescope (ACT) to a model that incorporates contributions from Saturn's rings to the planet's total flux density suggests a best fit value for the spectral index of Saturn's ring system of βring = 2.30 ± 0.03 over the 30-1000 GHz frequency range. Estimates of the polarization amplitude of the planets have also been made in the four bands that have polarization-sensitive detectors (100-353 GHz); this analysis provides a 95% confidence level upper limit on Mars's polarization of 1.8, 1.7, 1.2, and 1.7% at 100, 143, 217, and 353 GHz, respectively. The average ratio between the Planck-HFI measurements and the adopted model predictions for all five planets (excluding Jupiter observations for 353 GHz) is 1.004, 1.002, 1.021, and 1.033 for 100, 143, 217, and 353 GHz, respectively. Model predictions for planet thermodynamic temperatures are therefore consistent with the absolute calibration of Planck-HFI detectors at about the three-percent level. We compare our measurements with published results from recent cosmic microwave background experiments. In particular, we observe that the flux densities measured by Planck HFI and WMAP agree to within 2%. These results allow experiments operating in the mm

  7. Surface radiant flux densities inferred from LAC and GAC AVHRR data

    NASA Astrophysics Data System (ADS)

    Berger, F.; Klaes, D.

    To infer surface radiant flux densities from current (NOAA-AVHRR, ERS-1/2 ATSR) and future meteorological (Envisat AATSR, MSG, METOP) satellite data, the complex, modular analysis scheme SESAT (Strahlungs- und Energieflüsse aus Satellitendaten) could be developed (Berger, 2001). This scheme allows the determination of cloud types, optical and microphysical cloud properties as well as surface and TOA radiant flux densities. After testing of SESAT in Central Europe and the Baltic Sea catchment (more than 400scenes U including a detailed validation with various surface measurements) it could be applied to a large number of NOAA-16 AVHRR overpasses covering the globe.For the analysis, two different spatial resolutions U local area coverage (LAC) andwere considered. Therefore, all inferred results, like global area coverage (GAC) U cloud cover, cloud properties and radiant properties, could be intercompared. Specific emphasis could be made to the surface radiant flux densities (all radiative balance compoments), where results for different regions, like Southern America, Southern Africa, Northern America, Europe, and Indonesia, will be presented. Applying SESAT, energy flux densities, like latent and sensible heat flux densities could also be determined additionally. A statistical analysis of all results including a detailed discussion for the two spatial resolutions will close this study.

  8. Liquid behavior of cross-linked actin bundles.

    PubMed

    Weirich, Kimberly L; Banerjee, Shiladitya; Dasbiswas, Kinjal; Witten, Thomas A; Vaikuntanathan, Suriyanarayanan; Gardel, Margaret L

    2017-02-28

    The actin cytoskeleton is a critical regulator of cytoplasmic architecture and mechanics, essential in a myriad of physiological processes. Here we demonstrate a liquid phase of actin filaments in the presence of the physiological cross-linker, filamin. Filamin condenses short actin filaments into spindle-shaped droplets, or tactoids, with shape dynamics consistent with a continuum model of anisotropic liquids. We find that cross-linker density controls the droplet shape and deformation timescales, consistent with a variable interfacial tension and viscosity. Near the liquid-solid transition, cross-linked actin bundles show behaviors reminiscent of fluid threads, including capillary instabilities and contraction. These data reveal a liquid droplet phase of actin, demixed from the surrounding solution and dominated by interfacial tension. These results suggest a mechanism to control organization, morphology, and dynamics of the actin cytoskeleton.

  9. Spillage and flux density on a receiver aperture lip. [of solar thermal collector

    NASA Technical Reports Server (NTRS)

    Jaffe, L. D.

    1985-01-01

    In a dish-type point-focusing solar thermal collector, the spillage and the flux density on the receiver aperture lip are related in a very simple way, if the aperture is circular and centered on the optical axis. Specifically, the flux density on the lip is equal to the spillage times the peak flux density in the plane of the lip.

  10. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-02

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.

  11. Gas Flux and Density Surrounding a Cylindrical Aperture in the Free Molecular Flow Regime

    NASA Technical Reports Server (NTRS)

    Soulas, George C.

    2011-01-01

    The equations for rigorously calculating the particle flux and density surrounding a cylindrical aperture in the free molecular flow regime are developed and presented. The fundamental equations for particle flux and density from a reservoir and a diffusely reflecting surface will initially be developed. Assumptions will include a Maxwell-Boltzmann speed distribution, equal particle and wall temperatures, and a linear flux distribution along the cylindrical aperture walls. With this information, the equations for axial flux and density surrounding a cylindrical aperture will be developed. The cylindrical aperture will be divided into multiple volumes and regions to rigorously determine the surrounding axial flux and density, and appropriate limits of integration will be determined. The results of these equations will then be evaluated. The linear wall flux distribution assumption will be assessed. The axial flux and density surrounding a cylindrical aperture with a thickness-to-radius ratio of 1.25 will be presented. Finally, the equations determined in this study will be verified using multiple methods.

  12. Extracorporeal shock wave therapy with low-energy flux density inhibits hypertrophic scar formation in an animal model.

    PubMed

    Zhao, Jing-Chun; Zhang, Bo-Ru; Hong, Lei; Shi, Kai; Wu, Wei-Wei; Yu, Jia-Ao

    2018-04-01

    Hypertrophic scar is characterized by excessive deposits of collagen during skin wound healing, which could become a challenge to clinicians. This study assessed the effects of the extracorporeal shock wave therapy (ESWT) on hypertrophic scar formation and the underlying gene regu-lation. A rabbit ear hypertrophic scar model was generated and randomly divided into three groups: L-ESWT group to receive L-ESWT (energy flux density of 0.1 mJ/mm2), H-ESWT (energy flux density of 0.2 mJ/mm2) and sham ESWT group (S-ESWT). Hypertrophic scar tissues were then collected and stained with hematoxylin and eosin (H&E) and Masson's trichrome staining, respectively, to assess scar elevation index (SEI), fibroblast density and collagen fiber arrangement. Expression of cell proliferation marker proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were assessed using RT-PCR and immunohistochemistry in hypertrophic scar tissues. H&E staining sections showed significant reduction of SEI and fibroblast density in both ESWT treatment groups compared to S-ESWT, but there was no dramatic difference between L-ESWT and H-ESWT groups. Masson's trichrome staining showed that collagen fibers were more slender and broader and oriented in parallel to skin surface after administration of ESWT compared to control tissues. At the gene level, PCNA‑positive fibroblasts and α-SMA-positive myofibroblasts were significantly decreased after L-ESWT or H-ESWT compared to the controls. Furthermore, there was no significant difference in expression of PCNA mRNA between L-ESWT or H-ESWT and S-ESWT, whereas expression of α-SMA mRNA significantly decreased in L-ESWT compared to that of H-ESWT and S-ESWT (P=0.002 and P=0.030, respectively). In conclusion, L-ESWT could be effective on suppression of hypertrophic scar formation by inhibition of scar elevation index and fibroblast density as well as α-SMA expression in hypertrophic scar tissues of the rabbit model.

  13. Density variations of meteor flux along the Earth's orbit

    NASA Technical Reports Server (NTRS)

    Svetashkova, N. T.

    1987-01-01

    No model of distribution of meteor substance is known to explain the observed diurnal and annual variations of meteor rates, if that distribution is assumed to be constant during the year. Differences between the results of observations and the prediction of diurnal variation rates leads to the conclusion that the density of the orbits of meteor bodies changes with the motion of the Earth along its orbit. The distributions of the flux density over the celestial sphere are obtained by the method described previously by Svetashkova, 1984. The results indicate that the known seasonal and latitudinal variations of atmospheric conditions does not appear to significantly affect the value of the mean flux density of meteor bodies and the matter influx onto the Earth.

  14. Actinous enigma or enigmatic actin

    PubMed Central

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

    2014-01-01

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

  15. Magnetic flux density reconstruction using interleaved partial Fourier acquisitions in MREIT.

    PubMed

    Park, Hee Myung; Nam, Hyun Soo; Kwon, Oh In

    2011-04-07

    Magnetic resonance electrical impedance tomography (MREIT) has been introduced as a non-invasive modality to visualize the internal conductivity and/or current density of an electrically conductive object by the injection of current. In order to measure a magnetic flux density signal in MREIT, the phase difference approach in an interleaved encoding scheme cancels the systematic artifacts accumulated in phase signals and also reduces the random noise effect. However, it is important to reduce scan duration maintaining spatial resolution and sufficient contrast, in order to allow for practical in vivo implementation of MREIT. The purpose of this paper is to develop a coupled partial Fourier strategy in the interleaved sampling in order to reduce the total imaging time for an MREIT acquisition, whilst maintaining an SNR of the measured magnetic flux density comparable to what is achieved with complete k-space data. The proposed method uses two key steps: one is to update the magnetic flux density by updating the complex densities using the partially interleaved k-space data and the other is to fill in the missing k-space data iteratively using the updated background field inhomogeneity and magnetic flux density data. Results from numerical simulations and animal experiments demonstrate that the proposed method reduces considerably the scanning time and provides resolution of the recovered B(z) comparable to what is obtained from complete k-space data.

  16. Flux density calibration in diffuse optical tomographic systems.

    PubMed

    Biswas, Samir Kumar; Rajan, Kanhirodan; Vasu, Ram M

    2013-02-01

    The solution of the forward equation that models the transport of light through a highly scattering tissue material in diffuse optical tomography (DOT) using the finite element method gives flux density (Φ) at the nodal points of the mesh. The experimentally measured flux (Umeasured) on the boundary over a finite surface area in a DOT system has to be corrected to account for the system transfer functions (R) of various building blocks of the measurement system. We present two methods to compensate for the perturbations caused by R and estimate true flux density (Φ) from Umeasuredcal. In the first approach, the measurement data with a homogeneous phantom (Umeasuredhomo) is used to calibrate the measurement system. The second scheme estimates the homogeneous phantom measurement using only the measurement from a heterogeneous phantom, thereby eliminating the necessity of a homogeneous phantom. This is done by statistically averaging the data (Umeasuredhetero) and redistributing it to the corresponding detector positions. The experiments carried out on tissue mimicking phantom with single and multiple inhomogeneities, human hand, and a pork tissue phantom demonstrate the robustness of the approach.

  17. 3-D density imaging with muon flux measurements from underground galleries

    NASA Astrophysics Data System (ADS)

    Lesparre, N.; Cabrera, J.; Marteau, J.

    2017-03-01

    Atmospheric muon flux measurements provide information on subsurface density distribution. In this study, muon flux was measured underground, in the Tournemire experimental platform (France). The objective was to image the medium between the galleries and the surface and evaluate the feasibility to detect the presence of discontinuities, for example, produced by secondary subvertical faults or by karstic networks. Measurements were performed from three different sites with a partial overlap of muon trajectories, offering the possibility to seek density variations at different depths. The conversion of the measured muon flux to average density values showed global variations further analysed through a 3-D nonlinear inversion procedure. Main results are the presence of a very low density region at the level of the upper aquifer, compatible with the presence of a karstic network hosting local cavities, and the absence of secondary faults. We discuss the validity of the present results and propose different strategies to improve the accuracy of such measurements and analysis.

  18. Evaluation of Density Corrections to Methane Fluxes Measured by Open-Path Eddy Covariance over Contrasting Landscapes

    NASA Astrophysics Data System (ADS)

    Chamberlain, Samuel D.; Verfaillie, Joseph; Eichelmann, Elke; Hemes, Kyle S.; Baldocchi, Dennis D.

    2017-11-01

    Corrections accounting for air density fluctuations due to heat and water vapour fluxes must be applied to the measurement of eddy-covariance fluxes when using open-path sensors. Experimental tests and ecosystem observations have demonstrated the important role density corrections play in accurately quantifying carbon dioxide (CO2) fluxes, but less attention has been paid to evaluating these corrections for methane (CH4) fluxes. We measured CH4 fluxes with open-path sensors over a suite of sites with contrasting CH4 emissions and energy partitioning, including a pavement airfield, two negligible-flux ecosystems (drained alfalfa and pasture), and two high-flux ecosystems (flooded wetland and rice). We found that density corrections successfully re-zeroed fluxes in negligible-flux sites; however, slight overcorrection was observed above pavement. The primary impact of density corrections varied over negligible- and high-flux ecosystems. For negligible-flux sites, corrections led to greater than 100% adjustment in daily budgets, while these adjustments were only 3-10% in high-flux ecosystems. The primary impact to high-flux ecosystems was a change in flux diel patterns, which may affect the evaluation of relationships between biophysical drivers and fluxes if correction bias exists. Additionally, accounting for density effects to high-frequency CH4 fluctuations led to large differences in observed CH4 flux cospectra above negligible-flux sites, demonstrating that similar adjustments should be made before interpreting CH4 cospectra for comparable ecosystems. These results give us confidence in CH4 fluxes measured by open-path sensors, and demonstrate that density corrections play an important role in adjusting flux budgets and diel patterns across a range of ecosystems.

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

    PubMed Central

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

    2015-01-01

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

  20. Structural dynamics of F-actin: I. Changes in the C terminus.

    PubMed

    Orlova, A; Egelman, E H

    1995-02-03

    The biochemical properties of G-actin, and the kinetics of polymerization of G-actin into F-actin, are dependent upon whether Mg2+ or Ca2+ is bound at the high-affinity metal-binding site in actin. Three-dimensional reconstructions from electron micrographs show that a bridge of density, that we interpret as arising from a major shift of the C terminus, exists between the two strands of the filament in Ca(2+)-actin that is absent in Mg(2+)-actin. This bridge is also absent in models of F-actin built from an atomic structure of G-Ca(2+)-actin. The cleavage of the DNase I-binding loop in actin between residues 42 and 43, with the non-covalent association of the 42 cleaved residues with the remainder of the actin, induces an even larger bridge of density between the two strands. When the bridge is absent, the two C-terminal residues in F-actin are easily cleaved by trypsin, while these residues become increasingly resistant to tryptic cleavage as the bridge becomes more prominent. Conversely, cleavage of the two C-terminal residues leads to a conformational change in the DNase I-binding loop. Since both the DNase I-binding loop and the metal-binding site are quite distant from the C terminus, large allosteric effects must exist in F-actin. The conformational change in F-actin that results from the creation of this bridge may be induced by myosin binding, since this movement generates changes in actin's diffraction that are very similar to the changes in the muscle X-ray pattern during activation that are associated with the binding of myosin to the thin filament.

  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. Stoichiometry of Nck-dependent actin polymerization in living cells

    PubMed Central

    Ditlev, Jonathon A.; Michalski, Paul J.; Huber, Greg; Rivera, Gonzalo M.; Mohler, William A.

    2012-01-01

    Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott–Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation. We developed predictions for Nck-dependent actin polymerization using the Virtual Cell software system. In addition, we used antibody-induced aggregation of membrane-targeted Nck SH3 domains to test these predictions and to determine how the number of molecules in Nck aggregates and the density of aggregates affected localized actin polymerization in living cells. Our results indicate that the density of Nck molecules in aggregates is a critical determinant of actin polymerization. Furthermore, results from both computational simulations and experimentation support a model in which the Nck/N-WASp/Arp2/3 stoichiometry is 4:2:1. These results provide new insight into activities involving localized actin polymerization, including tumor cell invasion, microbial pathogenesis, and T cell activation. PMID:22613834

  3. Surface flux density distribution characteristics of bulk high- Tc superconductor in external magnetic field

    NASA Astrophysics Data System (ADS)

    Torii, S.; Yuasa, K.

    2004-10-01

    Various magnetic levitation systems using oxide superconductors are developed as strong pinning forces are obtained in melt-processed bulk. However, the trapped flux of superconductor is moved by flux creep and fluctuating magnetic field. Therefore, to examine the internal condition of superconductor, the authors measure the dynamic surface flux density distribution of YBCO bulk. Flux density measurement system has a structure with the air-core coil and the Hall sensors. Ten Hall sensors are arranged in series. The YBCO bulk, which has 25 mm diameter and 13 mm thickness, is field cooled by liquid nitrogen. After that, magnetic field is changed by the air-core coil. This paper describes about the measured results of flux density distribution of YBCO bulk in the various frequencies of air-core coils currents.

  4. Effect of Thermospheric Neutral Density upon Inner Trapped-belt Proton Flux

    NASA Technical Reports Server (NTRS)

    Wilson, Thomas L.; Lodhi, M. A. K.; Diaz, Abel B.

    2007-01-01

    We wish to point out that a secular change in the Earth's atmospheric neutral density alters charged-particle lifetime in the inner trapped radiation belts, in addition to the changes recently reported as produced by greenhouse gases. Heretofore, changes in neutral density have been of interest primarily because of their effect on the orbital drag of satellites. We extend this to include the orbital lifetime of charged particles in the lower radiation belts. It is known that the charged-belt population is coupled to the neutral density of the atmosphere through changes induced by solar activity, an effect produced by multiple scattering off neutral and ionized atoms along with ionization loss in the thermosphere where charged and neutral populations interact. It will be shown here that trapped-belt flux J is bivariant in energy E and thermospheric neutral density , as J(E,rho). One can conclude that proton lifetimes in these belts are also directly affected by secular changes in the neutral species populating the Earth s thermosphere. This result is a consequence of an intrinsic property of charged-particle flux, that flux is not merely a function of E but is dependent upon density rho when a background of neutrals is present.

  5. Flexibility of myosin attachment to surfaces influences F-actin motion.

    PubMed Central

    Winkelmann, D A; Bourdieu, L; Ott, A; Kinose, F; Libchaber, A

    1995-01-01

    We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement. Images FIGURE 1 FIGURE 4 FIGURE 8 PMID:7544167

  6. Optimization of magnetic flux density measurement using multiple RF receiver coils and multi-echo in MREIT.

    PubMed

    Jeong, Woo Chul; Chauhan, Munish; Sajib, Saurav Z K; Kim, Hyung Joong; Serša, Igor; Kwon, Oh In; Woo, Eung Je

    2014-09-07

    Magnetic Resonance Electrical Impedance Tomography (MREIT) is an MRI method that enables mapping of internal conductivity and/or current density via measurements of magnetic flux density signals. The MREIT measures only the z-component of the induced magnetic flux density B = (Bx, By, Bz) by external current injection. The measured noise of Bz complicates recovery of magnetic flux density maps, resulting in lower quality conductivity and current-density maps. We present a new method for more accurate measurement of the spatial gradient of the magnetic flux density gradient (∇ Bz). The method relies on the use of multiple radio-frequency receiver coils and an interleaved multi-echo pulse sequence that acquires multiple sampling points within each repetition time. The noise level of the measured magnetic flux density Bz depends on the decay rate of the signal magnitude, the injection current duration, and the coil sensitivity map. The proposed method uses three key steps. The first step is to determine a representative magnetic flux density gradient from multiple receiver coils by using a weighted combination and by denoising the measured noisy data. The second step is to optimize the magnetic flux density gradient by using multi-echo magnetic flux densities at each pixel in order to reduce the noise level of ∇ Bz and the third step is to remove a random noise component from the recovered ∇ Bz by solving an elliptic partial differential equation in a region of interest. Numerical simulation experiments using a cylindrical phantom model with included regions of low MRI signal to noise ('defects') verified the proposed method. Experimental results using a real phantom experiment, that included three different kinds of anomalies, demonstrated that the proposed method reduced the noise level of the measured magnetic flux density. The quality of the recovered conductivity maps using denoised ∇ Bz data showed that the proposed method reduced the conductivity

  7. 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

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

    PubMed Central

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

    2016-01-01

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

  9. Evaluation of hypoxia, angiogenesis, and lymphangiogenesis in actinic cheilitis.

    PubMed

    Barbosa, Natália G; Souza, Lélia B; Nonaka, Cassiano F W; Silveira, Ericka J D

    2016-11-01

    Actinic cheilitis is a potentially malignant condition caused mainly by chronic sun exposure. Here we aim to evaluate the role of hypoxia, angiogenesis, and lymphatic density in the clinical and morphological progression of a series of cases of actinic cheilitis. Immunohistochemistry was used to evaluate positivity to hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF)-C, and D2-40 in 40 cases of actinic cheilitis of the lower lip. The cases studied exhibited variable degrees of positivity to the markers. The median number of lymphatic vessels was 3.2, 2.4, and 3.0 in lesions showing no epithelial dysplasia (NED) and with mild (MED) and moderate (MOED) epithelial dysplasia, respectively. The median VEGF-C positivity index was 82.44% (NED), 92.74% (MED), and 82.83% (MOED), and the median HIF-1α positivity index was 11.57% (NED), 5.26% (MED), and 13.55% (MOED). No significant differences in lymphatic density or median VEGF-C and HIF-1α positivity indices were observed between histological grades or clinical presentations of actinic cheilitis (P > 0.05). Although representing early events in lip carcinogenesis, the present results suggest that hypoxia, angiogenesis, and lymphangiogenesis do not influence the morphological or clinical progression of actinic cheilitis. © 2016 The International Society of Dermatology.

  10. 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

  11. Double-cavity radiometer for high-flux density solar radiation measurements.

    PubMed

    Parretta, A; Antonini, A; Armani, M; Nenna, G; Flaminio, G; Pellegrino, M

    2007-04-20

    A radiometric method has been developed, suitable for both total power and flux density profile measurement of concentrated solar radiation. The high-flux density radiation is collected by a first optical cavity, integrated, and driven to a second optical cavity, where, attenuated, it is measured by a conventional radiometer operating under a stationary irradiation regime. The attenuation factor is regulated by properly selecting the aperture areas in the two cavities. The radiometer has been calibrated by a pulsed solar simulator at concentration levels of hundreds of suns. An optical model and a ray-tracing study have also been developed and validated, by which the potentialities of the radiometer have been largely explored.

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

    PubMed Central

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

    2014-01-01

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

  13. Mast cell desensitization inhibits calcium flux and aberrantly remodels actin

    PubMed Central

    Ang, W.X. Gladys; Church, Alison M.; Kulis, Mike; Choi, Hae Woong; Burks, A. Wesley

    2016-01-01

    Rush desensitization (DS) is a widely used and effective clinical strategy for the rapid inhibition of IgE-mediated anaphylactic responses. However, the cellular targets and underlying mechanisms behind this process remain unclear. Recent studies have implicated mast cells (MCs) as the primary target cells for DS. Here, we developed a murine model of passive anaphylaxis with demonstrated MC involvement and an in vitro assay to evaluate the effect of DS on MCs. In contrast with previous reports, we determined that functional IgE remains on the cell surface of desensitized MCs following DS. Despite notable reductions in MC degranulation following DS, the high-affinity IgE receptor FcεRI was still capable of transducing signals in desensitized MCs. Additionally, we found that displacement of the actin cytoskeleton and its continued association with FcεRI impede the capacity of desensitized MCs to evoke the calcium response that is essential for MC degranulation. Together, these findings suggest that reduced degranulation responses in desensitized MCs arise from aberrant actin remodeling, providing insights that may lead to improvement of DS treatments for anaphylactic responses. PMID:27669462

  14. A Mechanistic Model of the Actin Cycle

    PubMed Central

    Bindschadler, M.; Osborn, E. A.; Dewey, C. F.; McGrath, J. L.

    2004-01-01

    We have derived a broad, deterministic model of the steady-state actin cycle that includes its major regulatory mechanisms. Ours is the first model to solve the complete nucleotide profile within filaments, a feature that determines the dynamics and geometry of actin networks at the leading edges of motile cells, and one that has challenged investigators developing models to interpret steady-state experiments. We arrived at the nucleotide profile through analytic and numerical approaches that completely agree. Our model reproduces behaviors seen in numerous experiments with purified proteins, but allows a detailed inspection of the concentrations and fluxes that might exist in these experiments. These inspections provide new insight into the mechanisms that determine the rate of actin filament treadmilling. Specifically, we find that mechanisms for enhancing Pi release from the ADP·Pi intermediate on filaments, for increasing the off rate of ADP-bound subunits at pointed ends, and the multiple, simultaneous functions of profilin, make unique and essential contributions to increased treadmilling. In combination, these mechanisms have a theoretical capacity to increase treadmilling to levels limited only by the amount of available actin. This limitation arises because as the cycle becomes more dynamic, it tends toward the unpolymerized state. PMID:15111391

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

    PubMed Central

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

    2006-01-01

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

  16. Minnealloy: a new magnetic material with high saturation flux density and low magnetic anisotropy

    NASA Astrophysics Data System (ADS)

    Mehedi, Md; Jiang, Yanfeng; Suri, Pranav Kumar; Flannigan, David J.; Wang, Jian-Ping

    2017-09-01

    We are reporting a new soft magnetic material with high saturation magnetic flux density, and low magnetic anisotropy. The new material is a compound of iron, nitrogen and carbon, α‧-Fe8(NC), which has saturation flux density of 2.8  ±  0.15 T and magnetic anisotropy of 46 kJ m-3. The saturation flux density is 27% higher than pure iron, a widely used soft magnetic material. Soft magnetic materials are very important building blocks of motors, generators, inductors, transformers, sensors and write heads of hard disk. The new material will help in the miniaturization and efficiency increment of the next generation of electronic devices.

  17. Interactive Database of Pulsar Flux Density Measurements

    NASA Astrophysics Data System (ADS)

    Koralewska, O.; Krzeszowski, K.; Kijak, J.; Lewandowski, W.

    2012-12-01

    The number of astronomical observations is steadily growing, giving rise to the need of cataloguing the obtained results. There are a lot of databases, created to store different types of data and serve a variety of purposes, e. g. databases providing basic data for astronomical objects (SIMBAD Astronomical Database), databases devoted to one type of astronomical object (ATNF Pulsar Database) or to a set of values of the specific parameter (Lorimer 1995 - database of flux density measurements for 280 pulsars on the frequencies up to 1606 MHz), etc. We found that creating an online database of pulsar flux measurements, provided with facilities for plotting diagrams and histograms, calculating mean values for a chosen set of data, filtering parameter values and adding new measurements by the registered users, could be useful in further studies on pulsar spectra.

  18. Internal wave energy flux from density perturbations in nonlinear stratifications

    NASA Astrophysics Data System (ADS)

    Lee, Frank M.; Allshouse, Michael R.; Swinney, Harry L.; Morrison, P. J.

    2017-11-01

    Tidal flow over the topography at the bottom of the ocean, whose density varies with depth, generates internal gravity waves that have a significant impact on the energy budget of the ocean. Thus, understanding the energy flux (J = p v) is important, but it is difficult to measure simultaneously the pressure and velocity perturbation fields, p and v . In a previous work, a Green's-function-based method was developed to calculate the instantaneous p, v , and thus J , given a density perturbation field for a constant buoyancy frequency N. Here we extend the previous analytic Green's function work to include nonuniform N profiles, namely the tanh-shaped and linear cases, because background density stratifications that occur in the ocean and some experiments are nonlinear. In addition, we present a finite-difference method for the general case where N has an arbitrary profile. Each method is validated against numerical simulations. The methods we present can be applied to measured density perturbation data by using our MATLAB graphical user interface EnergyFlux. PJM was supported by the U.S. Department of Energy Contract DE-FG05-80ET-53088. HLS and MRA were supported by ONR Grant No. N000141110701.

  19. Membrane-associated actin from the microvillar membranes of ascites tumor cells

    PubMed Central

    1982-01-01

    A membrane fraction (MF2) has been purified from isolated microvilli of the MAT-C1 subline of the 13762 rat mammary ascites adenocarcinoma under conditions which cause F-actin depolymerization. This membrane preparation contains actin as a major component, although no filamentous structures are observed by transmission electron microscopy. Membranes were extracted with a Triton X-100-containing actin-stabilizing buffer (S buffer) or actin-destabilizing buffer (D buffer). In D buffer greater than 90% of metabolically labeled protein and glycoprotein was extracted, and 80-90% of these labeled species was extracted in S buffer. When S buffer extracts of MF2 were fractionated by either gel filtration on Sepharose 6 B or rate-zonal sucrose density gradient centrifugation, most of the actin was found to be intermediate in size between G- and F-actin. In D buffer most of the MF2 actin behaved as G-actin. Extraction and gel filtration of intact microvilli in S buffer also showed the presence of the intermediate form of actin, indicating that it did not arise during membrane preparation. When [35S]methionine-labeled G-actin from ascites cells was added to S buffer extracts of MF2 and chromatographed, all of the radioactivity chromatographed as G-actin, indicating that the intermediate form of actin did not result from an association of G-actin molecules during extraction or chromatography. The results of this study suggest that the microvillar membrane fraction is enriched in an intermediate form of actin smaller than F-actin and larger than G-actin. PMID:6890066

  20. Membrane-associated actin from the microvillar membranes of ascites tumor cells.

    PubMed

    Carraway, K L; Cerra, R F; Jung, G; Carraway, C A

    1982-09-01

    A membrane fraction (MF2) has been purified from isolated microvilli of the MAT-C1 subline of the 13762 rat mammary ascites adenocarcinoma under conditions which cause F-actin depolymerization. This membrane preparation contains actin as a major component, although no filamentous structures are observed by transmission electron microscopy. Membranes were extracted with a Triton X-100-containing actin-stabilizing buffer (S buffer) or actin-destabilizing buffer (D buffer). In D buffer greater than 90% of metabolically labeled protein and glycoprotein was extracted, and 80-90% of these labeled species was extracted in S buffer. When S buffer extracts of MF2 were fractionated by either gel filtration on Sepharose 6 B or rate-zonal sucrose density gradient centrifugation, most of the actin was found to be intermediate in size between G- and F-actin. In D buffer most of the MF2 actin behaved as G-actin. Extraction and gel filtration of intact microvilli in S buffer also showed the presence of the intermediate form of actin, indicating that it did not arise during membrane preparation. When [35S]methionine-labeled G-actin from ascites cells was added to S buffer extracts of MF2 and chromatographed, all of the radioactivity chromatographed as G-actin, indicating that the intermediate form of actin did not result from an association of G-actin molecules during extraction or chromatography. The results of this study suggest that the microvillar membrane fraction is enriched in an intermediate form of actin smaller than F-actin and larger than G-actin.

  1. 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.

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

    PubMed

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

    2000-10-01

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

  3. Estimation of electrical conductivity distribution within the human head from magnetic flux density measurement.

    PubMed

    Gao, Nuo; Zhu, S A; He, Bin

    2005-06-07

    We have developed a new algorithm for magnetic resonance electrical impedance tomography (MREIT), which uses only one component of the magnetic flux density to reconstruct the electrical conductivity distribution within the body. The radial basis function (RBF) network and simplex method are used in the present approach to estimate the conductivity distribution by minimizing the errors between the 'measured' and model-predicted magnetic flux densities. Computer simulations were conducted in a realistic-geometry head model to test the feasibility of the proposed approach. Single-variable and three-variable simulations were performed to estimate the brain-skull conductivity ratio and the conductivity values of the brain, skull and scalp layers. When SNR = 15 for magnetic flux density measurements with the target skull-to-brain conductivity ratio being 1/15, the relative error (RE) between the target and estimated conductivity was 0.0737 +/- 0.0746 in the single-variable simulations. In the three-variable simulations, the RE was 0.1676 +/- 0.0317. Effects of electrode position uncertainty were also assessed by computer simulations. The present promising results suggest the feasibility of estimating important conductivity values within the head from noninvasive magnetic flux density measurements.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2013-10-01

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

  6. Lights, camera, actin.

    PubMed

    Rubenstein, Peter A; Wen, Kuo-Kuang

    2005-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2006-09-01

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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25969948

  9. A model of heat transfer in sapwood and implications for sap flux density measurements using thermal dissipation probes

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

    Wullschleger, Stan D; Childs, Kenneth W; King, Anthony Wayne

    2011-01-01

    A variety of thermal approaches are used to estimate sap flux density in stems of woody plants. Models have proven valuable tools for interpreting the behavior of heat pulse, heat balance, and heat field deformation techniques, but have seldom been used to describe heat transfer dynamics for the heat dissipation method. Therefore, to better understand the behavior of heat dissipation probes, a model was developed that takes into account the thermal properties of wood, the physical dimensions and thermal characteristics of the probes, and the conductive and convective heat transfer that occurs due to water flow in the sapwood. Probesmore » were simulated as aluminum tubes 20 mm in length and 2 mm in diameter, whereas sapwood, heartwood, and bark each had a density and water fraction that determined their thermal properties. Base simulations assumed a constant sap flux density with sapwood depth and no wounding or physical disruption of xylem beyond the 2 mm diameter hole drilled for probe installation. Simulations across a range of sap flux densities showed that the dimensionless quantity k defined as ( Tm T)/ T where Tm is the temperature differential ( T) between the heated and unheated probe under zero flow conditions was dependent on the thermal conductivity of the sapwood. The relationship between sap flux density and k was also sensitive to radial gradients in sap flux density and to xylem disruption near the probe. Monte Carlo analysis in which 1000 simulations were conducted while simultaneously varying thermal conductivity and wound diameter revealed that sap flux density and k showed considerable departure from the original calibration equation used with this technique. The departure was greatest for abrupt patterns of radial variation typical of ring-porous species. Depending on the specific combination of thermal conductivity and wound diameter, use of the original calibration equation resulted in an 81% under- to 48% over-estimation of sap flux

  10. Nuclear Functions of Actin

    PubMed Central

    Visa, Neus; Percipalle, Piergiorgio

    2010-01-01

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

  11. Measurement of the photoneutron flux density distribution from cylindrical targets

    NASA Astrophysics Data System (ADS)

    Golovkov, V. M.; Basina, T. N.; Yakovlev, M. R.

    1989-09-01

    Measurements are performed of the density of photoneutron fluxes from cylindrical targets of2H2O (diameter 64 and height 86 mm), Be (outer diameter 70, inner diameter 40, height 100mm), and238U (diameter 44.5 mm, height 50 mm) under the action of braking radiation from electrons with energies of 4 to 8 MeV in order to determine the effect of target form and orientation relative to the detector upon the recorded photoneutron level. The fluxes were measured by an “all-wave” neutron detector based on an SNM-11 counter in a paraffin retarder at an angle of 90‡ to the axis of the braking radiation beam for various target orientations relative to the detector. Measurement results are compared to calculations. Photoneutron fluxes from heavy water and beryllium targets of the indicated dimensions were also measured for angles of 90, 135, and 167‡. An isotropic nature was noted in the photoneutron fluxes from both targets.

  12. Energy density and energy flux in the focus of an optical vortex: reverse flux of light energy.

    PubMed

    Kotlyar, Victor V; Kovalev, Alexey A; Nalimov, Anton G

    2018-06-15

    Using the Richards-Wolf formulas for an arbitrary circularly polarized optical vortex with an integer topological charge m, we obtain explicit expressions for all components of the electric and magnetic field strength vectors near the focus, as well as expressions for the intensity (energy density) and for the energy flux (components of the Poynting vector) in the focal plane of an aplanatic optical system. For m=2, from the obtained expressions it follows that the energy flux near the optical axis propagates in the reversed direction, rotating along a spiral around the optical axis. On the optical axis itself, the reversed flux is maximal and decays rapidly with the distance from the axis. For m=3, in contrast, the reversed energy flux in the focal plane is minimal (zero) on the optical axis and increases (until the first ring of the light intensity) as a squared distance from the axis.

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

    PubMed Central

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

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

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

    PubMed Central

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

    2015-01-01

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

  16. Cluster electric current density measurements within a magnetic flux rope in the plasma sheet

    NASA Technical Reports Server (NTRS)

    Slavin, J. A.; Lepping, R. P.; Gjerloev, J.; Goldstein, M. L.; Fairfield, D. H.; Acuna, M. H.; Balogh, A.; Dunlop, M.; Kivelson, M. G.; Khurana, K.

    2003-01-01

    On August 22, 2001 all 4 Cluster spacecraft nearly simultaneously penetrated a magnetic flux rope in the tail. The flux rope encounter took place in the central plasma sheet, Beta(sub i) approx. 1-2, near the leading edge of a bursty bulk flow. The "time-of-flight" of the flux rope across the 4 spacecraft yielded V(sub x) approx. 700 km/s and a diameter of approx.1 R(sub e). The speed at which the flux rope moved over the spacecraft is in close agreement with the Cluster plasma measurements. The magnetic field profiles measured at each spacecraft were first modeled separately using the Lepping-Burlaga force-free flux rope model. The results indicated that the center of the flux rope passed northward (above) s/c 3, but southward (below) of s/c 1, 2 and 4. The peak electric currents along the central axis of the flux rope predicted by these single-s/c models were approx.15-19 nA/sq m. The 4-spacecraft Cluster magnetic field measurements provide a second means to determine the electric current density without any assumption regarding flux rope structure. The current profile determined using the curlometer technique was qualitatively similar to those determined by modeling the individual spacecraft magnetic field observations and yielded a peak current density of 17 nA/m2 near the central axis of the rope. However, the curlometer results also showed that the flux rope was not force-free with the component of the current density perpendicular to the magnetic field exceeding the parallel component over the forward half of the rope, perhaps due to the pressure gradients generated by the collision of the BBF with the inner magnetosphere. Hence, while the single-spacecraft models are very successful in fitting flux rope magnetic field and current variations, they do not provide a stringent test of the force-free condition.

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

    NASA Astrophysics Data System (ADS)

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

    1990-05-01

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

  18. Abnormal changes in the density of thermal neutron flux in biocenoses near the earth surface.

    PubMed

    Plotnikova, N V; Smirnov, A N; Kolesnikov, M V; Semenov, D S; Frolov, V A; Lapshin, V B; Syroeshkin, A V

    2007-04-01

    We revealed an increase in the density of thermal neutron flux in forest biocenoses, which was not associated with astrogeophysical events. The maximum spike of this parameter in the biocenosis reached 10,000 n/(sec x m2). Diurnal pattern of the density of thermal neutron flux depended only on the type of biocenosis. The effects of biomodulation of corpuscular radiation for balneology are discussed.

  19. Effects of basic calponin on the flexural mechanics and stability of F-actin.

    PubMed

    Jensen, Mikkel Herholdt; Watt, James; Hodgkinson, Julie L; Gallant, Cynthia; Appel, Sarah; El-Mezgueldi, Mohammed; Angelini, Thomas E; Morgan, Kathleen G; Lehman, William; Moore, Jeffrey R

    2012-01-01

    The cellular actin cytoskeleton plays a central role in the ability of cells to properly sense, propagate, and respond to external stresses and other mechanical stimuli. Calponin, an actin-binding protein found both in muscle and non-muscle cells, has been implicated in actin cytoskeletal organization and regulation. In this work, we studied the mechanical and structural interaction of actin with basic calponin, a differentiation marker in smooth muscle cells, on a single filament level. We imaged fluorescently labeled thermally fluctuating actin filaments and found that at moderate calponin binding densities, actin filaments were more flexible, evident as a reduction in persistence length from 8.0 to 5.8 μm. When calponin-decorated actin filaments were subjected to shear, we observed a marked reduction of filament lengths after decoration with calponin, which we argue was due to shear-induced filament rupture rather than depolymerization. This increased shear susceptibility was exacerbated with calponin concentration. Cryo-electron microscopy results confirmed previously published negative stain electron microscopy results and suggested alterations in actin involving actin subdomain 2. A weakening of F-actin intermolecular association is discussed as the underlying cause of the observed mechanical perturbations. Copyright © 2011 Wiley Periodicals, Inc.

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

    PubMed Central

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

    1997-01-01

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

  1. One-point fitting of the flux density produced by a heliostat

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

    Collado, Francisco J.

    Accurate and simple models for the flux density reflected by an isolated heliostat should be one of the basic tools for the design and optimization of solar power tower systems. In this work, the ability and the accuracy of the Universidad de Zaragoza (UNIZAR) and the DLR (HFCAL) flux density models to fit actual energetic spots are checked against heliostat energetic images measured at Plataforma Solar de Almeria (PSA). Both the fully analytic models are able to acceptably fit the spot with only one-point fitting, i.e., the measured maximum flux. As a practical validation of this one-point fitting, the interceptmore » percentage of the measured images, i.e., the percentage of the energetic spot sent by the heliostat that gets the receiver surface, is compared with the intercept calculated through the UNIZAR and HFCAL models. As main conclusions, the UNIZAR and the HFCAL models could be quite appropriate tools for the design and optimization, provided the energetic images from the heliostats to be used in the collector field were previously analyzed. Also note that the HFCAL model is much simpler and slightly more accurate than the UNIZAR model. (author)« less

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

    PubMed Central

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

    2017-01-01

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

  3. Analysis of recoverable current from one component of magnetic flux density in MREIT and MRCDI.

    PubMed

    Park, Chunjae; Lee, Byung Il; Kwon, Oh In

    2007-06-07

    Magnetic resonance current density imaging (MRCDI) provides a current density image by measuring the induced magnetic flux density within the subject with a magnetic resonance imaging (MRI) scanner. Magnetic resonance electrical impedance tomography (MREIT) has been focused on extracting some useful information of the current density and conductivity distribution in the subject Omega using measured B(z), one component of the magnetic flux density B. In this paper, we analyze the map Tau from current density vector field J to one component of magnetic flux density B(z) without any assumption on the conductivity. The map Tau provides an orthogonal decomposition J = J(P) + J(N) of the current J where J(N) belongs to the null space of the map Tau. We explicitly describe the projected current density J(P) from measured B(z). Based on the decomposition, we prove that B(z) data due to one injection current guarantee a unique determination of the isotropic conductivity under assumptions that the current is two-dimensional and the conductivity value on the surface is known. For a two-dimensional dominating current case, the projected current density J(P) provides a good approximation of the true current J without accumulating noise effects. Numerical simulations show that J(P) from measured B(z) is quite similar to the target J. Biological tissue phantom experiments compare J(P) with the reconstructed J via the reconstructed isotropic conductivity using the harmonic B(z) algorithm.

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

    DOEpatents

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

    2011-10-18

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

  5. Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease.

    PubMed

    Kommaddi, Reddy Peera; Das, Debajyoti; Karunakaran, Smitha; Nanguneri, Siddharth; Bapat, Deepti; Ray, Ajit; Shaw, Eisha; Bennett, David A; Nair, Deepak; Ravindranath, Vijayalakshmi

    2018-01-31

    Dendritic spine loss is recognized as an early feature of Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD (APPswe/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1ΔE9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, Aβ load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression. SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimer's disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the

  6. Variability in radial sap flux density patterns and sapwood area among seven co-occurring temperate broad-leaved tree species.

    PubMed

    Gebauer, Tobias; Horna, Viviana; Leuschner, Christoph

    2008-12-01

    Forest transpiration estimates are frequently based on xylem sap flux measurements in the outer sections of the hydro-active stem sapwood. We used Granier's constant-heating technique with heating probes at various xylem depths to analyze radial patterns of sap flux density in the sapwood of seven broad-leaved tree species differing in wood density and xylem structure. Study aims were to (1) compare radial sap flux density profiles between diffuse- and ring-porous trees and (2) analyze the relationship between hydro-active sapwood area and stem diameter. In all investigated species except the diffuse-porous beech (Fagus sylvatica L.) and ring-porous ash (Fraxinus excelsior L.), sap flux density peaked at a depth of 1 to 4 cm beneath the cambium, revealing a hump-shaped curve with species-specific slopes. Beech and ash reached maximum sap flux densities immediately beneath the cambium in the youngest annual growth rings. Experiments with dyes showed that the hydro-active sapwood occupied 70 to 90% of the stem cross-sectional area in mature trees of diffuse-porous species, whereas it occupied only about 21% in ring-porous ash. Dendrochronological analyses indicated that vessels in the older sapwood may remain functional for 100 years or more in diffuse-porous species and for up to 27 years in ring-porous ash. We conclude that radial sap flux density patterns are largely dependent on tree species, which may introduce serious bias in sap-flux-derived forest transpiration estimates, if non-specific sap flux profiles are assumed.

  7. Optimization of magnetic flux density for fast MREIT conductivity imaging using multi-echo interleaved partial fourier acquisitions.

    PubMed

    Chauhan, Munish; Jeong, Woo Chul; Kim, Hyung Joong; Kwon, Oh In; Woo, Eung Je

    2013-08-27

    Magnetic resonance electrical impedance tomography (MREIT) has been introduced as a non-invasive method for visualizing the internal conductivity and/or current density of an electrically conductive object by externally injected currents. The injected current through a pair of surface electrodes induces a magnetic flux density distribution inside the imaging object, which results in additional magnetic flux density. To measure the magnetic flux density signal in MREIT, the phase difference approach in an interleaved encoding scheme cancels out the systematic artifacts accumulated in phase signals and also reduces the random noise effect by doubling the measured magnetic flux density signal. For practical applications of in vivo MREIT, it is essential to reduce the scan duration maintaining spatial-resolution and sufficient contrast. In this paper, we optimize the magnetic flux density by using a fast gradient multi-echo MR pulse sequence. To recover the one component of magnetic flux density Bz, we use a coupled partial Fourier acquisitions in the interleaved sense. To prove the proposed algorithm, we performed numerical simulations using a two-dimensional finite-element model. For a real experiment, we designed a phantom filled with a calibrated saline solution and located a rubber balloon inside the phantom. The rubber balloon was inflated by injecting the same saline solution during the MREIT imaging. We used the multi-echo fast low angle shot (FLASH) MR pulse sequence for MRI scan, which allows the reduction of measuring time without a substantial loss in image quality. Under the assumption of a priori phase artifact map from a reference scan, we rigorously investigated the convergence ratio of the proposed method, which was closely related with the number of measured phase encode set and the frequency range of the background field inhomogeneity. In the phantom experiment with a partial Fourier acquisition, the total scan time was less than 6 seconds to measure

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

    PubMed

    Thomasson, Maggie S; Macnaughtan, Megan A

    2013-12-15

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

  9. Cortactin binding to F-actin revealed by electron microscopy and 3D reconstruction.

    PubMed

    Pant, Kiran; Chereau, David; Hatch, Victoria; Dominguez, Roberto; Lehman, William

    2006-06-16

    Cortactin and WASP activate Arp2/3-mediated actin filament nucleation and branching. However, different mechanisms underlie activation by the two proteins, which rely on distinct actin-binding modules and modes of binding to actin filaments. It is generally thought that cortactin binds to "mother" actin filaments, while WASP donates actin monomers to Arp2/3-generated "daughter" filament branches. Interestingly, cortactin also binds WASP in addition to F-actin and the Arp2/3 complex. However, the structural basis for the role of cortactin in filament branching remains unknown, making interpretation difficult. Here, electron microscopy and 3D reconstruction were carried out on F-actin decorated with the actin-binding repeating domain of cortactin, revealing conspicuous density on F-actin attributable to cortactin that is located on a consensus-binding site on subdomain-1 of actin subunits. Strikingly, the binding of cortactin widens the gap between the two long-pitch filament strands. Although other proteins have been found to alter the structure of the filament, the cortactin-induced conformational change appears unique. The results are consistent with a mechanism whereby alterations of the F-actin structure may facilitate recruitment of the Arp2/3 complex to the "mother" filament in the cortex of cells. In addition, cortactin may act as a structural adapter protein, stabilizing nascent filament branches while mediating the simultaneous recruitment of Arp2/3 and WASP.

  10. Sensing magnetic flux density of artificial neurons with a MEMS device.

    PubMed

    Tapia, Jesus A; Herrera-May, Agustin L; García-Ramírez, Pedro J; Martinez-Castillo, Jaime; Figueras, Eduard; Flores, Amira; Manjarrez, Elías

    2011-04-01

    We describe a simple procedure to characterize a magnetic field sensor based on microelectromechanical systems (MEMS) technology, which exploits the Lorentz force principle. This sensor is designed to detect, in future applications, the spiking activity of neurons or muscle cells. This procedure is based on the well-known capability that a magnetic MEMS device can be used to sense a small magnetic flux density. In this work, an electronic neuron (FitzHugh-Nagumo) is used to generate controlled spike-like magnetic fields. We show that the magnetic flux density generated by the hardware of this neuron can be detected with a new MEMS magnetic field sensor. This microdevice has a compact resonant structure (700 × 600 × 5 μm) integrated by an array of silicon beams and p-type piezoresistive sensing elements, which need an easy fabrication process. The proposed microsensor has a resolution of 80 nT, a sensitivity of 1.2 V.T(-1), a resonant frequency of 13.87 kHz, low power consumption (2.05 mW), quality factor of 93 at atmospheric pressure, and requires a simple signal processing circuit. The importance of our study is twofold. First, because the artificial neuron can generate well-controlled magnetic flux density, we suggest it could be used to analyze the resolution and performance of different magnetic field sensors intended for neurobiological applications. Second, the introduced MEMS magnetic field sensor may be used as a prototype to develop new high-resolution biomedical microdevices to sense magnetic fields from cardiac tissue, nerves, spinal cord, or the brain.

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

    PubMed

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

    2011-12-06

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2014-05-14

    Cerebral hypoxia-ischemia damages synaptic proteins, resulting in cytoskeletal alterations, protein aggregation and neuronal death. In the previous works, we have shown neuronal and synaptic changes in rat neostriatum subjected to hypoxia that leads to ubi-protein accumulation. Recently, we also showed that, changes in F-actin organization could be related to early alterations induced by hypoxia in the Central Nervous System. However, little is known about effective treatment to diminish the damage. The main aim of this work is to study the effects of birth hypothermia on the actin cytoskeleton of neostriatal post-synaptic densities (PSD) in 60 days olds rats by immunohistochemistry, photooxidation and western blot. We used 2 different protocols of hypothermia: (a) intrahypoxic hypothermia at 15°C and (b) post-hypoxia hypothermia at 32°C. Consistent with previous data at 30 days, staining with phalloidin-Alexa(488) followed by confocal microscopy analysis showed an increase of F-actin fluorescent staining in the neostriatum of hypoxic animals. Correlative photooxidation electron microscopy confirmed these observations showing an increment in the number of mushroom-shaped F-actin staining spines in neostriatal excitatory synapses in rats subjected to hypoxia. In addition, western blot revealed β-actin increase in PSDs in hypoxic animals. The optic relative density measurement showed a significant difference between controls and hypoxic animals. When hypoxia was induced under hypothermic conditions, the changes observed in actin cytoskeleton were blocked. Post-hypoxic hypothermia showed similar answer but actin cytoskeleton modifications were not totally reverted as we observed at 15°C. These data suggest that the decrease of the body temperature decreases the actin modifications in dendritic spines preventing the neuronal death. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Evidence Coupling Increased Hexosamine Biosynthesis Pathway Activity to Membrane Cholesterol Toxicity and Cortical Filamentous Actin Derangement Contributing to Cellular Insulin Resistance†

    PubMed Central

    Bhonagiri, Padma; Pattar, Guruprasad R.; Habegger, Kirk M.; McCarthy, Alicia M.; Tackett, Lixuan

    2011-01-01

    Hyperinsulinemia is known to promote the progression/worsening of insulin resistance. Evidence reveals a hidden cost of hyperinsulinemia on plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated filamentous actin (F-actin) structure, components critical to the normal operation of the insulin-regulated glucose transport system. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes PIP2/F-actin dysregulation and subsequent insulin resistance. Increased glycosylation events were detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nm insulin; 12 h) and in cells in which HBP activity was amplified by 2 mm glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP2 and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP2 corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP2/F-actin structure, pharmacological inhibition of the rate-limiting HBP enzyme [glutamine-fructose-6-phosphate amidotransferase (GFAT)] restored PIP2-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of GFAT was associated with a loss of detectable PM PIP2 and insulin sensitivity. Even less invasive challenges with glucose, in the absence of insulin, also led to PIP2/F-actin dysregulation. Mechanistically we found that increased HBP activity increased PM cholesterol, the removal of which normalized PIP2/F-actin levels. Accordingly, these data suggest that glucose transporter-4 functionality, dependent on PIP2 and/or F-actin status, can be critically compromised by inappropriate HBP activity. Furthermore, these data are consistent with the PM cholesterol accrual/toxicity as a mechanistic basis of the HBP-induced defects in PIP2/F-actin

  15. Sliding movement of single actin filaments on one-headed myosin filaments

    NASA Astrophysics Data System (ADS)

    Harada, Yoshie; Noguchi, Akira; Kishino, Akiyoshi; Yanagida, Toshio

    1987-04-01

    The myosin molecule consists of two heads, each of which contains an enzymatic active site and an actin-binding site. The fundamental problem of whether the two heads function independently or cooperatively during muscle contraction has been studied by methods using an actomyosin thread1, superprecipitation2-4 and chemical modification of muscle fibres5. No clear conclusion has yet been reached. We have approached this question using an assay system in which sliding movements of fluorescently labelled single actin filaments along myosin filaments can be observed directly6,7. Here, we report direct measurement of the sliding of single actin filaments along one-headed myosin filaments in which the density of heads was varied over a wide range. Our results show that cooperative interaction between the two heads of myosin is not essential for inducing the sliding movement of actin filaments.

  16. Fast Radio Bursts’ Recipes for the Distributions of Dispersion Measures, Flux Densities, and Fluences

    NASA Astrophysics Data System (ADS)

    Niino, Yuu

    2018-05-01

    We investigate how the statistical properties of dispersion measure (DM) and apparent flux density/fluence of (nonrepeating) fast radio bursts (FRBs) are determined by unknown cosmic rate density history [ρ FRB(z)] and luminosity function (LF) of the transient events. We predict the distributions of DMs, flux densities, and fluences of FRBs taking account of the variation of the receiver efficiency within its beam, using analytical models of ρ FRB(z) and LF. Comparing the predictions with the observations, we show that the cumulative distribution of apparent fluences suggests that FRBs originate at cosmological distances and ρ FRB increases with redshift resembling the cosmic star formation history (CSFH). We also show that an LF model with a bright-end cutoff at log10 L ν (erg s‑1 Hz‑1) ∼ 34 are favored to reproduce the observed DM distribution if ρ FRB(z) ∝ CSFH, although the statistical significance of the constraints obtained with the current size of the observed sample is not high. Finally, we find that the correlation between DM and flux density of FRBs is potentially a powerful tool to distinguish whether FRBs are at cosmological distances or in the local universe more robustly with future observations.

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

    PubMed Central

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

    1997-01-01

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

  18. An analysis and implications of alternative methods of deriving the density (WPL) terms for eddy covariance flux measurements

    Treesearch

    W. J. Massman; J. -P. Tuovinen

    2006-01-01

    We explore some of the underlying assumptions used to derive the density or WPL terms (Webb et al. (1980) Quart J RoyMeteorol Soc 106:85-100) required for estimating the surface exchange fluxes by eddy covariance. As part of this effort we recast the origin of the density terms as an assumption regarding the density fluctuations rather than as a (dry air) flux...

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

    PubMed

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

    2016-03-04

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

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

    NASA Astrophysics Data System (ADS)

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

    1996-11-01

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

  1. Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization

    PubMed Central

    Domínguez-Iturza, Nuria; Calvo, María; Benoist, Marion; Esteban, José Antonio; Morales, Miguel

    2016-01-01

    Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine. PMID:26881098

  2. Intermittent electron density and temperature fluctuations and associated fluxes in the Alcator C-Mod scrape-off layer

    NASA Astrophysics Data System (ADS)

    Kube, R.; Garcia, O. E.; Theodorsen, A.; Brunner, D.; Kuang, A. Q.; LaBombard, B.; Terry, J. L.

    2018-06-01

    The Alcator C-Mod mirror Langmuir probe system has been used to sample data time series of fluctuating plasma parameters in the outboard mid-plane far scrape-off layer. We present a statistical analysis of one second long time series of electron density, temperature, radial electric drift velocity and the corresponding particle and electron heat fluxes. These are sampled during stationary plasma conditions in an ohmically heated, lower single null diverted discharge. The electron density and temperature are strongly correlated and feature fluctuation statistics similar to the ion saturation current. Both electron density and temperature time series are dominated by intermittent, large-amplitude burst with an exponential distribution of both burst amplitudes and waiting times between them. The characteristic time scale of the large-amplitude bursts is approximately 15 μ {{s}}. Large-amplitude velocity fluctuations feature a slightly faster characteristic time scale and appear at a faster rate than electron density and temperature fluctuations. Describing these time series as a superposition of uncorrelated exponential pulses, we find that probability distribution functions, power spectral densities as well as auto-correlation functions of the data time series agree well with predictions from the stochastic model. The electron particle and heat fluxes present large-amplitude fluctuations. For this low-density plasma, the radial electron heat flux is dominated by convection, that is, correlations of fluctuations in the electron density and radial velocity. Hot and dense blobs contribute only a minute fraction of the total fluctuation driven heat flux.

  3. Modification of ocean-estuary salt fluxes by density-driven advection of a headland eddy

    NASA Astrophysics Data System (ADS)

    Fram, J. P.; Stacey, M. T.

    2005-05-01

    Scalar exchange between San Francisco Bay and the coastal ocean is examined using shipboard observations made across the Golden Gate Channel. Ocean-estuary exchange is often described as a combination of two independent types of mechanisms: density-driven exchange such as gravitational circulation and tidal asymmetries such as tidal trapping. In this study we found that exchange is also governed by an interaction between these mechanisms. Tidally trapped eddies created in shallow shoals are mixed into the main channel earlier in the tidal cycle during the rainy season because the eddies are pushed seaward by gravitational circulation. This interaction increases the tidally averaged dispersive salt flux into the bay. The study consists of experiments during each of three 'seasons': winter/spring runoff (March 2002), summer upwelling (July 2003), and fall relaxation (October 2002). Within each experiment, transects across the channel were repeated approximately every 12 minutes for 25 hours during both spring tide and the following neap tide. Velocity was measured from a boat-mounted ADCP. Scalar concentrations were measured from a tow-yoed SeaSciences Acrobat. Salinity exchange over each spring-neap cycle is quantified with harmonic analysis. Harmonic results are decomposed into flux mechanisms using temporal and spatial correlations. The temporal correlation of cross-sectional averaged salinity and velocity (tidal pumping flux) is the largest part of the dispersive flux of salinity into the bay. From the tidal pumping portion of the dispersive flux, it is shown that there is less exchange than was found in earlier studies. Furthermore, tidal pumping flux scales strongly with flow due to density-driven movement of tidally trapped eddies and density-driven increases in ebb-flood frictional phasing. Complex bathymetry makes salinity exchange scale differently with flow than would be expected from simple tidal pumping and gravitational circulation models.

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

    PubMed

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

    2013-02-01

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

  5. Nucleation of actin polymerization by gelsolin.

    PubMed

    Ditsch, A; Wegner, A

    1994-08-15

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

  6. The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts

    PubMed Central

    Destaing, Olivier; Sanjay, Archana; Itzstein, Cecile; Horne, William C.; Toomre, Derek

    2008-01-01

    Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src−/− OCLs by Src inhibitors or by expressing dominant-negative SrcK295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization. PMID:17978100

  7. Optimization of multiply acquired magnetic flux density B(z) using ICNE-Multiecho train in MREIT.

    PubMed

    Nam, Hyun Soo; Kwon, Oh In

    2010-05-07

    The aim of magnetic resonance electrical impedance tomography (MREIT) is to visualize the electrical properties, conductivity or current density of an object by injection of current. Recently, the prolonged data acquisition time when using the injected current nonlinear encoding (ICNE) method has been advantageous for measurement of magnetic flux density data, Bz, for MREIT in the signal-to-noise ratio (SNR). However, the ICNE method results in undesirable side artifacts, such as blurring, chemical shift and phase artifacts, due to the long data acquisition under an inhomogeneous static field. In this paper, we apply the ICNE method to a gradient and spin echo (GRASE) multi-echo train pulse sequence in order to provide the multiple k-space lines during a single RF pulse period. We analyze the SNR of the measured multiple B(z) data using the proposed ICNE-Multiecho MR pulse sequence. By determining a weighting factor for B(z) data in each of the echoes, an optimized inversion formula for the magnetic flux density data is proposed for the ICNE-Multiecho MR sequence. Using the ICNE-Multiecho method, the quality of the measured magnetic flux density is considerably increased by the injection of a long current through the echo train length and by optimization of the voxel-by-voxel noise level of the B(z) value. Agarose-gel phantom experiments have demonstrated fewer artifacts and a better SNR using the ICNE-Multiecho method. Experimenting with the brain of an anesthetized dog, we collected valuable echoes by taking into account the noise level of each of the echoes and determined B(z) data by determining optimized weighting factors for the multiply acquired magnetic flux density data.

  8. NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability.

    PubMed

    Sun, Licui; Zheng, Junfang; Wang, Qiqi; Song, Ran; Liu, Hua; Meng, Ran; Tao, Tao; Si, Yang; Jiang, Wenguo; He, Junqi

    2016-02-01

    The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na(+)/H(+) exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresis-matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. α-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the α-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/α-actinin-4 interaction increased α-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified α-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1. © FASEB.

  9. Spectral flux from low-density photospheres - Numerical results

    NASA Technical Reports Server (NTRS)

    Hershkowitz, S.; Linder, E.; Wagoner, R. V.

    1986-01-01

    Radiative transfer through sharp, quasi-static atmospheres whose opacity is dominated by hydrogen is considered at densities low enough that scattering usually dominates absorption and radiative excitations usually dominate collisional excitations. Numerical results for the continuum spectral flux are obtained for effective temperatures T(e) = 6000-16,000 K and scale heights Delta-R = 10 to the 10th - 10 to the 14th cm. Spectra are significantly different than if LTE level populations were assumed. Comparison with observations of the Type II supernova 1980k tends to increase the value of the Hubble constant previously obtained by the Baade (1926) method.

  10. Modeling of the motion of the actin filament on the myosin motility assays

    NASA Astrophysics Data System (ADS)

    Young, Yuan; Shelley, Mike

    2007-11-01

    In motility assays, cytoskeletal actin filaments (actin filaments) glide over a surface coated with motor proteins, and the different modes of motion provide a simple measure of the force exerted by the motor proteins (Bourdieu, 1995). Motivated by these experiments, we consider the actin filament as a slender, elastic filament immersed in Stokesian flow, driven by a tangential forcing that mimics the force by the motor proteins. We find qualitative agreement on several points between our analysis and simulations and experimental observations. Furthermore, we study the correlation between filament transport and the characteristics of motion with the spatial pattern of motor protein density.

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

    PubMed Central

    Hong, Nan Hyung; Qi, Aidong

    2015-01-01

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

  12. Actin stress in cell reprogramming

    PubMed Central

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

    2014-01-01

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

  13. Treatment of Actinic Purpura

    PubMed Central

    2017-01-01

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

  14. Growing Actin Networks Form Lamellipodium and Lamellum by Self-Assembly

    PubMed Central

    Huber, Florian; Käs, Josef; Stuhrmann, Björn

    2008-01-01

    Many different cell types are able to migrate by formation of a thin actin-based cytoskeletal extension. Recently, it became evident that this extension consists of two distinct substructures, designated lamellipodium and lamellum, which differ significantly in their kinetic and kinematic properties as well as their biochemical composition. We developed a stochastic two-dimensional computer simulation that includes chemical reaction kinetics, G-actin diffusion, and filament transport to investigate the formation of growing actin networks in migrating cells. Model parameters were chosen based on experimental data or theoretical considerations. In this work, we demonstrate the system's ability to form two distinct networks by self-organization. We found a characteristic transition in mean filament length as well as a distinct maximum in depolymerization flux, both within the first 1–2 μm. The separation into two distinct substructures was found to be extremely robust with respect to initial conditions and variation of model parameters. We quantitatively investigated the complex interplay between ADF/cofilin and tropomyosin and propose a plausible mechanism that leads to spatial separation of, respectively, ADF/cofilin- or tropomyosin-dominated compartments. Tropomyosin was found to play an important role in stabilizing the lamellar actin network. Furthermore, the influence of filament severing and annealing on the network properties is explored, and simulation data are compared to existing experimental data. PMID:18708450

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

    PubMed Central

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

    2015-01-01

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

  16. The yeast actin cytoskeleton.

    PubMed

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

    2014-03-01

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

  17. Co-transcriptional nuclear actin dynamics

    PubMed Central

    Percipalle, Piergiorgio

    2013-01-01

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

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

    PubMed Central

    Case, Lindsay B.; Waterman, Clare M.

    2011-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  20. Nucleus-associated actin in Amoeba proteus.

    PubMed

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

    2016-10-01

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

  1. Bradykinin increases blood-tumor barrier permeability by down-regulating the expression levels of ZO-1, occludin, and claudin-5 and rearranging actin cytoskeleton.

    PubMed

    Liu, Li-Bo; Xue, Yi-Xue; Liu, Yun-Hui; Wang, Yi-Bao

    2008-04-01

    Bradykinin (BK) has been shown to open blood-tumor barrier (BTB) selectively and to increase permeability of the BTB transiently, but the mechanism is unclear. This study was performed to determine whether BK opens the BTB by affecting the tight junction (TJ)-associated proteins zonula occluden-1 (ZO-1), occludin, and caludin-5 and cytoskeleton protein filamentous actin (F-actin). In rat brain glioma model and BTB model in vitro, we find that the protein expression levels of ZO-1, occludin, and claudin-5 are attenuated by BK induction. Immunohistochemistry and immunofluorescence assays show that the attenuated expression of ZO-1, occludin, and claudin-5 and F-actin is most obvious in the smaller tumor capillaries (<20 microm) after BK infusion, and there is no change in the larger tumor capillaries (>20 microm). The redistribution of ZO-1, occludin, and claudin-5 and rearrangement of F-actin in brain microvascular endothelial cells are observed at the same time. Meanwhile, Evans blue assay shows that the permeability of BTB increases after BK infusion. Transmission electron microscopy indicates that TJ is opened and that pinocytotic vesicular density is increased. Transendothelial electrical resistance (TEER) and horseradish peroxidase flux assays also reveal that TJ is opened by BK induction. In addition, radioimmunity and Western blot assay reveal a significant decrease in expression levels of cAMP and catalytic subunit of protien kinase A (PKAcs) of tumor tissue. This study demonstrates that the increase of BK-mediated BTB permeability is associated with the down-regulation of ZO-1, occludin, and claudin-5 and the rearrangement of F-actin and that cAMP/PKA signal transduction system might be involved in the modulating process.

  2. 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.

  3. 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

  4. Simulation study of geometric shape factor approach to estimating earth emitted flux densities from wide field-of-view radiation measurements

    NASA Technical Reports Server (NTRS)

    Weaver, W. L.; Green, R. N.

    1980-01-01

    A study was performed on the use of geometric shape factors to estimate earth-emitted flux densities from radiation measurements with wide field-of-view flat-plate radiometers on satellites. Sets of simulated irradiance measurements were computed for unrestricted and restricted field-of-view detectors. In these simulations, the earth radiation field was modeled using data from Nimbus 2 and 3. Geometric shape factors were derived and applied to these data to estimate flux densities on global and zonal scales. For measurements at a satellite altitude of 600 km, estimates of zonal flux density were in error 1.0 to 1.2%, and global flux density errors were less than 0.2%. Estimates with unrestricted field-of-view detectors were about the same for Lambertian and non-Lambertian radiation models, but were affected by satellite altitude. The opposite was found for the restricted field-of-view detectors.

  5. What Does a Submillimeter Galaxy Selection Actually Select? The Dependence of Submillimeter Flux Density on Star Formation Rate and Dust Mass

    NASA Astrophysics Data System (ADS)

    Hayward, Christopher C.; Kereš, Dušan; Jonsson, Patrik; Narayanan, Desika; Cox, T. J.; Hernquist, Lars

    2011-12-01

    We perform three-dimensional dust radiative transfer (RT) calculations on hydrodynamic simulations of isolated and merging disk galaxies in order to quantitatively study the dependence of observed-frame submillimeter (submm) flux density on galaxy properties. We find that submm flux density and star formation rate (SFR) are related in dramatically different ways for quiescently star-forming galaxies and starbursts. Because the stars formed in the merger-induced starburst do not dominate the bolometric luminosity and the rapid drop in dust mass and more compact geometry cause a sharp increase in dust temperature during the burst, starbursts are very inefficient at boosting submm flux density (e.g., a >~ 16 × boost in SFR yields a <~ 2 × boost in submm flux density). Moreover, the ratio of submm flux density to SFR differs significantly between the two modes; thus one cannot assume that the galaxies with highest submm flux density are necessarily those with the highest bolometric luminosity or SFR. These results have important consequences for the bright submillimeter-selected galaxy (SMG) population. Among them are: (1) The SMG population is heterogeneous. In addition to merger-driven starbursts, there is a subpopulation of galaxy pairs, where two disks undergoing a major merger but not yet strongly interacting are blended into one submm source because of the large (gsim 15" or ~130 kpc at z = 2) beam of single-dish submm telescopes. (2) SMGs must be very massive (M sstarf >~ 6 × 1010 M ⊙). (3) The infall phase makes the SMG duty cycle a factor of a few greater than what is expected for a merger-driven starburst. Finally, we provide fitting functions for SCUBA and AzTEC submm flux densities as a function of SFR and dust mass and bolometric luminosity and dust mass; these should be useful for calculating submm flux density in semi-analytic models and cosmological simulations when performing full RT is computationally not feasible.

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

    PubMed

    Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

    2013-04-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2018-06-04

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

  9. Effects of charge density waves on flux dynamics in weak-pinning single crystals of NbSe2 : free flux flow, flux-core size effects, and unexpected doubling of Jc(H) `peak effect'

    NASA Astrophysics Data System (ADS)

    Favreau, Peter; Gapud, Albert A.; Moraes, Sunhee; Delong, Lance; Reyes, Arneil P.; Thompson, James R.; Christen, David K.

    2010-03-01

    The interaction of two different ordering schemes -- charge density waves (CDWs) and superconductivity -- is studied in high-quality samples of NbSe2, particularly in the motion of magnetic flux quanta. More specifically, the study is on the effect of ``switching off'' the CDW phase -- effected by doping with Ta -- on the magnetic-field H dependence of: (i) the Lorentz-force-driven free flux flow (FFF) resistivity ρf associated with the ordered motion of vortices, and (ii) critical current density Jc. FFF is achieved for the first time in this material. The field dependence of ρf deviates from traditional Bardeen-Stephen flux flow and is more consistent with effects of flux core size as predicted by Kogan and Zelezhina. However, the suppression of CDW's seems to have no significant effect on these properties. On the other hand, Jc(H) shows a surprising double peak for the CDW-suppressed sample --contrary to previous studies in which the Jc peak was shown to disappear. Possible mechanisms are discussed.

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

    PubMed

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

    2018-04-26

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

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

    PubMed Central

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

    2017-01-01

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

  12. A new empirical model to estimate hourly diffuse photosynthetic photon flux density

    NASA Astrophysics Data System (ADS)

    Foyo-Moreno, I.; Alados, I.; Alados-Arboledas, L.

    2018-05-01

    Knowledge of the photosynthetic photon flux density (Qp) is critical in different applications dealing with climate change, plant physiology, biomass production, and natural illumination in greenhouses. This is particularly true regarding its diffuse component (Qpd), which can enhance canopy light-use efficiency and thereby boost carbon uptake. Therefore, diffuse photosynthetic photon flux density is a key driving factor of ecosystem-productivity models. In this work, we propose a model to estimate this component, using a previous model to calculate Qp and furthermore divide it into its components. We have used measurements in urban Granada (southern Spain), of global solar radiation (Rs) to study relationships between the ratio Qpd/Rs with different parameters accounting for solar position, water-vapour absorption and sky conditions. The model performance has been validated with experimental measurements from sites having varied climatic conditions. The model provides acceptable results, with the mean bias error and root mean square error varying between - 0.3 and - 8.8% and between 9.6 and 20.4%, respectively. Direct measurements of this flux are very scarce so that modelling simulations are needed, this is particularly true regarding its diffuse component. We propose a new parameterization to estimate this component using only measured data of solar global irradiance, which facilitates its use for the construction of long-term data series of PAR in regions where continuous measurements of PAR are not yet performed.

  13. Sap flow measurements combining sap-flux density radial profiles with punctual sap-flux density measurements in oak trees (Quercus ilex and Quercus pyrenaica) - water-use implications in a water-limited savanna-

    NASA Astrophysics Data System (ADS)

    Reyes, J. Leonardo; Lubczynski1, Maciek W.

    2010-05-01

    Sap flow measurement is a key aspect for understanding how plants use water and their impacts on the ecosystems. A variety of sensors have been developed to measure sap flow, each one with its unique characteristics. When the aim of a research is to have accurate tree water use calculations, with high temporal and spatial resolution (i.e. scaled), a sensor with high accuracy, high measurement efficiency, low signal-to-noise ratio and low price is ideal, but such has not been developed yet. Granier's thermal dissipation probes (TDP) have been widely used in many studies and various environmental conditions because of its simplicity, reliability, efficiency and low cost. However, it has two major flaws when is used in semi-arid environments and broad-stem tree species: it is often affected by high natural thermal gradients (NTG), which distorts the measurements, and it cannot measure the radial variability of sap-flux density in trees with sapwood thicker than two centimeters. The new, multi point heat field deformation sensor (HFD) is theoretically not affected by NTG, and it can measure the radial variability of the sap flow at different depths. However, its high cost is a serious limitation when simultaneous measurements are required in several trees (e.g. catchment-scale studies). The underlying challenge is to develop a monitoring schema in which HFD and TDP are combined to satisfy the needs of measurement efficiency and accuracy in water accounting. To assess the level of agreement between TDP and HFD methods in quantifying sap flow rates and temporal patterns on Quercus ilex (Q.i ) and Quercus pyrenaica trees (Q.p.), three measurement schemas: standard TDP, TDP-NTG-corrected and HFD were compared in dry season at the semi-arid Sardon area, near Salamanca in Spain in the period from June to September 2009. To correct TDP measurements with regard to radial sap flow variability, a radial sap flux density correction factor was applied and tested by adjusting TDP

  14. Precision flux density measurements of the giant planets at 8420 MHz

    NASA Technical Reports Server (NTRS)

    Turegano, J. A.; Klein, M. J.

    1981-01-01

    Precision measurements of the 3.56 cm flux densities of Jupiter, Saturn, Uranus, and Neptune are reported. The results are compared with previously published measurements as a means of: (1) remotely sensing long-term changes in the microwave emission from the atmospheres of these planets; (2) measuring the effects of Saturn's rings on the disk temperature as observed from earth at different ring inclination angles.

  15. Precision flux density measurements of the giant planets at 8420 MHz

    NASA Technical Reports Server (NTRS)

    Turegano, J. A.; Klein, M. J.

    1981-01-01

    Precision measurements of the 3.56 cm flux densities of Jupiter, Saturn, Uranus, and Neptune are reported. The results are compared with previously published measurements as a means of: remotely sensing long-term changes in the microwave emission from the atmospheres of these planets and measuring the effects of Saturn's rings on the disk temperature as observed from earth at different ring inclination angles.

  16. WHAT DOES A SUBMILLIMETER GALAXY SELECTION ACTUALLY SELECT? THE DEPENDENCE OF SUBMILLIMETER FLUX DENSITY ON STAR FORMATION RATE AND DUST MASS

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

    Hayward, Christopher C.; Keres, Dusan; Jonsson, Patrik

    2011-12-20

    We perform three-dimensional dust radiative transfer (RT) calculations on hydrodynamic simulations of isolated and merging disk galaxies in order to quantitatively study the dependence of observed-frame submillimeter (submm) flux density on galaxy properties. We find that submm flux density and star formation rate (SFR) are related in dramatically different ways for quiescently star-forming galaxies and starbursts. Because the stars formed in the merger-induced starburst do not dominate the bolometric luminosity and the rapid drop in dust mass and more compact geometry cause a sharp increase in dust temperature during the burst, starbursts are very inefficient at boosting submm flux densitymore » (e.g., a {approx}> 16 Multiplication-Sign boost in SFR yields a {approx}< 2 Multiplication-Sign boost in submm flux density). Moreover, the ratio of submm flux density to SFR differs significantly between the two modes; thus one cannot assume that the galaxies with highest submm flux density are necessarily those with the highest bolometric luminosity or SFR. These results have important consequences for the bright submillimeter-selected galaxy (SMG) population. Among them are: (1) The SMG population is heterogeneous. In addition to merger-driven starbursts, there is a subpopulation of galaxy pairs, where two disks undergoing a major merger but not yet strongly interacting are blended into one submm source because of the large ({approx}> 15'' or {approx}130 kpc at z = 2) beam of single-dish submm telescopes. (2) SMGs must be very massive (M{sub *} {approx}> 6 Multiplication-Sign 10{sup 10} M{sub Sun }). (3) The infall phase makes the SMG duty cycle a factor of a few greater than what is expected for a merger-driven starburst. Finally, we provide fitting functions for SCUBA and AzTEC submm flux densities as a function of SFR and dust mass and bolometric luminosity and dust mass; these should be useful for calculating submm flux density in semi-analytic models and

  17. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

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

    2001-05-01

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

  18. Actin filaments as tension sensors.

    PubMed

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

    2012-02-07

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

  19. Optimization of Magneto-Rheological Damper for Maximizing Magnetic Flux Density in the Fluid Flow Gap Through FEA and GA Approaches

    NASA Astrophysics Data System (ADS)

    Krishna, Hemanth; Kumar, Hemantha; Gangadharan, Kalluvalappil

    2017-08-01

    A magneto rheological (MR) fluid damper offers cost effective solution for semiactive vibration control in an automobile suspension. The performance of MR damper is significantly depends on the electromagnetic circuit incorporated into it. The force developed by MR fluid damper is highly influenced by the magnetic flux density induced in the fluid flow gap. In the present work, optimization of electromagnetic circuit of an MR damper is discussed in order to maximize the magnetic flux density. The optimization procedure was proposed by genetic algorithm and design of experiments techniques. The result shows that the fluid flow gap size less than 1.12 mm cause significant increase of magnetic flux density.

  20. Calreticulin attenuated microwave radiation-induced human microvascular endothelial cell injury through promoting actin acetylation and polymerization.

    PubMed

    Xu, Feifei; Wang, You; Tao, Tianqi; Song, Dandan; Liu, Xiuhua

    2017-01-01

    Recent work reveals that actin acetylation modification has been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight the effects of calreticulin on actin acetylation and cell injury induced by microwave radiation in human microvascular endothelial cell (HMEC). HMEC injury was induced by high-power microwave of different power density (10, 30, 60, 100 mW/cm 2 , for 6 min) with or without exogenous recombinant calreticulin. The cell injury was assessed by lactate dehydrogenase (LDH) activity and Cell Counting Kit-8 in culture medium, migration ability, intercellular junction, and cytoskeleton staining in HMEC. Western blotting analysis was used to detected calreticulin expression in cytosol and nucleus and acetylation of globular actin (G-actin). We found that HMEC injury was induced by microwave radiation in a dose-dependent manner. Pretreatment HMEC with calreticulin suppressed microwave radiation-induced LDH leakage and increased cell viability and improved microwave radiation-induced decrease in migration, intercellular junction, and cytoskeleton. Meanwhile, pretreatment HMEC with exogenous calreticulin upregulated the histone acetyltransferase activity and the acetylation level of G-actin and increased the fibrous actin (F-actin)/G-actin ratio. We conclude that exogenous calreticulin protects HMEC against microwave radiation-induced injury through promoting actin acetylation and polymerization.

  1. Transition from Actin-Driven to Water-Driven Cell Migration Depends on External Hydraulic Resistance.

    PubMed

    Li, Yizeng; Sun, Sean X

    2018-06-19

    Cells in vivo can reside in diverse physical and biochemical environments. For example, epithelial cells typically live in a two-dimensional (2D) environment, whereas metastatic cancer cells can move through dense three-dimensional matrices. These distinct environments impose different kinds of mechanical forces on cells and thus potentially can influence the mechanism of cell migration. For example, cell movement on 2D flat surfaces is mostly driven by forces from focal adhesion and actin polymerization, whereas in confined geometries, it can be driven by water permeation. In this work, we utilize a two-phase model of the cellular cytoplasm in which the mechanics of the cytosol and the F-actin network are treated on an equal footing. Using conservation laws and simple force balance considerations, we are able to describe the contributions of water flux, actin polymerization and flow, and focal adhesions to cell migration both on 2D surfaces and in confined spaces. The theory shows how cell migration can seamlessly transition from a focal adhesion- and actin-based mechanism on 2D surfaces to a water-based mechanism in confined geometries. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    1990-09-11

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

  3. 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

  4. Scanning micro-Hall probe mapping of magnetic flux distributions and current densities in YBa2Cu3O7 thin films

    NASA Technical Reports Server (NTRS)

    Xing, W.; Heinrich, B.; Zhou, HU; Fife, A. A.; Cragg, A. R.; Grant, P. D.

    1995-01-01

    Mapping of the magnetic flux density B(sub z) (perpendicular to the film plane) for a YBa2Cu3O7 thin-film sample was carried out using a scanning micro-Hall probe. The sheet magnetization and sheet current densities were calculated from the B(sub z) distributions. From the known sheet magnetization, the tangential (B(sub x,y)) and normal components of the flux density B were calculated in the vicinity of the film. It was found that the sheet current density was mostly determined by 2B(sub x,y)/d, where d is the film thickness. The evolution of flux penetration as a function of applied field will be shown.

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

    PubMed Central

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

    1992-01-01

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

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

    PubMed

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

    1992-12-01

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

  7. Dynamics of photosynthetic photon flux density (PPFD) and estimates in coastal northern California

    USDA-ARS?s Scientific Manuscript database

    The seasonal trends and diurnal patterns of Photosynthetically Active Radiation (PAR) were investigated in the San Francisco Bay Area of Northern California from March through August in 2007 and 2008. During these periods, the daily values of PAR flux density (PFD), energy loading with PAR (PARE), a...

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

    PubMed Central

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

    2018-01-01

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

  9. Influence of field dependent critical current density on flux profiles in high Tc superconductors

    NASA Technical Reports Server (NTRS)

    Takacs, S.

    1990-01-01

    The field distribution for superconducting cylinders and slabs with field dependent critical current densities in combined DC and AC magnetic fields and the corresponding magnetic fluxes are calculated. It is shown that all features of experimental magnetic-field profile measurements can be explained in the framework of field dependent critical current density. Even the quantitative agreement between the experimental and theoretical results using Kim's model is very good.

  10. Statistics of actin-propelled trajectories in noisy environments

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

    Actin polymerization is ubiquitously utilized to power the locomotion of eukaryotic cells and pathogenic bacteria in living systems. Inevitably, actin polymerization and depolymerization proceed in a fluctuating environment that renders the locomotion stochastic. Previously, we have introduced a deterministic model that manages to reproduce actin-propelled trajectories in experiments, but not to address fluctuations around them. To remedy this, here we supplement the deterministic model with noise terms. It enables us to compute the effects of fluctuating actin density and forces on the trajectories. Specifically, the mean-squared displacement (MSD) of the trajectories is computed and found to show a super-ballistic scaling with an exponent 3 in the early stage, followed by a crossover to a normal, diffusive scaling of exponent 1 in the late stage. For open-end trajectories such as straights and S-shaped curves, the time of crossover matches the decay time of orientational order of the velocities along trajectories, suggesting that it is the spreading of velocities that leads to the crossover. We show that the super-ballistic scaling of MSD arises from the initial, linearly increasing correlation of velocities, before time translational symmetry is established. When the spreading of velocities reaches a steady state in the long-time limit, short-range correlation then yields a diffusive scaling in MSD. In contrast, close-loop trajectories like circles exhibit localized periodic motion, which inhibits spreading. The initial super-ballistic scaling of MSD arises from velocity correlation that both linearly increases and oscillates in time. Finally, we find that the above statistical features of the trajectories transcend the nature of noises, be it additive or multiplicative, and generalize to other self-propelled systems that are not necessarily actin based.

  11. Fascin regulates nuclear actin during Drosophila oogenesis

    PubMed Central

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

    2016-01-01

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

  12. The brightness temperature of Venus and the absolute flux-density scale at 608 MHz.

    NASA Technical Reports Server (NTRS)

    Muhleman, D. O.; Berge, G. L.; Orton, G. S.

    1973-01-01

    The disk temperature of Venus was measured at 608 MHz near the inferior conjunction of 1972, and a value of 498 plus or minus 33 K was obtained using a nominal CKL flux-density scale. The result is consistent with earlier measurements, but has a much smaller uncertainty. Our theoretical model prediction is larger by a factor of 1.21 plus or minus 0.09. This discrepancy has been noticed previously for frequencies below 1400 MHz, but was generally disregarded because of the large observational uncertainties. No way could be found to change the model to produce agreement without causing a conflict with well-established properties of Venus. Thus it is suggested that the flux-density scale may require an upward revision, at least near this frequency, in excess of what has previously been considered likely.

  13. Geometrical Determinants of Neuronal Actin Waves.

    PubMed

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

    2017-01-01

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

  14. Geometrical Determinants of Neuronal Actin Waves

    PubMed Central

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

    2017-01-01

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

  15. Actinic keratosis among seafarers.

    PubMed

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

    2013-11-01

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

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

    PubMed Central

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

    2001-01-01

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

  17. Bacterial Actins.

    PubMed

    Izoré, Thierry; van den Ent, Fusinita

    2017-01-01

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

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

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

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

    2013-04-04

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

  19. Surface-induced polymerization of actin.

    PubMed Central

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

    1999-01-01

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

  20. Contractile actin cables induced by Bacillus anthracis lethal toxin depend on the histone acetylation machinery.

    PubMed

    Rolando, Monica; Stefani, Caroline; Doye, Anne; Acosta, Maria I; Visvikis, Orane; Yevick, Hannah G; Buchrieser, Carmen; Mettouchi, Amel; Bassereau, Patricia; Lemichez, Emmanuel

    2015-10-01

    It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier. © 2015 Wiley Periodicals, Inc.

  1. Mesoscopic model of actin-based propulsion.

    PubMed

    Zhu, Jie; Mogilner, Alex

    2012-01-01

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

  2. Polycation induced actin bundles.

    PubMed

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

    2011-04-01

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

  3. A LOFAR census of non-recycled pulsars: average profiles, dispersion measures, flux densities, and spectra

    NASA Astrophysics Data System (ADS)

    Bilous, A. V.; Kondratiev, V. I.; Kramer, M.; Keane, E. F.; Hessels, J. W. T.; Stappers, B. W.; Malofeev, V. M.; Sobey, C.; Breton, R. P.; Cooper, S.; Falcke, H.; Karastergiou, A.; Michilli, D.; Osłowski, S.; Sanidas, S.; ter Veen, S.; van Leeuwen, J.; Verbiest, J. P. W.; Weltevrede, P.; Zarka, P.; Grießmeier, J.-M.; Serylak, M.; Bell, M. E.; Broderick, J. W.; Eislöffel, J.; Markoff, S.; Rowlinson, A.

    2016-06-01

    We present first results from a LOFAR census of non-recycled pulsars. The census includes almost all such pulsars known (194 sources) at declinations Dec > 8° and Galactic latitudes |Gb| > 3°, regardless of their expected flux densities and scattering times. Each pulsar was observed for ≥20 min in the contiguous frequency range of 110-188 MHz. Full-Stokes data were recorded. We present the dispersion measures, flux densities, and calibrated total intensity profiles for the 158 pulsars detected in the sample. The median uncertainty in census dispersion measures (1.5 × 10-3 pc cm-3) is ten times smaller, on average, than in the ATNF pulsar catalogue. We combined census flux densities with those in the literature and fitted the resulting broadband spectra with single or broken power-law functions. For 48 census pulsars such fits are being published for the first time. Typically, thechoice between single and broken power-laws, as well as the location of the spectral break, were highly influenced by the spectral coverage of the available flux density measurements. In particular, the inclusion of measurements below 100 MHz appears essential for investigating the low-frequency turnover in the spectra for most of the census pulsars. For several pulsars, we compared the spectral indices from different works and found the typical spread of values to be within 0.5-1.5, suggesting a prevailing underestimation of spectral index errors in the literature. The census observations yielded some unexpected individual source results, as we describe in the paper. Lastly, we will provide this unique sample of wide-band, low-frequency pulse profiles via the European Pulsar Network Database. Tables B.1-B.4 are only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/591/A134

  4. Algebraic reconstruction for 3D magnetic resonance-electrical impedance tomography (MREIT) using one component of magnetic flux density.

    PubMed

    Ider, Y Ziya; Onart, Serkan

    2004-02-01

    Magnetic resonance-electrical impedance tomography (MREIT) algorithms fall into two categories: those utilizing internal current density and those utilizing only one component of measured magnetic flux density. The latter group of algorithms have the advantage that the object does not have to be rotated in the magnetic resonance imaging (MRI) system. A new algorithm which uses only one component of measured magnetic flux density is developed. In this method, the imaging problem is formulated as the solution of a non-linear matrix equation which is solved iteratively to reconstruct resistivity. Numerical simulations are performed to test the algorithm both for noise-free and noisy cases. The uniqueness of the solution is monitored by looking at the singular value behavior of the matrix and it is shown that at least two current injection profiles are necessary. The method is also modified to handle region-of-interest reconstructions. In particular it is shown that, if the image of a certain xy-slice is sought for, then it suffices to measure the z-component of magnetic flux density up to a distance above and below that slice. The method is robust and has good convergence behavior for the simulation phantoms used.

  5. Electromagnetic potentials basis for energy density and power flux

    NASA Astrophysics Data System (ADS)

    Puthoff, H. E.

    2016-09-01

    In rounding out the education of students in advanced courses in applied electromagnetics it is incumbent on us as mentors to raise issues that encourage appreciation of certain subtle aspects that are often overlooked during first exposure to the field. One of these has to do with the interplay between fields and potentials, with the latter often seen as just a convenient mathematical artifice useful in solving Maxwell’s equations. Nonetheless, to those practiced in application it is well understood that various alternatives in the use of fields and potentials are available within electromagnetic (EM) theory for the definitions of energy density, momentum transfer, EM stress-energy tensor, and so forth. Although the various options are all compatible with the basic equations of electrodynamics (e.g., Maxwell’s equations, Lorentz force law, gauge invariance), nonetheless certain alternative formulations lend themselves to being seen as preferable to others with regard to the transparency of their application to physical problems of interest. Here we argue for the transparency of an energy density/power flux option based on the EM potentials alone.

  6. Nuclear positioning by actin cables and perinuclear actin

    PubMed Central

    Huelsmann, Sven; Brown, Nicholas H

    2014-01-01

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

  7. Non-Straub type actin from molluscan catch muscle

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

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

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

  8. Scanning micro-Hall probe mapping of magnetic flux distributions and current densities in YBa{sub 2}Cu{sub 3}O{sub 7}

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

    Xing, W.; Heinrich, B.; Zhou, H.

    1994-12-31

    Mapping of the magnetic flux density B{sub z} (perpendicular to the film plane) for a YBa{sub 2}Cu{sub 3}O{sub 7} thin-film sample was carried out using a scanning micro-Hall probe. The sheet magnetization and sheet current densities were calculated from the B{sub z} distributions. From the known sheet magnetization, the tangential (B{sub x,y}) and normal components of the flux density B were calculated in the vicinity of the film. It was found that the sheet current density was mostly determined by 2B{sub x,y}/d, where d is the film thickness. The evolution of flux penetration as a function of applied field willmore » be shown.« less

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

    PubMed Central

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

    2013-01-01

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

  10. Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire.

    PubMed

    Ferreira, Tiago; Ou, Yimiao; Li, Sally; Giniger, Edward; van Meyel, Donald J

    2014-02-01

    The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. Here, we describe a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actin-rich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. We found Spir to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. We conclude that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.

  11. Cargo crowding at actin-rich regions along axons causes local traffic jams.

    PubMed

    Sood, Parul; Murthy, Kausalya; Kumar, Vinod; Nonet, Michael L; Menon, Gautam I; Koushika, Sandhya P

    2018-03-01

    Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary up to several minutes. We examine the transport of precursors of synaptic vesicles (pre-SVs), endosomes and mitochondria in Caenorhabditis elegans touch receptor neurons, showing that stationary cargo are predominantly present at actin-rich regions along the neuronal process. Stationary vesicles at actin-rich regions increase the propensity of moving vesicles to stall at the same location, resulting in traffic jams arising from physical crowding. Such local traffic jams at actin-rich regions are likely to be a general feature of axonal transport since they also occur in Drosophila neurons. Repeated touch stimulation of C. elegans reduces the density of stationary pre-SVs, indicating that these traffic jams can act as both sources and sinks of vesicles. This suggests that vesicles trapped in actin-rich regions are functional reservoirs that may contribute to maintaining robust cargo flow in the neuron. A video abstract of this article can be found at: Video S1; Video S2. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    DOE PAGES

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

    2014-12-04

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

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

    PubMed

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

    2016-05-01

    Membrane deformations induced by attached BAR superfamily domains could trigger or facilitate the growth of plasma membrane protrusions. The BAR domain family consists of BAR, F-BAR and I-BAR domains, each enforcing a different local curvature when attached to the membrane surface. Our theoretical study mainly focuses on the role of I-BAR in the membrane tubular deformations generated or stabilised by actin filaments. The influence of the area density of membrane attached BAR domains and their intrinsic curvature on the closed membrane shapes (vesicles) was investigated numerically. We derived an analytical approximative expression for the critical relative area density of BARs at which the membrane tubular protrusions on vesicles are most prominent. We have shown that the BARs with a higher intrinsic curvature induce thinner and longer cylindrical protrusions. The average orientation of the membrane attached BARs is altered when the vesicle shape is subjected to external force of growing actin rod-like structure inside a vesicle. The average orientation angle of membrane attached BARs may indicate whether the actin filaments are just stabilising the protrusion or generating it by stretching the vesicle. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios

    2012-01-01

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

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

    PubMed Central

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

    2008-01-01

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

  17. 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

  18. 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.

  19. Actin filaments-A target for redox regulation.

    PubMed

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

    2016-10-01

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

  20. [Measurements of the flux densities of static magnetic fields generated by two types of dental magnetic attachments and their retentive forces].

    PubMed

    Xu, Chun; Chao, Yong-lie; Du, Li; Yang, Ling

    2004-05-01

    To measure and analyze the flux densities of static magnetic fields generated by two types of commonly used dental magnetic attachments and their retentive forces, and to provide guidance for the clinical application of magnetic attachments. A digital Gaussmeter was used to measure the flux densities of static magnetic fields generated by two types of magnetic attachments, under four circumstances: open-field circuit; closed-field circuit; keeper and magnet slid laterally for a certain distance; and existence of air gap between keeper and magnet. The retentive forces of the magnetic attachments in standard closed-field circuit, with the keeper and magnet sliding laterally for a certain distance or with a certain air gap between keeper and magnet were measured by a tensile testing machine. There were flux leakages under both the open-field circuit and closed-field circuit of the two types of magnetic attachments. The flux densities on the surfaces of MAGNEDISC 800 (MD800) and MAGFIT EX600W (EX600) magnetic attachments under open-field circuit were 275.0 mT and 147.0 mT respectively. The flux leakages under closed-field circuit were smaller than those under open-field circuit. The respective flux densities on the surfaces of MD800 and EX600 magnetic attachments decreased to 11.4 mT and 4.5 mT under closed-field circuit. The flux density around the magnetic attachment decreased as the distance from the surface of the attachment increased. When keeper and magnet slid laterally for a certain distance or when air gap existed between keeper and magnet, the flux leakage increased in comparison with that under closed-field circuit. Under the standard closed-field circuit, the two types of magnetic attachments achieved the largest retentive forces. The retentive forces of MD800 and EX600 magnetic attachments under the standard closed-field circuit were 6.20 N and 4.80 N respectively. The retentive forces decreased with the sliding distance or with the increase of air gap

  1. Actin dynamics in Amoeba proteus motility.

    PubMed

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

    2007-01-01

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

  2. Simplified Solar Modulation Model of Inner Trapped Belt Proton Flux As a Function of Atmospheric Density

    NASA Technical Reports Server (NTRS)

    Wilson, Thomas L.; Lodhi, M. A. K.; Diaz, Abel B.

    2005-01-01

    No simple algorithm seems to exist for calculating proton fluxes and lifetimes in the Earth's inner, trapped radiation belt throughout the solar cycle. Most models of the inner trapped belt in use depend upon AP8 which only describes the radiation environment at solar maximum and solar minimum in Cycle 20. One exception is NOAAPRO which incorporates flight data from the TIROS/NOAA polar orbiting spacecraft. The present study discloses yet another, simple formulation for approximating proton fluxes at any time in a given solar cycle, in particular between solar maximum and solar minimum. It is derived from AP8 using a regression algorithm technique from nuclear physics. From flux and its time integral fluence, one can then approximate dose rate and its time integral dose. It has already been published in this journal that the absorbed dose rate, D, in the trapped belts exhibits a power law relationship, D = A(rho)(sup -n), where A is a constant, rho is the atmospheric density, and the index n is weakly dependent upon shielding. However, that method does not work for flux and fluence. Instead, we extend this idea by showing that the power law approximation for flux J is actually bivariant in energy E as well as density rho. The resulting relation is J(E,rho)approx.(sum of)A(E(sup n))rho(sup -n), with A itself a power law in E. This provides another method for calculating approximate proton flux and lifetime at any time in the solar cycle. These in turn can be used to predict the associated dose and dose rate.

  3. A Systems-Biology Approach to Yeast Actin Cables

    PubMed Central

    Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios

    2011-01-01

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

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

    PubMed Central

    Bryan, Keith E.; Rubenstein, Peter A.

    2009-01-01

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

  5. Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders.

    PubMed

    George, Sudeep P; Wang, Yaohong; Mathew, Sijo; Srinivasan, Kamalakkannan; Khurana, Seema

    2007-09-07

    Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

  6. F-Actin Dynamics in Neurospora crassa ▿ †

    PubMed Central

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

    2010-01-01

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

  7. Actinic comedonal plaque.

    PubMed

    Eastern, J S; Martin, S

    1980-12-01

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

  8. Comparison between measured and computed magnetic flux density distribution of simulated transformer core joints assembled from grain-oriented and non-oriented electrical steel

    NASA Astrophysics Data System (ADS)

    Shahrouzi, Hamid; Moses, Anthony J.; Anderson, Philip I.; Li, Guobao; Hu, Zhuochao

    2018-04-01

    The flux distribution in an overlapped linear joint constructed in the central region of an Epstein Square was studied experimentally and results compared with those obtained using a computational magnetic field solver. High permeability grain-oriented (GO) and low permeability non-oriented (NO) electrical steels were compared at a nominal core flux density of 1.60 T at 50 Hz. It was found that the experimental results only agreed well at flux densities at which the reluctance of different paths of the flux are similar. Also it was revealed that the flux becomes more uniform when the working point of the electrical steel is close to the knee point of the B-H curve of the steel.

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

    PubMed

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

    2012-05-01

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

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

    PubMed

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

    2009-05-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. Actin expression in some Platyhelminthe species.

    PubMed

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

    1994-10-01

    Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.

  14. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

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

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143more » flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas

  15. Actin growth profile in clathrin-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Tweten, D. J.; Bayly, P. V.; Carlsson, A. E.

    2017-05-01

    Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000 -10 000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.

  16. Interactions of histatin-3 and histatin-5 with actin.

    PubMed

    Blotnick, Edna; Sol, Asaf; Bachrach, Gilad; Muhlrad, Andras

    2017-03-06

    Histatins are histidine rich polypeptides produced in the parotid and submandibular gland and secreted into the saliva. Histatin-3 and -5 are the most important polycationic histatins. They possess antimicrobial activity against fungi such as Candida albicans. Histatin-5 has a higher antifungal activity than histatin-3 while histatin-3 is mostly involved in wound healing in the oral cavity. We found that these histatins, like other polycationic peptides and proteins, such as LL-37, lysozyme and histones, interact with extracellular actin. Histatin-3 and -5 polymerize globular actin (G-actin) to filamentous actin (F-actin) and bundle F-actin filaments. Both actin polymerization and bundling by histatins is pH sensitive due to the high histidine content of histatins. In spite of the equal number of net positive charges and histidine residues in histatin-3 and -5, less histatin-3 is needed than histatin-5 for polymerization and bundling of actin. The efficiency of actin polymerization and bundling by histatins greatly increases with decreasing pH. Histatin-3 and -5 induced actin bundles are dissociated by 100 and 50 mM NaCl, respectively. The relatively low NaCl concentration required to dissociate histatin-induced bundles implies that the actin-histatin filaments bind to each other mainly by electrostatic forces. The binding of histatin-3 to F-actin is stronger than that of histatin-5 showing that hydrophobic forces have also some role in histatin-3- actin interaction. Histatins affect the fluorescence of probes attached to the D-loop of G-actin indicating histatin induced changes in actin structure. Transglutaminase cross-links histatins to actin. Competition and limited proteolysis experiments indicate that the main histatin cross-linking site on actin is glutamine-49 on the D-loop of actin. Both histatin-3 and -5 interacts with actin, however, histatin 3 binds stronger to actin and affects actin structure at lower concentration than histatin-5 due to the extra 8

  17. 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

  18. Distribution and dynamics of the cytoskeleton in graviresponding protonemata and rhizoids of characean algae: exclusion of microtubules and a convergence of actin filaments in the apex suggest an actin-mediated gravitropism.

    PubMed

    Braun, M; Wasteneys, G O

    1998-05-01

    The organization of the microtubule (MT) and actin microfilament (MF) cytoskeleton of tip-growing rhizoids and protonemata of characean green algae was examined by confocal laser scanning microscopy. This analysis included microinjection of fluorescent tubulin and phallotoxins into living cells, as well as immunofluorescence labeling of fixed material and fluorescent phallotoxin labeling of unfixed material. Although the morphologically very similar positively gravitropic (downward growing) rhizoids and negatively gravitropic (upward growing) protonemata show opposite gravitropic responses, no differences were detected in the extensive three-dimensional distribution of actin MFs and MTs in both cell types. Tubulin microinjection revealed that in contrast to internodal cells, fluorescent tubulin incorporated very slowly into the MT arrays of rhizoids, suggesting that MT dynamics are very different in tip-growing and diffusely expanding cells. Microtubules assembled from multiple sites at the plasma membrane in the basal zone, and a dense subapical array emerged from a diffuse nucleation centre on the basal side of the nuclear envelope. Immunofluorescence confirmed these distribution patterns but revealed more extensive MT arrays. In the basal zone, short branching clusters of MTs form two cortical hemicylinders. Subapical, axially oriented MTs are distributed in equal density throughout the peripheral and inner cytoplasm and are closely associated with subapical organelles. Microtubules, however, are completely absent from the apical zones of rhizoids and protonemata. Actin MFs were found in all zones of rhizoids and protonemata including the apex. Two files of axially oriented bundles of subcortical actin MFs and ring-like actin structures in the streaming endoplasm of rhizoids were detected in the basal zones by microinjection or rhodamine-phalloidin labeling. The subapical zone contains a dense array of mainly axially oriented actin MFs that co-distribute with

  19. Three’s company: The fission yeast actin cytoskeleton

    PubMed Central

    Kovar, David R.; Sirotkin, Vladimir; Lord, Matthew

    2010-01-01

    How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly vexing in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review, we explore recent studies in fission yeast that help unravel how different actin structures operate in cells. PMID:21145239

  20. The nature of the globular- to fibrous-actin transition.

    PubMed

    Oda, Toshiro; Iwasa, Mitsusada; Aihara, Tomoki; Maéda, Yuichiro; Narita, Akihiro

    2009-01-22

    Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

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

    PubMed

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

    2016-01-01

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

  2. Arrangement Analysis of Leaves Optimized on Photon Flux Density or Photosynthetic Rate

    NASA Astrophysics Data System (ADS)

    Obara, Shin'ya; Tanno, Itaru

    By clarifying a plant evolutive process, useful information may be obtained on engineering. Consequently, an analysis algorithm that investigates the optimal arrangement of plant leaves was developed. In the developed algorithm, the Monte Carlo method is introduced and sunlight is simulated. Moreover, the arrangement optimization of leaves is analyzed using a Genetic Algorithm (GA). The number of light quanta (photon flux density) that reaches leaves, or the average photosynthetic rate of the same was set as the objective function, and leaf models of a dogwood and a ginkgo tree were analyzed. The number of leaf models was set between two to four, and the position of the leaf was expressed in terms of the angle of direction, elevation angle, rotation angle, and the representative length of the branch of a leaf. The chromosome model introduced into GA consists of information concerning the position of the leaf. Based on the analysis results, the characteristics of the leaf of an actual plant could be simulated by ensuring the algorithm had multiple constrained conditions. The optimal arrangement of leaves differs in maximization of the photon flux density, and that of the average value of a photosynthetic rate. Furthermore, the leaf form affecting the optimal arrangement of leave and also having a significant influence also on a photosynthetic rate was shown.

  3. Using Solar Radio Burst Integrated Fluxes to Predict Energetic Proton Flux Increases.

    DTIC Science & Technology

    1982-08-31

    Energy Density, ET, of the radio burst, an integration across the frequency interval of the time-integrated radio fluxes at each frequency, was found to...integrated flux or energy at five frequencies in the 600- to 8800-MHz frequency interval and related them to the peak proton flux of the associated... energy of the burst normalized to its peak flux. One other characteristic of the radio burst to which Croom 13 referred was the total energy density, ET

  4. Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences.

    PubMed

    Bricheux, G; Brugerolle, G

    1997-08-01

    The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.

  5. Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics.

    PubMed

    Ono, Shoichiro; Ono, Kanako

    2002-03-18

    Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B-induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

  6. A first determination of the surface density of galaxy clusters at very low x-ray fluxes

    NASA Technical Reports Server (NTRS)

    Rosati, Piero; Della Ceca, Roberta; Burg, Richard; Norman, Colin; Giacconi, Riccardo

    1995-01-01

    We present the first results of a serendipitous search for clusters of galaxies in deep ROSAT position sensitive proportional counter (PSPC) pointed observations at high Galactic latitude. The survey is being carried out using a wavelet-based detection algorithm which is not biased against extended, low surface brightness sources. A new flux-diameter limited sample of 10 cluster candidates has been created from approximately 3 deg(exp 2) surveyed area. Preliminary CCD observations have revealed that a large fraction of these candidates correspond to a visible enhancement in the galaxy surface density, and several others have been identified from other surveys. We believe these sources to be either low- to moderate-redshift groups or intermediate- to high-redshift clusters. We show X-ray and optical images of some of the clusters identified to date. We present, for the first time, the derived number density of the galaxy clusters to a flux limit of 1 x 10(exp -14) ergs cm(exp -2) s(exp -1) (0.5-2.0 keV). This extends the log N-log S of previous cluster surveys by more than one decade in flux. Results are compared to theoretical predictions for cluster number counts.

  7. Managing actinic keratosis in primary care.

    PubMed

    Salmon, Nicola; Tidman, Michael J

    2016-10-01

    Actinic, or solar, keratosis is caused by chronic ultraviolet-induced damage to the epidermis. In the UK, 15-23% of individuals have actinic keratosis lesions. Risk factors include: advanced age; male gender; cumulative sun exposure or phototherapy; Fitzpatrick skin phototypes I-II; long-term immuno-suppression and genetic syndromes e.g. xeroderma pigmentosum and albinism. Actinic keratoses are regarded by some authorities as premalignant lesions that may transform into invasive squamous cell carcinoma (SCC) and by others as in situ SCC that may progress to an invasive stage. The risk of malignant change appears low; up to 0.5% per lesion per year. Up to 20-30% of lesions may spontaneously regress but in the absence of any reliable prognostic clinical indicators regarding malignant potential active treatment is considered appropriate. Actinic keratosis lesions may present as discrete hyperkeratotic papules, cutaneous horns, or more subtle flat lesions on sun-exposed areas of skin. The single most helpful diagnostic sign is an irregularly roughened surface texture: a sandpaper-like feel almost always indicates actinic damage. Dermatoscopy can be helpful in excluding signs of basal cell carcinoma when actinic keratosis is non-keratotic. It is always important to consider the possibility of SCC. The principal indication for referral to secondary care is the possibility of cutaneous malignancy. However, widespread and severe actinic damage in patients who are immunosuppressed is also a reason for referral.

  8. Two new methods used to simulate the circumferential solar flux density concentrated on the absorber of a parabolic trough solar collector

    NASA Astrophysics Data System (ADS)

    Guo, Minghuan; Wang, Zhifeng; Sun, Feihu

    2016-05-01

    The optical efficiencies of a solar trough concentrator are important to the whole thermal performance of the solar collector, and the outer surface of the tube absorber is a key interface of energy flux. So it is necessary to simulate and analyze the concentrated solar flux density distributions on the tube absorber of a parabolic trough solar collector for various sun beam incident angles, with main optical errors considered. Since the solar trough concentrators are linear focusing, it is much of interest to investigate the solar flux density distribution on the cross-section profile of the tube absorber, rather than the flux density distribution along the focal line direction. Although a few integral approaches based on the "solar cone" concept were developed to compute the concentrated flux density for some simple trough concentrator geometries, all those integral approaches needed special integration routines, meanwhile, the optical parameters and geometrical properties of collectors also couldn't be changed conveniently. Flexible Monte Carlo ray trace (MCRT) methods are widely used to simulate the more accurate concentrated flux density distribution for compound parabolic solar trough concentrators, while generally they are quite time consuming. In this paper, we first mainly introduce a new backward ray tracing (BRT) method combined with the lumped effective solar cone, to simulate the cross-section flux density on the region of interest of the tube absorber. For BRT, bundles of rays are launched at absorber-surface points of interest, directly go through the glass cover of the absorber, strike on the uniformly sampled mirror segment centers in the close-related surface region of the parabolic reflector, and then direct to the effective solar cone around the incident sun beam direction after the virtual backward reflection. All the optical errors are convoluted into the effective solar cone. The brightness distribution of the effective solar cone is supposed

  9. Bioinformatics study of the mangrove actin genes

    NASA Astrophysics Data System (ADS)

    Basyuni, M.; Wasilah, M.; Sumardi

    2017-01-01

    This study describes the bioinformatics methods to analyze eight actin genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, subcellular localization, similarity, and phylogenetic. The physical and chemical properties of eight mangroves showed variation among the genes. The percentage of the secondary structure of eight mangrove actin genes followed the order of a helix > random coil > extended chain structure for BgActl, KcActl, RsActl, and A. corniculatum Act. In contrast to this observation, the remaining actin genes were random coil > extended chain structure > a helix. This study, therefore, shown the prediction of secondary structure was performed for necessary structural information. The values of chloroplast or signal peptide or mitochondrial target were too small, indicated that no chloroplast or mitochondrial transit peptide or signal peptide of secretion pathway in mangrove actin genes. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove actin genes. To clarify the relationship among the mangrove actin gene, a phylogenetic tree was constructed. Three groups of mangrove actin genes were formed, the first group contains B. gymnorrhiza BgAct and R. stylosa RsActl. The second cluster which consists of 5 actin genes the largest group, and the last branch consist of one gene, B. sexagula Act. The present study, therefore, supported the previous results that plant actin genes form distinct clusters in the tree.

  10. Kinetics of Binding of Caldesmon to Actin*

    PubMed Central

    Chalovich, Joseph M.; Chen, Yi-der; Dudek, Ronald; Luo, Hai

    2005-01-01

    The time course of interaction of caldesmon with actin may be monitored by fluorescence changes that occur upon the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or to acrylodan actin. The concentration dependence of the observed rate of caldesmon-actin binding was analyzed to a first approximation as a single-step reaction using a Monte Carlo simulation. The derived association and dissociation rates were 107 m−1 s−1 and 18.2 s−1, respectively. Smooth muscle tropomyosin enhances the binding of caldesmon to actin, and this was found to be due to a reduction in the rate of dissociation to 6.3 s −1. There is no evidence from this study for a different mechanism of binding in the presence of tropomyosin. The fluorescence changes that occurred with the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or actin-tropomyosin were reversed by the addition of myosin subfragment 1 as predicted by a competitive binding mechanism. PMID:7730374

  11. Are non-muscle actin isoforms functionally equivalent?

    PubMed

    Simiczyjew, Aleksandra; Pietraszek-Gremplewicz, Katarzyna; Mazur, Antonina Joanna; Nowak, Dorota

    2017-11-01

    Actin is highly conserved and it is the most widespread protein in eukaryotic cells. One of the most important features of actin, which allows it to have many different functions, is its ability to polymerize and interact with many other proteins. Actins are the major constituent of the actin cytoskeleton, which is an important system that is involved in various aspects of cell function, including cell motility, structure, integrity, regulation of signal transduction and transcription. Six mammal actin isoforms are highly conserved and share common functions. Two of them, β and γ non-muscle actin isoforms, which differ only by four amino acids located at the N-terminus of the polypeptide chain, are required for survival and proper cell functioning. We also summarized data about actbl2, which is suggested to be a newly discovered isoactin. Here, we review the current knowledge about tissue-specific expression of the non-muscle actin isoforms and possible functional differences between them. We also discuss molecular tools, which in recent years have allowed for a better understanding of the role of these proteins in cell functioning.

  12. [Photodynamic therapy for actinic cheilitis].

    PubMed

    Castaño, E; Comunión, A; Arias, D; Miñano, R; Romero, A; Borbujo, J

    2009-12-01

    Actinic cheilitis is a subtype of actinic keratosis that mainly affects the lower lip and has a higher risk of malignant transformation. Its location on the labial mucosa influences the therapeutic approach. Vermilionectomy requires local or general anesthetic and is associated with a risk of an unsightly scar, and the treatment with 5-fluorouracil or imiquimod lasts for several weeks and the inflammatory reaction can be very intense. A number of authors have used photodynamic therapy as an alternative to the usual treatments. We present 3 patients with histologically confirmed actinic cheilitis treated using photodynamic therapy with methyl aminolevulinic acid as the photosensitizer and red light at 630 nm. The clinical response was good, with no recurrences after 3 to 6 months of follow-up. Our experience supports the use of photodynamic therapy as a good alternative for the treatment of actinic cheilitis.

  13. 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.

  14. Actin cable dynamics in budding yeast

    PubMed Central

    Yang, Hyeong-Cheol; Pon, Liza A.

    2002-01-01

    Actin cables, bundles of actin filaments that align along the long axis of budding yeast, are crucial for establishment of cell polarity. We fused green fluorescent protein (GFP) to actin binding protein 140 (Abp140p) and visualized actin cable dynamics in living yeast. We detected two populations of actin cables: (i) bud-associated cables, which extend from the bud along the mother-bud axis, and (ii) randomly oriented cables, which are relatively short. Time-lapse imaging of Abp140p–GFP revealed an apparent increase in the length of bud-associated actin cables. Analysis of movement of Abp140p–GFP fiduciary marks on bud-associated cables and fluorescence loss in photobleaching experiments revealed that this apparent elongation occurs by assembly of new material at the end of the cable within the bud and movement of the opposite end of the cable toward the tip of the mother cell distal to the bud. The rate of extension of the tip of an elongating actin cable is 0.29 ± 0.08 μm/s. Latrunculin A (Lat-A) treatment completely blocked this process. We also observed movement of randomly oriented cables around the cortex of cells at a rate of 0.59 ± 0.14 μm/s. Mild treatment with Lat-A did not affect the velocity of movement of randomly oriented cables. However, Lat-A treatment did increase the number of randomly oriented, motile cables per cell. Our observations suggest that establishment of bud-associated actin cables during the cell cycle is accomplished not by realignment of existing cables but by assembly of new cables within the bud or bud neck, followed by elongation. PMID:11805329

  15. A high-resolution optical measurement system for rapid acquisition of radiation flux density maps

    NASA Astrophysics Data System (ADS)

    Thelen, Martin; Raeder, Christian; Willsch, Christian; Dibowski, Gerd

    2017-06-01

    To identify the power and flux density of concentrated solar radiation the Institute of Solar Research at the German Aerospace Center (DLR - Deutsches Zentrum für Luft-und Raumfahrt e. V.) has used the camera-based measurement system FATMES (Flux and Temperature Measurement System) since 1995. The disadvantages of low resolution, difficult handling and poor computing power required a revision of the existing measurement system. The measurement system FMAS (Flux Mapping Acquisition system) is equipped with state-of-the-art-hardware, is compatible with computers off-the-shelf and is programmed in LabView. The expenditure of time for an image evaluation is reduced by the factor 60 compared to FATMES. The new measurement system is no longer associated with the facilities Solar Furnace and High Flux Solar Simulator at the DLR in Cologne but is also applicable as a mobile system. The data and the algorithms are transparent throughout the complete process. The measurement accuracy of FMAS is determined to at most ±3 % until now. The error of measurement of FATMES is at least 2 % higher according to the conducted comparison tests.

  16. From Cytoskeleton to Gene Expression: Actin in the Nucleus.

    PubMed

    Viita, Tiina; Vartiainen, Maria K

    2017-01-01

    Although most people still associate actin mainly with the cytoskeleton, several lines of evidence, with the earliest studies dating back to decades ago, have emphasized the importance of actin also inside the cell nucleus. Actin has been linked to many gene expression processes from gene activation to chromatin remodeling, but also to maintenance of genomic integrity and intranuclear movement of chromosomes and chromosomal loci. Recent advances in visualizing different forms and dynamic properties of nuclear actin have clearly advanced our understanding of the basic concepts by which actin operates in the nucleus. In this chapter we address the different breakthroughs in nuclear actin studies, as well as discuss the regulation nuclear actin and the importance of nuclear actin dynamics in relation to its different nuclear functions. Our aim is to highlight the fact that actin should be considered as an essential component of the cell nucleus, and its nuclear actions should be taken into account also in experiments on cytoplasmic actin networks.

  17. A Continuum Model of Actin Waves in Dictyostelium discoideum

    PubMed Central

    Khamviwath, Varunyu; Hu, Jifeng; Othmer, Hans G.

    2013-01-01

    Actin waves are complex dynamical patterns of the dendritic network of filamentous actin in eukaryotes. We developed a model of actin waves in PTEN-deficient Dictyostelium discoideum by deriving an approximation of the dynamics of discrete actin filaments and combining it with a signaling pathway that controls filament branching. This signaling pathway, together with the actin network, contains a positive feedback loop that drives the actin waves. Our model predicts the structure, composition, and dynamics of waves that are consistent with existing experimental evidence, as well as the biochemical dependence on various protein partners. Simulation suggests that actin waves are initiated when local actin network activity, caused by an independent process, exceeds a certain threshold. Moreover, diffusion of proteins that form a positive feedback loop with the actin network alone is sufficient for propagation of actin waves at the observed speed of . Decay of the wave back can be caused by scarcity of network components, and the shape of actin waves is highly dependent on the filament disassembly rate. The model allows retraction of actin waves and captures formation of new wave fronts in broken waves. Our results demonstrate that a delicate balance between a positive feedback, filament disassembly, and local availability of network components is essential for the complex dynamics of actin waves. PMID:23741312

  18. Actin filaments – a target for redox regulation

    PubMed Central

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

    2016-01-01

    Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through non-covalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates – the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL – and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. PMID:27309342

  19. Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

    PubMed Central

    Al Tanoury, Ziad; Schaffner-Reckinger, Elisabeth; Halavatyi, Aliaksandr; Hoffmann, Céline; Moes, Michèle; Hadzic, Ermin; Catillon, Marie; Yatskou, Mikalai; Friederich, Evelyne

    2010-01-01

    Background Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. Methodology/Principal Findings To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-δ isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. Conclusions/Significance Altogether these findings quantitatively

  20. 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).

  1. A modified thermal conductivity for low density plasma magnetic flux tubes

    NASA Technical Reports Server (NTRS)

    Comfort, R. H.; Craven, P. D.; Richards, P. G.

    1995-01-01

    In response to inconsistencies which have arisen in results from a hydrodynamic model in simulation of high ion temperature (1-2 eV) observed in low density, outer plasmasphere flux tubes, we postulate a reduced thermal conductivity coefficient in which only particles in the loss cone of the quasi-collisionless plasma contribute to the thermal conduction. Other particles are assumed to magnetically mirror before they reach the topside ionosphere and therefore not to remove thermal energy from the plasmasphere. This concept is used to formulate a mathematically simple, but physically limiting model for a modified thermal conductivity coefficient. When this modified coefficient is employed in the hydrodynamic model in a case study, the inconsistencies between simulation results and observations are largely resolved. The high simulated ion temperatures are achieved with significantly lower ion temperatures in the topside ionosphere. We suggest that this mechanism may be operative under the limited low density, refilling conditions in which high ion temperatures are observed.

  2. Electrostatic interaction map reveals a new binding position for tropomyosin on F-actin.

    PubMed

    Rynkiewicz, Michael J; Schott, Veronika; Orzechowski, Marek; Lehman, William; Fischer, Stefan

    2015-12-01

    Azimuthal movement of tropomyosin around the F-actin thin filament is responsible for muscle activation and relaxation. Recently a model of αα-tropomyosin, derived from molecular-mechanics and electron microscopy of different contractile states, showed that tropomyosin is rather stiff and pre-bent to present one specific face to F-actin during azimuthal transitions. However, a new model based on cryo-EM of troponin- and myosin-free filaments proposes that the interacting-face of tropomyosin can differ significantly from that in the original model. Because resolution was insufficient to assign tropomyosin side-chains, the interacting-face could not be unambiguously determined. Here, we use structural analysis and energy landscapes to further examine the proposed models. The observed bend in seven crystal structures of tropomyosin is much closer in direction and extent to the original model than to the new model. Additionally, we computed the interaction map for repositioning tropomyosin over the F-actin surface, but now extended over a much larger surface than previously (using the original interacting-face). This map shows two energy minima-one corresponding to the "blocked-state" as in the original model, and the other related by a simple 24 Å translation of tropomyosin parallel to the F-actin axis. The tropomyosin-actin complex defined by the second minimum fits perfectly into the recent cryo-EM density, without requiring any change in the interacting-face. Together, these data suggest that movement of tropomyosin between regulatory states does not require interacting-face rotation. Further, they imply that thin filament assembly may involve an interplay between initially seeded tropomyosin molecules growing from distinct binding-site regions on actin.

  3. A methodology for mapping forest latent heat flux densities using remote sensing

    NASA Technical Reports Server (NTRS)

    Pierce, Lars L.; Congalton, Russell G.

    1988-01-01

    Surface temperatures and reflectances of an upper elevation Sierran mixed conifer forest were monitored using the Thematic Mapper Simulator sensor during the summer of 1985 in order to explore the possibility of using remote sensing to determine the distribution of solar energy on forested watersheds. The results show that the method is capable of quantifying the relative energy allocation relationships between the two cover types defined in the study. It is noted that the method also has the potential to map forest latent heat flux densities.

  4. Concerning the measurement of atmospheric trace gas fluxes with open- and closed-path eddy covariance systems: The density terms and spectral attenuation [Chapter 7

    Treesearch

    W. J. Massman

    2004-01-01

    Atmospheric trace gas fluxes measured with an eddy covariance sensor that detects a constituent's density fluctuations within the in situ air need to include terms resulting from concurrent heat and moisture fluxes, the so called 'density' or 'WPL corrections' (Webb et al. 1980). The theory behind these additional terms is well established. But...

  5. Xenopus egg cytoplasm with intact actin.

    PubMed

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

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

  6. Capillary pericytes express α-smooth muscle actin, which requires prevention of filamentous-actin depolymerization for detection.

    PubMed

    Alarcon-Martinez, Luis; Yilmaz-Ozcan, Sinem; Yemisci, Muge; Schallek, Jesse; Kılıç, Kıvılcım; Can, Alp; Di Polo, Adriana; Dalkara, Turgay

    2018-03-21

    Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at -20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling. © 2018, Alarcon-Martinez et al.

  7. 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.

  8. Resistance of Actin to Cleavage during Apoptosis

    NASA Astrophysics Data System (ADS)

    Song, Qizhong; Wei, Tie; Lees-Miller, Susan; Alnemri, Emad; Watters, Dianne; Lavin, Martin F.

    1997-01-01

    A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1β -converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.

  9. Live-cell imaging of G-actin dynamics using sequential FDAP

    PubMed Central

    Kiuchi, Tai; Nagai, Tomoaki; Ohashi, Kazumasa; Watanabe, Naoki; Mizuno, Kensaku

    2011-01-01

    Various microscopic techniques have been developed to understand the mechanisms that spatiotemporally control actin filament dynamics in live cells. Kinetic data on the processes of actin assembly and disassembly on F-actin have been accumulated. However, the kinetics of cytoplasmic G-actin, a key determinant for actin polymerization, has remained unclear because of a lack of appropriate methods to measure the G-actin concentration quantitatively. We have developed two new microscopic techniques based on the fluorescence decay after photoactivation (FDAP) time-lapse imaging of photoswitchable Dronpa-labeled actin. These techniques, sequential FDAP (s-FDAP) and multipoint FDAP, were used to measure the time-dependent changes in and spatial distribution of the G-actin concentration in live cells. Use of s-FDAP provided data on changes in the G-actin concentration with high temporal resolution; these data were useful for the model analysis of actin assembly processes in live cells. The s-FDAP analysis also provided evidence that the cytoplasmic G-actin concentration substantially decreases after cell stimulation and that the extent of stimulus-induced actin assembly and cell size extension are linearly correlated with the G-actin concentration before cell stimulation. The advantages of using s-FDAP and multipoint FDAP to measure spatiotemporal G-actin dynamics and the roles of G-actin concentration and ADF/cofilin in stimulus-induced actin assembly and lamellipodium extension in live cells are discussed. PMID:22754616

  10. 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.

  11. Mechanics model for actin-based motility.

    PubMed

    Lin, Yuan

    2009-02-01

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

  12. Diverse roles of actin in C. elegans early embryogenesis

    PubMed Central

    Velarde, Nathalie; Gunsalus, Kristin C; Piano, Fabio

    2007-01-01

    Background The actin cytoskeleton plays critical roles in early development in Caenorhabditis elegans. To further understand the complex roles of actin in early embryogenesis we use RNAi and in vivo imaging of filamentous actin (F-actin) dynamics. Results Using RNAi, we found processes that are differentially sensitive to levels of actin during early embryogenesis. Mild actin depletion shows defects in cortical ruffling, pseudocleavage, and establishment of polarity, while more severe depletion shows defects in polar body extrusion, cytokinesis, chromosome segregation, and eventually, egg production. These defects indicate that actin is required for proper oocyte development, fertilization, and a wide range of important events during early embryogenesis, including proper chromosome segregation. In vivo visualization of the cortical actin cytoskeleton shows dynamics that parallel but are distinct from the previously described myosin dynamics. Two distinct types of actin organization are observed at the cortex. During asymmetric polarization to the anterior, or the establishment phase (Phase I), actin forms a meshwork of microfilaments and focal accumulations throughout the cortex, while during the anterior maintenance phase (Phase II) it undergoes a morphological transition to asymmetrically localized puncta. The proper asymmetric redistribution is dependent on the PAR proteins, while both asymmetric redistribution and morphological transitions are dependent upon PFN-1 and NMY-2. Just before cytokinesis, actin disappears from most of the cortex and is only found around the presumptive cytokinetic furrow. Finally, we describe dynamic actin-enriched comets in the early embryo. Conclusion During early C. elegans embryogenesis actin plays more roles and its organization is more dynamic than previously described. Morphological transitions of F-actin, from meshwork to puncta, as well as asymmetric redistribution, are regulated by the PAR proteins. Results from this study

  13. Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*

    PubMed Central

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

    2013-01-01

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

  14. 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

  15. 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.

  16. 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.

  17. Characterization of magnetic flux density in passive sources used in magnetic stimulation

    NASA Astrophysics Data System (ADS)

    Torres, J.; Hincapie, E.; Gilart, F.

    2018-03-01

    The spatial distribution of the magnetic flux density (B) was determined for the passive sources of magnetic field most used in magnetic stimulation of biological systems, toroidal dipole magnets and cylindrical dipole magnets, in order to find the spatial characteristics of the magnetic field within the volumes of interest for the treatment of biological systems. The perpendicular and parallel components of B regarding the polar surface of the magnets were measured, for which a FW Bell 5180 digital teslameter was used with longitudinal and transverse probes and a two-dimensional positioning system with millimeter scale. It was found that the magnets of this type, which are the most used, present a strong variation of the magnitude and direction of the magnetic flux density for spaces specified in millimeters, reason why the homogeneity of the magnetic field in the regions of interest was found to be relatively low, which makes them elements with a strong applicability for the stimulation of biological systems in which magnetic field gradients up to mT/mm are required in the case of cylindrical magnets, and up to tens of mT/mm in the case of toroidal magnets. Finally, it is concluded that a high percentage of experiments reported in the literature on magnetic treatment of biological systems may be presenting values of B in their doses with deviations of more than 100% of the real value, which raises an incongruence in the cause-effect proposed relation.

  18. 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

  19. Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

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

    Wu, Shenping; Liu, Jun; Reedy, Mary C.

    2010-10-22

    Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filamentmore » density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the 'target zone', situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77{sup o}/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127{sup o} range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very

  20. The Dynamic Actin Cytoskeleton in Smooth Muscle.

    PubMed

    Tang, Dale D

    2018-01-01

    Smooth muscle contraction requires both myosin activation and actin cytoskeletal remodeling. Actin cytoskeletal reorganization facilitates smooth muscle contraction by promoting force transmission between the contractile unit and the extracellular matrix (ECM), and by enhancing intercellular mechanical transduction. Myosin may be viewed to serve as an "engine" for smooth muscle contraction whereas the actin cytoskeleton may function as a "transmission system" in smooth muscle. The actin cytoskeleton in smooth muscle also undergoes restructuring upon activation with growth factors or the ECM, which controls smooth muscle cell proliferation and migration. Abnormal smooth muscle contraction, cell proliferation, and motility contribute to the development of vascular and pulmonary diseases. A number of actin-regulatory proteins including protein kinases have been discovered to orchestrate actin dynamics in smooth muscle. In particular, Abelson tyrosine kinase (c-Abl) is an important molecule that controls actin dynamics, contraction, growth, and motility in smooth muscle. Moreover, c-Abl coordinates the regulation of blood pressure and contributes to the pathogenesis of airway hyperresponsiveness and vascular/airway remodeling in vivo. Thus, c-Abl may be a novel pharmacological target for the development of new therapy to treat smooth muscle diseases such as hypertension and asthma. © 2018 Elsevier Inc. All rights reserved.

  1. The Bacterial Actin MamK

    PubMed Central

    Ozyamak, Ertan; Kollman, Justin; Agard, David A.; Komeili, Arash

    2013-01-01

    It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general. PMID:23204522

  2. Probing actin polymerization by intermolecular cross-linking.

    PubMed

    Millonig, R; Salvo, H; Aebi, U

    1988-03-01

    We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant

  3. Estimation of transient heat flux density during the heat supply of a catalytic wall steam methane reformer

    NASA Astrophysics Data System (ADS)

    Settar, Abdelhakim; Abboudi, Saïd; Madani, Brahim; Nebbali, Rachid

    2018-02-01

    Due to the endothermic nature of the steam methane reforming reaction, the process is often limited by the heat transfer behavior in the reactors. Poor thermal behavior sometimes leads to slow reaction kinetics, which is characterized by the presence of cold spots in the catalytic zones. Within this framework, the present work consists on a numerical investigation, in conjunction with an experimental one, on the one-dimensional heat transfer phenomenon during the heat supply of a catalytic-wall reactor, which is designed for hydrogen production. The studied reactor is inserted in an electric furnace where the heat requirement of the endothermic reaction is supplied by electric heating system. During the heat supply, an unknown heat flux density, received by the reactive flow, is estimated using inverse methods. In the basis of the catalytic-wall reactor model, an experimental setup is engineered in situ to measure the temperature distribution. Then after, the measurements are injected in the numerical heat flux estimation procedure, which is based on the Function Specification Method (FSM). The measured and estimated temperatures are confronted and the heat flux density which crosses the reactor wall is determined.

  4. Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro

    PubMed Central

    Grantham, Julie; Ruddock, Lloyd W.; Roobol, Anne; Carden, Martin J.

    2002-01-01

    We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament “seeds” and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers. PMID:12482199

  5. The effects of nonuniform magnetic field strength on density flux and test particle transport in drift wave turbulence

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

    Dewhurst, J. M.; Hnat, B.; Dendy, R. O.

    2009-07-15

    The extended Hasegawa-Wakatani equations generate fully nonlinear self-consistent solutions for coupled density n and vorticity {nabla}{sup 2}{phi}, where {phi} is electrostatic potential, in a plasma with background density inhomogeneity {kappa}=-{partial_derivative} ln n{sub 0}/{partial_derivative}x and magnetic field strength inhomogeneity C=-{partial_derivative} ln B/{partial_derivative}x. Finite C introduces interchange effects and {nabla}B drifts into the framework of drift turbulence through compressibility of the ExB and diamagnetic drifts. This paper addresses the direct computation of the radial ExB density flux {gamma}{sub n}=-n{partial_derivative}{phi}/{partial_derivative}y, tracer particle transport, the statistical properties of the turbulent fluctuations that drive {gamma}{sub n} and tracer motion, and analytical underpinnings. Systematic trends emergemore » in the dependence on C of the skewness of the distribution of pointwise {gamma}{sub n} and in the relative phase of density-velocity and density-potential pairings. It is shown how these effects, together with conservation of potential vorticity {pi}={nabla}{sup 2}{phi}-n+({kappa}-C)x, account for much of the transport phenomenology. Simple analytical arguments yield a Fickian relation {gamma}{sub n}=({kappa}-C)D{sub x} between the radial density flux {gamma}{sub n} and the radial tracer diffusivity D{sub x}, which is shown to explain key trends in the simulations.« less

  6. 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.

  7. Simulating the effect of high column density absorbers on the one-dimensional Lyman α forest flux power spectrum

    NASA Astrophysics Data System (ADS)

    Rogers, Keir K.; Bird, Simeon; Peiris, Hiranya V.; Pontzen, Andrew; Font-Ribera, Andreu; Leistedt, Boris

    2018-03-01

    We measure the effect of high column density absorbing systems of neutral hydrogen (H I) on the one-dimensional (1D) Lyman α forest flux power spectrum using cosmological hydrodynamical simulations from the Illustris project. High column density absorbers (which we define to be those with H I column densities N(H I) > 1.6 × 10^{17} atoms cm^{-2}) cause broadened absorption lines with characteristic damping wings. These damping wings bias the 1D Lyman α forest flux power spectrum by causing absorption in quasar spectra away from the location of the absorber itself. We investigate the effect of high column density absorbers on the Lyman α forest using hydrodynamical simulations for the first time. We provide templates as a function of column density and redshift, allowing the flexibility to accurately model residual contamination, i.e. if an analysis selectively clips out the largest damping wings. This flexibility will improve cosmological parameter estimation, for example, allowing more accurate measurement of the shape of the power spectrum, with implications for cosmological models containing massive neutrinos or a running of the spectral index. We provide fitting functions to reproduce these results so that they can be incorporated straightforwardly into a data analysis pipeline.

  8. Liquid droplets of cross-linked actin filaments

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Banerjee, Shiladitya; Dasbiswas, Kinjal; Vaikuntanathan, Suriyanarayan; Gardel, Margaret

    Soft materials constructed from biomolecules self-assemble into a myriad of structures that work in concert to support cell physiology. One critical soft material is the actin cytoskeleton, a viscoelastic gel composed of cross-linked actin filaments. Although actin networks are primarily known for their elastic properties, which are crucial to regulating cell mechanics, the viscous behavior has been theorized to enable shape changes and flows. We experimentally demonstrate a fluid phase of cross-linked actin, where cross-linker condenses dilute short actin filaments into spindle-shaped droplets, or tactoids. Tactoids have shape dynamics consistent with a continuum model of liquid crystal droplets. The cross-linker, which acts as a long range attractive interaction, analogous to molecular cohesion, controls the tactoid shape and dynamics, which reports on the liquid's interfacial tension and viscosity. We investigate how the cross-linker properties and filament length influence the liquid properties. These results demonstrate a novel mechanism to control organization of the actin cytoskeleton and provide insight into design principles for complex, macromolecular liquid phases.

  9. Sarcomeric Pattern Formation by Actin Cluster Coalescence

    PubMed Central

    Friedrich, Benjamin M.; Fischer-Friedrich, Elisabeth; Gov, Nir S.; Safran, Samuel A.

    2012-01-01

    Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells. PMID:22685394

  10. Drosophila Spire is an actin nucleation factor.

    PubMed

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

    2005-01-27

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

  11. Measurement and Analysis of in vitro Actin Polymerization

    PubMed Central

    Doolittle, Lynda K.; Rosen, Michael K.; Padrick, Shae B.

    2014-01-01

    Summary The polymerization of actin underlies force generation in numerous cellular processes. While actin polymerization can occur spontaneously, cells maintain control over this important process by preventing actin filament nucleation and then allowing stimulated polymerization and elongation by several regulated factors. Actin polymerization, regulated nucleation and controlled elongation activities can be reconstituted in vitro, and used to probe the signaling cascades cells use to control when and where actin polymerization occurs. Introducing a pyrene fluorophore allows detection of filament formation by an increase in pyrene fluorescence. This method has been used for many years and continues to be broadly used, owing to its simplicity and flexibility. Here we describe how to perform and analyze these in vitro actin polymerization assays, with an emphasis on extracting useful descriptive parameters from kinetic data. PMID:23868594

  12. Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes

    PubMed Central

    Balasubramanian, Sundaravadivel; Mani, Santhosh K.; Kasiganesan, Harinath; Baicu, Catalin C.; Kuppuswamy, Dhandapani

    2010-01-01

    The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While α-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of β-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, β-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of β-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of β-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of β-actin. To determine the localization and dynamics of β-actin, we adenovirally expressed GFP-tagged β-actin in isolated adult cardiomyocytes. The ectopically expressed β-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of β-actin dynamics revealed that β-actin at the Z-discs is constantly being exchanged with β-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while β-actin overexpression improved cardiomyocyte contractility, immunoneutralization of β-actin resulted in a reduced contractility suggesting that β-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of β-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during

  13. Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

    PubMed

    Balasubramanian, Sundaravadivel; Mani, Santhosh K; Kasiganesan, Harinath; Baicu, Catalin C; Kuppuswamy, Dhandapani

    2010-07-12

    The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac

  14. [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

  15. 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

  16. Nuclear positioning by actin cables and perinuclear actin: Special and general?

    PubMed

    Huelsmann, Sven; Brown, Nicholas H

    2014-01-01

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

  17. Korean VLBI Network Calibrator Survey (KVNCS). 1. Source Catalog of KVN Single-dish Flux Density Measurement in the K and Q Bands

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

    Lee, Jeong Ae; Sohn, Bong Won; Jung, Taehyun

    We present the catalog of the KVN Calibrator Survey (KVNCS). This first part of the KVNCS is a single-dish radio survey simultaneously conducted at 22 ( K band) and 43 GHz ( Q band) using the Korean VLBI Network (KVN) from 2009 to 2011. A total of 2045 sources are selected from the VLBA Calibrator Survey with an extrapolated flux density limit of 100 mJy at the K  band. The KVNCS contains 1533 sources in the K band with a flux density limit of 70 mJy and 553 sources in the Q band with a flux density limit of 120more » mJy; it covers the whole sky down to −32.°5 in decl. We detected 513 sources simultaneously in the K and Q bands; ∼76% of them are flat-spectrum sources (−0.5 ≤ α ≤ 0.5). From the flux–flux relationship, we anticipated that most of the radiation of many of the sources comes from the compact components. The sources listed in the KVNCS therefore are strong candidates for high-frequency VLBI calibrators.« less

  18. IFT88 influences chondrocyte actin organization and biomechanics.

    PubMed

    Wang, Z; Wann, A K T; Thompson, C L; Hassen, A; Wang, W; Knight, M M

    2016-03-01

    Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

    PubMed

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

    2014-07-15

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

  20. Biphasic interactions between a cationic dendrimer and actin.

    PubMed

    Ruenraroengsak, Pakatip; Florence, Alexander T

    2010-12-01

    Gene delivery systems face the problem not only of the route toward the cell and tissues in question, but also of the molecularly crowded environment of both the cytoplasm and the nucleus itself. One of the physical barriers in the cytoplasm for diffusing nanoparticles is an actin network. Here, we describe the finding that a self-fluorescent sixth generation cationic dendrimer (6 nm in diameter) interacts reversibly and possibly electrostatically with actin filaments in vitro. Not only does this interaction slow the diffusion of the dendrimer but it also affects actin polymerization in a biphasic manner. At low concentrations the dendrimer behaves like a G-binding actin protein, retarding actin polymerization, whereas at high concentrations the dendrimer acts as a nucleating protein accelerating the polymerization. Thus in vivo the diffusion of a dendrimer carrier such as this has both physical and chemical elements: by decreasing polymerization it might accelerate its own transport, and by enhancing actin polymerization retard it. This finding suggests that such a dendrimer may have a role as an anticancer agent through its inhibitory effect on actin polymerization.

  1. Actin genes and their expression in pacific white shrimp, Litopenaeus vannamei.

    PubMed

    Zhang, Xiaoxi; Zhang, Xiaojun; Yuan, Jianbo; Du, Jiangli; Li, Fuhua; Xiang, Jianhai

    2018-04-01

    Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.

  2. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor.

    PubMed

    Freeman, Spencer A; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E; Wong, Harikesh S; Abraham, Libin; Graves, Marcia L; Coombs, Daniel; Roskelley, Calvin D; Das, Raibatak; Grinstein, Sergio; Gold, Michael R

    2015-02-03

    Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

  3. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor

    PubMed Central

    Freeman, Spencer A.; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E.; Wong, Harikesh S.; Abraham, Libin; Graves, Marcia L.; Coombs, Daniel; Roskelley, Calvin D.; Das, Raibatak; Grinstein, Sergio; Gold, Michael R.

    2015-01-01

    Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection. PMID:25644899

  4. Soft Listeria: actin-based propulsion of liquid drops.

    PubMed

    Boukellal, Hakim; Campás, Otger; Joanny, Jean-François; Prost, Jacques; Sykes, Cécile

    2004-06-01

    We study the motion of oil drops propelled by actin polymerization in cell extracts. Drops deform and acquire a pearlike shape under the action of the elastic stresses exerted by the actin comet, a tail of cross-linked actin filaments. We solve this free boundary problem and calculate the drop shape taking into account the elasticity of the actin gel and the variation of the polymerization velocity with normal stress. The pressure balance on the liquid drop imposes a zero propulsive force if gradients in surface tension or internal pressure are not taken into account. Quantitative parameters of actin polymerization are obtained by fitting theory to experiment.

  5. Actin Hydrophobic Loop (262-274) and Filament Nucleation and Elongation

    PubMed Central

    Shvetsov, Alexander; Galkin, Vitold E.; Orlova, Albina; Phillips, Martin; Bergeron, Sarah E.; Rubenstein, Peter A.; Egelman, Edward H.; Reisler, Emil

    2014-01-01

    Summary The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently using a yeast actin mutant, L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked G-actin does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin - to assist with actin nucleation - and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not alone, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin the helical twist of F-actin changes by ~ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin, and a change of twist by ~ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or a competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics to both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors. PMID:18037437

  6. Theory of flux cutting and flux transport at the critical current of a type-II superconducting cylindrical wire

    NASA Astrophysics Data System (ADS)

    Clem, John R.

    2011-06-01

    I introduce a critical-state theory incorporating both flux cutting and flux transport to calculate the magnetic-field and current-density distributions inside a type-II superconducting cylinder at its critical current in a longitudinal applied magnetic field. The theory is an extension of the elliptic critical-state model introduced by Romero-Salazar and Pérez-Rodríguez. The vortex dynamics depend in detail on two nonlinear effective resistivities for flux cutting (ρ∥) and flux flow (ρ⊥), and their ratio r=ρ∥/ρ⊥. When r<1, the low relative efficiency of flux cutting in reducing the magnitude of the internal magnetic-flux density leads to a paramagnetic longitudinal magnetic moment. As a model for understanding the experimentally observed interrelationship between the critical currents for flux cutting and depinning, I calculate the forces on a helical vortex arc stretched between two pinning centers when the vortex is subjected to a current density of arbitrary angle ϕ. Simultaneous initiation of flux cutting and flux transport occurs at the critical current density Jc(ϕ) that makes the vortex arc unstable.

  7. 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.

  8. Correlative nanoscale imaging of actin filaments and their complexes

    NASA Astrophysics Data System (ADS)

    Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil; Gimzewski, James K.

    2013-06-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

  9. Simulation study of a geometric shape factor technique for estimating earth-emitted radiant flux densities from wide-field-of-view radiation measurements

    NASA Technical Reports Server (NTRS)

    Weaver, W. L.; Green, R. N.

    1980-01-01

    Geometric shape factors were computed and applied to satellite simulated irradiance measurements to estimate Earth emitted flux densities for global and zonal scales and for areas smaller than the detector field of view (FOV). Wide field of view flat plate detectors were emphasized, but spherical detectors were also studied. The radiation field was modeled after data from the Nimbus 2 and 3 satellites. At a satellite altitude of 600 km, zonal estimates were in error 1.0 to 1.2 percent and global estimates were in error less than 0.2 percent. Estimates with unrestricted field of view (UFOV) detectors were about the same for Lambertian and limb darkening radiation models. The opposite was found for restricted field of view detectors. The UFOV detectors are found to be poor estimators of flux density from the total FOV and are shown to be much better as estimators of flux density from a circle centered at the FOV with an area significantly smaller than that for the total FOV.

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. Myosin isoform determines the conformational dynamics and cooperativity of actin filaments in the strongly bound actomyosin complex

    PubMed Central

    Prochniewicz, Ewa; Chin, Harvey F.; Henn, Arnon; Hannemann, Diane E.; Olivares, Adrian O.; Thomas, David D.; De La Cruz, Enrique M.

    2010-01-01

    SUMMARY We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dynamics of erythrosin iodoacetamide (ErIA)-labeled actin strongly bound to single-headed fragments of muscle myosin (muscle S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at sub-stoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects; muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly, and muscle myosin more slowly, to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound ErIA; while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex is determined by the isoform of the bound myosin, in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells. PMID:19962990

  13. Design of PCB search coils for AC magnetic flux density measurement

    NASA Astrophysics Data System (ADS)

    Ulvr, Michal

    2018-04-01

    This paper presents single-layer, double-layer and ten-layer planar square search coils designed for AC magnetic flux density amplitude measurement up to 1 T in the low frequency range in a 10 mm air gap. The printed-circuit-board (PCB) method was used for producing the search coils. Special attention is given to a full characterization of the PCB search coils including a comparison between the detailed analytical design method and the finite integration technique method (FIT) on the one hand, and experimental results on the other. The results show very good agreement in the resistance, inductance and search coil constant values (the area turns) and also in the frequency dependence of the search coil constant.

  14. Magnetic-flux pump

    NASA Technical Reports Server (NTRS)

    Hildebrandt, A. F.; Elleman, D. D.; Whitmore, F. C. (Inventor)

    1966-01-01

    A magnetic flux pump is described for increasing the intensity of a magnetic field by transferring flux from one location to the magnetic field. The device includes a pair of communicating cavities formed in a block of superconducting material, and a piston for displacing the trapped magnetic flux into the secondary cavity producing a field having an intense flux density.

  15. Optimizing Power Density and Efficiency of a Double-Halbach Array Permanent-Magnet Ironless Axial-Flux Motor

    NASA Technical Reports Server (NTRS)

    Duffy, Kirsten P.

    2016-01-01

    NASA Glenn Research Center is investigating hybrid electric and turboelectric propulsion concepts for future aircraft to reduce fuel burn, emissions, and noise. Systems studies show that the weight and efficiency of the electric system components need to be improved for this concept to be feasible. This effort aims to identify design parameters that affect power density and efficiency for a double-Halbach array permanent-magnet ironless axial flux motor configuration. These parameters include both geometrical and higher-order parameters, including pole count, rotor speed, current density, and geometries of the magnets, windings, and air gap.

  16. Oligomerization of coronin: Implication on actin filament length in Leishmania.

    PubMed

    Srivastava, Rashmi; Prasadareddy Kajuluri, Lova; Pathak, Neelam; Gupta, Chhitar M; Sahasrabuddhe, Amogh A

    2015-12-01

    Coronin proteins bind with actin filaments and participate in regulation of actin-dependent processes. These proteins contain a coiled-coil domain at their C-terminus, which is responsible for their dimeric or trimeric forms. However, the functional significance of these oligomeric configurations in organizing the actin cytoskeleton is obscure. Here, we report that the Leishmania coronin exists in a higher oligomeric form through its coiled-coil domain, the truncation of which ablates the ability of Leishmania coronin to assist actin-filament formation. F-actin co-sedimentation assay using purified proteins shows that the coiled-coil domain does not interact with actin-filaments and its absence does not abrogate actin-coronin interaction. Furthermore, it was shown that unlike other coronins, Leishmania coronin interacts with actin-filaments through its unique region. These results provided important insights into the role of coronin oligomerization in modulating actin-network. © 2015 Wiley Periodicals, Inc.

  17. Coactosin accelerates cell dynamism by promoting actin polymerization.

    PubMed

    Hou, Xubin; Katahira, Tatsuya; Ohashi, Kazumasa; Mizuno, Kensaku; Sugiyama, Sayaka; Nakamura, Harukazu

    2013-07-01

    During development, cells dynamically move or extend their processes, which are achieved by actin dynamics. In the present study, we paid attention to Coactosin, an actin binding protein, and studied its role in actin dynamics. Coactosin was associated with actin and Capping protein in neural crest cells and N1E-115 neuroblastoma cells. Accumulation of Coactosin to cellular processes and its association with actin filaments prompted us to reveal the effect of Coactosin on cell migration. Coactosin overexpression induced cellular processes in cultured neural crest cells. In contrast, knock-down of Coactosin resulted in disruption of actin polymerization and of neural crest cell migration. Importantly, Coactosin was recruited to lamellipodia and filopodia in response to Rac signaling, and mutated Coactosin that cannot bind to F-actin did not react to Rac signaling, nor support neural crest cell migration. It was also shown that deprivation of Rac signaling from neural crest cells by dominant negative Rac1 (DN-Rac1) interfered with neural crest cell migration, and that co-transfection of DN-Rac1 and Coactosin restored neural crest cell migration. From these results we have concluded that Coactosin functions downstream of Rac signaling and that it is involved in neurite extension and neural crest cell migration by actively participating in actin polymerization. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Mutant Profilin Suppresses Mutant Actin-dependent Mitochondrial Phenotype in Saccharomyces cerevisiae*

    PubMed Central

    Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Rubenstein, Peter A.

    2011-01-01

    In the Saccharomyces cerevisiae actin-profilin interface, Ala167 of the actin barbed end W-loop and His372 near the C terminus form a clamp around a profilin segment containing residue Arg81 and Tyr79. Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function. PMID:21956104

  19. How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

    PubMed Central

    1988-01-01

    We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099

  20. 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.

  1. Thymosin-beta(4) changes the conformation and dynamics of actin monomers.

    PubMed Central

    De La Cruz, E M; Ostap, E M; Brundage, R A; Reddy, K S; Sweeney, H L; Safer, D

    2000-01-01

    Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) of the actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange. PMID:10777749

  2. A turnkey data logger program for field-scale energy flux density measurements using eddy covariance and surface renewal

    USDA-ARS?s Scientific Manuscript database

    Micrometeorological methods and ecosystem-scale energy and mass flux density measurements have become increasingly important in soil, agricultural, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Eddy cov...

  3. Treatment options for actinic keratoses.

    PubMed

    McIntyre, William J; Downs, Michael R; Bedwell, Sondra A

    2007-09-01

    Actinic keratoses are rough, scaly lesions that commonly occur on sun-exposed areas of the skin. The prevalence of the condition increases with age. Actinic keratoses are thought to be carcinomas in situ, which can progress to squamous cell carcinomas. The decision to treat can be based on cosmetic reasons; symptom relief; or, most importantly, the prevention of malignancy and metastasis. Treatment options include ablative (destructive) therapies such as cryosurgery, curettage with electrosurgery, and photodynamic therapy. Topical therapies are used in patients with multiple lesions. Fluorouracil has been the traditional topical treatment for actinic keratoses, although imiquimod 5% cream and diclofenac 3% gel are effective alternative therapies. There are too few controlled trials comparing treatment modalities for physicians to make sound, evidence-based treatment decisions.

  4. Side-binding proteins modulate actin filament dynamics

    PubMed Central

    Crevenna, Alvaro H; Arciniega, Marcelino; Dupont, Aurélie; Mizuno, Naoko; Kowalska, Kaja; Lange, Oliver F; Wedlich-Söldner, Roland; Lamb, Don C

    2015-01-01

    Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics. DOI: http://dx.doi.org/10.7554/eLife.04599.001 PMID:25706231

  5. 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

  6. Spatial control of actin polymerization during neutrophil chemotaxis.

    PubMed

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

    1999-06-01

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

  7. Placed in a steady magnetic field, the flux density inside a permalloy-shielded volume decreases over hours and days

    NASA Astrophysics Data System (ADS)

    Feinberg, Benedict; Gould, Harvey

    2018-03-01

    Following the application of an external magnetic field to a thin-walled demagnetized Permalloy cylinder, the magnetic flux density at the center of the shielded volume decreases by roughly 20% over periods of hours to days. We measured this effect for applied magnetic fields from 0.48 A/m to 16 A/m, the latter being comparable to the Earths magnetic field at its weakest point. Delayed changes in magnetic flux density are also observed following alternating current demagnetization. We attribute these effects to delayed changes in magnetization, which have previously been observed in thin Permalloy films and small bulk samples of ferromagnetic materials. Phenomenological models of thermal activation are discussed. Some possible effects on experiments that rely on static shielding are noted.

  8. 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

  9. Solar Radiation Measurements Onboard the Research Aircraft HALO

    NASA Astrophysics Data System (ADS)

    Lohse, I.; Bohn, B.; Werner, F.; Ehrlich, A.; Wendisch, M.

    2014-12-01

    Airborne measurements of the separated upward and downward components of solar spectral actinic flux densities for the determination of photolysis frequencies and of upward nadir spectral radiance were performed with the HALO Solar Radiation (HALO-SR) instrument package onboard the High Altitude and Long Range Research Aircraft (HALO). The instrumentation of HALO-SR is characterized and first measurement data from the Next-generation Aircraft Remote-Sensing for Validation Studies (NARVAL) campaigns in 2013 and 2014 are presented. The measured data are analyzed in the context of the retrieved microphysical and optical properties of clouds which were observed underneath the aircraft. Detailed angular sensitivities of the two optical actinic flux receivers were determined in the laboratory. The effects of deviations from the ideal response are investigated using radiative transfer calculations of atmospheric radiance distributions under various atmospheric conditions and different ground albedos. Corresponding correction factors are derived. Example photolysis frequencies are presented, which were sampled in the free troposphere and lower stratosphere over the Atlantic Ocean during the 2013/14 HALO NARVAL campaigns. Dependencies of photolysis frequencies on cloud cover, flight altitude and wavelength range of the photolysis process are investigated. Calculated actinic flux densities in the presence of clouds benefit from the measured spectral radiances. Retrieved cloud optical thicknesses and effective droplet radii are used as model input for the radiative transfer calculations. By comparison with the concurrent measurements of actinic flux densities the retrieval approach is validated. Acknowledgements: Funding by the Deutsche Forschungsgemeinschaft within the priority program HALO (BO 1580/4-1, WE 1900/21-1) is gratefully acknowledged.

  10. The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

    PubMed Central

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

  11. Joint-inversion of gravity data and cosmic ray muon flux to detect shallow subsurface density structure beneath volcanoes: Testing the method at a well-characterized site

    NASA Astrophysics Data System (ADS)

    Roy, M.; Lewis, M.; George, N. K.; Johnson, A.; Dichter, M.; Rowe, C. A.; Guardincerri, E.

    2016-12-01

    The joint-inversion of gravity data and cosmic ray muon flux measurements has been utilized by a number of groups to image subsurface density structure in a variety of settings, including volcanic edifices. Cosmic ray muons are variably-attenuated depending upon the density structure of the material they traverse, so measuring muon flux through a region of interest provides an independent constraint on the density structure. Previous theoretical studies have argued that the primary advantage of combining gravity and muon data is enhanced resolution in regions not sampled by crossing muon trajectories, e.g. in sensing deeper structure or structure adjacent to the region sampled by muons. We test these ideas by investigating the ability of gravity data alone and the joint-inversion of gravity and muon flux to image subsurface density structure, including voids, in a well-characterized field location. Our study area is a tunnel vault located at the Los Alamos National Laboratory within Quaternary ash-flow tuffs on the Pajarito Plateau, flanking the Jemez Volcano in New Mexico. The regional geology of the area is well-characterized (with density measurements in nearby wells) and the geometry of the tunnel and the surrounding terrain is known. Gravity measurements were made using a Lacoste and Romberg D meter and the muon detector has a conical acceptance region of 45 degrees from the vertical and track resolution of several milliradians. We obtain individual and joint resolution kernels for gravity and muon flux specific to our experimental design and plan to combine measurements of gravity and muon flux both within and above the tunnel to infer density structure. We plan to compare our inferred density structure against the expected densities from the known regional hydro-geologic framework.

  12. Theory of flux cutting and flux transport at the critical current of a type-II superconducting cylindrical wire

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

    Clem, John R

    2011-02-17

    I introduce a critical-state theory incorporating both flux cutting and flux transport to calculate the magnetic-field and current-density distributions inside a type-II superconducting cylinder at its critical current in a longitudinal applied magnetic field. The theory is an extension of the elliptic critical-state model introduced by Romero-Salazar and Pérez-Rodríguez. The vortex dynamics depend in detail on two nonlinear effective resistivities for flux cutting (ρ{sub ∥}) and flux flow (ρ{sub ⊥}), and their ratio r=ρ{sub ∥}/ρ{sub ⊥}. When r<1, the low relative efficiency of flux cutting in reducing the magnitude of the internal magnetic-flux density leads to a paramagnetic longitudinal magneticmore » moment. As a model for understanding the experimentally observed interrelationship between the critical currents for flux cutting and depinning, I calculate the forces on a helical vortex arc stretched between two pinning centers when the vortex is subjected to a current density of arbitrary angle Φ. Simultaneous initiation of flux cutting and flux transport occurs at the critical current density J{sub c}(Φ) that makes the vortex arc unstable.« less

  13. Theory of flux cutting and flux transport at the critical current of a type-II superconducting cylindrical wire

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

    Clem, John R.

    2011-02-17

    I introduce a critical-state theory incorporating both flux cutting and flux transport to calculate the magnetic-field and current-density distributions inside a type-II superconducting cylinder at its critical current in a longitudinal applied magnetic field. The theory is an extension of the elliptic critical-state model introduced by Romero-Salazar and Perez-Rodriguez. The vortex dynamics depend in detail on two nonlinear effective resistivities for flux cutting ({rho}{parallel}) and flux flow ({rho}{perpendicular}), and their ratio r = {rho}{parallel}/{rho}{perpendicular}. When r < 1, the low relative efficiency of flux cutting in reducing the magnitude of the internal magnetic-flux density leads to a paramagnetic longitudinal magneticmore » moment. As a model for understanding the experimentally observed interrelationship between the critical currents for flux cutting and depinning, I calculate the forces on a helical vortex arc stretched between two pinning centers when the vortex is subjected to a current density of arbitrary angle {phi}. Simultaneous initiation of flux cutting and flux transport occurs at the critical current density J{sub c}({phi}) that makes the vortex arc unstable.« less

  14. Nucleotide-dependent conformational states of actin

    PubMed Central

    Pfaendtner, Jim; Branduardi, Davide; Parrinello, Michele; Pollard, Thomas D.; Voth, Gregory A.

    2009-01-01

    The influence of the state of the bound nucleotide (ATP, ADP-Pi, or ADP) on the conformational free-energy landscape of actin is investigated. Nucleotide-dependent folding of the DNase-I binding (DB) loop in monomeric actin and the actin trimer is carried out using all-atom molecular dynamics (MD) calculations accelerated with a multiscale implementation of the metadynamics algorithm. Additionally, an investigation of the opening and closing of the actin nucleotide binding cleft is performed. Nucleotide-dependent free-energy profiles for all of these conformational changes are calculated within the framework of metadynamics. We find that in ADP-bound monomer, the folded and unfolded states of the DB loop have similar relative free-energy. This result helps explain the experimental difficulty in obtaining an ordered crystal structure for this region of monomeric actin. However, we find that in the ADP-bound actin trimer, the folded DB loop is stable and in a free-energy minimum. It is also demonstrated that the nucleotide binding cleft favors a closed conformation for the bound nucleotide in the ATP and ADP-Pi states, whereas the ADP state favors an open confirmation, both in the monomer and trimer. These results suggest a mechanism of allosteric interactions between the nucleotide binding cleft and the DB loop. This behavior is confirmed by an additional simulation that shows the folding free-energy as a function of the nucleotide cleft width, which demonstrates that the barrier for folding changes significantly depending on the value of the cleft width. PMID:19620726

  15. Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

    PubMed Central

    1991-01-01

    The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide- stimulated cells was examined. F-actin was quantified by the TRITC- labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar. PMID:1918158

  16. Actinic cheilitis in dental practice.

    PubMed

    Savage, N W; McKay, C; Faulkner, C

    2010-06-01

    Actinic cheilitis is a potentially premalignant condition involving predominantly the vermilion of the lower lip. The aim of the current paper was to review the clinical presentation of actinic cheilitis and demonstrate the development of management plans using a series of cases. These are designed to provide immediate treatment where required but also to address the medium and long-term requirements of the patient. The authors suggest that the clinical examination of lips and the assessment of actinic cheilitis and other lip pathology become a regular part of the routine soft tissue examination undertaken as a part of the periodic examination of dental patients. Early recognition of actinic cheilitis can allow the development of strategies for individual patients that prevent progression. These are based on past sun exposure, future lifestyle changes and the daily use of emollient sunscreens, broad-brimmed hats and avoidance of sun exposure during the middle of the day. This is a service that is not undertaken as a matter of routine in general medical practice as patients are not seen with the regularity of dental patients and generally not under the ideal examination conditions available in the dental surgery.

  17. Regulation of the Pollen-Specific Actin-Depolymerizing Factor LlADF1

    PubMed Central

    Allwood, Ellen G.; Anthony, Richard G.; Smertenko, Andrei P.; Reichelt, Stefanie; Drobak, Bjorn K.; Doonan, John H.; Weeds, Alan G.; Hussey, Patrick J.

    2002-01-01

    Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by ∼60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy. PMID:12417710

  18. Protein Kinase Cζ Mediates Insulin-induced Glucose Transport through Actin Remodeling in L6 Muscle Cells

    PubMed Central

    Liu, Li-Zhong; Zhao, Hai-Lu; Zuo, Jin; Ho, Stanley K.S.; Chan, Juliana C.N.; Meng, Yan; Fang, Fu-De; Tong, Peter C.Y.

    2006-01-01

    Protein kinase C (PKC) ζ has been implicated in insulin-induced glucose uptake in skeletal muscle cell, although the underlying mechanism remains unknown. In this study, we investigated the effect of PKCζ on actin remodeling and glucose transport in differentiated rat L6 muscle cells expressing myc-tagged glucose transporter 4 (GLUT4). On insulin stimulation, PKCζ translocated from low-density microsomes to plasma membrane accompanied by increase in GLUT4 translocation and glucose uptake. Z-scan confocal microscopy revealed a spatial colocalization of relocated PKCζ with the small GTPase Rac-1, actin, and GLUT4 after insulin stimulation. The insulin-mediated colocalization, PKCζ distribution, GLUT4 translocation, and glucose uptake were inhibited by wortmannin and cell-permeable PKCζ pseudosubstrate peptide. In stable transfected cells, overexpression of PKCζ caused an insulin-like effect on actin remodeling accompanied by a 2.1-fold increase in GLUT4 translocation and 1.7-fold increase in glucose uptake in the absence of insulin. The effects of PKCζ overexpression were abolished by cell-permeable PKCζ pseudosubstrate peptide, but not wortmannin. Transient transfection of constitutively active Rac-1 recruited PKCζ to new structures resembling actin remodeling, whereas dominant negative Rac-1 prevented the insulin-mediated PKCζ translocation. Together, these results suggest that PKCζ mediates insulin effect on glucose transport through actin remodeling in muscle cells. PMID:16525020

  19. Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy.

    PubMed

    Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O

    2010-12-22

    The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

  20. 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

  1. Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins.

    PubMed

    Aktories, K; Wegner, A

    1992-10-01

    Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.

  2. The evolution of compositionally and functionally distinct actin filaments.

    PubMed

    Gunning, Peter W; Ghoshdastider, Umesh; Whitaker, Shane; Popp, David; Robinson, Robert C

    2015-06-01

    The actin filament is astonishingly well conserved across a diverse set of eukaryotic species. It has essentially remained unchanged in the billion years that separate yeast, Arabidopsis and man. In contrast, bacterial actin-like proteins have diverged to the extreme, and many of them are not readily identified from sequence-based homology searches. Here, we present phylogenetic analyses that point to an evolutionary drive to diversify actin filament composition across kingdoms. Bacteria use a one-filament-one-function system to create distinct filament systems within a single cell. In contrast, eukaryotic actin is a universal force provider in a wide range of processes. In plants, there has been an expansion of the number of closely related actin genes, whereas in fungi and metazoa diversification in tropomyosins has increased the compositional variety in actin filament systems. Both mechanisms dictate the subset of actin-binding proteins that interact with each filament type, leading to specialization in function. In this Hypothesis, we thus propose that different mechanisms were selected in bacteria, plants and metazoa, which achieved actin filament compositional variation leading to the expansion of their functional diversity. © 2015. Published by The Company of Biologists Ltd.

  3. Cell-cycle regulation of formin-mediated actin cable assembly

    PubMed Central

    Miao, Yansong; Wong, Catherine C. L.; Mennella, Vito; Michelot, Alphée; Agard, David A.; Holt, Liam J.; Yates, John R.; Drubin, David G.

    2013-01-01

    Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation. PMID:24133141

  4. Mechanism of Cdc42-induced actin polymerization in neutrophil extracts.

    PubMed

    Zigmond, S H; Joyce, M; Yang, C; Brown, K; Huang, M; Pring, M

    1998-08-24

    Cdc42, activated with GTPgammaS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool. Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 micron in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 micron. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.

  5. 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.

  6. Course 6: Physics of Composite Cell Membrane and Actin Based Cytoskeleton

    NASA Astrophysics Data System (ADS)

    Sackmann, E.; Bausch, A. R.; Vonna, L.

    1 Architecture of composite cell membranes 1.1 The lipid/protein bilayer is a multicomponent smectic phase with mosaic like architecture 1.2 The spectrin/actin cytoskeleton as hyperelastic cell stabilizer 1.3 The actin cortex: Architecture and function 2 Physics of the actin based cytoskeleton 2.1 Actin is a living semiflexible polymer 2.2 Actin network as viscoelastic body 2.3 Correlation between macroscopic viscoelasticity and molecular 3 Heterogeneous actin gels in cells and biological function 3.1 Manipulation of actin gels 3.2 Control of organization and function of actin cortex by cell signalling 4 Micromechanics and microrheometry of cells 5 Activation of endothelial cells: On the possibility of formation of stress fibers as phase transition of actin-network triggered by cell signalling pathways 6 On cells as adaptive viscoplastic bodies 7 Controll of cellular protrusions controlled by actin/myosin cortex

  7. eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1

    PubMed Central

    García-Ortiz, Almudena; Martín-Cofreces, Noa B.; Ibiza, Sales; Ortega, Ángel; Izquierdo-Álvarez, Alicia; Trullo, Antonio; Victor, Víctor M.; Calvo, Enrique; Sot, Begoña; Martínez-Ruiz, Antonio; Vázquez, Jesús; Sánchez-Madrid, Francisco

    2017-01-01

    The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS. PMID:28394935

  8. Cations Modulate Actin Bundle Mechanics, Assembly Dynamics, and Structure.

    PubMed

    Castaneda, Nicholas; Zheng, Tianyu; Rivera-Jacquez, Hector J; Lee, Hyun-Ju; Hyun, Jaekyung; Balaeff, Alexander; Huo, Qun; Kang, Hyeran

    2018-04-12

    Actin bundles are key factors in the mechanical support and dynamic reorganization of the cytoskeleton. High concentrations of multivalent counterions promote bundle formation through electrostatic attraction between actin filaments that are negatively charged polyelectrolytes. In this study, we evaluate how physiologically relevant divalent cations affect the mechanical, dynamic, and structural properties of actin bundles. Using a combination of total internal reflection fluorescence microscopy, transmission electron microscopy, and dynamic light scattering, we demonstrate that divalent cations modulate bundle stiffness, length distribution, and lateral growth. Molecular dynamics simulations of an all-atom model of the actin bundle reveal specific actin residues coordinate cation-binding sites that promote the bundle formation. Our work suggests that specific cation interactions may play a fundamental role in the assembly, structure, and mechanical properties of actin bundles.

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

    PubMed

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

    2015-02-20

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

  10. Structural basis for profilin-mediated actin nucleotide exchange

    PubMed Central

    Porta, Jason C.; Borgstahl, Gloria E.O.

    2015-01-01

    Actin is a ubiquitous eukaryotic protein that is responsible for cellular scaffolding, motility and division. The ability of actin to form a helical filament is the driving force behind these cellular activities. Formation of a filament is dependent the successful exchange of actin’s ADP for ATP. Mammalian profilin is a small actin binding protein that catalyzes the exchange of nucleotide and facilitates the addition of an actin monomer to a growing filament. Here, crystal structures of profilin:actin have been determined showing an actively exchanging ATP. The structural analysis shows how the binding of profilin to the barbed end of actin causes a rotation of the small domain relative to the large domain. This conformational change is propagated to the ATP site and causes a shift in the nucleotide loops which in turn causes a repositioning of Ca2+ to its canonical position as the cleft closes around ATP. Reversing the solvent exposure of Trp-356 is also involved in cleft closure. In addition, secondary calcium binding sites were identified. PMID:22366544

  11. Actin motility: formin a SCAry tail.

    PubMed

    Alberts, Art; Way, Michael

    2011-01-11

    A new biochemical analysis has revealed that the Rickettsia bacterial protein Sca2--recently shown to be essential for virulence and actin-dependent motility--assembles actin filaments using a mechanism that functionally resembles the processive elongation tactics used by formins. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Mechanism of Cdc42-induced Actin Polymerization in Neutrophil Extracts

    PubMed Central

    Zigmond, Sally H.; Joyce, Michael; Yang, Changsong; Brown, Kevin; Huang, Minzhou; Pring, Martin

    1998-01-01

    Cdc42, activated with GTPγS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool. Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 μm in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 μm. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends. PMID:9722612

  13. Actin-based propulsion of a microswimmer.

    PubMed

    Leshansky, A M

    2006-07-01

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

  14. Traveling waves in actin dynamics and cell motility

    PubMed Central

    Allard, Jun; Mogilner, Alex

    2012-01-01

    Much of current understanding of cell motility arose from studying steady treadmilling of actin arrays. Recently, there have been a growing number of observations of a more complex, non-steady, actin behavior, including self-organized waves. It is becoming clear that these waves result from activation and inhibition feedbacks in actin dynamics acting on different scales, but the exact molecular nature of these feedbacks and respective roles of biomechanics and biochemistry are still unclear. Here, we review recent advances achieved in experimental and theoretical studies of actin waves and discuss mechanisms and physiological significance of wavy protrusions. PMID:22985541

  15. A structural study of F-actin - filamin networks

    NASA Astrophysics Data System (ADS)

    Ahrens-Braunstein, Ashley; Nguyen, Lam; Hirst, Linda

    2010-03-01

    The cell's ability to move and contract is attributed to the semi-flexible filamentous protein, F -actin, one of the three filaments in the cytoskeleton. Actin bundling can be formed by a cross-linking actin binding protein (ABP) filamin. By examining filamin's cross-linking abilities at different concentrations and molar ratios, we can study the flexibility, structure and multiple network formations created when cross-linking F-actin with this protein. We have studied the phase diagram of this protein system using fluorescence microscopy, analyzing the network structures observed in the context of a coarse grained molecular dynamics simulation carried out by our group.

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

    PubMed

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

    2010-01-01

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

  17. A state-space modeling approach to estimating canopy conductance and associated uncertainties from sap flux density data

    Treesearch

    David M. Bell; Eric J. Ward; A. Christopher Oishi; Ram Oren; Paul G. Flikkema; James S. Clark; David Whitehead

    2015-01-01

    Uncertainties in ecophysiological responses to environment, such as the impact of atmospheric and soil moisture conditions on plant water regulation, limit our ability to estimate key inputs for ecosystem models. Advanced statistical frameworks provide coherent methodologies for relating observed data, such as stem sap flux density, to unobserved processes, such as...

  18. An investigation into the torque density capabilities of flux-focusing magnetic gearboxes

    NASA Astrophysics Data System (ADS)

    Uppalapati, Krishna Kiran

    Wind and many rotary based ocean energy conversion devices rely on a mechanical gearbox to increase their speed so as to match the requirements of the electromagnetic generator. However, mechanical gearboxes have a number of disadvantages such as the need for gear lubrication, no overload protection and the creation of acoustic noise. Frequently direct-drive generators are employed to overcome these issues, wherein the gearbox is removed and the shaft of the turbine is directly connected to the synchronous generator, either with an electrically excited or permanent magnet rotor. If the input speed to the generator is very low the torque must be very high in order to generate the necessary power. However, as the electrical loading of a synchronous generator is thermally limited, the size of the generator will become excessively large at high power levels. An alternative to these technologies is to consider replacing the mechanical gearbox with a magnetic gear. A magnetic gear can create speed change without any physical contact. It has inherent overload protection, and its non-contact operation offers the potential for high reliability. Despite significant progress, existing magnetic gear designs do not achieve torque densities that are competitive with mechanical gearboxes. This research has focused on designing a coaxial magnetic gear that can operate at a volumetric torque density that is comparable to a mechanical gearbox. A flux-focusing rotor topology also called spoke-type rotor magnet arrangement was adopted to improve the air-gap magnetic flux density which in turn improves the torque transferred between the rotors. Finite element analysis was utilized to conduct a parameter sweep analysis of the different geometric parameters of the magnetic gear. A sub-scale magnetic gear with a diameter of 110 mm and a scaled-up magnetic gear with a diameter of 228 mm was designed, constructed and experimentally evaluated. The torque and torque density of sub

  19. Characterization of actin filament severing by actophorin from Acanthamoeba castellanii

    PubMed Central

    1991-01-01

    Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem. 261:477-485). Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1:1 covalent complex of actin and actophorin. Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steady-state actin polymer concentration gave a Kd of 0.2 microM for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration. The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate. PMID:1757465

  20. Momentum transport and nonlocality in heat-flux-driven magnetic reconnection in high-energy-density plasmas.

    PubMed

    Liu, Chang; Fox, William; Bhattacharjee, Amitava; Thomas, Alexander G R; Joglekar, Archis S

    2017-10-01

    Recent theory has demonstrated a novel physics regime for magnetic reconnection in high-energy-density plasmas where the magnetic field is advected by heat flux via the Nernst effect. Here we elucidate the physics of the electron dissipation layer in this regime. Through fully kinetic simulation and a generalized Ohm's law derived from first principles, we show that momentum transport due to a nonlocal effect, the heat-flux-viscosity, provides the dissipation mechanism for magnetic reconnection. Scaling analysis, and simulations show that the reconnection process comprises a magnetic field compression stage and quasisteady reconnection stage, and the characteristic width of the current sheet in this regime is several electron mean-free paths. These results show the important interplay between nonlocal transport effects and generation of anisotropic components to the distribution function.

  1. INTERPRETING FLUX FROM BROADBAND PHOTOMETRY

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

    Brown, Peter J.; Breeveld, Alice; Roming, Peter W. A.

    2016-10-01

    We discuss the transformation of observed photometry into flux for the creation of spectral energy distributions (SED) and the computation of bolometric luminosities. We do this in the context of supernova studies, particularly as observed with the Swift spacecraft, but the concepts and techniques should be applicable to many other types of sources and wavelength regimes. Traditional methods of converting observed magnitudes to flux densities are not very accurate when applied to UV photometry. Common methods for extinction and the integration of pseudo-bolometric fluxes can also lead to inaccurate results. The sources of inaccuracy, though, also apply to other wavelengths.more » Because of the complicated nature of translating broadband photometry into monochromatic flux densities, comparison between observed photometry and a spectroscopic model is best done by forward modeling the spectrum into the count rates or magnitudes of the observations. We recommend that integrated flux measurements be made using a spectrum or SED which is consistent with the multi-band photometry rather than converting individual photometric measurements to flux densities, linearly interpolating between the points, and integrating. We also highlight some specific areas where the UV flux can be mischaracterized.« less

  2. Ion-dependent Polymerization Differences between Mammalian β- and γ-Nonmuscle Actin Isoforms*

    PubMed Central

    Bergeron, Sarah E.; Zhu, Mei; Thiem, Suzanne M.; Friderici, Karen H.; Rubenstein, Peter A.

    2010-01-01

    β- and γ-nonmuscle actins differ by 4 amino acids at or near the N terminus and distant from polymerization interfaces. β-Actin contains an Asp1-Asp2-Asp3 and Val10 whereas γ-actin has a Glu1-Glu2-Glu3 and Ile10. Despite these small changes, conserved across mammals, fish, and birds, their differential localization in the same cell suggests they may play different roles reflecting differences in their biochemical properties. To test this hypothesis, we established a baculovirus-driven expression system for producing these actins in isoform-pure populations although contaminated with 20–25% insect actin. Surprisingly, Ca-γ-actin exhibits a slower monomeric nucleotide exchange rate, a much longer nucleation phase, and a somewhat slower elongation rate than β-actin. In the Mg-form, this difference between the two is much smaller. Ca-γ-actin depolymerizes half as fast as does β-actin. Mixing experiments with Ca-actins reveal the two will readily co-polymerize. In the Ca-form, phosphate release from polymerizing β-actin occurs much more rapidly and extensively than polymerization, whereas phosphate release lags behind polymerization with γ-actin. Phosphate release during treadmilling is twice as fast with β- as with γ-actin. With Mg-actin in the initial stages, phosphate release for both actins correlates much more closely with polymerization. Calcium bound in the high affinity binding site of γ-actin may cause a selective energy barrier relative to β-actin that retards the equilibration between G- and F-monomer conformations resulting in a slower polymerizing actin with greater filament stability. This difference may be particularly important in sites such as the γ-actin-rich cochlear hair cell stereocilium where local mm calcium concentrations may exist. PMID:20308063

  3. 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.

  4. Microheterogeneity of actin gels formed under controlled linear shear.

    PubMed

    Cortese, J D; Frieden, C

    1988-10-01

    The diffusion coefficients and fluorescence polarization properties of actin subjected to a known shear have been determined both during and after polymerization, using a modification of a cone-plate Wells-Brookfield rheometer that allows monitoring of samples with an epifluorescence microscope. Fluorescence polarization and fluorescence photobleaching recovery experiments using rhodamine-labeled actin as a tracer showed that under conditions of low shear (shear rates of 0.05 s-1), a spatial heterogeneity of polymerized actin was observed with respect to fluorescence intensity and the diffusion coefficients with actin mobility becoming quite variable in different regions of the sample. In addition, complex changes in fluorescence polarization were noted after stopping the shear. Actin filaments of controlled length were obtained using plasma gelsolin (gelsolin/actin molar ratios of 1:50 to 1:300). At ratios of 1:50, neither spatial heterogeneity nor changes in polarization were observed on subjecting the polymerized actin to shear. At ratios of approximately 1:100, a decrease on the intensity of fluorescence polarization occurs on stopping the shear. Longer filaments exhibit spatial micro-heterogeneity and complex changes in fluorescence polarization. In addition, at ratios of 1:100 or 1:300, the diffusion coefficient decreases as the total applied shear increased. This behavior is interpreted as bundling of filaments aligned under shear. We also find that the F-actin translational diffusion coefficients decrease as the total applied shear increases (shear rates between 0.05 and 12.66 s-1), as expected for a cumulative process. When chicken gizzard filamin was added to gelsolin-actin filaments (at filamin/actin molar ratios of 1:300 to 1:10), a similar decrease in the diffusion coefficients was observed for unsheared samples. Spatial microheterogeneity might be related to the effects of the shear field in the alignment of filaments, and the balance between a three

  5. Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

    PubMed Central

    Muller, Jean; Mehlen, André; Vetter, Guillaume; Yatskou, Mikalai; Muller, Arnaud; Chalmel, Frédéric; Poch, Olivier; Friederich, Evelyne; Vallar, Laurent

    2007-01-01

    Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our data demonstrate that

  6. Actin kinetics shapes cortical network structure and mechanics

    PubMed Central

    Fritzsche, Marco; Erlenkämper, Christoph; Moeendarbary, Emad; Charras, Guillaume; Kruse, Karsten

    2016-01-01

    The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs. PMID:27152338

  7. Actin kinetics shapes cortical network structure and mechanics.

    PubMed

    Fritzsche, Marco; Erlenkämper, Christoph; Moeendarbary, Emad; Charras, Guillaume; Kruse, Karsten

    2016-04-01

    The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

  8. Actin dynamics, architecture, and mechanics in cell motility.

    PubMed

    Blanchoin, Laurent; Boujemaa-Paterski, Rajaa; Sykes, Cécile; Plastino, Julie

    2014-01-01

    Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

  9. Maleimidobenzoyl-G-actin: Structural properties and interaction with skeletal myosin subfragment-1

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

    Bettache, N.; Bertrand, R.; Kassab, R.

    1990-09-25

    The authors have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt-and myosin subfragment 1 (S-1)--induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain. The far-ultraviolet CD spectrum and {alpha}-helix content of the MBS-actin were identical with those displayed by native G-actin. {sup 45}Ca{sup 2+} measurements showed the same content of tightly bound Ca{sup 2+} in MBS-actin as in G-actin and the EDTA treatment of the modified protein promotedmore » the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl+5 mM MgCl{sub 2} led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6mg/mL, at 25{degree}C, pH 8.0. The MBS-F-actin formed activated the Mg{sup 2+}-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was to be at all affected by its specific covalent conjugation to S-1. This finding led them to isolate, for the first time, by gel filtration, a ternary complex comprising DNase I tightly bound to MBS-actin cross-linked to the S-1 heavy chain, demonstrating that S-1 and DNase I bind at distinct sites on G-actin. Collectively, the data illustrate further the nativeness of the MBS-G-actin and its potential use in solution studies of the actin-myosin head interactions.« less

  10. The magnitude of the snow-sourced reactive nitrogen flux to the boundary layer in the Uintah Basin, Utah, USA

    NASA Astrophysics Data System (ADS)

    Zatko, Maria; Erbland, Joseph; Savarino, Joel; Geng, Lei; Easley, Lauren; Schauer, Andrew; Bates, Timothy; Quinn, Patricia K.; Light, Bonnie; Morison, David; Osthoff, Hans D.; Lyman, Seth; Neff, William; Yuan, Bin; Alexander, Becky

    2016-11-01

    Reactive nitrogen (Nr = NO, NO2, HONO) and volatile organic carbon emissions from oil and gas extraction activities play a major role in wintertime ground-level ozone exceedance events of up to 140 ppb in the Uintah Basin in eastern Utah. Such events occur only when the ground is snow covered, due to the impacts of snow on the stability and depth of the boundary layer and ultraviolet actinic flux at the surface. Recycling of reactive nitrogen from the photolysis of snow nitrate has been observed in polar and mid-latitude snow, but snow-sourced reactive nitrogen fluxes in mid-latitude regions have not yet been quantified in the field. Here we present vertical profiles of snow nitrate concentration and nitrogen isotopes (δ15N) collected during the Uintah Basin Winter Ozone Study 2014 (UBWOS 2014), along with observations of insoluble light-absorbing impurities, radiation equivalent mean ice grain radii, and snow density that determine snow optical properties. We use the snow optical properties and nitrate concentrations to calculate ultraviolet actinic flux in snow and the production of Nr from the photolysis of snow nitrate. The observed δ15N(NO3-) is used to constrain modeled fractional loss of snow nitrate in a snow chemistry column model, and thus the source of Nr to the overlying boundary layer. Snow-surface δ15N(NO3-) measurements range from -5 to 10 ‰ and suggest that the local nitrate burden in the Uintah Basin is dominated by primary emissions from anthropogenic sources, except during fresh snowfall events, where remote NOx sources from beyond the basin are dominant. Modeled daily averaged snow-sourced Nr fluxes range from 5.6 to 71 × 107 molec cm-2 s-1 over the course of the field campaign, with a maximum noontime value of 3.1 × 109 molec cm-2 s-1. The top-down emission estimate of primary, anthropogenic NOx in Uintah and Duchesne counties is at least 300 times higher than the estimated snow NOx emissions presented in this study. Our results suggest

  11. Modeling and Analysis of High Torque Density Transverse Flux Machines for Direct-Drive Applications

    NASA Astrophysics Data System (ADS)

    Hasan, Iftekhar

    Commercially available permanent magnet synchronous machines (PMSM) typically use rare-earth-based permanent magnets (PM). However, volatility and uncertainty associated with the supply and cost of rare-earth magnets have caused a push for increased research into the development of non-rare-earth based PM machines and reluctance machines. Compared to other PMSM topologies, the Transverse Flux Machine (TFM) is a promising candidate to get higher torque densities at low speed for direct-drive applications, using non-rare-earth based PMs. The TFMs can be designed with a very small pole pitch which allows them to attain higher force density than conventional radial flux machines (RFM) and axial flux machines (AFM). This dissertation presents the modeling, electromagnetic design, vibration analysis, and prototype development of a novel non-rare-earth based PM-TFM for a direct-drive wind turbine application. The proposed TFM addresses the issues of low power factor, cogging torque, and torque ripple during the electromagnetic design phase. An improved Magnetic Equivalent Circuit (MEC) based analytical model was developed as an alternative to the time-consuming 3D Finite Element Analysis (FEA) for faster electromagnetic analysis of the TFM. The accuracy and reliability of the MEC model were verified, both with 3D-FEA and experimental results. The improved MEC model was integrated with a Particle Swarm Optimization (PSO) algorithm to further enhance the capability of the analytical tool for performing rigorous optimization of performance-sensitive machine design parameters to extract the highest torque density for rated speed. A novel concept of integrating the rotary transformer within the proposed TFM design was explored to completely eliminate the use of magnets from the TFM. While keeping the same machine envelope, and without changing the stator or rotor cores, the primary and secondary of a rotary transformer were embedded into the double-sided TFM. The proposed

  12. Load Adaptation of Lamellipodial Actin Networks.

    PubMed

    Mueller, Jan; Szep, Gregory; Nemethova, Maria; de Vries, Ingrid; Lieber, Arnon D; Winkler, Christoph; Kruse, Karsten; Small, J Victor; Schmeiser, Christian; Keren, Kinneret; Hauschild, Robert; Sixt, Michael

    2017-09-21

    Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Effect of phosphorylation of myelin basic protein by MAPK on its interactions with actin and actin binding to a lipid membrane in vitro.

    PubMed

    Boggs, Joan M; Rangaraj, Godha; Gao, Wen; Heng, Yew-Meng

    2006-01-17

    Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isomers of varying charge, some resulting from phosphorylation at several sites by different kinases, including mitogen-activated protein kinase (MAPK). Phosphorylation of MBP in oligodendrocytes occurs in response to various extracellular stimuli. Phosphorylation/dephosphorylation of MBP also occurs in the myelin sheath in response to electrical activity in the brain. Here we investigate the effect of phosphorylation of MBP on its interaction with actin in vitro by phosphorylating the most highly charged unmodified isomer, C1, at two sites with MAPK. Phosphorylation decreased the ability of MBP to polymerize actin and to bundle actin filaments but had no effect on the dissociation constant of the MBP-actin complex or on the ability of Ca2+-calmodulin to dissociate the complex. The most significant effect of phosphorylation on the MBP-actin complex was a dramatic reduction in its ability to bind to negatively charged lipid bilayers. The effect was much greater than that reported earlier for another charge isomer of MBP, C8, in which six arginines were deiminated to citrulline, resulting in a reduction of net positive charge of 6. These results indicate that although average electrostatic forces are the primary determinant of the interaction of MBP with actin, phosphorylation may have an additional effect due to a site-specific electrostatic effect or to a conformational change. Thus, phosphorylation of MBP, which occurs in response to various extracellular signals in both myelin and oligodendrocytes, attenuates the ability of MBP to polymerize and bundle actin and to

  14. QUENCHING STAR FORMATION AT INTERMEDIATE REDSHIFTS: DOWNSIZING OF THE MASS FLUX DENSITY IN THE GREEN VALLEY

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

    Goncalves, Thiago S.; Menendez-Delmestre, Karin; Martin, D. Christopher

    2012-11-01

    The bimodality in galaxy properties has been observed at low and high redshifts, with a clear distinction between star-forming galaxies in the blue cloud and passively evolving objects in the red sequence; the absence of galaxies with intermediate properties indicates that the quenching of star formation and subsequent transition between populations must happen rapidly. In this paper, we present a study of over 100 transiting galaxies in the so-called green valley at intermediate redshifts (z {approx} 0.8). By using very deep spectroscopy with the DEIMOS instrument at the Keck telescope we are able to infer the star formation histories ofmore » these objects and measure the stellar mass flux density transiting from the blue cloud to the red sequence when the universe was half its current age. Our results indicate that the process happened more rapidly and for more massive galaxies in the past, suggesting a top-down scenario in which the massive end of the red sequence is forming first. This represents another aspect of downsizing, with the mass flux density moving toward smaller galaxies in recent times.« less

  15. Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

    PubMed Central

    Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

    2012-01-01

    Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

  16. Distinct structural changes detected by X-ray fiber diffraction in stabilization of F-actin by lowering pH and increasing ionic strength.

    PubMed

    Oda, T; Makino, K; Yamashita, I; Namba, K; Maéda, Y

    2001-02-01

    Lowering pH or raising salt concentration stabilizes the F-actin structure by increasing the free energy change associated with its polymerization. To understand the F-actin stabilization mechanism, we studied the effect of pH, salt concentration, and cation species on the F-actin structure. X-ray fiber diffraction patterns recorded from highly ordered F-actin sols at high density enabled us to detect minute changes of diffraction intensities and to precisely determine the helical parameters. F-actin in a solution containing 30 mM NaCl at pH 8 was taken as the control. F-actin at pH 8, 30 to 90 mM NaCl or 30 mM KCl showed a helical symmetry of 2.161 subunits per turn of the 1-start helix (12.968 subunits/6 turns). Lowering pH from 8 to 6 or replacing NaCl by LiCl altered the helical symmetry to 2.159 subunits per turn (12.952/6). The diffraction intensity associated with the 27-A meridional layer-line increased as the pH decreased but decreased as the NaCl concentration increased. None of the solvent conditions tested gave rise to significant changes in the pitch of the left-handed 1-start helix (approximately 59.8 A). The present results indicate that the two factors that stabilize F-actin, relatively low pH and high salt concentration, have distinct effects on the F-actin structure. Possible mechanisms will be discussed to understand how F-actin is stabilized under these conditions.

  17. 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.

  18. Stability of actin-lysozyme complexes formed in cystic fibrosis disease.

    PubMed

    Mohammadinejad, Sarah; Ghamkhari, Behnoush; Abdolmaleki, Sarah

    2016-08-21

    Finding the conditions for destabilizing actin-lysozyme complexes is of biomedical importance in preventing infections in cystic fibrosis. In this manuscript, the effects of different charge-mutants of lysozyme and salt concentration on the stability of actin-lysozyme complexes are studied using Langevin dynamics simulation. A coarse-grained model of F-actin is used in which both its twist and bending rigidities are considered. We observe that the attraction between F-actins is stronger in the presence of wild-type lysozymes relative to the mutated lysozymes of lower charges. By calculating the potential of mean force between F-actins, we conclude that the stability of actin-lysozyme complexes is decreased by reducing the charge of lysozyme mutants. The distributions of different lysozyme charge-mutants show that wild-type (+9e) lysozymes are mostly accumulated in the center of triangles formed by three adjacent F-actins, while lysozyme mutants of charges +7e and +5e occupy the bridging regions between F-actins. Low-charge mutants of lysozyme (+3e) distribute uniformly around F-actins. A rough estimate of the electrostatic energy for these different distributions proves that the distribution in which lysozymes reside in the center of triangles leads to more stable complexes. Also our results in the presence of a salt suggest that at physiological salt concentration of airway, F-actin complexes are not formed by charge-reduced mutants of lysozyme. The findings are interesting because if we can design charge-reduced lysozyme mutants with considerable antibacterial activity, they are not sequestered inside F-actin aggregates and can play their role as antibacterial agents against airway infection.

  19. Organization and function of the actin cytoskeleton in developing root cells.

    PubMed

    Blancaflor, Elison B; Wang, Yuh-Shuh; Motes, Christy M

    2006-01-01

    The actin cytoskeleton is a highly dynamic structure, which mediates various cellular functions in large part through accessory proteins that tilt the balance between monomeric G-actin and filamentous actin (F-actin) or by facilitating interactions between actin and the plasma membrane, microtubules, and other organelles. Roots have become an attractive model to study actin in plant development because of their simple anatomy and accessibility of some root cell types such as root hairs for microscopic analyses. Roots also exhibit a remarkable developmental plasticity and possess a delicate sensory system that is easily manipulated, so that one can design experiments addressing a range of important biological questions. Many facets of root development can be regulated by the diverse actin network found in the various root developmental regions. Various molecules impinge on this actin scaffold to define how a particular root cell type grows or responds to a specific environmental signal. Although advances in genomics are leading the way toward elucidating actin function in roots, more significant strides will be realized when such tools are combined with improved methodologies for accurately depicting how actin is organized in plant cells.

  20. POLAMI: Polarimetric Monitoring of Active Galactic Nuclei at Millimetre Wavelengths - III. Characterization of total flux density and polarization variability of relativistic jets

    NASA Astrophysics Data System (ADS)

    Agudo, Iván; Thum, Clemens; Ramakrishnan, Venkatessh; Molina, Sol N.; Casadio, Carolina; Gómez, José L.

    2018-01-01

    We report on the first results of the POLAMI (Polarimetric Monitoring of AGNs with Millimetre Wavelengths) programme, a simultaneous 3.5 and 1.3 mm full-Stokes-polarization monitoring of a sample of 36 of the brightest active galactic nuclei in the northern sky with the IRAM 30 m telescope. Through a systematic statistical study of data taken from 2006 October (from 2009 December for the case of the 1.3 mm observations) to 2014 August, we characterize the variability of the total flux density and linear polarization. We find that all sources in the sample are highly variable in total flux density at both 3.5 and 1.3 mm, as well as in spectral index, which (except in particularly prominent flares) is found to be optically thin between these two wavelengths. The total flux-density variability at 1.3 mm is found, in general, to be faster, and to have larger fractional amplitude and flatter power-spectral-density slopes than at 3.5 mm. The polarization degree is on average larger at 1.3 mm than at 3.5 mm, by a factor of 2.6. The variability of linear polarization degree is faster and has higher fractional amplitude than for total flux density, with the typical time-scales during prominent polarization peaks being significantly faster at 1.3 mm than at 3.5 mm. The polarization angle at both 3.5 and 1.3 mm is highly variable. Most of the sources show one or two excursions of >180° on time-scales from a few weeks to about a year during the course of our observations. The 3.5 and 1.3 mm polarization angle evolution follows each other rather well, although the 1.3 mm data show a clear preference to more prominent variability on the short time-scales, i.e. weeks. The data are compatible with multizone models of conical jets involving smaller emission regions for the shortest-wavelength emitting sites. Such smaller emitting regions should also be more efficient in energising particle populations, as implied by the coherent evolution of the spectral index and the total flux

  1. 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

  2. Triggering signaling pathways using F-actin self-organization.

    PubMed

    Colin, A; Bonnemay, L; Gayrard, C; Gautier, J; Gueroui, Z

    2016-10-04

    The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity.

  3. Triggering signaling pathways using F-actin self-organization

    PubMed Central

    Colin, A.; Bonnemay, L.; Gayrard, C.; Gautier, J.; Gueroui, Z.

    2016-01-01

    The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity. PMID:27698406

  4. Bacterial subversion of host actin dynamics at the plasma membrane.

    PubMed

    Carabeo, Rey

    2011-10-01

    Invasion of non-phagocytic cells by a number of bacterial pathogens involves the subversion of the actin cytoskeletal remodelling machinery to produce actin-rich cell surface projections designed to engulf the bacteria. The signalling that occurs to induce these actin-rich structures has considerable overlap among a diverse group of bacteria. The molecular organization within these structures act in concert to internalize the invading pathogen. This dynamic process could be subdivided into three acts - actin recruitment, engulfment, and finally, actin disassembly/internalization. This review will present the current state of knowledge of the molecular processes involved in each stage of bacterial invasion, and provide a perspective that highlights the temporal and spatial control of actin remodelling that occurs during bacterial invasion. © 2011 Blackwell Publishing Ltd.

  5. A nucleator arms race: cellular control of actin assembly.

    PubMed

    Campellone, Kenneth G; Welch, Matthew D

    2010-04-01

    For over a decade, the actin-related protein 2/3 (ARP2/3) complex, a handful of nucleation-promoting factors and formins were the only molecules known to directly nucleate actin filament formation de novo. However, the past several years have seen a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASP and SCAR homologue (WASH), WASP homologue associated with actin, membranes and microtubules (WHAMM), and junction-mediating regulatory protein (JMY), stimulate ARP2/3 activity at distinct cellular locations. Formin nucleators with additional biochemical and cellular activities have also been uncovered. Finally, the Spire, cordon-bleu and leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymerization.

  6. Communication: On the calculation of time-dependent electron flux within the Born-Oppenheimer approximation: A flux-flux reflection principle

    NASA Astrophysics Data System (ADS)

    Albert, Julian; Hader, Kilian; Engel, Volker

    2017-12-01

    It is commonly assumed that the time-dependent electron flux calculated within the Born-Oppenheimer (BO) approximation vanishes. This is not necessarily true if the flux is directly determined from the continuity equation obeyed by the electron density. This finding is illustrated for a one-dimensional model of coupled electronic-nuclear dynamics. There, the BO flux is in perfect agreement with the one calculated from a solution of the time-dependent Schrödinger equation for the coupled motion. A reflection principle is derived where the nuclear BO flux is mapped onto the electronic flux.

  7. Momentum transport and nonlocality in heat-flux-driven magnetic reconnection in high-energy-density plasmas

    DOE PAGES

    Liu, Chang; Fox, William; Bhattacharjee, Amitava; ...

    2017-10-06

    Recent theory has demonstrated a novel physics regime for magnetic reconnection in high-energy-density plasmas where the magnetic field is advected by heat flux via the Nernst effect. In this paper, we elucidate the physics of the electron dissipation layer in this regime. Through fully kinetic simulation and a generalized Ohm's law derived from first principles, we show that momentum transport due to a nonlocal effect, the heat-flux-viscosity, provides the dissipation mechanism for magnetic reconnection. Scaling analysis, and simulations show that the reconnection process comprises a magnetic field compression stage and quasisteady reconnection stage, and the characteristic width of the currentmore » sheet in this regime is several electron mean-free paths. Finally, these results show the important interplay between nonlocal transport effects and generation of anisotropic components to the distribution function.« less

  8. 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

  9. Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation.

    PubMed

    Zenker, Jennifer; White, Melanie D; Gasnier, Maxime; Alvarez, Yanina D; Lim, Hui Yi Grace; Bissiere, Stephanie; Biro, Maté; Plachta, Nicolas

    2018-04-19

    Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. The unusual dynamics of parasite actin result from isodesmic polymerization

    PubMed Central

    Skillman, Kristen M.; Ma, Christopher I.; Fremont, Daved H.; Diraviyam, Karthikeyan; Cooper, John A.; Sept, David; Sibley, L. David

    2013-01-01

    Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here, we re-examine the polymerization properties of actin in Toxoplasma gondii (TgACTI), unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. TgACTI polymerization kinetics lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly, and the size distribution of TgACTI filaments in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers. PMID:23921463

  11. Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.

    PubMed

    Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

    2010-01-01

    The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

  12. Filamentous actin organization in the unfertilized sea urchin egg cortex.

    PubMed

    Henson, J H; Begg, D A

    1988-06-01

    We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2-3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.

  13. Management of actinic keratosis.

    PubMed

    2013-07-01

    Actinic keratoses are common, often multiple, epidermal lesions found mainly on the sun-exposed skin of fair-skinned middle-aged and older people.(1) Over time, lesions may remain unchanged or may proliferate, regress, reappear or develop into squamous cell carcinoma (SCC).(2) Detectable (spot) lesions are often associated with alteration of the surrounding skin (field) where subclinical lesions might be present.(2) Interventions may target individual or multiple lesions or a whole field.(2) Here, we update our previous review(3) on the prevention and treatment of actinic keratoses, focusing on the licensed treatments most commonly used in the UK and recommended in UK guidelines.

  14. Nonequilibrium stabilization of an RNA/protein droplet emulsion by nuclear actin

    NASA Astrophysics Data System (ADS)

    Brangwynne, Clifford

    2013-03-01

    Actin plays a structural role in the cytoplasm. However, actin takes on new functions and structures in the nucleus that are poorly understood. The nuclei of the large oocytes of the frog X. laevisspecifically accumulate actin to reach high concentrations; however, it remains unclear if this actin polymerizes into a network, and what, if any, structural role such an actin network might play. Here, we use microrheological and confocal imaging techniques to probe the local architecture and mechanics of the nucleus. Our data show that actin forms a weak network that spatially organizes the nucleus by kinetically stabilizing embedded liquid-like RNA/protein bodies which are important for cell growth. In actin-disrupted nuclei this RNA/protein droplet emulsion is destabilized leading to homotypic coalescence into single large droplets. Our data provide intriguing new insights into why large cell nuclei require an actin-based structural scaffold.

  15. The cell wall of Arabidopsis thaliana influences actin network dynamics.

    PubMed

    Tolmie, Frances; Poulet, Axel; McKenna, Joseph; Sassmann, Stefan; Graumann, Katja; Deeks, Michael; Runions, John

    2017-07-20

    In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Surfing pathogens and the lessons learned for actin polymerization.

    PubMed

    Frischknecht, F; Way, M

    2001-01-01

    A number of unrelated bacterial species as well as vaccinia virus (ab)use the process of actin polymerization to facilitate and enhance their infection cycle. Studies into the mechanism by which these pathogens hijack and control the actin cytoskeleton have provided many interesting insights into the regulation of actin polymerization in migrating cells. This review focuses on what we have learnt from the actin-based motilities of Listeria, Shigella and vaccinia and discusses what we would still like to learn from our nasty friends, including enteropathogenic Escherichia coli and Rickettsia

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

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Gardel, Margaret

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

  18. Actin Engine in Immunological Synapse

    PubMed Central

    Piragyte, Indre

    2012-01-01

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

  19. Actin in Mung Bean Mitochondria and Implications for Its Function[W][OA

    PubMed Central

    Lo, Yih-Shan; Cheng, Ning; Hsiao, Lin-June; Annamalai, Arunachalam; Jauh, Guang-Yuh; Wen, Tuan-Nan; Dai, Hwa; Chiang, Kwen-Sheng

    2011-01-01

    Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin–green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography–tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed. PMID:21984697

  20. A critical comparison of the current view of Ca signaling with the novel concept of F-actin-based Ca signaling.

    PubMed

    Lange, Klaus; Gartzke, Joachim

    2006-01-01

    A detailed comparative survey on the current idea of Ca signaling and the alternative concept of F-actin-based Ca signaling is given. The two hypotheses differ in one central aspect - the mechanism of Ca storage. The current theory rests on the assumption of Ca-accumulating vesicles derived from the endoplasmic/ sarcoplasmic reticulum, which are equipped with an ATP-dependent Ca pump and IP3- or ryanodine-sensitive Ca-release channels/receptors. The alternative hypothesis proceeds from the idea of Ca storage at the high-affinity binding sites of F-actin subunits. Several prominent features of Ca signaling, which are not adequately described by the current concept, are inherent properties of the F-actin system and its dynamic state of treadmilling. F-actin is the only known biological Ca-binding system that has been proven by in vitro experiments to work within the physiological range of Ca concentrations and the only system that meets all necessary conditions to function as receptor-operated Ca store and as a coupling device between the Ca store and the store-operated Ca influx pathway. The most important properties of Ca signaling, such as store-channel coupling, quantal Ca release, spiking and oscillations, biphasic and "phasic" uptake kinetics, and Ca-induced Ca release, turn out to be systematic features of the new concept but remain unexplained by the classical vesicle storage hypothesis. A number of novel findings, specifically recent reports about direct effects of actin-specific toxins on Ca stores, have strengthened the new concept. The concept of F-actin-based Ca signaling combined with the notion of microvillar regulation of ion and substrate fluxes opens new aspects and far-reaching consequences, not only for cellular Ca signaling but also for various other cell functions, and represents an opportunity to connect several fields of cell physiology on the basis of a common mechanism.

  1. A peek into tropomyosin binding and unfolding on the actin filament.

    PubMed

    Singh, Abhishek; Hitchcock-Degregori, Sarah E

    2009-07-24

    Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism). This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding

  2. The Revised Electromagnetic Fields Directive and Worker Exposure in Environments With High Magnetic Flux Densities

    PubMed Central

    Stam, Rianne

    2014-01-01

    Some of the strongest electromagnetic fields (EMF) are found in the workplace. A European Directive sets limits to workers’ exposure to EMF. This review summarizes its origin and contents and compares magnetic field exposure levels in high-risk workplaces with the limits set in the revised Directive. Pubmed, Scopus, grey literature databases, and websites of organizations involved in occupational exposure measurements were searched. The focus was on EMF with frequencies up to 10 MHz, which can cause stimulation of the nervous system. Selected studies had to provide individual maximum exposure levels at the workplace, either in terms of the external magnetic field strength or flux density or as induced electric field strength or current density. Indicative action levels and the corresponding exposure limit values for magnetic fields in the revised European Directive will be higher than those in the previous version. Nevertheless, magnetic flux densities in excess of the action levels for peripheral nerve stimulation are reported for workers involved in welding, induction heating, transcranial magnetic stimulation, and magnetic resonance imaging (MRI). The corresponding health effects exposure limit values for the electric fields in the worker’s body can be exceeded for welding and MRI, but calculations for induction heating and transcranial magnetic stimulation are lacking. Since the revised European Directive conditionally exempts MRI-related activities from the exposure limits, measures to reduce exposure may be necessary for welding, induction heating, and transcranial nerve stimulation. Since such measures can be complicated, there is a clear need for exposure databases for different workplace scenarios with significant EMF exposure and guidance on good practices. PMID:24557933

  3. The revised electromagnetic fields directive and worker exposure in environments with high magnetic flux densities.

    PubMed

    Stam, Rianne

    2014-06-01

    Some of the strongest electromagnetic fields (EMF) are found in the workplace. A European Directive sets limits to workers' exposure to EMF. This review summarizes its origin and contents and compares magnetic field exposure levels in high-risk workplaces with the limits set in the revised Directive. Pubmed, Scopus, grey literature databases, and websites of organizations involved in occupational exposure measurements were searched. The focus was on EMF with frequencies up to 10 MHz, which can cause stimulation of the nervous system. Selected studies had to provide individual maximum exposure levels at the workplace, either in terms of the external magnetic field strength or flux density or as induced electric field strength or current density. Indicative action levels and the corresponding exposure limit values for magnetic fields in the revised European Directive will be higher than those in the previous version. Nevertheless, magnetic flux densities in excess of the action levels for peripheral nerve stimulation are reported for workers involved in welding, induction heating, transcranial magnetic stimulation, and magnetic resonance imaging (MRI). The corresponding health effects exposure limit values for the electric fields in the worker's body can be exceeded for welding and MRI, but calculations for induction heating and transcranial magnetic stimulation are lacking. Since the revised European Directive conditionally exempts MRI-related activities from the exposure limits, measures to reduce exposure may be necessary for welding, induction heating, and transcranial nerve stimulation. Since such measures can be complicated, there is a clear need for exposure databases for different workplace scenarios with significant EMF exposure and guidance on good practices. © The Author 2014. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.

  4. Diversification of caldesmon-linked actin cytoskeleton in cell motility

    PubMed Central

    Mayanagi, Taira

    2011-01-01

    The actin cytoskeleton plays a key role in regulating cell motility. Caldesmon (CaD) is an actin-linked regulatory protein found in smooth muscle and non-muscle cells that is conserved among a variety of vertebrates. It binds and stabilizes actin filaments, as well as regulating actin-myosin interaction in a calcium (Ca2+)/calmodulin (CaM)- and/or phosphorylation-dependent manner. CaD function is regulated qualitatively by Ca2+/CaM and by its phosphorylation state and quantitatively at the mRNA level, by three different transcriptional regulation of the CALD1 gene. CaD has numerous functions in cell motility, such as migration, invasion and proliferation, exerted via the reorganization of the actin cytoskeleton. Here we will outline recent findings regarding CaD's structural features and functions. PMID:21350330

  5. Single-Molecule Studies of Actin Assembly and Disassembly Factors

    PubMed Central

    Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

    2014-01-01

    The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

  6. Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

    PubMed Central

    Vitriol, Eric A.; McMillen, Laura M.; Kapustina, Maryna; Gomez, Shawn M.; Vavylonis, Dimitrios; Zheng, James Q.

    2015-01-01

    Summary Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer binding protein thymosin β4 (Tβ4) for optimal leading edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it doesn’t interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions. PMID:25865895

  7. Automated Detection of Actinic Keratoses in Clinical Photographs

    PubMed Central

    Hames, Samuel C.; Sinnya, Sudipta; Tan, Jean-Marie; Morze, Conrad; Sahebian, Azadeh; Soyer, H. Peter; Prow, Tarl W.

    2015-01-01

    Background Clinical diagnosis of actinic keratosis is known to have intra- and inter-observer variability, and there is currently no non-invasive and objective measure to diagnose these lesions. Objective The aim of this pilot study was to determine if automatically detecting and circumscribing actinic keratoses in clinical photographs is feasible. Methods Photographs of the face and dorsal forearms were acquired in 20 volunteers from two groups: the first with at least on actinic keratosis present on the face and each arm, the second with no actinic keratoses. The photographs were automatically analysed using colour space transforms and morphological features to detect erythema. The automated output was compared with a senior consultant dermatologist’s assessment of the photographs, including the intra-observer variability. Performance was assessed by the correlation between total lesions detected by automated method and dermatologist, and whether the individual lesions detected were in the same location as the dermatologist identified lesions. Additionally, the ability to limit false positives was assessed by automatic assessment of the photographs from the no actinic keratosis group in comparison to the high actinic keratosis group. Results The correlation between the automatic and dermatologist counts was 0.62 on the face and 0.51 on the arms, compared to the dermatologist’s intra-observer variation of 0.83 and 0.93 for the same. Sensitivity of automatic detection was 39.5% on the face, 53.1% on the arms. Positive predictive values were 13.9% on the face and 39.8% on the arms. Significantly more lesions (p<0.0001) were detected in the high actinic keratosis group compared to the no actinic keratosis group. Conclusions The proposed method was inferior to assessment by the dermatologist in terms of sensitivity and positive predictive value. However, this pilot study used only a single simple feature and was still able to achieve sensitivity of detection of 53

  8. Biallelic truncating mutations in FMN2, encoding the actin-regulatory protein Formin 2, cause nonsyndromic autosomal-recessive intellectual disability.

    PubMed

    Law, Rosalind; Dixon-Salazar, Tracy; Jerber, Julie; Cai, Na; Abbasi, Ansar A; Zaki, Maha S; Mittal, Kirti; Gabriel, Stacey B; Rafiq, Muhammad Arshad; Khan, Valeed; Nguyen, Maria; Ali, Ghazanfar; Copeland, Brett; Scott, Eric; Vasli, Nasim; Mikhailov, Anna; Khan, Muhammad Nasim; Andrade, Danielle M; Ayaz, Muhammad; Ansar, Muhammad; Ayub, Muhammad; Vincent, John B; Gleeson, Joseph G

    2014-12-04

    Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  9. Altered Actin Centripetal Retrograde Flow in Physically Restricted Immunological Synapses

    PubMed Central

    Yu, Cheng-han; Wu, Hung-Jen; Kaizuka, Yoshihisa; Vale, Ronald D.; Groves, Jay T.

    2010-01-01

    Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3ε on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network. PMID:20686692

  10. Actin Filament Polymerization Regulates Gliding Motility by Apicomplexan ParasitesV⃞

    PubMed Central

    Wetzel, D.M.; Håkansson, S.; Hu, K.; Roos, D.; Sibley, L.D.

    2003-01-01

    Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric. In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites. To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin. JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane. Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility. However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry. Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells. These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa. PMID:12589042

  11. Multiple forms of Spire-actin complexes and their functional consequences.

    PubMed

    Chen, Christine K; Sawaya, Michael R; Phillips, Martin L; Reisler, Emil; Quinlan, Margot E

    2012-03-23

    Spire is a WH2 domain-containing actin nucleator essential for establishing an actin mesh during oogenesis. In vitro, in addition to nucleating filaments, Spire can sever them and sequester actin monomers. Understanding how Spire is capable of these disparate functions and which are physiologically relevant is an important goal. To study severing, we examined the effect of Drosophila Spire on preformed filaments in bulk and single filament assays. We observed rapid depolymerization of actin filaments by Spire, which we conclude is largely due to its sequestration activity and enhanced by its weak severing activity. We also studied the solution and crystal structures of Spire-actin complexes. We find structural and functional differences between constructs containing four WH2 domains (Spir-ABCD) and two WH2 domains (Spir-CD) that may provide insight into the mechanisms of nucleation and sequestration. Intriguingly, we observed lateral interactions between actin monomers associated with Spir-ABCD, suggesting that the structures built by these four tandem WH2 domains are more complex than originally imagined. Finally, we propose that Spire-actin mixtures contain both nuclei and sequestration structures.

  12. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

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

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

    PubMed

    Wang, Ruizhe; Carlsson, Anders E

    2015-11-25

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

  14. Antenna Mechanism of Length Control of Actin Cables

    PubMed Central

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

    2015-01-01

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

  15. Antenna Mechanism of Length Control of Actin Cables.

    PubMed

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

    2015-06-01

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

  16. Isolation of a 5-Kilodalton Actin-Sequestering Peptide from Human Blood Platelets

    NASA Astrophysics Data System (ADS)

    Safer, Daniel; Golla, Rajasree; Nachmias, Vivianne T.

    1990-04-01

    Resting human platelets contain ≈0.3 mM unpolymerized actin. When freshly drawn and washed platelets are treated with saponin, 85-90% of the unpolymerized actin diffuses out. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions shows that the bulk of this unpolymerized actin migrates with a higher mobility than does pure G-actin, profilactin, or actin-gelsolin complex. When muscle G-actin is added to fresh or boiled saponin extract, the added muscle actin is shifted to the high-mobility form. The saponin extract contains an acidic peptide having a molecular mass in the range of 5 kDa, which has been purified to homogeneity by reverse-phase HPLC. This peptide also shifts muscle actin to the high-mobility form. Addition of either boiled saponin extract or the purified peptide to muscle G-actin also strongly and stoichiometrically inhibits salt-induced polymerization, as assayed by falling-ball viscometry and by sedimentation. We conclude that this peptide binds to the bulk of the unpolymerized actin in platelets and prevents it from polymerizing.

  17. Toward the Structure of Dynamic Membrane-Anchored Actin Networks

    PubMed Central

    Weber, Igor

    2007-01-01

    In the cortex of a motile cell, membrane-anchored actin filaments assemble into structures of varying shape and function. Filopodia are distinguished by a core of bundled actin filaments within finger-like extensions of the membrane. In a recent paper by Medalia et al1 cryo-electron tomography has been used to reconstruct, from filopodia of Dictyostelium cells, the 3-dimensional organization of actin filaments in connection with the plasma membrane. A special arrangement of short filaments converging toward the filopod's tip has been called a “terminal cone”. In this region force is applied for protrusion of the membrane. Here we discuss actin organization in the filopodia of Dictyostelium in the light of current views on forces that are generated by polymerizing actin filaments, and on the resistance of membranes against deformation that counteracts these forces. PMID:19262130

  18. Monoubiquitination Inhibits the Actin Bundling Activity of Fascin*

    PubMed Central

    Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

    2016-01-01

    Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys247 and Lys250, two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. PMID:27879315

  19. Monoubiquitination Inhibits the Actin Bundling Activity of Fascin.

    PubMed

    Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

    2016-12-30

    Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys 247 and Lys 250 , two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC 50 , delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. A single charge in the actin binding domain of fascin can independently tune the linear and non-linear response of an actin bundle network.

    PubMed

    Maier, M; Müller, K W; Heussinger, C; Köhler, S; Wall, W A; Bausch, A R; Lieleg, O

    2015-05-01

    Actin binding proteins (ABPs) not only set the structure of actin filament assemblies but also mediate the frequency-dependent viscoelastic moduli of cross-linked and bundled actin networks. Point mutations in the actin binding domain of those ABPs can tune the association and dissociation dynamics of the actin/ABP bond and thus modulate the network mechanics both in the linear and non-linear response regime. We here demonstrate how the exchange of a single charged amino acid in the actin binding domain of the ABP fascin triggers such a modulation of the network rheology. Whereas the overall structure of the bundle networks is conserved, the transition point from strain-hardening to strain-weakening sensitively depends on the cross-linker off-rate and the applied shear rate. Our experimental results are consistent both with numerical simulations of a cross-linked bundle network and a theoretical description of the bundle network mechanics which is based on non-affine bending deformations and force-dependent cross-link dynamics.

  1. Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation.

    PubMed

    Naresh, S; Atreja, S K

    2015-12-01

    In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa. © 2015 Blackwell Verlag GmbH.

  2. The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

    PubMed Central

    Torreno-Pina, Juan A.; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C.; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S.; Cerundolo, Vincenzo

    2016-01-01

    Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

  3. Development of a vector-tensor system to measure the absolute magnetic flux density and its gradient in magnetically shielded rooms.

    PubMed

    Voigt, J; Knappe-Grüneberg, S; Gutkelch, D; Haueisen, J; Neuber, S; Schnabel, A; Burghoff, M

    2015-05-01

    Several experiments in fundamental physics demand an environment of very low, homogeneous, and stable magnetic fields. For the magnetic characterization of such environments, we present a portable SQUID system that measures the absolute magnetic flux density vector and the gradient tensor. This vector-tensor system contains 13 integrated low-critical temperature (LTc) superconducting quantum interference devices (SQUIDs) inside a small cylindrical liquid helium Dewar with a height of 31 cm and 37 cm in diameter. The achievable resolution depends on the flux density of the field under investigation and its temporal drift. Inside a seven-layer mu-metal shield, an accuracy better than ±23 pT for the components of the static magnetic field vector and ±2 pT/cm for each of the nine components of the gradient tensor is reached by using the shifting method.

  4. Human spire interacts with the barbed end of the actin filament.

    PubMed

    Ito, Takuto; Narita, Akihiro; Hirayama, Tasuku; Taki, Masayasu; Iyoshi, Shohei; Yamamoto, Yukio; Maéda, Yuichiro; Oda, Toshiro

    2011-04-22

    Spire is an actin nucleator that initiates actin polymerization at a specific place in the cell. Similar to the Arp2/3 complex, spire was initially considered to bind to the pointed end of the actin filament when it generates a new actin filament. Subsequently, spire was reported to be associated with the barbed end (B-end); thus, there is still no consensus regarding the end with which spire interacts. Here, we report direct evidence that spire binds to the B-end of the actin filament, under conditions where spire accelerates actin polymerization. Using electron microscopy, we visualized the location of spire bound to the filament by gold nanoparticle labeling of the histidine-tagged spire, and the polarity of the actin filament was determined by image analysis. In addition, our results suggest that multiple spires, linked through one gold nanoparticle, enhance the acceleration of actin polymerization. The B-end binding of spire provides the basis for understanding its functional mechanism in the cell. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

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

    1999-10-01

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

  6. Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry*

    PubMed Central

    Stokasimov, Ema; Rubenstein, Peter A.

    2009-01-01

    Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G- and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts. PMID:19605362

  7. A model for heliospheric flux-ropes

    NASA Astrophysics Data System (ADS)

    Nieves-Chinchilla, T.; Linton, M.; Vourlidas, A.; Hidalgo, M. A. U.

    2017-12-01

    This work is presents an analytical flux-rope model, which explores different levels of complexity starting from a circular-cylindrical geometry. The framework of this series of models was established by Nieves-Chinchilla et al. 2016 with the circular-cylindrical analytical flux rope model. The model attempts to describe the magnetic flux rope topology with distorted cross-section as a possible consequence of the interaction with the solar wind. In this model, the flux rope is completely described in a non-orthogonal geometry. The Maxwell equations are solved using tensor calculus consistent with the geometry chosen, invariance along the axial direction, and with the assumption of no radial current density. The model is generalized in terms of the radial and azimuthal dependence of the poloidal current density component and axial current density component. The misalignment between current density and magnetic field is studied in detail for several example profiles of the axial and poloidal current density components. This theoretical analysis provides a map of the force distribution inside of the flux-rope. For reconstruction of the heliospheric flux-ropes, the circular-cylindrical reconstruction technique has been adapted to the new geometry and applied to in situ ICMEs with a flux-rope entrained and tested with cases with clear in situ signatures of distortion. The model adds a piece in the puzzle of the physical-analytical representation of these magnetic structures that should be evaluated with the ultimate goal of reconciling in-situ reconstructions with imaging 3D remote sensing CME reconstructions. Other effects such as axial curvature and/or expansion could be incorporated in the future to fully understand the magnetic structure.

  8. Z-disc-associated, Alternatively Spliced, PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy*

    PubMed Central

    Lin, Xiaoyan; Ruiz, Janelle; Bajraktari, Ilda; Ohman, Rachel; Banerjee, Soojay; Gribble, Katherine; Kaufman, Joshua D.; Wingfield, Paul T.; Griggs, Robert C.; Fischbeck, Kenneth H.; Mankodi, Ami

    2014-01-01

    The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. PMID:24668811

  9. F-actin distribution and function during sexual development in Eimeria maxima.

    PubMed

    Frölich, Sonja; Wallach, Michael

    2015-06-01

    To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

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

    PubMed

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

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Experimental results for 2D magnetic resonance electrical impedance tomography (MR-EIT) using magnetic flux density in one direction.

    PubMed

    Birgül, Ozlem; Eyüboğlu, B Murat; Ider, Y Ziya

    2003-11-07

    Magnetic resonance electrical impedance tomography (MR-EIT) is an emerging imaging technique that reconstructs conductivity images using magnetic flux density measurements acquired employing MRI together with conventional EIT measurements. In this study, experimental MR-EIT images from phantoms with conducting and insulator objects are presented. The technique is implemented using the 0.15 T Middle East Technical University MRI system. The dc current method used in magnetic resonance current density imaging is adopted. A reconstruction algorithm based on the sensitivity matrix relation between conductivity and only one component of magnetic flux distribution is used. Therefore, the requirement for object rotation is eliminated. Once the relative conductivity distribution is found, it is scaled using the peripheral voltage measurements to obtain the absolute conductivity distribution. Images of several insulator and conductor objects in saline filled phantoms are reconstructed. The L2 norm of relative error in conductivity values is found to be 13%, 17% and 14% for three different conductivity distributions.

  12. Density of Gadolinium Nitrate Solutions for the High Flux Isotope Reactor

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

    Taylor, Paul Allen; Lee, Denise L

    2009-05-01

    In late 1992, the High Flux Isotope Reactor (HFIR) was planning to switch the solution contained in the poison injection tank from cadmium nitrate to gadolinium nitrate. The poison injection system is an emergency system used to shut down the reactor by adding a neutron poison to the cooling water. This system must be able to supply a minimum of 69 pounds of gadolinium to the reactor coolant system in order to guarantee that the reactor would become subcritical. A graph of the density of gadolinium nitrate solutions over a concentration range of 5 to 30 wt% and a temperaturemore » range of 15 to 40{sup o}C was prepared. Routine density measurements of the solution in the poison injection tank are made by HFIR personnel, and an adaptation of the original graph is used to determine the gadolinium nitrate concentration. In late 2008, HFIR personnel decided that the heat tracing that was present on the piping for the poison injection system could be removed without any danger of freezing the solution; however, the gadolinium nitrate solution might get as cold as 5{sup o}C. This was outside the range of the current density-concentration correlation, so the range needed to be expanded. This report supplies a new density-concentration correlation that covers the extended temperature range. The correlation is given in new units, which greatly simplifies the calculation that is required to determine the pounds of gadolinium in the tank solution. The procedure for calculating the amount of gadolinium in the HFIR poison injection system is as follows: (1) Calculate the usable volume in the system; (2) Measure the density of the solution; (3) Calculate the gadolinium concentration using the following equation: Gd(lb/ft{sup 3}) = measured density (g/mL) x 34.681 - 34.785; (4) Calculate the amount of gadolinium in the system using the following equation: Amount of Gd(lb) = Gd concentration (lb/ft{sup 3}) x usable volume (ft{sup 3}). The equation in step 3 is exact for a

  13. The effects of near-UV radiation on elasmobranch lens cytoskeletal actin.

    PubMed

    Zigman, S; Rafferty, N S; Scholz, D L; Lowe, K

    1992-08-01

    The role of near-UV radiation as a cytoskeletal actin-damaging agent was investigated. Two procedures were used to analyse fresh smooth dogfish (Mustelus canis) eye lenses that were incubated for up to 22 hr in vitro, with elasmobranch Ringer's medium, and with or without exposure to a near-UV lamp (emission principally at 365 nm; irradiance of 2.5 mW cm-2). These were observed histologically using phalloidin-rhodamine specific staining and by transmission electron microscopy. In addition, solutions of purified polymerized rabbit muscle actin were exposed to the same UV conditions and depolymerization was assayed by ultracentrifugation and high-pressure liquid chromatography. While the two actins studied do differ very slightly in some amino acid sequences, they would react physically nearly identically. The results showed that dogfish lenses developed superficial opacities due to near-UV exposure. Whole mounts of lens epithelium exhibited breakdown of actin filaments in the basal region of the cells within 18 hr of UV exposure. TEM confirmed the breakdown of actin filaments due to UV exposure. SDS-PAGE and immunoblotting positively identified actin in these cells. Direct exposure of purified polymerized muscle actin in polymerizing buffer led to an increase in actin monomer of approximately 25% in the UV-exposed solutions within 3-18 hr, whether assayed by ultracentrifugation or HPLC. The above indicates that elasmobranch lens epithelial cells contain UV-labile actin filaments, and that near-UV radiation, as is present in the sunlit environment, can break down the actin structure in these cells. Furthermore, breakdown of purified polymerized muscle actin does occur due to near-UV light exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Hypotonic activation of short ClC3 isoform is modulated by direct interaction between its cytosolic C-terminal tail and subcortical actin filaments.

    PubMed

    McCloskey, Diana T; Doherty, Lynda; Dai, Yan-Ping; Miller, Lisa; Hume, Joseph R; Yamboliev, Ilia A

    2007-06-08

    Short ClC3 isoform (sClC3) functions as a volume-sensitive outwardly rectifying anion channel (VSOAC) in some cell types. In previous studies, we have shown that the hypotonic activation of sClC3 is linked to cell swelling-mediated remodeling of the actin cytoskeleton. In the present study, we have tested the hypothesis that the cytosolic tails of sClC3 bind to actin directly and that binding modulates the hypotonic activation of the channel. Co-sedimentation assays in vitro demonstrated a strong binding between the glutathione S-transferase-fused cytosolic C terminus of sClC3 (GST-sClC3-CT) to filamentous actin (F-actin) but not to globular monomeric actin (G-actin). The GST-fused N terminus (GST-sClC3-NT) exhibited low binding affinity to both G- and F-actin. Co-sedimentation experiments with progressively truncated GST-sClC3-CT indicated that the F-actin binding region is located between amino acids 690 and 760 of sClC3. Two synthetic peptides mapping basic clusters of the cytosolic sClC3-CT (CTP2, isoleucine 716 to leucine 734; and CTP3, proline 688 to proline 709) prevented binding of GST-sClC3-CT to F-actin in vitro. Dialysis into NIH/3T3 cells of these two peptides (but not of synthetic peptide CTP1 (isoleucine 737 to glutamine 748)) reduced the maximal current density by 60 and 38%, respectively. Based on these results, we have concluded that, by direct interaction with subcortical actin filaments, sClC3 contributes to the hypotonic stress-induced VSOACs in NIH/3T3 cells.

  15. Rab8 Regulates the Actin-based Movement of MelanosomesV⃞

    PubMed Central

    Chabrillat, Marion L.; Wilhelm, Claire; Wasmeier, Christina; Sviderskaya, Elena V.; Louvard, Daniel; Coudrier, Evelyne

    2005-01-01

    Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes. PMID:15673612

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

    PubMed

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

    2016-12-06

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

  17. Cloning and characterization of an abalone (Haliotis discus hannai) actin gene

    NASA Astrophysics Data System (ADS)

    Ma, Hongming; Xu, Wei; Mai, Kangsen; Liufu, Zhiguo; Chen, Hong

    2004-10-01

    An actin encoding gene was cloned by using RT-PCR, 3‧ RACE and 5‧ RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3‧ untranslated region of 307 base pairs and 79 base pairs of 5‧ untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin (β-actin) than to human muscle actin.

  18. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

    2014-01-01

    STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by

  19. Retinoids for prevention and treatment of actinic keratosis*

    PubMed Central

    Ianhez, Mayra; Fleury, Luiz Fernando Fróes; Miot, Hélio Amante; Bagatin, Edileia

    2013-01-01

    Actinic keratosis is a common cause of dermatological consultations and it presents a strong association with squamous cell carcinoma. Many substances are used for treatment and prevention, such as retinoids. Nevertheless, many studies on retinoids emphasize their application in treating and preventing non melanoma skin cancers. In this article, we reviewed studies about systemic and topical retinoids used with immunocompetent patients and organ transplant recipients with actinic keratosis, as primary or secondary outcomes. The majority of these papers pointed to a reduction in actinic keratosis count after treatment with retinoids. However, studies need to be better-defined in order to address the lack of a standardized dose, the absence of control groups, the low number of patients and short follow-up periods. Blind, randomized and controlled clinical trials with adequate sample sizes, specifically focused on actinic keratosis, are needed to clarify the real benefit of topical and/or oral retinoids. Comparison of efficacy and safety between oral and topical retinoids in the prevention and treatment of non-melanoma skin cancers and actinic keratosis is an essential pre requisite to establish new strategies to control these conditions. PMID:24068130

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

    PubMed Central

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

    2014-01-01

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

  1. Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization

    PubMed Central

    Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M.; Crippa, Massimo P.; Scita, Giorgio

    2009-01-01

    We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of “elongating” RNAPII, Prep1, β-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes. PMID:19477923

  2. Induction of HoxB transcription by retinoic acid requires actin polymerization.

    PubMed

    Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M; Crippa, Massimo P; Scita, Giorgio; Blasi, Francesco

    2009-08-01

    We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of "elongating" RNAPII, Prep1, beta-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes.

  3. 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. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

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

    Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

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

  5. Actinic Keratosis Pathogenesis Update and New Patents.

    PubMed

    Cantisani, Carmen; Paolino, Giovanni; Melis, Marcello; Faina, Valentina; Romaniello, Federico; Didona, Dario; Cardone, Michele; Calvieri, Stefano

    2016-01-01

    Actinic keratosis is a common premalignant skin lesion. Because of its increasing incidence, several efforts have been made to earlier detectection and to improve knowledge on photocarcinogenic pathways of keratinocytes. As a consequence, recently new discoveries have been done in this field. Starting from our previous review on actinic keratosis, we reviewed the literature focusing on pathogenesis and new patents in order to highlight the most recent progresses in diagnosis and therapeutic approach. Although several efforts have been done in the field of photodamaged skin, new upgrades in diagnosis and therapy are needed to detect superficial actinic keratosis earlier, to improve the disease free survival of patient and to better treat the field cancerization.

  6. Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

    PubMed Central

    Kiuchi, Tai; Ohashi, Kazumasa; Kurita, Souichi; Mizuno, Kensaku

    2007-01-01

    Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers. PMID:17470633

  7. Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny

    PubMed Central

    Tondeleir, Davina; Noelanders, Rivka; Bakkali, Karima; Ampe, Christophe

    2014-01-01

    The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11. PMID:24409333

  8. An atomic model of the tropomyosin cable on F-actin.

    PubMed

    Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan; Lehman, William

    2014-08-05

    Tropomyosin regulates a wide variety of actin filament functions and is best known for the role that it plays together with troponin in controlling muscle activity. For effective performance on actin filaments, adjacent 42-nm-long tropomyosin molecules are joined together by a 9- to 10-residue head-to-tail overlapping domain to form a continuous cable that wraps around the F-actin helix. Yet, despite the apparent simplicity of tropomyosin's coiled-coil structure and its well-known periodic association with successive actin subunits along F-actin, the structure of the tropomyosin cable on actin is uncertain. This is because the conformation of the overlap region that joins neighboring molecules is poorly understood, thus leaving a significant gap in our understanding of thin-filament structure and regulation. However, recent molecular-dynamics simulations of overlap segments defined their overall shape and provided unique and sufficient cues to model the whole actin-tropomyosin filament assembly in atomic detail. In this study, we show that these MD structures merge seamlessly onto the ends of tropomyosin coiled-coils. Adjacent tropomyosin molecules can then be joined together to provide a comprehensive model of the tropomyosin cable running continuously on F-actin. The resulting complete model presented here describes for the first time (to our knowledge) an atomic-level structure of αα-striated muscle tropomyosin bound to an actin filament that includes the critical overlap domain. Thus, the model provides a structural correlate to evaluate thin-filament mechanics, self-assembly mechanisms, and the effect of disease-causing mutations. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. Kindlin-2 directly binds actin and regulates integrin outside-in signaling

    PubMed Central

    Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid; Vaynberg, Julia; Pluskota, Elzbieta

    2016-01-01

    Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2+/− mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK47/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK47/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK47/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses. PMID:27044892

  10. Changes in actin dynamics are involved in salicylic acid signaling pathway.

    PubMed

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  11. Cortactin promotes exosome secretion by controlling branched actin dynamics

    PubMed Central

    Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Seiki, Motoharu; Tyska, Matthew J.

    2016-01-01

    Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. PMID:27402952

  12. Cortactin promotes exosome secretion by controlling branched actin dynamics.

    PubMed

    Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Kirkbride, Kellye C; Grega-Larson, Nathan E; Seiki, Motoharu; Tyska, Matthew J; Weaver, Alissa M

    2016-07-18

    Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. © 2016 Sinha et al.

  13. Contribution of actin filaments to the global compressive properties of fibroblasts.

    PubMed

    Ujihara, Yoshihiro; Nakamura, Masanori; Miyazaki, Hiroshi; Wada, Shigeo

    2012-10-01

    Actin filaments are often regarded as tension-bearing components. Here, we examined the effects of actin filaments on global compressive properties of cells experimentally and numerically. Fibroblasts were harvested from the patellar tendon of a mature Japanese white rabbit and treated with cytochalasin D to depolymerize the actin filaments. Intact cells and cells with disrupted actin filaments were subjected to the compressive tests. Each floating cell was held between the cantilever and compressive plates and compressed by moving the compressive plate with a linear actuator to obtain a load-deformation curve under quasi-static conditions. The experimental results demonstrated that the initial stiffness of a cell with disrupted actin filaments decreased by 51%. After the experiments, we simulated the compressive test of cells with/without bundles of actin filaments. A bundle of actin filaments was modeled as a tension-bearing component that generates a force based on Hooke's law only when it was elongated. By contrast, if it was shortened, it was assumed to exert no force. The computational results revealed that the alignment of bundles of actin filaments significantly affected the cell stiffness. In addition, the passive reorientation of bundles of actin filaments perpendicular to the compression induced an increase in the resistance to the vertical elongation of a cell and thereby increased the cell stiffness. These results clearly indicated that bundles of actin filaments contribute to the compressive properties of a cell, even if they are tension-bearing components. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Cofilin1-dependent actin dynamics control DRP1-mediated mitochondrial fission

    PubMed Central

    Rehklau, Katharina; Hoffmann, Lena; Gurniak, Christine B; Ott, Martin; Witke, Walter; Scorrano, Luca; Culmsee, Carsten; Rust, Marco B

    2017-01-01

    Mitochondria form highly dynamic networks in which organelles constantly fuse and divide. The relevance of mitochondrial dynamics is evident from its implication in various human pathologies, including cancer or neurodegenerative, endocrine and cardiovascular diseases. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission that oligomerizes at the mitochondrial outer membrane and hydrolyzes GTP to drive mitochondrial fragmentation. Previous studies demonstrated that DRP1 recruitment and mitochondrial fission is promoted by actin polymerization at the mitochondrial surface, controlled by the actin regulatory proteins inverted formin 2 (INF2) and Spire1C. These studies suggested the requirement of additional actin regulatory activities to control DRP1-mediated mitochondrial fission. Here we show that the actin-depolymerizing protein cofilin1, but not its close homolog actin-depolymerizing factor (ADF), is required to maintain mitochondrial morphology. Deletion of cofilin1 caused mitochondrial DRP1 accumulation and fragmentation, without altering mitochondrial function or other organelles’ morphology. Mitochondrial morphology in cofilin1-deficient cells was restored upon (i) re-expression of wild-type cofilin1 or a constitutively active mutant, but not of an actin-binding-deficient mutant, (ii) pharmacological destabilization of actin filaments and (iii) genetic depletion of DRP1. Our work unraveled a novel function for cofilin1-dependent actin dynamics in mitochondrial fission, and identified cofilin1 as a negative regulator of mitochondrial DRP1 activity. We conclude that cofilin1 is required for local actin dynamics at mitochondria, where it may balance INF2/Spire1C-induced actin polymerization. PMID:28981113

  15. Absolute flux density calibrations of radio sources: 2.3 GHz

    NASA Technical Reports Server (NTRS)

    Freiley, A. J.; Batelaan, P. D.; Bathker, D. A.

    1977-01-01

    A detailed description of a NASA/JPL Deep Space Network program to improve S-band gain calibrations of large aperture antennas is reported. The program is considered unique in at least three ways; first, absolute gain calibrations of high quality suppressed-sidelobe dual mode horns first provide a high accuracy foundation to the foundation to the program. Second, a very careful transfer calibration technique using an artificial far-field coherent-wave source was used to accurately obtain the gain of one large (26 m) aperture. Third, using the calibrated large aperture directly, the absolute flux density of five selected galactic and extragalactic natural radio sources was determined with an absolute accuracy better than 2 percent, now quoted at the familiar 1 sigma confidence level. The follow-on considerations to apply these results to an operational network of ground antennas are discussed. It is concluded that absolute gain accuracies within + or - 0.30 to 0.40 db are possible, depending primarily on the repeatability (scatter) in the field data from Deep Space Network user stations.

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

    NASA Astrophysics Data System (ADS)

    Braun, Markus; Hauslage, Jens; Limbach, Christoph

    2003-08-01

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

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

    DOE PAGES

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

    2017-01-27

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

  18. Latrunculin B-induced plant dwarfism: Plant cell elongation is F-actin-dependent.

    PubMed

    Baluska, F; Jasik, J; Edelmann, H G; Salajová, T; Volkmann, D

    2001-03-01

    Marine macrolides latrunculins are highly specific toxins which effectively depolymerize actin filaments (generally F-actin) in all eukaryotic cells. We show that latrunculin B is effective on diverse cell types in higher plants and describe the use of this drug in probing F-actin-dependent growth and in plant development-related processes. In contrast to other eukaryotic organisms, cell divisions occurs in plant cells devoid of all actin filaments. However, the alignment of the division planes is often distorted. In addition to cell division, postembryonic development and morphogenesis also continue in the absence of F-actin. These experimental data suggest that F-actin is of little importance in the morphogenesis of higher plants, and that plants can develop more or less normally without F-actin. In contrast, F-actin turns out to be essential for cell elongation. When latrunculin B was added during germination, morphologically normal Arabidopsis and rye seedlings developed but, as a result of the absence of cell elongation, these were stunted, resembling either genetic dwarfs or environmental bonsai plants. In conclusion, F-actin is essential for the plant cell elongation, while this F-actin-dependent cell elongation is not an essential feature of plant-specific developmental programs.

  19. 14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote Giardia lamblia

    PubMed Central

    Xu, Jennifer; Steele-Ogus, Melissa; Alas, Germain C. M.

    2017-01-01

    ABSTRACT The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin; this function has been previously attributed to sequestration of phosphorylated cofilin. 14-3-3 was identified as an actin-associated protein in the deep-branching eukaryote Giardia lamblia; however, Giardia lacks cofilin and all other canonical actin-binding proteins (ABPs). Thus, the role of G. lamblia 14-3-3 (Gl-14-3-3) in actin regulation was unknown. Gl-14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia’s sole Rho family GTPase, Gl-Rac, modulates Gl-14-3-3’s association with actin, providing the first connection between Gl-Rac and the actin cytoskeleton in Giardia. Giardia actin (Gl-actin) contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a regulator of actin; however, the presence of multiple 14-3-3–actin complexes suggests a more complex regulatory relationship than might be expected for a minimalistic parasite. IMPORTANCE Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex

  20. Development of a vector-tensor system to measure the absolute magnetic flux density and its gradient in magnetically shielded rooms

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

    Voigt, J.; Knappe-Grüneberg, S.; Gutkelch, D.

    2015-05-15

    Several experiments in fundamental physics demand an environment of very low, homogeneous, and stable magnetic fields. For the magnetic characterization of such environments, we present a portable SQUID system that measures the absolute magnetic flux density vector and the gradient tensor. This vector-tensor system contains 13 integrated low-critical temperature (LTc) superconducting quantum interference devices (SQUIDs) inside a small cylindrical liquid helium Dewar with a height of 31 cm and 37 cm in diameter. The achievable resolution depends on the flux density of the field under investigation and its temporal drift. Inside a seven-layer mu-metal shield, an accuracy better than ±23more » pT for the components of the static magnetic field vector and ±2 pT/cm for each of the nine components of the gradient tensor is reached by using the shifting method.« less

  1. Cofilin-Linked Changes in Actin Filament Flexibility Promote Severing

    PubMed Central

    McCullough, Brannon R.; Grintsevich, Elena E.; Chen, Christine K.; Kang, Hyeran; Hutchison, Alan L.; Henn, Arnon; Cao, Wenxiang; Suarez, Cristian; Martiel, Jean-Louis; Blanchoin, Laurent; Reisler, Emil; De La Cruz, Enrique M.

    2011-01-01

    The actin regulatory protein, cofilin, increases the bending and twisting elasticity of actin filaments and severs them. It has been proposed that filaments partially decorated with cofilin accumulate stress from thermally driven shape fluctuations at bare (stiff) and decorated (compliant) boundaries, thereby promoting severing. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. Here, we test this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale with the flexural rigidities determined from analysis of shape fluctuations. Yeast actin filaments are more compliant in bending than vertebrate actin filaments. Severing activities of cofilactin isoforms correlate with changes in filament flexibility. Vertebrate cofilin binds but does not increase the yeast actin filament flexibility, and does not sever them. Imaging of filament thermal fluctuations reveals that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully decorated filaments. These measurements support a cofilin-severing mechanism in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments, analogous to failure of some nonprotein materials. PMID:21723825

  2. Two-dimensional periodic texture of actin filaments formed upon drying

    PubMed Central

    Honda, Hajime; Ishiwata, Shin’ichi

    2011-01-01

    We found that a solution of actin filaments can form a periodic texture in the process of drying on a flat glass surface in the air; the periodic texture was composed of smooth meandering bundles of actin filaments. We also found that a branched salt crystal grows in the space between the meandering bundles of actin filaments. The distance between the adjacent striae (striation period) in the resulting dried two-dimensional pattern of striation decreased from about 50 to 2 μm, as the ambient temperature was increased from 4 to 40°C at 1 mg/ml actin, and showed an increasing tendency from a few to several tens μm with the increase in the initial concentration of actin filaments from 0.6 to 2.0mg/ml at room temperature. As the speed of drying is increased at a certain temperature, the striation period was also found to decrease. We propose that the formation of the two-dimensional striation pattern of bundles of actin filaments is the result of condensation of proteins due to dehydration, and suggest that the solvent flow from the center to the periphery of the sample causes the meandering of actin filaments. PMID:27857588

  3. Ionisation in turbulent magnetic molecular clouds. I. Effect on density and mass-to-flux ratio structures

    NASA Astrophysics Data System (ADS)

    Bailey, Nicole D.; Basu, Shantanu; Caselli, Paola

    2017-05-01

    Context. Previous studies show that the physical structures and kinematics of a region depend significantly on the ionisation fraction. These studies have only considered these effects in non-ideal magnetohydrodynamic simulations with microturbulence. The next logical step is to explore the effects of turbulence on ionised magnetic molecular clouds and then compare model predictions with observations to assess the importance of turbulence in the dynamical evolution of molecular clouds. Aims: In this paper, we extend our previous studies of the effect of ionisation fractions on star formation to clouds that include both non-ideal magnetohydrodynamics and turbulence. We aim to quantify the importance of a treatment of the ionisation fraction in turbulent magnetised media and investigate the effect of the turbulence on shaping the clouds and filaments before star formation sets in. In particular, here we investigate how the structure, mass and width of filamentary structures depend on the amount of turbulence in ionised media and the initial mass-to-flux ratio. Methods: To determine the effects of turbulence and mass-to-flux ratio on the evolution of non-ideal magnetised clouds with varying ionisation profiles, we have run two sets of simulations. The first set assumes different initial turbulent Mach values for a fixed initial mass-to-flux ratio. The second set assumes different initial mass-to-flux ratio values for a fixed initial turbulent Mach number. Both sets explore the effect of using one of two ionisation profiles: step-like (SL) or cosmic ray only (CR-only). We compare the resulting density and mass-to-flux ratio structures both qualitatively and quantitatively via filament and core masses and filament fitting techniques (Gaussian and Plummer profiles). Results: We find that even with almost no turbulence, filamentary structure still exists although at lower density contours. Comparison of simulations shows that for turbulent Mach numbers above 2, there is

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

    PubMed Central

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

    2016-01-01

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

  5. Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization

    NASA Astrophysics Data System (ADS)

    Yi, Jinsoo; Schmidt, Jacob; Chien, Aichi; Montemagno, Carlo D.

    2009-02-01

    We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

  6. Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization.

    PubMed

    Yi, Jinsoo; Schmidt, Jacob; Chien, Aichi; Montemagno, Carlo D

    2009-02-25

    We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

  7. Energetics and kinetics of cooperative cofilin-actin filament interactions.

    PubMed

    Cao, Wenxiang; Goodarzi, Jim P; De La Cruz, Enrique M

    2006-08-11

    We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to actin filaments, accounting for contributions of ion-linked equilibria, and determined the kinetic basis of cooperative cofilin binding. Ions weaken non-contiguous (isolated, non-cooperative) cofilin binding to an actin filament without affecting cooperative filament interactions. Non-contiguous cofilin binding is coupled to the dissociation of approximately 1.7 thermodynamically bound counterions. Counterion dissociation contributes approximately 40% of the total cofilin binding free energy (in the presence of 50 mM KCl). The non-contiguous and cooperative binding free energies are driven entirely by large, positive entropy changes, consistent with a cofilin-mediated increase in actin filament structural dynamics. The rate constant for cofilin binding to an isolated site on an actin filament is slow and likely to be limited by filament breathing. Cooperative cofilin binding arises from an approximately tenfold more rapid association rate constant and an approximately twofold slower dissociation rate constant. The more rapid association rate constant is presumably a consequence of cofilin-dependent changes in the average orientation of subdomain 2, subunit angular disorder and filament twist, which increase the accessibility of a neighboring cofilin-binding site on an actin filament. Cooperative association is more rapid than binding to an isolated site, but still slow for a second-order reaction, suggesting that cooperative binding is limited also by binding site accessibility. We suggest that the dissociation of actin-associated ions weakens intersubunit interactions in the actin filament lattice that enhance cofilin-binding site accessibility, favor cooperative binding and promote filament severing.

  8. Clarin-1, encoded by the Usher Syndrome III causative gene, forms a membranous microdomain: possible role of clarin-1 in organizing the actin cytoskeleton.

    PubMed

    Tian, Guilian; Zhou, Yun; Hajkova, Dagmar; Miyagi, Masaru; Dinculescu, Astra; Hauswirth, William W; Palczewski, Krzysztof; Geng, Ruishuang; Alagramam, Kumar N; Isosomppi, Juha; Sankila, Eeva-Marja; Flannery, John G; Imanishi, Yoshikazu

    2009-07-10

    Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actin-enriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1(-/-) mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products.

  9. Effects of oxidative stress-induced changes in the actin cytoskeletal structure on myoblast damage under compressive stress: confocal-based cell-specific finite element analysis.

    PubMed

    Yao, Yifei; Lacroix, Damien; Mak, Arthur F T

    2016-12-01

    Muscle cells are frequently subjected to both mechanical and oxidative stresses in various physiological and pathological situations. To explore the mechanical mechanism of muscle cell damage under loading and oxidative stresses, we experimentally studied the effects of extrinsic hydrogen peroxides on the actin cytoskeletal structure in C2C12 myoblasts and presented a finite element (FE) analysis of how such changes in the actin cytoskeletal structure affected a myoblast's capability to resist damage under compression. A confocal-based cell-specific FE model was built to parametrically study the effects of stress fiber density, fiber cross-sectional area, fiber tensile prestrain, as well as the elastic moduli of the stress fibers, actin cortex, nucleus and cytoplasm. The results showed that a decrease in the elastic moduli of both the stress fibers and actin cortex could increase the average tensile strain on the actin cortex-membrane structure and reduce the apparent cell elastic modulus. Assuming the cell would die when a certain percentage of membrane elements were strained beyond a threshold, a lower elastic modulus of actin cytoskeleton would compromise the compressive resistance of a myoblast and lead to cell death more readily. This model was used with a Weibull distribution function to successfully describe the extent of myoblasts damaged in a monolayer under compression.

  10. 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)

  11. Identification of Actin-Binding Proteins from Maize Pollen

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

    Staiger, C.J.

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPsmore » (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.« less

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

    PubMed Central

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

    2016-01-01

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

  13. Rapid Glucose Depletion Immobilizes Active Myosin-V on Stabilized Actin Cables

    PubMed Central

    Xu, Li; Bretscher, Anthony

    2014-01-01

    Summary Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton [1]. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles [2, 3]. This is mediated by myosin-Vs transporting cargos along F-actin bundles known as actin cables [4]. Actin cables are dynamic structures regulated by assembly, stabilization and disassembly [5]. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage [6]. Thus upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase [7, 8], translation [9] and phosphoinositide metabolism [10]. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin-V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton resulting in disassembly of actin patches with concomitant inhibition of endocytosis, and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin-V in a newly described state on selectively stabilized actin cables. PMID:25308080

  14. A state-space modeling approach to estimating canopy conductance and associated uncertainties from sap flux density data.

    PubMed

    Bell, David M; Ward, Eric J; Oishi, A Christopher; Oren, Ram; Flikkema, Paul G; Clark, James S

    2015-07-01

    Uncertainties in ecophysiological responses to environment, such as the impact of atmospheric and soil moisture conditions on plant water regulation, limit our ability to estimate key inputs for ecosystem models. Advanced statistical frameworks provide coherent methodologies for relating observed data, such as stem sap flux density, to unobserved processes, such as canopy conductance and transpiration. To address this need, we developed a hierarchical Bayesian State-Space Canopy Conductance (StaCC) model linking canopy conductance and transpiration to tree sap flux density from a 4-year experiment in the North Carolina Piedmont, USA. Our model builds on existing ecophysiological knowledge, but explicitly incorporates uncertainty in canopy conductance, internal tree hydraulics and observation error to improve estimation of canopy conductance responses to atmospheric drought (i.e., vapor pressure deficit), soil drought (i.e., soil moisture) and above canopy light. Our statistical framework not only predicted sap flux observations well, but it also allowed us to simultaneously gap-fill missing data as we made inference on canopy processes, marking a substantial advance over traditional methods. The predicted and observed sap flux data were highly correlated (mean sensor-level Pearson correlation coefficient = 0.88). Variations in canopy conductance and transpiration associated with environmental variation across days to years were many times greater than the variation associated with model uncertainties. Because some variables, such as vapor pressure deficit and soil moisture, were correlated at the scale of days to weeks, canopy conductance responses to individual environmental variables were difficult to interpret in isolation. Still, our results highlight the importance of accounting for uncertainty in models of ecophysiological and ecosystem function where the process of interest, canopy conductance in this case, is not observed directly. The StaCC modeling

  15. Actin cytoskeleton and exocytosis in rat melanotrophs.

    PubMed

    Chowdhury, Helana H; Popoff, Michel R; Zorec, Robert

    2000-01-01

    We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (C m ). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1μM [Ca 2+ ] i . Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.

  16. Actin cytoskeleton and exocytosis in rat melanotrophs.

    PubMed

    Chowdhury, H H; Popoff, M R; Zorec, R

    2000-01-01

    We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (Cm). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1 microM [Ca2+]i. Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.

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

    PubMed Central

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

    1996-01-01

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

  18. Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner.

    PubMed

    Yokota, Etsuo; Tominaga, Motoki; Mabuchi, Issei; Tsuji, Yasunori; Staiger, Christopher J; Oiwa, Kazuhiro; Shimmen, Teruo

    2005-10-01

    From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).

  19. Topical Imiquimod in the Treatment of Conjunctival Actinic Keratosis.

    PubMed

    Rowlands, Megan A; Giacometti, Joseph N; Servat, Javier; Materin, Miguel A; Levin, Flora

    Conjunctival actinic keratosis is rare and difficult to treat, as recurrences are common. Imiquimod, an immune response modulator, is currently Food and Drug Administration-approved for cutaneous actinic keratosis and superficial basal cell carcinomas. Emerging reports have shown it to be effective in treating some periocular and conjunctival lesions. The authors present a case of a 68-year-old white man with recurrent actinic keratosis involving the pretarsal conjunctiva, which was successfully treated with 5% topical imiquimod following previous failure with cryotherapy and interferon α-2b. The patient had ocular irritation that resolved on cessation of treatment. To the authors' knowledge, this is the first report of conjunctival actinic keratosis being treated with and successfully eradicated by topical imiquimod.

  20. Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

    PubMed Central

    Westendorf, Christian; Negrete, Jose; Bae, Albert J.; Sandmann, Rabea; Bodenschatz, Eberhard; Beta, Carsten

    2013-01-01

    The rapid reorganization of the actin cytoskeleton in response to external stimuli is an essential property of many motile eukaryotic cells. Here, we report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. When averaging the actin response of many cells to a short pulse of the chemoattractant cAMP, we observed a transient accumulation of cortical actin reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. To substantiate that an oscillatory mechanism governs the actin dynamics in these cells, we systematically exposed a large number of cells to periodic pulse trains of different frequencies. Our results indicate a resonance peak at a stimulation period of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the regulatory network of the actin system. To test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and actin-interacting protein 1, as well as knockout mutants that lack Coronin and actin-interacting protein 1. These actin-binding proteins enhance the disassembly of actin filaments and thus allow us to estimate the delay time in the regulatory feedback loop. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed in our periodic stimulation experiments. PMID:23431176

  1. PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility

    PubMed Central

    Luo, Weibo; Lin, Benjamin; Wang, Yingfei; Zhong, Jun; O'Meally, Robert; Cole, Robert N.; Pandey, Akhilesh; Levchenko, Andre; Semenza, Gregg L.

    2014-01-01

    Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins. PMID:25079693

  2. Actin grips: circular actin-rich cytoskeletal structures that mediate the wrapping of polymeric microfibers by endothelial cells.

    PubMed

    Jones, Desiree; Park, DoYoung; Anghelina, Mirela; Pécot, Thierry; Machiraju, Raghu; Xue, Ruipeng; Lannutti, John J; Thomas, Jessica; Cole, Sara L; Moldovan, Leni; Moldovan, Nicanor I

    2015-06-01

    Interaction of endothelial-lineage cells with three-dimensional substrates was much less studied than that with flat culture surfaces. We investigated the in vitro attachment of both mature endothelial cells (ECs) and of less differentiated EC colony-forming cells to poly-ε-capro-lactone (PCL) fibers with diameters in 5-20 μm range ('scaffold microfibers', SMFs). We found that notwithstanding the poor intrinsic adhesiveness to PCL, both cell types completely wrapped the SMFs after long-term cultivation, thus attaining a cylindrical morphology. In this system, both EC types grew vigorously for more than a week and became increasingly more differentiated, as shown by multiplexed gene expression. Three-dimensional reconstructions from multiphoton confocal microscopy images using custom software showed that the filamentous (F) actin bundles took a conspicuous ring-like organization around the SMFs. Unlike the classical F-actin-containing stress fibers, these rings were not associated with either focal adhesions or intermediate filaments. We also demonstrated that plasma membrane boundaries adjacent to these circular cytoskeletal structures were tightly yet dynamically apposed to the SMFs, for which reason we suggest to call them 'actin grips'. In conclusion, we describe a particular form of F-actin assembly with relevance for cytoskeletal organization in response to biomaterials, for endothelial-specific cell behavior in vitro and in vivo, and for tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Clinical Response to Ingenol Mebutate in Patients With Actinic Keratoses.

    PubMed

    Batalla, A; Flórez, Á; Feal, C; Peón, G; Abalde, M T; Salgado-Boquete, L; de la Torre, C

    2015-12-01

    Cryotherapy is the most common treatment for actinic keratosis, but its effect is limited to individual lesions. Several topical drugs, however, are available that, in addition to treating individual actinic keratoses, target field cancerization and thereby act on subclinical lesions. Examples are 5-fluorouracil, imiquimod, diclofenac, and ingenol mebutate. We report on 17 patients with actinic keratoses treated with ingenol mebutate and describe our findings on treatment effectiveness, adherence, and tolerance. Complete and partial response rates were 35% and 53%, respectively. Ninety-four percent of patients fully adhered to treatment and 18% developed severe local reactions. Ingenol mebutate is an effective treatment for actinic keratosis. Although it has a similar rate of local reactions to other treatments available for actinic keratosis, its short treatment regimen favors better adherence. Copyright © 2014 Elsevier España, S.L.U. y AEDV. All rights reserved.

  4. Interaction of aldolase with actin-containing filaments. Structural studies.

    PubMed Central

    Stewart, M; Morton, D J; Clarke, F M

    1980-01-01

    Electron micrographs of the paracrystals formed when fructose bisphosphate aldolase (EC 4.1.2.13) is added to actin-containing filaments were analysed by computer methods so that ultrastructural changes could be correlated with the various stoicheiometries of binding determined in the preceding paper [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98]. Paracrystals formed with aldolase and either F-actin or F-actin-tropomyosin have a single light transverse band every 38 nm, which is due to aldolase molecules cross-linking the filaments. In contrast, the paracrystals formed between aldolase and F-actin-tropomyosin-troponin filaments show two transverse bands every 38 nm: a major band, interpreted as aldolase binding to troponin, and a minor band, interpreted as aldolase cross-linking the filaments. The intensity of the minor band varies with Ca2+ concentration, being greatest when the Ca2+ concentration is low. A model for the different paracrystal structures which relates the various patterns and binding stoicheiometries to structural changes in the actin-containing filaments is proposed. Images PLATE 1 PMID:6892771

  5. 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.

  6. Actin-based gravity-sensing mechanisms in unicellular plant model systems

    NASA Astrophysics Data System (ADS)

    Braun, Markus; Limbach, Christoph

    2005-08-01

    Considerable progress has been made in the understanding of the molecular and cellular mechanisms underlying gravity sensing and gravity-oriented polarized growth in single-celled rhizoids and protonemata of the characean algae. It is well known that the actin cytoskeleton plays a key role in these processes. Numerous actin-binding proteins control apical actin polymerization and the dynamic remodeling of the actin arrangement. An actomyosin-based system mediates the delivery and incorporation of secretory vesicles at the growing tip and coordinates the tip-high gradient of cytoplasmic free calcium which is required for local exocytosis. Additionally, the actomyosin system precisely controls the position of statoliths and, upon a change in orientation relative to the gravity vector, directs sedimenting statoliths to the confined graviperception sites of the plasma membrane where gravitropic signalling is initiated. The upward growth response of protonemata is preceded by an actin-dependent relocalization of the Ca2+-gradient to the upper flank. The downward growth response of rhizoids, however, is caused by differential growth of the opposite flankes due to a local reduction of cytoplasmic free calcium limited to the plasma membrane area where statoliths are sedimented. Thus, constant actin polymerization in the growing tip and the spatiotemporal control of actin remodeling are essential for gravity sensing and gravity-oriented polarized growth of characean rhizoids and protonemata.

  7. The actinic UV-radiation budget during the ESCOMPTE campaign 2001: results of airborne measurements with the microlight research aircraft D-MIFU

    NASA Astrophysics Data System (ADS)

    Junkermann, Wolfgang

    2005-03-01

    During the ESCOMPTE campaign 2001, the vertical distribution of ultraviolet actinic radiation was investigated with concurrent measurements of ozone, aerosol size distributions, and scattering coefficients using a microlight aircraft as airborne platform. Three-dimensional (3D) measurements were performed on a regional scale in the area between Avignon, Aix-en-Provence, and Marseille up to an altitude of 4000 m a.s.l. The results show a pronounced dependence of the vertical actinic flux distribution on aerosol load and stratification while horizontally no significant variability was observed. Furthermore, investigations under cloudy conditions and in the vicinity of cumulus clouds were performed allowing comparisons with one-dimensional and recently published three-dimensional model results. Cloud effects of scattered convective clouds were often found to be masked by aerosols and the aerosol content was generally the dominating factor controlling radiation transfer.

  8. Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks

    PubMed Central

    1981-01-01

    The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X- 100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed. PMID:6799521

  9. Flux density measurement of radial magnetic bearing with a rotating rotor based on fiber Bragg grating-giant magnetostrictive material sensors.

    PubMed

    Ding, Guoping; Zhang, Songchao; Cao, Hao; Gao, Bin; Zhang, Biyun

    2017-06-10

    The rotational magnetic field of radial magnetic bearings characterizes remarkable time and spatial nonlinearity due to the eddy current and induced electromagnetic field. It is significant to experimentally obtain the features of the rotational magnetic field of the radial magnetic bearings to validate the theoretical analysis and reveal the discipline of a rotational magnetic field. This paper developed thin-slice fiber Bragg grating-giant magnetostrictive material (FBG-GMM) magnetic sensors to measure air-gap flux density of a radial magnetic bearing with a rotating rotor; a radial magnetic bearing test rig was constructed and the rotational magnetic field with different rotation speed was measured. Moreover, the finite element method (FEM) was used to simulate the rotational magnetic field; the measurement results and FEM results were investigated, and it was concluded that the FBG-GMM sensors were capable of measuring the radial magnetic bearing's air gap flux density with a rotating rotor, and the measurement results showed a certain degree of accuracy.

  10. Dual effect of pseudorabies virus growth factor (PRGF) displayed on actin cytoskeleton.

    PubMed

    Urbancíková, M; Vozárová, G; Lesko, J; Golais, F

    1999-10-01

    Pseudorabies virus growth factor (PRGF) was shown to possess transforming activity as well as transformation repressing activity in in vitro systems. In order to better understand these phenomena we studied actin cytoskeleton and its alterations induced by PRGF using normal human fibroblasts VH-10 and transformed cell line HeLa. For specific detection of filamentous actin cells were stained with phalloidin conjugated with fluorescein isothiocyanate (FITC)-phalloidin. PRGF was applied to VH-10 cells for various length of time from 10 min up to 48 h. The effect was very fast and changes in actin filament composition could be detected already after 10 min. In comparison to untreated cells the staining of treated cells was more diffuse and a number of actin microfilaments in individual stress fibers became reduced. After 30 min thick short actin bundles appeared in the perinuclear region. A 24-h exposure resulted in a large reduction of actin bundles. After additional 24 h a partial restoration of actin cytoskeleton in cells was observed. In transformed HeLa cells PRGF induced opposite process than in normal cells: the number of filamentous actin structures increased. We hypothesise that PRGF may act as a transcription-like factor and may initiate changes in gene expression which consequently result in actin cytoskeleton alterations.

  11. Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

  12. Long non-coding RNA CRYBG3 blocks cytokinesis by directly binding G-actin.

    PubMed

    Pei, Hailong; Hu, Wentao; Guo, Ziyang; Chen, Huaiyuan; Ma, Ji; Mao, Weidong; Li, Bingyan; Wang, Aiqing; Wan, Jianmei; Zhang, Jian; Nie, Jing; Zhou, Guangming; Hei, Tom K

    2018-06-22

    The dynamic interchange between monomeric globular actin (G-actin) and polymeric filamentous actin filaments (F-actin) is fundamental and essential to many cellular processes including cytokinesis and maintenance of genomic stability. Here we report that the long non-coding RNA LNC CRYBG3 directly binds G-actin to inhibit its polymerization and formation of contractile rings, resulting in M-Phase cell arrest. Knockdown of LNC CRYBG3 in tumor cells enhanced their malignant phenotypes. Nucleotide sequence 228-237 of the full-length LNC CRYBG3 and the ser14 domain of beta-actin are essential for their interaction, and mutation of either of these sites abrogated binding of LNC CRYBG3 to G-actin. Binding of LNC CRYBG3 to G-actin blocked nuclear localization of MAL, which consequently kept serum response factor (SRF) away from the promoter region of several immediate early genes, including JUNB and Arp3, which are necessary for cellular proliferation, tumor growth, adhesion, movement, and metastasis. These findings reveal a novel lncRNA-actin-MAL-SRF pathway and highlight LNC CRYBG3 as a means to block cytokinesis and treat cancer by targeting the actin cytoskeleton. Copyright ©2018, American Association for Cancer Research.

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

    PubMed

    Winterhoff, Moritz; Faix, Jan

    2015-06-01

    Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Optogenetics to target actin-mediated synaptic loss in Alzheimer's

    NASA Astrophysics Data System (ADS)

    Zahedi, Atena; DeFea, Kathryn; Ethell, Iryna

    2013-03-01

    Numerous studies in Alzheimer's Disease (AD) animal models show that overproduction of Aβ peptides and their oligomerization can distort dendrites, damage synapses, and decrease the number of dendritic spines and synapses. Aβ may trigger synapse loss by modulating activity of actin-regulating proteins, such as Rac1 and cofilin. Indeed, Aβ1-42 oligomers can activate actin severing protein cofilin through calcineurin-mediated activation of phosphatase slingshot and inhibit an opposing pathway that suppresses cofilin phosphorylation through Rac-mediated activation of LIMK1. Excessive activation of actin-severing protein cofilin triggers the formation of a non-dynamic actin bundles, called rods that are found in AD brains and cause loss of synapses. Hence, regulation of these actin-regulating proteins in dendritic spines could potentially provide useful tools for preventing the synapse/spine loss associated with earlier stages of AD neuropathology. However, lack of spatiotemporal control over their activity is a key limitation. Recently, optogenetic advancements have provided researchers with convenient light-activating proteins such as photoactivatable Rac (PARac). Here, we transfected cultured primary hippocampal neurons and human embryonic kidney (HEK) cells with a PARac/ mCherry-containing plasmid and the mCherry-positive cells were identified and imaged using an inverted fluorescence microscope. Rac1 activation was achieved by irradiation with blue light (480nm) and live changes in dendritic spine morphology were observed using mCherry (587nm). Rac activation was confirmed by immunostaining for phosphorylated form of effector proteinP21 protein-activated kinase 1 (PAK1) and reorganization of actin. Thus, our studies confirm the feasibility of using the PA-Rac construct to trigger actin re-organization in the dendritic spines.

  15. Assembly kinetics determine the architecture of α-actinin crosslinked F-actin networks.

    PubMed

    Falzone, Tobias T; Lenz, Martin; Kovar, David R; Gardel, Margaret L

    2012-05-29

    The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and crosslinking determine the architecture of reconstituted actin networks formed with α-actinin crosslinks. Crosslink-mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semiflexible biopolymer networks.

  16. Brownian dynamics simulations of interactions between aldolase and G- or F-actin.

    PubMed Central

    Ouporov, I V; Knull, H R; Thomasson, K A

    1999-01-01

    Compartmentation of proteins in cells is important to proper cell function. Interactions of F-actin and glycolytic enzymes is one mechanism by which glycolytic enzymes can compartment. Brownian dynamics (BD) simulations of the binding of the muscle form of the glycolytic enzyme fructose-1,6-bisphosphate aldolase (aldolase) to F- or G-actin provide first-encounter snapshots of these interactions. Using x-ray structures of aldolase, G-actin, and three-dimensional models of F-actin, the electrostatic potential about each protein was predicted by solving the linearized Poisson-Boltzmann equation for use in BD simulations. The BD simulations provided solution complexes of aldolase with F- or G-actin. All complexes demonstrate the close contacts between oppositely charged regions of the protein surfaces. Positively charged surface regions of aldolase (residues Lys 13, 27, 288, 293, and 341 and Arg 257) are attracted to the negatively charged amino terminus (Asp 1 and Glu 2 and 4) and other patches (Asp 24, 25, and 363 and Glu 361, 364, 99, and 100) of actin subunits. According to BD results, the most important factor for aldolase binding to actin is the quaternary structure of aldolase and actin. Two pairs of adjacent aldolase subunits greatly add to the positive electrostatic potential of each other creating a region of attraction for the negatively charged subdomain 1 of the actin subunit that is exposed to solvent in the quaternary F-actin structure. PMID:9876119

  17. F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain.

    PubMed

    Zhao, Yongtong; Shapiro, Sandor S; Eto, Masumi

    2016-01-01

    Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants W148R and E227K than the wild-type protein to the cytoskeleton. Ectopic expression of FLNB-W148R and, to a lesser extent, FLNB-E227K induced prominent F-actin accumulations and the consequent rearrangement of focal adhesions, myosin II, and septin filaments and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal rearrangement was partially attenuated by the inhibition of myosin II, p21-activated protein kinase, or Rho-associated protein kinase. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization, preventing normal skeletal development. Copyright © 2016 the American Physiological Society.

  18. Simulations of the Cleft Ion Fountain outflows resulting from the passage of Storm Enhanced Density (SED) plasma flux tubes through the dayside cleft auroral processes region

    NASA Astrophysics Data System (ADS)

    Horwitz, James; Zeng, Wen

    2007-10-01

    Foster et al. [2002] reported elevated ionospheric density regions convected from subauroral plasmaspheric regions toward noon, in association with convection of plasmaspheric tails. These Storm Enhanced Density (SED) regions could supply cleft ion fountain outflows. Here, we will utilize our Dynamic Fluid Kinetic (DyFK) model to simulate the entry of a high-density ``plasmasphere-like'' flux tube entering the cleft region and subjected to an episode of wave-driven transverse ion heating. It is found that the O^+ ion density at higher altitudes increases and the density at lower altitudes decreases, following this heating episode, indicating increased fluxes of O^+ ions from the ionospheric source gain sufficient energy to reach higher altitudes after the effects of transverse wave heating. Foster, J. C., P. J. Erickson, A. J. Coster, J. Goldstein, and F. J. Rich, Ionospheric signatures of plasmaspheric tails, Geophys. Res. Lett., 29(13), 1623, doi:10.1029/2002GL015067, 2002.

  19. 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

  20. A dynamo theory prediction for solar cycle 22: Sunspot number, radio flux, exospheric temperature, and total density at 400 km

    NASA Technical Reports Server (NTRS)

    Schatten, K. H.; Hedin, A. E.

    1986-01-01

    Using the dynamo theory method to predict solar activity, a value for the smoothed sunspot number of 109 + or - 20 is obtained for solar cycle 22. The predicted cycle is expected to peak near December, 1990 + or - 1 year. Concommitantly, F(10.7) radio flux is expected to reach a smoothed value of 158 + or - 18 flux units. Global mean exospheric temperature is expected to reach 1060 + or - 50 K and global total average total thermospheric density at 400 km is expected to reach 4.3 x 10 to the -15th gm/cu cm + or - 25 percent.

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

    PubMed

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

    2018-05-03

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

  2. Actin filaments participate in West Nile (Sarafend) virus maturation process.

    PubMed

    Chu, J J H; Choo, B G H; Lee, J W M; Ng, M L

    2003-11-01

    West Nile (Sarafend) virus has previously been shown to egress by budding at the plasma membrane of infected cells, but relatively little is known about the mechanism involved in this mode of release. During the course of this study, it was discovered that actin filaments take part in the virus maturation process. Using dual-labeled immunofluorescence and immunoelectron microscopy at late infection (10 hr p.i.), co-localization of viral structural (envelope and capsid) proteins with actin filaments was confirmed. The virus structural proteins were also immunoprecipitated with anti-actin antibody, further demonstrating the strong association between the two components. Perturbation of actin filaments by cytochalasin B strongly inhibited the release of West Nile virus (approximately 10,000-fold inhibition) when compared with the untreated cells. Infectious virus particles were recovered after the removal of cytochalasin B. Further confirmation was obtained when nucleocapsid particles were found associated with disrupted actin filaments at the periphery of cytochalasin B-treated cells. Together, these results showed that actin filaments do indeed have a key role in the release of West Nile (Sarafend) virions. Copyright 2003 Wiley-Liss, Inc.

  3. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division.

    PubMed

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-08-25

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

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

    PubMed Central

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

    2016-01-01

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

  5. Actin Is Crucial for All Kinetically Distinguishable Forms of Endocytosis at Synapses.

    PubMed

    Wu, Xin-Sheng; Lee, Sung Hoon; Sheng, Jiansong; Zhang, Zhen; Zhao, Wei-Dong; Wang, Dongsheng; Jin, Yinghui; Charnay, Patrick; Ervasti, James M; Wu, Ling-Gang

    2016-12-07

    Mechanical force is needed to mediate endocytosis. Whether actin, the most abundant force-generating molecule, is essential for endocytosis is highly controversial in mammalian cells, particularly synapses, likely due to the use of actin blockers, the efficiency and specificity of which are often unclear in the studied cell. Here we addressed this issue using a knockout approach combined with measurements of membrane capacitance and fission pore conductance, imaging of vesicular protein endocytosis, and electron microscopy. We found that two actin isoforms, β- and γ-actin, are crucial for slow, rapid, bulk, and overshoot endocytosis at large calyx-type synapses, and for slow endocytosis and bulk endocytosis at small hippocampal synapses. Polymerized actin provides mechanical force to form endocytic pits. Actin also facilitates replenishment of the readily releasable vesicle pool, likely via endocytic clearance of active zones. We conclude that polymerized actin provides mechanical force essential for all kinetically distinguishable forms of endocytosis at synapses. Published by Elsevier Inc.

  6. Actin is crucial for all kinetically distinguishable forms of endocytosis at synapses

    PubMed Central

    Wu, Xin-Sheng; Lee, Sunghoon; Sheng, Jiansong; Zhang, Zhen; Zhao, Weidong; Wang, Dongsheng; Jin, Yinghui; Charnay, Patrick; Ervasti, James M.; Wu, Ling-Gang

    2016-01-01

    Summary Mechanical force is needed to mediate endocytosis. Whether actin, the most abundant force-generating molecule, is essential for endocytosis is highly controversial in mammalian cells, particularly synapses, likely due to the use of actin blockers, the efficiency and specificity of which are often unclear in the studied cell. Here we addressed this issue using knockout approach combined with measurements of membrane capacitance and fission pore conductance, imaging of vesicular protein endocytosis, and electron microscopy. We found that two actin isoforms, β- and γ-actin, are crucial for slow, rapid, bulk, and overshoot endocytosis at large calyx-type synapses, and for slow endocytosis and bulk endocytosis at small hippocampal synapses. Polymerized actin provides mechanical force to form endocytic pits. Actin also facilitates replenishment of the readily releasable vesicle pool, likely via endocytic clearance of active zones. We conclude that polymerized actin provides mechanical force essential for all kinetically distinguishable forms of endocytosis at synapses. PMID:27840001

  7. Endocytosis-dependent coordination of multiple actin regulators is required for wound healing

    PubMed Central

    Matsubayashi, Yutaka; Coulson-Gilmer, Camilla

    2015-01-01

    The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. In this paper, using Drosophila melanogaster embryos, we demonstrate that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, we show that endocytosis is essential for wound edge actin assembly and wound closure. We identify adherens junctions (AJs) as a key target of endocytosis during wound healing and propose that endocytic remodeling of AJs is required to form “signaling centers” along the wound edge that control actin assembly. We conclude that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing. PMID:26216900

  8. Assembly Kinetics Determine the Architecture of α-actinin Crosslinked F-actin Networks

    PubMed Central

    Falzone, Tobias T.; Lenz, Martin; Kovar, David R.; Gardel, Margaret L.

    2013-01-01

    The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and cross-linking determine the architecture of reconstituted actin networks formed with α-actinin cross-links. Cross-link mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semi-flexible biopolymer networks. PMID:22643888

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

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

  10. Oral acetylsalicylic acid and prevalence of actinic keratosis.

    PubMed

    Schmitt, Juliano; Miot, Hélio

    2014-01-01

    To investigate the influence of a regular oral use of acetylsalicylic acid in the prevalence of actinic keratosis. A case-control study with dermatologic outpatients above 50 years of age assessed between 2009 and 2011. Cases were defined as those who had been under regular use of oral acetylsalicylic acid for more than six consecutive months. The assessment focused on: age, sex, skin-type, tobacco smoking, use of medication, occurrence of individual or family skin cancer, and sunscreen and sun exposure habits. Actinic keratoses were counted in the medial region of the face and upper limbs. Counts were adjusted by co-variables based on a generalized linear model. A total of 74 cases and 216 controls were assessed. The median time of acetylsalicylic acid use was 36 months. Cases differed from controls as to the highest age, highest prevalence of use of angiotensin-converting enzyme inhibitors and fewer keratosis on the face and on the upper limbs (p<0.05). The multivariate model showed that the use of acetylsalicylic acid was associated to lower counts of face actinic keratosis and upper-limb erythematous actinic keratosis (p<0.05), regardless of other risk factors. The regular use of oral acetylsalicylic acid for more than six months was associated to a lower prevalence of actinic keratosis, especially facial and erythematous ones.

  11. The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin

    PubMed Central

    Haag, Natja; Kessels, Michael M.; Qualmann, Britta

    2015-01-01

    Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca2+ and multiple, direct associations of the Ca2+ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca2+ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca2+/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca2+/CaM and reveal that the Ca2+/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca2+/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca2+ signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation. PMID

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2014-01-01

    Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells. PMID:25140420

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  16. Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

    PubMed Central

    Huang, Junqi; Huang, Yinyi; Yu, Haochen; Subramanian, Dhivya; Padmanabhan, Anup; Thadani, Rahul; Tao, Yaqiong; Tang, Xie; Wedlich-Soldner, Roland

    2012-01-01

    In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly. PMID:23185032

  17. Addition of electrophilic lipids to actin alters filament structure

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

    Gayarre, Javier; Sanchez, David; Sanchez-Gomez, Francisco J.

    2006-11-03

    Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-{delta}{sup 12,14}-PGJ{sub 2} (15d-PGJ{sub 2}) and PGA{sub 1} in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA{sub 1} and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is importantmore » for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ{sub 2} or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ{sub 2} at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.« less

  18. DNA vaccine encoding Haemonchus contortus actin induces partial protection in goats.

    PubMed

    Yan, Ruofeng; Wang, Jingjing; Xu, Lixin; Song, Xiaokai; Li, Xiangrui

    2014-10-01

    Actin is a globular multi-functional protein that forms microfilaments, and participates in many important cellular processes. Previous study found that Haemonchus contortus actin could be recognized by the serum of goats infected with the homology parasite. This indicated that H. contortus actin could be a potential candidate for vaccine. In this study, DNA vaccine encoding H. contortus actin was tested for protection against experimental H. contortus infections in goats. Fifteen goats were allocated into three trial groups. The animals of Actin group were vaccinated with the DNA vaccine on day 0 and 14, and challenged with 5000 infective H. contortus third stage larval (L3) on day 28. An unvaccinated positive control group was challenged with L3 at the same time. An unvaccinated negative control group was not challenged with L3. The results showed that DNA vaccine were transcribed at local injection sites and expressed in vivo post immunizations respectively. For goats in Actin vaccinated group, higher levels of serum IgG, serum IgA and mucosal IgA were produced, the percentages of CD4(+) T lymphocytes, CD8(+) T lymphocytes and B lymphocytes and the concentrations of TGF-β were increased significantly (P<0.05). Following L3 challenge, the mean eggs per gram feces (EPG) and worm burdens of Actin group were reduced by 34.4% and 33.1%, respectively. This study suggest that recombinant H. contortus Actin DNA vaccine induced partial immune response and has protective potential against goat haemonchosis.

  19. State transitions of actin cortices in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Tan, Tzer Han; Keren, Kinneret; Mackintosh, Fred; Schmidt, Christoph; Fakhri, Nikta

    Most animal cells are enveloped by a thin layer of actin cortex which governs the cell mechanics. A functional cortex must be rigid to provide mechanical support while being flexible to allow for rapid restructuring events such as cell division. To satisfy these requirements, the actin cortex is highly dynamic with fast actin turnover and myosin-driven contractility. The regulatory mechanism responsible for the transition between a mechanically stable state and a restructuring state is not well understood. Here, we develop a technique to map the dynamics of reconstituted actin cortices in emulsion droplets using IR fluorescent single-walled carbon nanotubes (SWNTs). By increasing crosslinker concentration, we find that a homogeneous cortex transitions to an intermediate state with broken rotational symmetry and a globally contractile state which further breaks translational symmetry. We apply this new dynamic mapping technique to cortices of live starfish oocytes in various developmental stages. To identify the regulatory mechanism for steady state transitions, we subject the oocytes to actin and myosin disrupting drugs.

  20. Cortical actin nanodynamics determines nitric oxide release in vascular endothelium.

    PubMed

    Fels, Johannes; Jeggle, Pia; Kusche-Vihrog, Kristina; Oberleithner, Hans

    2012-01-01

    The release of the main vasodilator nitric oxide (NO) by the endothelial NO synthase (eNOS) is a hallmark of endothelial function. We aim at elucidating the underlying mechanism how eNOS activity depends on cortical stiffness (К(cortex)) of living endothelial cells. It is hypothesized that cortical actin dynamics determines К(cortex) and directly influences eNOS activity. By combined atomic force microscopy and fluorescence imaging we generated mechanical and optical sections of single living cells. This approach allows the discrimination between К(cortex) and bulk cell stiffness (К(bulk)) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that К(cortex) softens when cortical F-actin depolymerizes and that this shift from a gel-like stiff cortex to a soft G-actin rich layer, triggers the stiffness-sensitive eNOS activity. The results implicate that stiffness changes in the ∼100 nm phase of the submembranous actin web, without affecting К(bulk), regulate NO release and thus determines endothelial function.

  1. Chronophin activation is necessary in Doxorubicin-induced actin cytoskeleton alteration.

    PubMed

    Lee, Su Jin; Park, Jeen Woo; Kang, Beom Sik; Lee, Dong-Seok; Lee, Hyun-Shik; Choi, Sooyoung; Kwon, Oh-Shin

    2017-06-01

    Although doxorubicin (Dox)-induced oxidative stress is known to be associated with cytotoxicity, the precise mechanism remains unclear. Genotoxic stress not only generates free radicals, but also affects actin cytoskeleton stability. We showed that Dox-induced RhoA signaling stimulated actin cytoskeleton alterations, resulting in central stress fiber disruption at early time points and cell periphery cortical actin formation at a later stage, in HeLa cells. Interestingly, activation of a cofilin phosphatase, chronophin (CIN), was initially evoked by Dox-induced RhoA signaling, resulting in a rapid phosphorylated cofilin turnover leading to actin cytoskeleton remodeling. In addition, a novel interaction between CIN and 14-3-3ζ was detected in the absence of Dox treatment. We demonstrated that CIN activity is quite contrary to 14-3-3ζ binding, and the interaction leads to enhanced phosphorylated cofilin levels. Therefore, initial CIN activation regulation could be critical in Dox-induced actin cytoskeleton remodeling through RhoA/cofilin signaling. [BMB Reports 2017; 50(6): 335-340].

  2. Oxidation of F-actin controls the terminal steps of cytokinesis

    PubMed Central

    Frémont, Stéphane; Hammich, Hussein; Bai, Jian; Wioland, Hugo; Klinkert, Kerstin; Rocancourt, Murielle; Kikuti, Carlos; Stroebel, David; Romet-Lemonne, Guillaume; Pylypenko, Olena; Houdusse, Anne; Echard, Arnaud

    2017-01-01

    Cytokinetic abscission, the terminal step of cell division, crucially depends on the local constriction of ESCRT-III helices after cytoskeleton disassembly. While the microtubules of the intercellular bridge are cut by the ESCRT-associated enzyme Spastin, the mechanism that clears F-actin at the abscission site is unknown. Here we show that oxidation-mediated depolymerization of actin by the redox enzyme MICAL1 is key for ESCRT-III recruitment and successful abscission. MICAL1 is recruited to the abscission site by the Rab35 GTPase through a direct interaction with a flat three-helix domain found in MICAL1 C terminus. Mechanistically, in vitro assays on single actin filaments demonstrate that MICAL1 is activated by Rab35. Moreover, in our experimental conditions, MICAL1 does not act as a severing enzyme, as initially thought, but instead induces F-actin depolymerization from both ends. Our work reveals an unexpected role for oxidoreduction in triggering local actin depolymerization to control a fundamental step of cell division. PMID:28230050

  3. Initiation of DNA replication requires actin dynamics and formin activity.

    PubMed

    Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

    2017-11-02

    Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

  4. Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

    PubMed Central

    Defacque, Hélène; Bos, Evelyne; Garvalov, Boyan; Barret, Cécile; Roy, Christian; Mangeat, Paul; Shin, Hye-Won; Rybin, Vladimir; Griffiths, Gareth

    2002-01-01

    Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P2-binding proteins ezrin and/or moesin were essential for this process (Defacque et al., 2000b). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P2, and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P2-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane. PMID:11950931

  5. Actin Out: Regulation of the Synaptic Cytoskeleton

    PubMed Central

    Spence, Erin F.; Soderling, Scott H.

    2015-01-01

    The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes that encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease. PMID:26453304

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

    PubMed Central

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

    2015-01-01

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

  7. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

    PubMed Central

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-01-01

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division. DOI: http://dx.doi.org/10.7554/eLife.08828.001 PMID:26305500

  8. Interactions between globular proteins and F-actin in isotonic saline solution.

    PubMed

    Lakatos, S; Minton, A P

    1991-10-05

    Solutions of each of three different globular proteins (cytochrome c, chromophorically labeled serum albumin, and chromophorically labeled aldolase), mixed with another unlabeled globular protein or with fibrous actin, were prepared in pH 8.0 Tris-HCl buffer containing 0.15 M NaCl. Each solution was centrifuged at low speed, at 5 degrees C, until unassociated globular protein in solution achieved sedimentation equilibrium. Individual absorbance gradients of both macrosolutes in the mixtures subsequent to centrifugation were obtained via optical scans of the centrifuge tubes at two wavelengths. The gradients of each macrosolute in mixtures of two globular proteins revealed no association of globular proteins under the conditions of these experiments, but perturbation of the gradients of serum albumin, aldolase, and cytochrome c in the presence of F-actin indicated association of all three globular proteins with F-actin. Perturbation of actin gradients in the presence of serum albumin and aldolase suggested partial depolymerization of the F-actin by the globular protein. Analysis of the data with a simple phenomenological model relating free globular protein, bound globular protein, and total actin concentration provided estimates of the respective equilibrium constants for association of serum albumin and aldolase with F-actin, under the conditions of these experiments, of the order of 0.1 microM-1.

  9. A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F-actin.

    PubMed

    Araiza-Olivera, Daniela; Chiquete-Felix, Natalia; Rosas-Lemus, Mónica; Sampedro, José G; Peña, Antonio; Mujica, Adela; Uribe-Carvajal, Salvador

    2013-08-01

    In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO₂ molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies. © 2013 FEBS.

  10. On the Maximum and Characteristic Curvature of Current Density of High tc Superconductor Ybco in Flux Relaxation

    NASA Astrophysics Data System (ADS)

    Ye, Jiping; Sun, Lei; Dai, Xianxi; Dai, Jixin

    The flux relaxation is one of important topics in the studies of high Tc superconductivity, because it is related to the energy loss in practical applications. There are many mechanisms, theories and relaxation laws suggested in the literatures. It is very interesting to test them according to the characters and compare them with the experiments. Some people think that the characters of the famous theories are their negative curvature. According our inversion solution, the relaxation ArcG law and experimental data analysis, the relaxation law has both positive and negative signs. This prediction is hopeful to be checked by experiments in future. The current densities of many high Tc superconductors decrease very rapidly in the early time in the relaxation. People do not know what their maximums are. In this work, a theory to determine these maximums of the current densities is presented. The theory is concretely realized by inversion for some real data of the YBCO and their maximum current densities are obtained.

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

    PubMed Central

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

    2015-01-01

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

  12. The assembly of MreB, a prokaryotic homolog of actin.

    PubMed

    Esue, Osigwe; Cordero, Maria; Wirtz, Denis; Tseng, Yiider

    2005-01-28

    MreB, a major component of the bacterial cytoskeleton, exhibits high structural homology to its eukaryotic counterpart actin. Live cell microscopy studies suggest that MreB molecules organize into large filamentous spirals that support the cell membrane and play a key shape-determining function. However, the basic properties of MreB filament assembly remain unknown. Here, we studied the assembly of Thermotoga maritima MreB triggered by ATP in vitro and compared it to the well-studied assembly of actin. These studies show that MreB filament ultrastructure and polymerization depend crucially on temperature as well as the ions present on solution. At the optimal growth temperature of T. maritima, MreB assembly proceeded much faster than that of actin, without nucleation (or nucleation is highly favorable and fast) and with little or no contribution from filament end-to-end annealing. MreB exhibited rates of ATP hydrolysis and phosphate release similar to that of F-actin, however, with a critical concentration of approximately 3 nm, which is approximately 100-fold lower than that of actin. Furthermore, MreB assembled into filamentous bundles that have the ability to spontaneously form ring-like structures without auxiliary proteins. These findings suggest that despite high structural homology, MreB and actin display significantly different assembly properties.

  13. Effect of Profilin on Actin Critical Concentration: A Theoretical Analysis

    PubMed Central

    Yarmola, Elena G.; Dranishnikov, Dmitri A.; Bubb, Michael R.

    2008-01-01

    To explain the effect of profilin on actin critical concentration in a manner consistent with thermodynamic constraints and available experimental data, we built a thermodynamically rigorous model of actin steady-state dynamics in the presence of profilin. We analyzed previously published mechanisms theoretically and experimentally and, based on our analysis, suggest a new explanation for the effect of profilin. It is based on a general principle of indirect energy coupling. The fluctuation-based process of exchange diffusion indirectly couples the energy of ATP hydrolysis to actin polymerization. Profilin modulates this coupling, producing two basic effects. The first is based on the acceleration of exchange diffusion by profilin, which indicates, paradoxically, that a faster rate of actin depolymerization promotes net polymerization. The second is an affinity-based mechanism similar to the one suggested in 1993 by Pantaloni and Carlier although based on indirect rather than direct energy coupling. In the model by Pantaloni and Carlier, transformation of chemical energy of ATP hydrolysis into polymerization energy is regulated by direct association of each step in the hydrolysis reaction with a corresponding step in polymerization. Thus, hydrolysis becomes a time-limiting step in actin polymerization. In contrast, indirect coupling allows ATP hydrolysis to lag behind actin polymerization, consistent with experimental results. PMID:18835900

  14. Lifeact-mEGFP Reveals a Dynamic Apical F-Actin Network in Tip Growing Plant Cells

    PubMed Central

    Hepler, Peter K.; Bezanilla, Magdalena

    2009-01-01

    Background Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. Methodology/Principal Findings In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. Conclusions/Significance Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells. PMID:19478943

  15. Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells

    PubMed Central

    Vasicova, Pavla; Rinnerthaler, Mark; Haskova, Danusa; Novakova, Lenka; Malcova, Ivana; Breitenbach, Michael; Hasek, Jiri

    2016-01-01

    Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae. This pattern has been shown to be depolarized in glucose-depleted cells after formaldehyde fixation and staining with rhodamine-tagged phalloidin. Loss of actin cables in mother cells was remarkable. Here we extend our knowledge on actin in live glucose-depleted cells co-expressing the marker of actin patches (Abp1-RFP) with the marker of actin cables (Abp140-GFP). Glucose depletion resulted in appearance of actin patches also in mother cells. However, even after 80 min of glucose deprivation these cells showed a clear network of actin cables labeled with Abp140-GFP in contrast to previously published data. In live cells with a mitochondrial dysfunction (rho0 cells), glucose depletion resulted in almost immediate appearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches in mother cells. Residual actin cables were clustered in patch-associated bundles. A similar overlapping “patchy” pattern of both actin markers was observed upon treatment of glucose-deprived rho+ cells with FCCP (the inhibitor of oxidative phosphorylation) and upon treatment with formaldehyde. While the formaldehyde-targeted process stays unknown, our results indicate that published data on yeast actin cytoskeleton obtained from glucose-depleted cells after fixation should be considered with caution. PMID:28357356

  16. A master equation approach to actin polymerization applied to endocytosis in yeast.

    PubMed

    Wang, Xinxin; Carlsson, Anders E

    2017-12-01

    We present a Master Equation approach to calculating polymerization dynamics and force generation by branched actin networks at membranes. The method treats the time evolution of the F-actin distribution in three dimensions, with branching included as a directional spreading term. It is validated by comparison with stochastic simulations of force generation by actin polymerization at obstacles coated with actin "nucleation promoting factors" (NPFs). The method is then used to treat the dynamics of actin polymerization and force generation during endocytosis in yeast, using a model in which NPFs form a ring around the endocytic site, centered by a spot of molecules attaching the actin network strongly to the membrane. We find that a spontaneous actin filament nucleation mechanism is required for adequate forces to drive the process, that partial inhibition of branching and polymerization lead to different characteristic responses, and that a limited range of polymerization-rate values provide effective invagination and obtain correct predictions for the effects of mutations in the active regions of the NPFs.

  17. A master equation approach to actin polymerization applied to endocytosis in yeast

    PubMed Central

    Wang, Xinxin

    2017-01-01

    We present a Master Equation approach to calculating polymerization dynamics and force generation by branched actin networks at membranes. The method treats the time evolution of the F-actin distribution in three dimensions, with branching included as a directional spreading term. It is validated by comparison with stochastic simulations of force generation by actin polymerization at obstacles coated with actin “nucleation promoting factors” (NPFs). The method is then used to treat the dynamics of actin polymerization and force generation during endocytosis in yeast, using a model in which NPFs form a ring around the endocytic site, centered by a spot of molecules attaching the actin network strongly to the membrane. We find that a spontaneous actin filament nucleation mechanism is required for adequate forces to drive the process, that partial inhibition of branching and polymerization lead to different characteristic responses, and that a limited range of polymerization-rate values provide effective invagination and obtain correct predictions for the effects of mutations in the active regions of the NPFs. PMID:29240771

  18. Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex

    PubMed Central

    Tsurumura, Toshiharu; Tsumori, Yayoi; Qiu, Hao; Oda, Masataka; Sakurai, Jun; Nagahama, Masahiro; Tsuge, Hideaki

    2013-01-01

    Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD+ analog βTAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD+-bound form (NAD+-Ia-actin) and the ADP ribosylated form [Ia-ADP ribosylated (ADPR)-actin] remain unclear. Accidentally, we found that ethylene glycol as cryo-protectant inhibits ADP ribosylation and crystallized the NAD+-Ia-actin complex. Here we report high-resolution structures of NAD+-Ia-actin and Ia-ADPR-actin obtained by soaking apo-Ia-actin crystal with NAD+ under different conditions. The structures of NAD+-Ia-actin and Ia-ADPR-actin represent the pre- and postreaction states, respectively. By assigning the βTAD-Ia-actin structure to the transition state, the strain-alleviation model of ADP ribosylation, which we proposed previously, is experimentally confirmed and improved. Moreover, this reaction mechanism appears to be applicable not only to Ia but also to other ADP ribosyltransferases. PMID:23382240

  19. Assembly and Function of the Actin Cytoskeleton of Yeast: Relationships between Cables and Patches

    PubMed Central

    Karpova, Tatiana S.; McNally, James G.; Moltz, Samuel L.; Cooper, John A.

    1998-01-01

    Actin in eukaryotic cells is found in different pools, with filaments being organized into a variety of supramolecular assemblies. To investigate the assembly and functional relationships between different parts of the actin cytoskeleton in one cell, we studied the morphology and dynamics of cables and patches in yeast. The fine structure of actin cables and the manner in which cables disassemble support a model in which cables are composed of a number of overlapping actin filaments. No evidence for intrinsic polarity of cables was found. To investigate to what extent different parts of the actin cytoskeleton depend on each other, we looked for relationships between cables and patches. Patches and cables were often associated, and their polarized distributions were highly correlated. Therefore, patches and cables do appear to depend on each other for assembly and function. Many cell types show rearrangements of the actin cytoskeleton, which can occur via assembly or movement of actin filaments. In our studies, dramatic changes in actin polarization did not include changes in filamentous actin. In addition, the concentration of actin patches was relatively constant as cells grew. Therefore, cells do not have bursts of activity in which new parts of the actin cytoskeleton are created. PMID:9744880

  20. Loss of cytokeratin 10 indicates malignant transformation in actinic cheilitis.

    PubMed

    Garcia, Natália Galvão; Oliveira, Denise Tostes; Lauris, José Roberto Pereira; Domingues, Maria Aparecida Custódio; Minicucci, Eliana Maria; Soares, Cléverson Teixeira

    2016-05-01

    The aim of this study was to investigate the relationship the expression of cytokeratins (CK10 and CK13) and the cell proliferation index determined by Ki-67 of lip squamous cell carcinoma and actinic cheilitis with different degrees of dysplasia. Forty-five paraffin-embedded actinic cheilitis with and without dysplasia and 20 lip squamous cell carcinoma were analyzed by immunohistochemistry using anti-human anti-CK10, anti-CK13, and anti-Ki-67 antibodies. The majority of actinic cheilitis showed immunopositivity for CK10 and CK13 with decrease or loss of expression in dysplastic areas. In lip squamous cell carcinoma of the lip, heterogeneous expression of CK13 and immunonegativity for CK10 were observed. There was a statistically significant difference between CK10 expression in lip squamous cell carcinoma and in actinic cheilitis with or without dysplasia (p < 0.001). The cell proliferation index was higher in actinic cheilitis with dysplasia and lip squamous cell carcinoma than in actinic cheilitis without epithelial dysplasia. A significant correlation was found between the intensity of the epithelial dysplasia and the cell proliferation index (p < 0.001). These results provide evidence that there is a downregulation of CK10 expression in dysplastic areas of patients with actinic cheilitis and in those with lip squamous cell carcinoma (LSCC) and that the index of cell proliferation, determined by Ki-67, is directly correlated with the intensity of the epithelial dysplasia. Altogether, these results suggest that CK10 expression and the epithelial cell proliferation index can help to identify malignant transformation in the lip region.