Science.gov

Sample records for institucionalizada actos fallidos

  1. ACToR - Aggregated Computational Toxicology Resource

    SciTech Connect

    Judson, Richard Richard, Ann; Dix, David; Houck, Keith; Elloumi, Fathi; Martin, Matthew; Cathey, Tommy; Transue, Thomas R.; Spencer, Richard; Wolf, Maritja

    2008-11-15

    ACToR (Aggregated Computational Toxicology Resource) is a database and set of software applications that bring into one central location many types and sources of data on environmental chemicals. Currently, the ACToR chemical database contains information on chemical structure, in vitro bioassays and in vivo toxicology assays derived from more than 150 sources including the U.S. Environmental Protection Agency (EPA), Centers for Disease Control (CDC), U.S. Food and Drug Administration (FDA), National Institutes of Health (NIH), state agencies, corresponding government agencies in Canada, Europe and Japan, universities, the World Health Organization (WHO) and non-governmental organizations (NGOs). At the EPA National Center for Computational Toxicology, ACToR helps manage large data sets being used in a high-throughput environmental chemical screening and prioritization program called ToxCast{sup TM}.

  2. ACToR: Aggregated Computational Toxicology Resource (T)

    EPA Science Inventory

    The EPA Aggregated Computational Toxicology Resource (ACToR) is a set of databases compiling information on chemicals in the environment from a large number of public and in-house EPA sources. ACToR has 3 main goals: (1) The serve as a repository of public toxicology information ...

  3. ACToR A Aggregated Computational Toxicology Resource

    EPA Science Inventory

    We are developing the ACToR system (Aggregated Computational Toxicology Resource) to serve as a repository for a variety of types of chemical, biological and toxicological data that can be used for predictive modeling of chemical toxicology.

  4. ACToR A Aggregated Computational Toxicology Resource (S)

    EPA Science Inventory

    We are developing the ACToR system (Aggregated Computational Toxicology Resource) to serve as a repository for a variety of types of chemical, biological and toxicological data that can be used for predictive modeling of chemical toxicology.

  5. More Evidence Diabetes Drug Actos Raises Bladder Cancer Risk a Bit

    MedlinePlus

    ... Johnsen, head of the company's Global Product and Pipeline Communications. Actos and Avandia are thiazolidinediones, a class ... City; Elissa J. Johnsen, head, Global Product and Pipeline Communications, Takeda Pharmaceuticals; Robert Courgi, M.D., endocrinologist, ...

  6. The role of catch-bonds in acto-myosin mechanics and cell mechano-sensitivity

    NASA Astrophysics Data System (ADS)

    Akalp, Umut; Vernerey, Franck J.

    Contraction and spreading of adherent cells are important phenomena in range of cellular processes such as differentiation, morphogenesis, and healing. In this presentation, we propose a novel mechanism of adherent cell mechano-sensing, based on the idea that the contractile acto-myosin machinery behaves as a catch-bond. For this, we construct a simplified model of the acto-myosin structure that constitute the building block of stress fibers and express the stability of cross-bridges in terms of the force-dependent bonding energy of the acto-myosin bond. Consistent with experimental measurements, we then consider that the energy barrier of the acto-myosin bond increases for tension and show that this response is enough to explain the force-induced stabilization of an SF. The resulting model eventually takes the form of a force-sensitive, active visco-elastic material, powered by ATP hydrolysis. The model is used to investigate the organization and contraction of the actin cytoskeleton of cells laying on arrays of microposts. Upon comparison with experimental observations and measurements, simulations show that the catch-bond hypothesis is satisfactory to predict the sensitivity of adherent cells to substrate stiffness as well as the complex organization of the actin cytoskeleton.

  7. Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos.

    PubMed

    Spiró, Zoltán; Thyagarajan, Kalyani; De Simone, Alessandro; Träger, Sylvain; Afshar, Katayoun; Gönczy, Pierre

    2014-07-01

    Regulation of centrosome and spindle positioning is crucial for spatial cell division control. The one-cell Caenorhabditis elegans embryo has proven attractive for dissecting the mechanisms underlying centrosome and spindle positioning in a metazoan organism. Previous work revealed that these processes rely on an evolutionarily conserved force generator complex located at the cell cortex. This complex anchors the motor protein dynein, thus allowing cortical pulling forces to be exerted on astral microtubules emanating from microtubule organizing centers (MTOCs). Here, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain proper levels of CHC-1. We demonstrate that CHC-1 is necessary for normal organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network in otherwise wild-type embryos results in excess centrosome movements resembling those in chc-1(RNAi) embryos. We developed a 2D computational model to simulate cortical rigidity-dependent pulling forces, which recapitulates the experimental data and further demonstrates that excess centrosome movements are produced at medium cortical rigidity values. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension. PMID:24961801

  8. Cross-helix separation of tropomyosin molecules in acto-tropomyosin as determined by neutron scattering.

    PubMed Central

    Bivin, D B; Stone, D B; Schneider, D K; Mendelson, R A

    1991-01-01

    The cross-helix separation of Tm molecules in acto-tropomyosin has been determined using neutron scattering. Deuterated Dictyostelium discoideum actin was density matched in a 93% D2O buffer so that effectively only the protonated tropomyosin was "visible" to neutrons. Analysis of the solution scattering pattern in the region of the first oscillation yielded a value for the cross-helix separation of 7.9 +/- 0.3 nm. The implications of this value for the mechanism of the regulation of muscle contraction are discussed in light of recent results by others. Images FIGURE 2 PMID:1829644

  9. Real space refinement of acto-myosin structures from sectioned muscle.

    PubMed

    Chen, L F; Blanc, E; Chapman, M S; Taylor, K A

    2001-01-01

    We have adapted a real space refinement protocol originally developed for high-resolution crystallographic analysis for use in fitting atomic models of actin filaments and myosin subfragment 1 (S1) to 3-D images of thin-sectioned, plastic-embedded whole muscle. The rationale for this effort is to obtain a refinement protocol that will optimize the fit of the model to the density obtained by electron microscopy and correct for poor geometry introduced during the manual fitting of a high-resolution atomic model into a lower resolution 3-D image. The starting atomic model consisted of a rigor acto-S1 model obtained by X-ray crystallography and helical reconstruction of electron micrographs. This model was rebuilt to fit 3-D images of rigor insect flight muscle at a resolution of 7 nm obtained by electron tomography and image averaging. Our highly constrained real space refinement resulted in modest improvements in the agreement of model and reconstruction but reduced the number of conflicting atomic contacts by 70% without loss of fit to the 3-D density. The methodology seems to be well suited to the derivation of stereochemically reasonable atomic models that are consistent with experimentally determined 3-D reconstructions computed from electron micrographs. PMID:11472093

  10. ACToR Chemical Structure processing using Open Source ChemInformatics Libraries (FutureToxII)

    EPA Science Inventory

    ACToR (Aggregated Computational Toxicology Resource) is a centralized database repository developed by the National Center for Computational Toxicology (NCCT) at the U.S. Environmental Protection Agency (EPA). Free and open source tools were used to compile toxicity data from ove...

  11. Millisecond time resolution electron cryo-microscopy of the M-ATP transient kinetic state of the acto-myosin ATPase.

    PubMed Central

    Walker, M; Trinick, J; White, H

    1995-01-01

    The structure of the AM-ATP transient kinetic state of the acto-myosin ATPase cycle has been examined by electron microscopy using frozen-hydrated specimens prepared in low ionic strength. By spraying grids layered with the acto-S1 complex with ATP immediately before freezing, it was possible to examine the structure of the ternary complex with a time resolution of 10 ms. Disordered binding of the S1 was observed, suggesting more than one attachment geometry. This could be due to the presence of more than one biochemical intermediate, or to a single intermediate binding in more than one conformation. Images FIGURE 2 PMID:7787114

  12. Two-Phase Acto-Cytosolic Fluid Flow in a Moving Keratocyte: A 2D Continuum Model.

    PubMed

    Nikmaneshi, M R; Firoozabadi, B; Saidi, M S

    2015-09-01

    The F-actin network and cytosol in the lamellipodia of crawling cells flow in a centripetal pattern and spout-like form, respectively. We have numerically studied this two-phase flow in the realistic geometry of a moving keratocyte. Cytosol has been treated as a low viscosity Newtonian fluid flowing through the high viscosity porous medium of F-actin network. Other involved phenomena including myosin activity, adhesion friction, and interphase interaction are also discussed to provide an overall view of this problem. Adopting a two-phase coupled model by myosin concentration, we have found new accurate perspectives of acto-cytosolic flow and pressure fields, myosin distribution, as well as the distribution of effective forces across the lamellipodia of a keratocyte with stationary shape. The order of magnitude method is also used to determine the contribution of forces in the internal dynamics of lamellipodia. PMID:26403420

  13. An acto-myosin II constricting ring initiates the fission of activity-dependent bulk endosomes in neurosecretory cells.

    PubMed

    Gormal, Rachel S; Nguyen, Tam H; Martin, Sally; Papadopulos, Andreas; Meunier, Frederic A

    2015-01-28

    Activity-dependent bulk endocytosis allows neurons to internalize large portions of the plasma membrane in response to stimulation. However, whether this critical type of compensatory endocytosis is unique to neurons or also occurs in other excitable cells is currently unknown. Here we used fluorescent 70 kDa dextran to demonstrate that secretagogue-induced bulk endocytosis also occurs in bovine chromaffin cells. The relatively large size of the bulk endosomes found in this model allowed us to investigate how the neck of the budding endosomes constricts to allow efficient recruitment of the fission machinery. Using time-lapse imaging of Lifeact-GFP-transfected chromaffin cells in combination with fluorescent 70 kDa dextran, we detected acto-myosin II rings surrounding dextran-positive budding endosomes. Importantly, these rings were transient and contracted before disappearing, suggesting that they might be involved in restricting the size of the budding endosome neck. Based on the complete recovery of dextran fluorescence after photobleaching, we demonstrated that the actin ring-associated budding endosomes were still connected with the extracellular fluid. In contrast, no such recovery was observed following the constriction and disappearance of the actin rings, suggesting that these structures were pinched-off endosomes. Finally, we showed that the rings were initiated by a circular array of phosphatidylinositol(4,5)bisphosphate microdomains, and that their constriction was sensitive to both myosin II and dynamin inhibition. The acto-myosin II rings therefore play a key role in constricting the neck of budding bulk endosomes before dynamin-dependent fission from the plasma membrane of neurosecretory cells. PMID:25632116

  14. Aggregating data for computational toxicology applications: The U.S. Environmental Protection Agency (EPA) Aggregated Computational Toxicology Resource (ACToR) System.

    PubMed

    Judson, Richard S; Martin, Matthew T; Egeghy, Peter; Gangwal, Sumit; Reif, David M; Kothiya, Parth; Wolf, Maritja; Cathey, Tommy; Transue, Thomas; Smith, Doris; Vail, James; Frame, Alicia; Mosher, Shad; Cohen Hubal, Elaine A; Richard, Ann M

    2012-01-01

    Computational toxicology combines data from high-throughput test methods, chemical structure analyses and other biological domains (e.g., genes, proteins, cells, tissues) with the goals of predicting and understanding the underlying mechanistic causes of chemical toxicity and for predicting toxicity of new chemicals and products. A key feature of such approaches is their reliance on knowledge extracted from large collections of data and data sets in computable formats. The U.S. Environmental Protection Agency (EPA) has developed a large data resource called ACToR (Aggregated Computational Toxicology Resource) to support these data-intensive efforts. ACToR comprises four main repositories: core ACToR (chemical identifiers and structures, and summary data on hazard, exposure, use, and other domains), ToxRefDB (Toxicity Reference Database, a compilation of detailed in vivo toxicity data from guideline studies), ExpoCastDB (detailed human exposure data from observational studies of selected chemicals), and ToxCastDB (data from high-throughput screening programs, including links to underlying biological information related to genes and pathways). The EPA DSSTox (Distributed Structure-Searchable Toxicity) program provides expert-reviewed chemical structures and associated information for these and other high-interest public inventories. Overall, the ACToR system contains information on about 400,000 chemicals from 1100 different sources. The entire system is built using open source tools and is freely available to download. This review describes the organization of the data repository and provides selected examples of use cases. PMID:22408426

  15. Muscle weakness in TPM3-myopathy is due to reduced Ca2+-sensitivity and impaired acto-myosin cross-bridge cycling in slow fibres.

    PubMed

    Yuen, Michaela; Cooper, Sandra T; Marston, Steve B; Nowak, Kristen J; McNamara, Elyshia; Mokbel, Nancy; Ilkovski, Biljana; Ravenscroft, Gianina; Rendu, John; de Winter, Josine M; Klinge, Lars; Beggs, Alan H; North, Kathryn N; Ottenheijm, Coen A C; Clarke, Nigel F

    2015-11-15

    Dominant mutations in TPM3, encoding α-tropomyosinslow, cause a congenital myopathy characterized by generalized muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in 12 TPM3-myopathy patients. We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant α-tropomyosinslow was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant α-tropomyosinslow likely impacts actin-tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant α-tropomyosinslow (R168C) to filamentous actin. Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca(2+)] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca(2+)-sensitivity, at sub-saturating [Ca(2+)] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres. Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca(2+)], and impaired acto-myosin cross-bridge cycling kinetics. Fast myofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca(2+)-sensitivity in TPM3-myopathy patients suggests Ca(2+)-sensitizing drugs may represent a useful treatment for this condition. PMID:26307083

  16. Non-muscle myosin-II-B filament regulation of paracellular resistance in cervical epithelial cells is associated with modulation of the cortical acto-myosin

    PubMed Central

    Li, Xin; Gorodeski, George

    2007-01-01

    Objective To understand myosin regulation of epithelial permeability. Methods Experimental study, using human cervical epithelial cells CaSki. Endpoints were paracellular permeability (determined in terms of transepithelial electrical resistance); non-muscle myosin-II-B (NMM-II-B) cellular localization; NMM-II-B phosphorylation status; NMM-II-B – actin interaction (determined in-vitro by the immunoprecipitation-immunoreactivity method); and NMM-II-B filamentation (determined in-vitro using purified NMM-II-B filaments in terms of filaments disassembly / assembly ratios. Results Treatment of cells with the ROCK inhibitor Y-27632 or with the phosphatase inhibitor okadaic acid decreased the Resistance of the Lateral Intercellular Space (RLIS), and increased phosphorylation of non-muscle myosin-II-B (NMM-II-B) on threonine and serine residues. Y-27632 induced disorganization of the cortical acto-myosin and decreased co-immunoprecipitation of actin with NMM-II-B. Homodimerization assays using NMM-II-B filaments from cells treated with Y-27632 or okadaic acid revealed decreased filamentation compared to control cells. However, okadaic acid blocked Y-27632 decreased filamentation. Treatment with DRB, CK2 inhibitor, induced opposing effects to those of Y-27632 and okadaic acid. Treatment with DRB did not involve modulation of actin depolymerization, suggesting that NMM-II-B regulation of the RLIS was independent of actin polymerization status. Exposure of NMM-II-B filaments to CK2 increased filamentation, regardless of prior treatments in-vivo with Y-27632, okadaic acid, or DRB. Conclusions The results suggest that NMM-II-B filaments are in steady-state equilibrium of phosphorylation-dephosphorylation mediated by CK2 and by ROCK-regulated myosin heavy chain phosphatase, respectively. Increased phosphorylation would tend to inhibit assembly of NMM-II-B filaments and lead to decreased actin-myosin interaction, which would tend to decrease the RLIS and increase the

  17. Structural determinants of cooperativity in acto-myosin interactions.

    PubMed

    Moraczewska, Joanna

    2002-01-01

    Regulation of muscle contraction is a very cooperative process. The presence of tropomyosin on the thin filament is both necessary and sufficient for cooperativity to occur. Data recently obtained with various tropomyosin isoforms and mutants help us to understand better the structural requirements in the thin filament for cooperative protein interactions. Forming an end-to-end overlap between neighboring tropomyosin molecules is not necessary for the cooperativity of the thin filament activation. When direct contacts between tropomyosin molecules are disrupted, the conformational changes in the filament are most probably transmitted cooperatively through actin subunits, although the exact nature of these changes is not known. The function of tropomyosin ends, alternatively expressed in various isoforms, is to confer specific actin affinity. Tropomyosin's affinity or actin is directly related to the size of the apparent cooperative unit defined as the number of actin subunits turned into the active state by binding of one myosin head. Inner sequences of tropomyosin, particularly actin-binding periods 3 to 5, play crucial role in myosin-induced activation of the thin filament. A plausible mechanism of tropomyosin function in this process is that inner tropomyosin regions are either specifically recognized by myosin or they define the right actin conformation required for tropomyosin movement from its blocking position. PMID:12545187

  18. Pioglitazone (Actos) and bladder cancer: Legal system triumphs over the evidence.

    PubMed

    Davidson, Mayer B

    2016-08-01

    In preclinical studies, pioglitazone was associated with bladder cancer in male rats (but not in female rats, mice dogs or monkeys). Because of this association, the Federal Drug Administration requested a large 10year epidemiological study to evaluate whether there was an association between bladder cancer and exposure to pioglitazone in patients. A 5-year interim report published in 2011 showed no significant association between ever vs never exposure to the drug but a significant association in patients exposed to pioglitazone for >2years. Importantly, the final 10year report did not confirm the 5year interim report finding no association between bladder cancer and pioglitazone, even after >4years of exposure to the drug. However, as would be expected, following the 5-year interim report, many epidemiological studies were carried out and civil litigation lawsuits began to be filed. Of the 23 epidemiological studies that have been published to date, 18 showed no association between bladder cancer and pioglitazone (5 with a combination of rosiglitazone and pioglitazone). Of the five that did show a significant association with pioglitazone, three could not be confirmed in the same population and in one of them there were significantly more risk factors for bladder cancer in the patients exposed to pioglitazone. In the fourth one, a significant association became non-significant when patients >79years were included. In the fifth one, detection bias was a major flaw. Currently, >11,000 legal cases have been filed, many of which claim emotional distress due to the fear of bladder cancer. To limit their legal costs, the pharmaceutical company has established a 2.4 billion dollar settlement pool. So much for evidence-based medicine. PMID:27133452

  19. Non-equilibrium phase transition in reconstituted acto-myosin cortices

    NASA Astrophysics Data System (ADS)

    Fakhri, Nikta; Abu Shah, Enas; Malik-Garbi, Maya; Mackintosh, Fred C.; Keren, Kinneret; Schmidt, Christoph F.

    2015-03-01

    The cortical actin cytoskeleton is a quasi 2-D active material in which dynamics are dominated by rapid actin turnover and myosin-driven contractility. Here we present a reconstituted model system that emulates these processes in artificial cell-like compartments. By tuning physical and chemical parameters, we induce a non-equilibrium phase transition. We characterize the local dynamics of these reconstituted cortices by tracking embedded single-walled carbon nanotubes (SWNTs). We create high-resolution maps of the contractile actomyosin flows in a homogenous and during transition to an inhomogeneous steady state. We find evidence that connectivity percolation drives the non-equilibrium phase transition.

  20. Cell-sized liposome doublets reveal active tension build-up driven by acto-myosin dynamics.

    PubMed

    Caorsi, V; Lemière, J; Campillo, C; Bussonnier, M; Manzi, J; Betz, T; Plastino, J; Carvalho, K; Sykes, C

    2016-07-20

    Cells modulate their shape to fulfill specific functions, mediated by the cell cortex, a thin actin shell bound to the plasma membrane. Myosin motor activity, together with actin dynamics, contributes to cortical tension. Here, we examine the individual contributions of actin polymerization and myosin activity to tension increase with a non-invasive method. Cell-sized liposome doublets are covered with either a stabilized actin cortex of preformed actin filaments, or a dynamic branched actin network polymerizing at the membrane. The addition of myosin II minifilaments in both cases triggers a change in doublet shape that is unambiguously related to a tension increase. Preformed actin filaments allow us to evaluate the effect of myosin alone while, with dynamic actin cortices, we examine the synergy of actin polymerization and myosin motors in driving shape changes. Our assay paves the way for a quantification of tension changes triggered by various actin-associated proteins in a cell-sized system. PMID:27378156

  1. Single molecule investigations of DNA looping using the tethered particle method and translocation by acto-myosin using polarized total internal reflection fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Beausang, John F.

    Single molecule biophysics aims to understand biological processes by studying them at the single molecule level in real time. The proteins and nucleic acids under investigation typically exist in an aqueous environment within ˜ ten degrees of room temperature. These seemingly benign conditions are actually quite chaotic at the nanoscale, where single bio-molecules perform their function. As a result, sensitive experiments and statistical analyses are required to separate the weak single molecule signal from its background. Protein-DNA interactions were investigated by monitoring DNA looping events in tethered particle experiments. A new analysis technique, called the Diffusive hidden Markov method, was developed to extract kinetic rate constants from experimental data without any filtering of the raw data; a common step that improves the signal to noise ratio, but at the expense of lower time resolution. In the second system, translocation of the molecular motor myosin along its actin filament track was studied using polarized total internal reflection (polTIRF) microscopy, a technique that determines the orientation and wobble of a single fluorophore attached to the bio-molecule of interest. The range of resolvable angles was increased 4-fold to include a hemisphere of possible orientations. As a result, the handedness of actin filament twirling as it translocated along a myosin-coated surface was determined to be left-handed. The maximum time resolution of a polTIRF setup was increased 50-fold, in part by recording the arrival times and polarization state of single photons using a modified time-correlated single photon counting device. A new analysis, the Multiple Intensity Change Point algorithm, was developed to detect changes in molecular orientation and wobble using the raw time-stamped data with no user-defined bins or thresholds. The analysis objectively identified changes in the orientation of a bifunctional-rhodamine labeled calmodulin that was attached to a myosin V molecule translocating along an actin filament. Long intervals corresponding to stable positions between tilting motions of the lever arm during each step were routinely observed. Substeps in the cycle that preceded these long dwells were measured, but only occasionally most likely because of the low number of photons detected during these rapid events.

  2. Aggregating Data for Computational Toxicology Applications: The U.S. Environmental Protection Agency (EPA) Aggregated Computational Toxicology Resource (ACToR) System

    EPA Science Inventory

    Computational toxicology combines data from high-throughput test methods, chemical structure analyses and other biological domains (e.g., genes, proteins, cells, tissues) with the goals of predicting and understanding the underlying mechanistic causes of chemical toxicity and for...

  3. Phentermine and Topiramate

    MedlinePlus

    ... pressure, irritable bowel disease, mental illness, motion sickness, Parkinson's disease, ulcers, or urinary problems; medications for seizures such as carbamazepine (Carbatrol, Equetro, Tegretol), phenytoin (Dilantin), or valproic acid (Stavzor, Depakene); pioglitazone (Actos, ...

  4. Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.

    PubMed Central

    Kim, E; Miller, C J; Motoki, M; Seguro, K; Muhlrad, A; Reisler, E

    1996-01-01

    Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin. Images FIGURE 7 FIGURE 8 PMID:8785300

  5. Kinetics of muscle contraction and actomyosin NTP hydrolysis from rabbit using a series of metal–nucleotide substrates

    PubMed Central

    Burton, Kevin; White, Howard; Sleep, John

    2005-01-01

    Mechanical properties of skinned single fibres from rabbit psoas muscle have been correlated with biochemical steps in the cross-bridge cycle using a series of metal–nucleotide (Me·NTP) substrates (Mn2+ or Ni2+ substituted for Mg2+; CTP or ITP for ATP) and inorganic phosphate. Measurements were made of the rate of force redevelopment following (1) slack tests in which force recovery followed a period of unloaded shortening, or (2) ramp shortening at low load terminated by a rapid restretch. The form and rate of force recovery were described as the sum of two exponential functions. Actomyosin-Subfragment 1 (acto-S1) Me·NTPase activity and Me·NDP release were monitored under the same conditions as the fibre experiments. Mn·ATP and Mg·CTP both supported contraction well and maintained good striation order. Relative to Mg·ATP, they increased the rates and Me·NTPase activity of cross-linked acto-S1 and the fast component of a double-exponential fit to force recovery by ∼50% and 10–35%, respectively, while shortening velocity was moderately reduced (by 20–30%). Phosphate also increased the rate of the fast component of force recovery. In contrast to Mn2+ and CTP, Ni·ATP and Mg·ITP did not support contraction well and caused striations to become disordered. The rates of force recovery and Me·NTPase activity were less than for Mg·ATP (by 40–80% and 50–85%, respectively), while shortening velocity was greatly reduced (by ∼80%). Dissociation of ADP from acto-S1 was little affected by Ni2+, suggesting that Ni·ADP dissociation does not account for the large reduction in shortening velocity. The different effects of Ni2+ and Mn2+ were also observed during brief activations elicited by photolytic release of ATP. These results confirm that at least one rate-limiting step is shared by acto-S1 ATPase activity and force development. Our results are consistent with a dual rate-limitation model in which the rate of force recovery is limited by both NTP

  6. Roles of paxillin family members in adhesion and ECM degradation coupling at invadosomes.

    PubMed

    Petropoulos, Christos; Oddou, Christiane; Emadali, Anouk; Hiriart-Bryant, Edwige; Boyault, Cyril; Faurobert, Eva; Vande Pol, Scott; Kim-Kaneyama, Joo-Ri; Kraut, Alexandra; Coute, Yohann; Block, Marc; Albiges-Rizo, Corinne; Destaing, Olivier

    2016-06-01

    Invadosomes are acto-adhesive structures able to both bind the extracellular matrix (ECM) and digest it. Paxillin family members-paxillin, Hic-5, and leupaxin-are implicated in mechanosensing and turnover of adhesion sites, but the contribution of each paxillin family protein to invadosome activities is unclear. We use genetic approaches to show that paxillin and Hic-5 have both redundant and distinctive functions in invadosome formation. The essential function of paxillin-like activity is based on the coordinated activity of LD motifs and LIM domains, which support invadosome assembly and morphology, respectively. However, paxillin preferentially regulates invadosome assembly, whereas Hic-5 regulates the coupling between ECM degradation and acto-adhesive functions. Mass spectrometry analysis revealed new partners that are important for paxillin and Hic-5 specificities: paxillin regulates the acto-adhesive machinery through janus kinase 1 (JAK1), whereas Hic-5 controls ECM degradation via IQGAP1. Integrating the redundancy and specificities of paxillin and Hic-5 in a functional complex provides insights into the coupling between the acto-adhesive and ECM-degradative machineries in invadosomes. PMID:27269065

  7. DSSTOX PROJECT UPDATE: SUPPORTING IMPROVED TOXICO-CHEMOINFORMATICS CAPABILITIES.

    EPA Science Inventory

    DSSTox is serving as a source of high quality structure-annotated toxicity and EPA data files for the new Aggregated Computational Toxicology Resource (ACToR) data repository, under development within the EPA NCCT, which will house in an integrated platform multiple domains of to...

  8. The Barrio Endowment to American Literature.

    ERIC Educational Resources Information Center

    Lint, Robert G.

    The emergence in Chicano poetry of the language experiments and folk wisdom of the oral traditions of barrio cuentos, corridos, dichos, and actos reveals that this literature is neither fleeting nor novel. Jose Montoya's "Resonant Valley" exemplifies the Chicano's preservation of ancient wisdom and practice in new formal experiments and…

  9. 48 CFR 252.229-7005 - Tax exemptions (Spain).

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Transfer Tax). (4) Impuesto Sobre el Lujo (Luxury Tax). (5) Actos Juridocos Documentados (Legal Official Transactions). (6) Impuesto Sobre el Trafico de Empresas (Business Trade Tax). (7) Impuestos Especiales de Fabricacion (Special Products Tax). (8) Impuesto Sobre el Petroleo y Derivados (Tax on Petroleum and its...

  10. ToxRefDB - Release user-friendly web-based tool for mining ToxRefDB

    EPA Science Inventory

    ACToR links to a chemical toxicity reference database called ToxRefDB (http://actor.epa.gov/toxrefdb) which allows scientists and the interested public to search and download thousands of toxicity testing results on hundreds of chemicals. ToxRefDB provides detailed chemical toxic...

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

  12. The Toxicity Data Landscape for Environmental Chemicals

    PubMed Central

    Judson, Richard; Richard, Ann; Dix, David J.; Houck, Keith; Martin, Matthew; Kavlock, Robert; Dellarco, Vicki; Henry, Tala; Holderman, Todd; Sayre, Philip; Tan, Shirlee; Carpenter, Thomas; Smith, Edwin

    2009-01-01

    Objective Thousands of chemicals are in common use, but only a portion of them have undergone significant toxicologic evaluation, leading to the need to prioritize the remainder for targeted testing. To address this issue, the U.S. Environmental Protection Agency (EPA) and other organizations are developing chemical screening and prioritization programs. As part of these efforts, it is important to catalog, from widely dispersed sources, the toxicology information that is available. The main objective of this analysis is to define a list of environmental chemicals that are candidates for the U.S. EPA screening and prioritization process, and to catalog the available toxicology information. Data sources We are developing ACToR (Aggregated Computational Toxicology Resource), which combines information for hundreds of thousands of chemicals from > 200 public sources, including the U.S. EPA, National Institutes of Health, Food and Drug Administration, corresponding agencies in Canada, Europe, and Japan, and academic sources. Data extraction ACToR contains chemical structure information; physical–chemical properties; in vitro assay data; tabular in vivo data; summary toxicology calls (e.g., a statement that a chemical is considered to be a human carcinogen); and links to online toxicology summaries. Here, we use data from ACToR to assess the toxicity data landscape for environmental chemicals. Data synthesis We show results for a set of 9,912 environmental chemicals being considered for analysis as part of the U.S. EPA ToxCast screening and prioritization program. These include high-and medium-production-volume chemicals, pesticide active and inert ingredients, and drinking water contaminants. Conclusions Approximately two-thirds of these chemicals have at least limited toxicity summaries available. About one-quarter have been assessed in at least one highly curated toxicology evaluation database such as the U.S. EPA Toxicology Reference Database, U.S. EPA Integrated

  13. Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin, ATM1*

    PubMed Central

    Haraguchi, Takeshi; Tominaga, Motoki; Matsumoto, Rie; Sato, Kei; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2014-01-01

    Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg2+-ATPase activity (Vmax = 4 s−1), although their affinities for actin were high (Kactin = 4 μm). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s−1, respectively). Physiological concentrations of free Mg2+ modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis. PMID:24637024

  14. The multi-faceted role of the actin cap in cellular mechanosensation and mechanotransduction.

    PubMed

    Kim, Dong-Hwee; Chambliss, Allison B; Wirtz, Denis

    2013-06-21

    The perinuclear actin cap (or actin cap) is a recently characterized cytoskeletal organelle composed of thick, parallel, and highly contractile acto-myosin filaments that are specifically anchored to the apical surface of the interphase nucleus. The actin cap is present in a wide range of adherent eukaryotic cells, but is disrupted in several human diseases, including laminopathies and cancer. Through its large terminating focal adhesions and anchorage to the nuclear lamina and nuclear envelope through LINC complexes, the perinuclear actin cap plays a critical role both in mechanosensation and mechanotransduction, the ability of cells to sense changes in matrix compliance and to respond to mechanical forces, respectively. PMID:23930135

  15. Changing the calculus of pediatric product development: narrowing the too-big gap between need and solution in a small market.

    PubMed

    Campbell, Sarah

    2015-01-01

    First, Deliece Hofen drops the pill into hot water to soften the outside coating. Then, she slices through the center of the pill with an X-ACTO knife and squeezes the isotretinoin inside into a syringe. With the drug in liquid form, she can now administer it to her ten-year-old son Brandon. ?The problem is...some of [the isotretinoin] is still left inside of the capsule and I?m not getting an exact dosage,? admits Hofen. PMID:25974908

  16. Model for Probing Membrane-Cortex Adhesion by Micropipette Aspiration and Fluctuation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Alert, Ricard; Casademunt, Jaume; Brugués, Jan; Sens, Pierre

    2015-04-01

    We propose a model for membrane-cortex adhesion which couples membrane deformations, hydrodynamics and kinetics of membrane-cortex ligands. In its simplest form, the model gives explicit predictions for the critical pressure for membrane detachment and for the value of adhesion energy. We show that these quantities exhibit a significant dependence on the active acto-myosin stresses. The model provides a simple framework to access quantitative information on cortical activity by means of micropipette experiments. We also extend the model to incorporate fluctuations and show that detailed information on the stability of membrane-cortex coupling can be obtained by a combination of micropipette aspiration and fluctuation spectroscopy measurements.

  17. BIOÉTICA EN NICARAGUA

    PubMed Central

    Gonzálezy, Armando Ulloa; Monge, Melba de la Cruz Barrantes

    2009-01-01

    Este trabajo describe la situación de la bioética en Nicaragua, caracterizando las circunstancias y el contexto de las actividades de educación médica y las unidades prestadoras de servicios de salud. El desarrollo de un nuevo modelo de atención integral en salud, la implementación de políticas de salud que garanticen a la población el mayor acceso y gratuidad a los servicios, y los cambios acontecidos en los cuidados médicos, debidos en parte al reconocimiento creciente de una mayor autonomía de los pacientes y al uso creciente de nuevas tecnologías médicas, hace que se presenten algunas limitantes y dilemas en las unidades asistenciales y entre el personal de salud. La bioética en Nicaragua tiene un desarrollo incipiente: no está institucionalizada ni se han previsto los mecanismos formales que permitan resolver los problemas éticamente complejos, por lo tanto, constituye un gran reto por parte de las instituciones educativas y rectoras de la salud. PMID:20352016

  18. Evidence against essential roles for subdomain 1 of actin in actomyosin sliding movements

    SciTech Connect

    Siddique, Md. Shahjahan P.; Miyazaki, Takashi; Katayama, Eisaku; Uyeda, Taro Q.P.; Suzuki, Makoto . E-mail: msuzuki@material.tohoku.ac.jp

    2005-07-01

    We have engineered acto-S1chimera proteins carrying the entire actin inserted in loop 2 of the motor domain of Dictyostelium myosin II with 24 or 18 residue-linkers (CP24 and CP18, respectively). These proteins were capable of self-polymerization as well as copolymerization with skeletal actin and exhibited rigor-like structures. The MgATPase rate of CP24-skeletal actin copolymer was 1.06 s{sup -1}, which is slightly less than the V {sub max} of Dictyostelium S1. Homopolymer filaments of skeletal actin, CP24, and CP18 moved at 4.7 {+-} 0.6, 2.9 {+-} 0.6, and 4.1 {+-} 0.8 {mu}m/s (mean {+-} SD), respectively, on coverslips coated with skeletal myosin at 27 deg C. Statistically thermodynamic considerations suggest that the S1 portion of chimera protein mostly resides on subdomain 1 (SD-1) of the actin portion even in the presence of ATP. This and the fact that filaments of CP18 with shorter linkers moved faster than CP24 filaments suggest that SD-1 might not be as essential as conventionally presumed for actomyosin sliding interactions.

  19. Synthetic polyamines: new compounds specific to actin dynamics for mammalian cell and fission yeast.

    PubMed

    Riveline, Daniel; Thiagarajan, Raghavan; Lehn, Jean-Marie; Carlier, Marie-France

    2014-01-01

    Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research. PMID:25664996

  20. Synthetic polyamines: new compounds specific to actin dynamics for mammalian cell and fission yeast

    PubMed Central

    Riveline, Daniel; Thiagarajan, Raghavan; Lehn, Jean-Marie; Carlier, Marie-France

    2014-01-01

    Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research. PMID:25664996

  1. Del sujeto que ha intentado suicidarse y el Otro: la Institución Psiquiátrica

    PubMed Central

    Liliana, Mondragón B.; Miguel Ángel, Caballero G.

    2009-01-01

    El hospital psiquiátrico se ha constituido como un lugar donde se posibilita legitimar la exclusión y la radicalidad de ese otro “que no es igual”, como es el caso del sujeto que atenta contra su propia vida. En consecuencia, el intento de suicidio desde el pensamiento foucaultiano, es una resistencia que desmantela la estructura de dominación a través de un acto de poder, el cual se ejerce sobre el propio cuerpo. Así, la intención de este texto es mostrar que la relación Otro-otro es un lugar en la estructura subjetiva, que se deposita en la institución psiquiátrica, la cual representa el poder, la ley, y es aquello a lo que se le quiere agredir, resistir, abatir con un intento de suicidio. Para demostrar como se materializan estos hechos, se exponen los testimonios de tres adolescentes atendidas en un hospital psiquiátrico por intentos suicidas, en los cuales se señalan los diferentes significados que le atribuyen a la institución psiquiátrica en tanto que representa un Otro en sus actos autoinfligidos. PMID:25400324

  2. Cell shape, spreading symmetry, and the polarization of stress-fibers in cells

    NASA Astrophysics Data System (ADS)

    Zemel, A.; Rehfeldt, F.; Brown, A. E. X.; Discher, D. E.; Safran, S. A.

    2010-05-01

    The active regulation of cellular forces during cell adhesion plays an important role in the determination of cell size, shape, and internal structure. While on flat, homogeneous and isotropic substrates some cells spread isotropically, others spread anisotropically and assume elongated structures. In addition, in their native environment as well as in vitro experiments, the cell shape and spreading asymmetry can be modulated by the local distribution of adhesive molecules and topography of the environment. We present a simple elastic model and experiments on stem cells to explain the variation of cell size with the matrix rigidity. In addition, we predict the experimental consequences of two mechanisms of acto-myosin polarization and focus here on the effect of the cell spreading asymmetry on the regulation of the stress-fiber alignment in the cytoskeleton. We show that when cell spreading is sufficiently asymmetric the alignment of acto-myosin forces in the cell increases monotonically with the matrix rigidity; however, in general this alignment is non-monotonic, as shown previously. These results highlight the importance of the symmetry characteristics of cell spreading in the regulation of cytoskeleton structure and suggest a mechanism by which different cell types may acquire different morphologies and internal structures in different mechanical environments.

  3. Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants.

    PubMed

    Zhou, Zhiqun; Huang, Wenrui; Liang, Jingsheng; Szczesna-Cordary, Danuta

    2016-01-01

    The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1(+∕+)-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca(2+) sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady

  4. Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants

    PubMed Central

    Zhou, Zhiqun; Huang, Wenrui; Liang, Jingsheng; Szczesna-Cordary, Danuta

    2016-01-01

    The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1+∕+-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca2+ sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady-state and

  5. Live cell tracking of symmetry break in actin cytoskeleton triggered by abrupt changes in micromechanical environments.

    PubMed

    Inoue, S; Frank, V; Hörning, M; Kaufmann, S; Yoshikawa, H Y; Madsen, J P; Lewis, A L; Armes, S P; Tanaka, M

    2015-12-01

    With the aid of stimulus-responsive hydrogel substrates composed of ABA triblock copolymer micelles, we monitored the morphological dynamics of myoblast (C2C12) cells in response to an abrupt change in the substrate elasticity by live cell imaging. The remodeling of actin cytoskeletons could be monitored by means of transient transfection with LifeAct-GFP. Dynamic changes in the orientational order of actin filaments were characterized by an order parameter, which enables one to generalize the mechanically induced actin cytoskeletons as a break of symmetry. The critical role that acto-myosin complexes play in the morphological transition was verified by the treatment of cells with myosin II inhibitor (blebbistatin) and the fluorescence localization of focal adhesion contacts. Such dynamically tunable hydrogels can be utilized as in vitro cellular micro-environments that can exert time-dependent stimuli to mechanically regulate target cells. PMID:26347909

  6. RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells

    PubMed Central

    Chia, Joanne; Goh, Germaine; Racine, Victor; Ng, Susanne; Kumar, Pankaj; Bard, Frederic

    2012-01-01

    The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome- and phosphatome-wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto-myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells. PMID:23212246

  7. Cell and tissue mechanics in cell migration.

    PubMed

    Lange, Janina R; Fabry, Ben

    2013-10-01

    Migrating cells generate traction forces to counteract the movement-resisting forces arising from cell-internal stresses and matrix adhesions. In the case of collective migration in a cell colony, or in the case of 3-dimensional migration through connective tissue, movement-resisting forces arise also from external stresses. Although the deformation of a stiffer cell or matrix causes larger movement-resisting forces, at the same time a larger stiffness can also promote cell migration due to a feedback between forces, deformations, and deformation speed that is mediated by the acto-myosin contractile machinery of cells. This mechanical feedback is also important for stiffness sensing, durotaxis, plithotaxis, and collective migration in cell colonies. PMID:23664834

  8. Modeling traction forces in collective cell migration

    NASA Astrophysics Data System (ADS)

    Zimmermann, Juliane; Basan, Markus; Hayes, Ryan L.; Rappel, Wouter-Jan; Levine, Herbert

    2015-03-01

    Collective cell migration is an important process in embryonic development, wound healing, and cancer metastasis. We have developed a particle-based simulation for collective cell migration that describes flow patterns and finger formation at the tissue edge observed in wound healing experiments. We can apply methods for calculating intercellular stress to our simulation model, and have thereby provided evidence for the validity of a stress reconstitution method from traction forces used in experiments. To accurately capture experimentally measured traction forces and stresses in the tissue, which are mostly tensile, we have to include intracellular acto-myosin contraction into our simulation. We can then reproduce the experimentally observed behavior of cells moving around a circular obstacle, and suggest underlying mechanisms for cell-cell alignment and generation of traction force patterns.

  9. Contractile activity is required for Z-disc sarcomere maturation in vivo

    PubMed Central

    Geach, Timothy J; Hirst, Elizabeth MA; Zimmerman, Lyle B

    2015-01-01

    Sarcomere structure underpins structural integrity, signaling, and force transmission in the muscle. In embryos of the frog Xenopus tropicalis, muscle contraction begins even while sarcomerogenesis is ongoing. To determine whether contractile activity plays a role in sarcomere formation in vivo, chemical tools were used to block acto-myosin contraction in embryos of the frog X. tropicalis, and Z-disc assembly was characterized in the paralyzed dicky ticker mutant. Confocal and ultrastructure analysis of paralyzed embryos showed delayed Z-disc formation and defects in thick filament organization. These results suggest a previously undescribed role for contractility in sarcomere maturation in vivo. genesis 53:299–307, 2015. © 2015 The Authors. Genesis Published by Wiley Periodicals, Inc. PMID:25845369

  10. Growth, collapse, and stalling in a mechanical model for neurite motility

    NASA Astrophysics Data System (ADS)

    Recho, Pierre; Jerusalem, Antoine; Goriely, Alain

    2016-03-01

    Neurites, the long cellular protrusions that form the routes of the neuronal network, are capable of actively extending during early morphogenesis or regenerating after trauma. To perform this task, they rely on their cytoskeleton for mechanical support. In this paper, we present a three-component active gel model that describes neurites in the three robust mechanical states observed experimentally: collapsed, static, and motile. These states arise from an interplay between the physical forces driven by growth of the microtubule-rich inner core of the neurite and the acto-myosin contractility of its surrounding cortical membrane. In particular, static states appear as a mechanical traction or compression balance of these two parallel structures. The model predicts how the response of a neurite to a towing force depends on the force magnitude and recovers the response of neurites to several drug treatments that modulate the cytoskeleton active and passive properties.

  11. Still and rotating myosin clusters determine cytokinetic ring constriction

    PubMed Central

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

    2016-01-01

    The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo. PMID:27363521

  12. Elasticity of adherent active cells on a compliant substrate

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya; Mertz, Aaron F.; Dufresne, Eric R.; Marchetti, M. Cristina

    2012-02-01

    We present a continuum mechanical model of rigidity sensing by livings cells adhering to a compliant substrate. The cell or cell colony is modeled as an elastic active gel, adapting recently developed continuum theories of active viscoelastic fluids. The coupling to the substrate enters as a boundary condition that relates the cell's deformation field to local stress gradients. In the presence of activity, the substrate induces spatially inhomogeneous contractile stresses and deformations, with a power law dependence of the total traction forces on cell or colony size. This is in agreement with recent experiments on keratinocyte colonies adhered to fibronectin coated surfaces. In the presence of acto-myosin activity, the substrate also enhances the cell polarization, breaking the cell's front-rear symmetry. Maximal polarization is observed when the substrate stiffness matches that of the cell, in agreement with experiments on stem cells.

  13. Macrophage engulfment of a cell or nanoparticle is regulated by unavoidable opsonization, a species-specific ‘Marker of Self’ CD47, and target physical properties

    PubMed Central

    Sosale, Nisha G; Spinler, Kyle R; Alvey, Cory; Discher, Dennis E

    2015-01-01

    Professional phagocytes of the mononuclear phagocyte system (MPS), especially ubiquitous macrophages, are commonly thought to engulf or not a target based strictly on ‘eat me’ molecules such as Antibodies. The target might be a viable ‘self’ cell or a drug-delivering nanoparticle, or it might be a cancer cell or a microbe. ‘Marker of Self’ CD47 signals into a macrophage to inhibit the acto-myosin cytoskeleton that makes engulfment efficient. In adhesion of any cell, the same machinery is generally activated by rigidity of target surfaces, and recent results confirm phagocytosis is likewise driven by the rigidity typical of microbes and many synthetics. Basic insights are already being applied in order to make macrophages eat cancer or to delay nanoparticle clearance for better drug delivery and imaging. PMID:26172292

  14. Still and rotating myosin clusters determine cytokinetic ring constriction.

    PubMed

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

    2016-01-01

    The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo. PMID:27363521

  15. Model for probing membrane-cortex adhesion by micropipette aspiration and fluctuation spectroscopy.

    PubMed

    Alert, Ricard; Casademunt, Jaume; Brugués, Jan; Sens, Pierre

    2015-04-21

    We propose a model for membrane-cortex adhesion that couples membrane deformations, hydrodynamics, and kinetics of membrane-cortex ligands. In its simplest form, the model gives explicit predictions for the critical pressure for membrane detachment and for the value of adhesion energy. We show that these quantities exhibit a significant dependence on the active acto-myosin stresses. The model provides a simple framework to access quantitative information on cortical activity by means of micropipette experiments. We also extend the model to incorporate fluctuations and show that detailed information on the stability of membrane-cortex coupling can be obtained by a combination of micropipette aspiration and fluctuation spectroscopy measurements. PMID:25902428

  16. Cell and tissue mechanics in cell migration

    PubMed Central

    Lange, Janina R.; Fabry, Ben

    2013-01-01

    Migrating cells generate traction forces to counteract the movement-resisting forces arising from cell-internal stresses and matrix adhesions. In the case of collective migration in a cell colony, or in the case of 3-dimensional migration through connective tissue, movement-resisting forces arise also from external stresses. Although the deformation of a stiffer cell or matrix causes larger movement-resisting forces, at the same time a larger stiffness can also promote cell migration due to a feedback between forces, deformations, and deformation speed that is mediated by the acto-myosin contractile machinery of cells. This mechanical feedback is also important for stiffness sensing, durotaxis, plithotaxis, and collective migration in cell colonies. PMID:23664834

  17. Molecular control of fission yeast cytokinesis.

    PubMed

    Rincon, Sergio A; Paoletti, Anne

    2016-05-01

    Cytokinesis gives rise to two independent daughter cells at the end of the cell division cycle. The fission yeast Schizosaccharomyces pombe has emerged as one of the most powerful systems to understand how cytokinesis is controlled molecularly. Like in most eukaryotes, fission yeast cytokinesis depends on an acto-myosin based contractile ring that assembles at the division site under the control of spatial cues that integrate information on cell geometry and the position of the mitotic apparatus. Cytokinetic events are also tightly coordinated with nuclear division by the cell cycle machinery. These spatial and temporal regulations ensure an equal cleavage of the cytoplasm and an accurate segregation of the genetic material in daughter cells. Although this model system has specificities, the basic mechanisms of contractile ring assembly and function deciphered in fission yeast are highly valuable to understand how cytokinesis is controlled in other organisms that rely on a contractile ring for cell division. PMID:26806637

  18. Auxiliary control systems for Pachmarhi array of Cverenkov telescopes

    NASA Astrophysics Data System (ADS)

    Gothe, K. S.; Acharya, B. S.; Bhat, P. N.; Chitnis, V. R.; D'Souza, A. I.; Francis, P. J.; John, A. V.; Joshi, S. R.; Majumdar, P.; Nagesh, B. K.; Pose, M. S.; Purohit, P. N.; Rahman, M. A.; Rao, K. K.; Rao, S. K.; Sharma, S. K.; Singh, B. B.; Stanislaus, A. J.; Sudershanan, P. V.; Upadhya, S. S.; Venkateshmurthy, B. L.; Vishwanath, P. R.

    2002-03-01

    Pachmarhi Array of Cverenkov Telescopes (PACT) consists of 25 Telescopes deployed over an area of 100m x 80m. The experiment is based on atmospheric Cverenkov technique to detect Very High Energy celestial gamma-rays using wavefront sampling method. Each telescope consists of 7 large area parabolic mirrors mounted para-axially on an equatorial mount and a fast photo-multiplier tube at the focus of each mirror. For efficient operation of the experiment 3 automated control systems were developed and installed, viz. Automated Computerized Telescope Orientation System (ACTOS) to control the pointing and tracking of individual telescopes, Automatic Photo-multiplier Exposure System (APES) to facilitate the exposure of photo-tubes only during observations, and Computerized Automated Rate Adjustment and Monitoring System (CARAMS) to ensure uniform gains for all the phototube - mirror systems. The design features and performance of each of these systems are discussed.

  19. Continuous Binary Ink Jets: New Output Choice For Computer Imaging Systems.

    NASA Astrophysics Data System (ADS)

    Thompson, Tad

    1987-04-01

    Significant advances in system reliability and droplet control, resulting in improved image quality, have opened up new opportunities for continuous-flow ink jet technology -a relatively mature non-impact printing technique. These opportunities exist within the graphic arts industry--an industry experiencing an upheaval in its fundamental methods of operation because of the increasing use of powerful, highly sophisticated computer systems for both the design and creation of images and the electronic manipulation of images and text in preparation for high volume printing. High resolution computer systems are increasingly supplanting much of the manual, crafts-oriented nature of the business. Skilled graphic design and production personnel are exchanging keyboards, function menus and electronic pucks for drawing tables, T-squares, and X-acto knives.

  20. Local mechanical properties of bladder cancer cells measured by AFM as a signature of metastatic potential

    NASA Astrophysics Data System (ADS)

    Abidine, Y.; Laurent, V. M.; Michel, R.; Duperray, A.; Verdier, C.

    2015-10-01

    The rheological properties of bladder cancer cells of different invasivities have been investigated using a microrheological technique well adapted in the range [1-300Hz] of interest to understand local changes in the cytoskeleton microstructure, in particular actin fibres. Drugs disrupting actin and acto-myosin functions were used to study the resistance of such cancer cells. Results on a variety of cell lines were fitted with a model revealing the importance of two parameters, the elastic shear plateau modulus G N 0 as well as the glassy transition frequency f T. These parameters are good markers for invasiveness, with the notable exception of the cell periphery, which is stiffer for less invasive cells, and could be of importance in cancer metastasis.

  1. NOS1 induces NADPH oxidases and impairs contraction kinetics in aged murine ventricular myocytes.

    PubMed

    Villmow, Marten; Klöckner, Udo; Heymes, Christophe; Gekle, Michael; Rueckschloss, Uwe

    2015-09-01

    Nitric oxide (NO) modulates calcium transients and contraction of cardiomyocytes. However, it is largely unknown whether NO contributes also to alterations in the contractile function of cardiomyocytes during aging. Therefore, we analyzed the putative role of nitric oxide synthases and NO for the age-related alterations of cardiomyocyte contraction. We used C57BL/6 mice, nitric oxide synthase 1 (NOS1)-deficient mice (NOS1(-/-)) and mice with cardiomyocyte-specific NOS1-overexpression to analyze contractions, calcium transients (Indo-1 fluorescence), acto-myosin ATPase activity (malachite green assay), NADPH oxidase activity (lucigenin chemiluminescence) of isolated ventricular myocytes and cardiac gene expression (Western blots, qPCR). In C57BL/6 mice, cardiac expression of NOS1 was upregulated by aging. Since we found a negative regulation of NOS1 expression by cAMP in isolated cardiomyocytes, we suggest that reduced efficacy of β-adrenergic signaling that is evident in aged hearts promotes upregulation of NOS1. Shortening and relengthening of cardiomyocytes from aged C57BL/6 mice were decelerated, but were normalized by pharmacological inhibition of NOS1/NO. Cardiomyocytes from NOS1(-/-) mice displayed no age-related changes in contraction, calcium transients or acto-myosin ATPase activity. Aging increased cardiac expression of NADPH oxidase subunits NOX2 and NOX4 in C57BL/6 mice, but not in NOS1(-/-) mice. Similarly, cardiac expression of NOX2 and NOX4 was upregulated in a murine model with cardiomyocyte-specific overexpression of NOS1. We conclude that age-dependently upregulated NOS1, putatively via reduced efficacy of β-adrenergic signaling, induces NADPH oxidases. By increasing nitrosative and oxidative stress, both enzyme systems act synergistically to decelerate contraction of aged cardiomyocytes. PMID:26173391

  2. The integrity of the cytokinesis machinery under stress conditions requires the glucan synthase Bgs1p and its regulator Cfh3p.

    PubMed

    Sharifmoghadam, Mohammad Reza; Curto, M-Ángeles; Hoya, Marta; de León, Nagore; Martin-Garcia, Rebeca; Doncel, Cristina; Valdivieso, M-Henar

    2012-01-01

    In yeast, cytokinesis requires coordination between nuclear division, acto-myosin ring contraction, and septum synthesis. We studied the role of the Schizosaccharomyces pombe Bgs1p and Cfh3p proteins during cytokinesis under stress conditions. Cfh3p formed a ring in the septal area that contracted during mitosis; Cfh3p colocalized and co-immunoprecipitated with Cdc15p, showing that Cfh3p interacted with the contractile acto-myosin ring. In a wild-type strain, a significant number of contractile rings collapsed under stress conditions and this number increased dramatically in the cfh3Δ, bgs1cps1-191, and cfh3Δ bgs1/cps1-191. Our results show that after osmotic shock Cfh3p is essential for the stability of the (1,3) glucan synthase Bgs1p in the septal area, but not at the cell poles. Finally, cells adapted to stress; they repaired their contractile rings and re-localized Bgs1p to the cell surface some time after osmotic shock. A detailed analysis of the cytokinesis machinery in the presence of KCl revealed that the actomyosin ring collapsed before Bgs1p was internalized, and that it was repaired before Bgs1p re-localized to the cell surface. In the cfh3Δ, bgs1/cps1-191, and cfh3Δ bgs1/cps1-191 mutants, which have reduced glucan synthesis, the damage produced to the ring had stronger consequences, suggesting that an intact primary septum contributes to ring stability. The results show that the contractile actomyosin ring is very sensitive to stress, and that cells have efficient mechanisms to remedy the damage produced in this structure. PMID:22905165

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

    PubMed Central

    Borejdo, J; Ortega, H

    1989-01-01

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

  4. Advancing Edge Speeds of Epithelial Monolayers Depend on Their Initial Confining Geometry.

    PubMed

    Kollimada, Somanna A; Kulkarni, Ankur H; Ravan, Aniket; Gundiah, Namrata

    2016-01-01

    Collective cell migrations are essential in several physiological processes and are driven by both chemical and mechanical cues. The roles of substrate stiffness and confinement on collective migrations have been investigated in recent years, however few studies have addressed how geometric shapes influence collective cell migrations. Here, we address the hypothesis that the relative position of a cell within the confinement influences its motility. Monolayers of two types of epithelial cells--MCF7, a breast epithelial cancer cell line, and MDCK, a control epithelial cell line--were confined within circular, square, and cross-shaped stencils and their migration velocities were quantified upon release of the constraint using particle image velocimetry. The choice of stencil geometry allowed us to investigate individual cell motility within convex, straight and concave boundaries. Cells located in sharp, convex boundaries migrated at slower rates than those in concave or straight edges in both cell types. The overall cluster migration occurred in three phases: an initial linear increase with time, followed by a plateau region and a subsequent decrease in cluster speeds. An acto-myosin contractile ring, present in the MDCK but absent in MCF7 monolayer, was a prominent feature in the emergence of leader cells from the MDCK clusters which occurred every ~125 μm from the vertex of the cross. Further, coordinated cell movements displayed vorticity patterns in MDCK which were absent in MCF7 clusters. We also used cytoskeletal inhibitors to show the importance of acto-myosin bounding cables in collective migrations through translation of local movements to create long range coordinated movements and the creation of leader cells within ensembles. To our knowledge, this is the first demonstration of how bounding shapes influence long-term migratory behaviours of epithelial cell monolayers. These results are important for tissue engineering and may also enhance our

  5. Advancing Edge Speeds of Epithelial Monolayers Depend on Their Initial Confining Geometry

    PubMed Central

    Kollimada, Somanna A.; Kulkarni, Ankur H.; Ravan, Aniket; Gundiah, Namrata

    2016-01-01

    Collective cell migrations are essential in several physiological processes and are driven by both chemical and mechanical cues. The roles of substrate stiffness and confinement on collective migrations have been investigated in recent years, however few studies have addressed how geometric shapes influence collective cell migrations. Here, we address the hypothesis that the relative position of a cell within the confinement influences its motility. Monolayers of two types of epithelial cells—MCF7, a breast epithelial cancer cell line, and MDCK, a control epithelial cell line—were confined within circular, square, and cross-shaped stencils and their migration velocities were quantified upon release of the constraint using particle image velocimetry. The choice of stencil geometry allowed us to investigate individual cell motility within convex, straight and concave boundaries. Cells located in sharp, convex boundaries migrated at slower rates than those in concave or straight edges in both cell types. The overall cluster migration occurred in three phases: an initial linear increase with time, followed by a plateau region and a subsequent decrease in cluster speeds. An acto-myosin contractile ring, present in the MDCK but absent in MCF7 monolayer, was a prominent feature in the emergence of leader cells from the MDCK clusters which occurred every ~125 μm from the vertex of the cross. Further, coordinated cell movements displayed vorticity patterns in MDCK which were absent in MCF7 clusters. We also used cytoskeletal inhibitors to show the importance of acto-myosin bounding cables in collective migrations through translation of local movements to create long range coordinated movements and the creation of leader cells within ensembles. To our knowledge, this is the first demonstration of how bounding shapes influence long-term migratory behaviours of epithelial cell monolayers. These results are important for tissue engineering and may also enhance our

  6. Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain.

    PubMed

    Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D

    2015-04-14

    We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders. PMID:25825773

  7. The exposure data landscape for manufactured chemicals.

    PubMed

    Egeghy, Peter P; Judson, Richard; Gangwal, Sumit; Mosher, Shad; Smith, Doris; Vail, James; Cohen Hubal, Elaine A

    2012-01-01

    The U.S. Environmental Protection Agency is developing chemical screening and prioritization programs to evaluate environmental chemicals for potential risk to human health in a rapid and efficient manner. As part of these efforts, it is important to catalog available information on chemical toxicity and exposure from widely dispersed sources. The main objective of this analysis is to define important aspects of the exposure space and to catalog the available exposure information for chemicals being considered for analysis as part of the U.S. EPA ToxCast™ screening and prioritization program. Publicly available exposure data have been extracted into ACToR (Aggregated Computational Toxicology Resource), which combines information for hundreds of thousands of chemicals from >600 public sources. We use data from ACToR to assess the exposure data landscape for environmental chemicals. Of the roughly 100,000 chemicals that have at least limited toxicity information available, less than one-fifth also have exposure information - and for most of these the information is of limited utility (e.g., production volume). Readily accessible data on concentrations in exposure-related media are only available for a much smaller fraction. Among these, the largest number of chemicals is measured in water with over 1150 unique compounds, followed by 788 substances measured in soil, and 670 in air. These small numbers clearly reflect a focus of resources on those substances previously identified as possibly posing a hazard to human health. Exposure to a much broader number of chemicals will need to be measured in order to fully realize the envisioned goal of using exposure information to guide toxicity testing. PMID:22104386

  8. Two independent mechanical events in the interaction cycle of skeletal muscle myosin with actin.

    PubMed

    Capitanio, M; Canepari, M; Cacciafesta, P; Lombardi, V; Cicchi, R; Maffei, M; Pavone, F S; Bottinelli, R

    2006-01-01

    During skeletal muscle contraction, regular arrays of actin and myosin filaments slide past each other driven by the cyclic ATP-dependent interaction of the motor protein myosin II (the cross-bridge) with actin. The rate of the cross-bridge cycle and its load-dependence, defining shortening velocity and energy consumption at the molecular level, vary widely among different isoforms of myosin II. However, the underlying mechanisms remain poorly understood. We have addressed this question by applying a single-molecule approach to rapidly ( approximately 300 mus) and precisely ( approximately 0.1 nm) detect acto-myosin interactions of two myosin isoforms having large differences in shortening velocity. We show that skeletal myosin propels actin filaments, performing its conformational change (working stroke) in two steps. The first step ( approximately 3.4-5.2 nm) occurs immediately after myosin binding and is followed by a smaller step ( approximately 1.0-1.3 nm), which occurs much faster in the fast myosin isoform than in the slow one, independently of ATP concentration. On the other hand, the rate of the second phase of the working stroke, from development of the latter step to dissociation of the acto-myosin complex, is very similar in the two isoforms and depends linearly on ATP concentration. The finding of a second mechanical event in the working stroke of skeletal muscle myosin provides the molecular basis for a simple model of actomyosin interaction. This model can account for the variation, in different fiber types, of the rate of the cross-bridge cycle and provides a common scheme for the chemo-mechanical transduction within the myosin family. PMID:16371472

  9. Head-neck domain of Arabidopsis myosin XI, MYA2, fused with GFP produces F-actin patterns that coincide with fast organelle streaming in different plant cells

    PubMed Central

    Walter, Nadine; Holweg, Carola L

    2008-01-01

    Background The cytoskeletal mechanisms that underlie organelle transport in plants are intimately linked to acto-myosin function. This function is mediated by the attachment of myosin heads to F-actin and the binding of cargo to the tails. Acto-myosin also powers vigorous cytoplasmic streaming in plant cells. Class XI myosins exhibit strikingly fast velocities and may have extraordinary roles in cellular motility. Studies of the structural basis of organelle transport have focused on the cargo-binding tails of myosin XI, revealing a close relationship with the transport of peroxisomes, mitochondria, and Golgi-vesicles. Links between myosin heads and F-actin-based motility have been less investigated. To address this function, we performed localization studies using the head-neck domain of AtMYA2, a myosin XI from Arabidopsis. Results We expressed the GFP-fused head-neck domain of MYA2 in epidermal cells of various plant species and found that it associated with F-actin. By comparison to other markers such as fimbrin and talin, we revealed that the myosin-labeled F-actin was of a lower quality and absent from the fine microfilament arrays at the cell cortex. However, it colocalized with cytoplasmic (transvacuolar) F-actin in areas coinciding with the tracks of fast organelles. This observation correlates well with the proposed function of myosin XI in organelle trafficking. The fact that organelle streaming was reduced in cells expressing the GFP-MYA2-head6IQ indicated that the functionless motor protein inhibits endogenous myosins. Furthermore, co-expression of the GFP-MYA2-head6IQ with other F-actin markers disrupted its attachment to F-actin. In nuclei, the GFP-myosin associated with short bundles of F-actin. Conclusion The localization of the head of MYA2 in living plant cells, as investigated here for the first time, suggests a close linkage between this myosin XI and cytoplasmic microfilaments that support the rapid streaming of organelles such as peroxisomes

  10. Single-headed binding of a spin-labeled-HMM-ADP complex to F-actin. Saturation transfer electron paramagnetic resonance and sedimentation studies.

    PubMed Central

    Manuck, B A; Seidel, J C; Gergely, J

    1986-01-01

    The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl-imidodiphosphate (AMPPNP) to determine the contributions of single and double-headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results. PMID:3017466

  11. Collisions of deformable cells lead to collective migration

    NASA Astrophysics Data System (ADS)

    Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

    2015-03-01

    Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility - acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignment of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility.

  12. Mechanical Coupling of Smooth Muscle Cells Using Microengineered Substrates and Local Stimulation

    NASA Astrophysics Data System (ADS)

    Copeland, Craig; Hunter, David; Tung, Leslie; Chen, Christopher; Reich, Daniel

    2013-03-01

    Mechanical stresses directly affect many cellular processes, including signal transduction, growth, differentiation, and survival. Cells can themselves generate such stresses by activating myosin to contract the actin cytoskeleton, which in turn can regulate both cell-substrate and cell-cell interactions. We are studying mechanical forces at cell-cell and cell-substrate interactions using arrays of selectively patterned flexible PDMS microposts combined with the ability to apply local chemical stimulation. Micropipette ``spritzing'', a laminar flow technique, uses glass micropipettes mounted on a microscope stage to deliver drugs to controlled regions within a cellular construct while cell traction forces are recorded via the micropost array. The pipettes are controlled by micromanipulators allowing for rapid and precise movement across the array and the ability to treat multiple constructs within a sample. This technique allows for observing the propagation of a chemically induced mechanical stimulus through cell-cell and cell-substrate interactions. We have used this system to administer the acto-myosin inhibitors Blebbistatin and Y-27632 to single cells and observed the subsequent decrease in cell traction forces. Experiments using trypsin-EDTA have shown this system to be capable of single cell manipulation through removal of one cell within a pair configuration while leaving the other cell unaffected. This project is supported in part by NIH grant HL090747

  13. Modification of Cellular Cholesterol Content Affects Traction Force, Adhesion and Cell Spreading

    PubMed Central

    Norman, Leann L.; Oetama, Ratna J.; Dembo, Micah; Byfield, F.; Hammer, Daniel A.; Levitan, Irena; Aranda-Espinoza, Helim

    2011-01-01

    Cellular cholesterol is a critical component of the plasma membrane, and plays a key role in determining the physical properties of the lipid bilayer, such as elasticity, viscosity, and permeability. Surprisingly, it has been shown that cholesterol depletion increases cell stiffness, not due to plasma membrane stiffening, but rather, due to the interaction between the actin cytoskeleton and the plasma membrane. This indicates that traction stresses of the acto-myosin complex likely increase during cholesterol depletion. Here we use force traction microscopy to quantify the forces individual cells are exerting on the substrate, and total internal reflection fluorescence microscopy as well as interference reflection microscopy to observe cell–substrate adhesion and spreading. We show that single cells depleted of cholesterol produce larger traction forces and have large focal adhesions compared to untreated or cholesterol-enriched cells. Cholesterol depletion also causes a decrease in adhesion area for both single cells and monolayers. Spreading experiments illustrate a decrease in spreading area for cholesterol-depleted cells, and no effect on cholesterol-enriched cells. These results demonstrate that cholesterol plays an important role in controlling and regulating the cell–substrate interactions through the actin–plasma membrane complex, cell–cell adhesion, and spreading. PMID:21461187

  14. Development and optimization of novel controlled-release pioglitazone provesicular powders using 3² factorial design.

    PubMed

    Shukr, Marwa H; Eltablawy, Nadia A

    2015-02-01

    This work aimed at studying a novel controlled drug delivery proniosomal formulation of pioglitazone for treatment of diabetes type-2. The effects of independent variables like type of surfactant and ratio of surfactants/cholesterol were studied using 3(2) factorial design. The provesicular powders were characterized regarding their encapsulation efficiency, vesicle size, morphology, and in vitro drug release. The revealed optimal provesicular powder was exposed to stability testing and in vivo performance evaluation. Results showed that F6 was selected as the optimal formulation, and its in vivo hypoglycemic effect on normal healthy and STZ-induced diabetic albino rats was investigated. F6 proniosomal formulation exhibited a significantly higher % decrease (56.18 % for STZ-induced diabetic albino rats) of blood glucose level (BGL) than Actos® (32. % for STZ-induced diabetic albino rats). Higher % decrease of BGL with longer t max and lower AUC0-24 confirms the development of a successful proniosomal pioglitazone formulation. PMID:25787339

  15. Frequency analysis of amyloplast movement in Arabidopsis suggests dynamic gravity sensing

    NASA Astrophysics Data System (ADS)

    Hasenstein, K.; Gilroy, S.

    We investigated the motility of amyloplasts in root caps of Arabidopsis thaliana to analyze the interaction between the acto-myosin system and plastids In addition to sedimentation cytoskeletal activity appears to lift amyloplasts and may cause impinging of sedimenting amyloplasts onto the membrane system This activity may represent graviperception We analyzed the frequency of amyloplast motion by Fourier analysis based upon the position of amyloplasts in image sequences taken at 6 second intervals The frequency analysis showed a maximum at an average cycle of about 16 seconds 0 06 Hz for vertical and lateral displacement However amyloplast velocity was frequency independent Application of the actin-depolymerizing agent Latrunculin B 0 1 mu M reduced the maximum displacement but did not change the frequency DMSO-treated roots showed a similar reduction The frequency dependency was similar for cell from different positions within the root cap The data suggest that the saltatory motion of amyloplasts is a continuous process that is mediated by cytoskeletal events and is an integral part of gravisensing Therefore gravisensing may depend on the dynamic motion of statoliths rather than simple aggregation onto the lower cell membrane Supported by NASA grants NAG2-1423 NAG10-0190 KHH and NAG2-1594 NSF MCB 02-12099 SG

  16. Mechanical properties of metastatic breast cancer cells invading into collagen I matrices

    NASA Astrophysics Data System (ADS)

    Ros, Robert

    2014-03-01

    Mechanical interactions between cells and the extracellular matrix (ECM) are critical to the metastasis of cancer cells. To investigate the mechanical interplay between the cells and ECM during invasion, we created thin bovine collagen I hydrogels ranging from 0.1-5 kPa in Young's modulus that were seeded with highly metastatic MDA-MB-231 breast cancer cells. Significant population fractions invaded the matrices either partially or fully within 24 h. We then combined confocal fluorescence microscopy and indentation with an atomic force microscope to determine the Young's moduli of individual embedded cells and the pericellular matrix using novel analysis methods for heterogeneous samples. In partially embedded cells, we observe a statistically significant correlation between the degree of invasion and the Young's modulus, which was up to an order of magnitude greater than that of the same cells measured in 2D. ROCK inhibition returned the cells' Young's moduli to values similar to 2D and diminished but did not abrogate invasion. This provides evidence that Rho/ROCK-dependent acto-myosin contractility is employed for matrix reorganization during initial invasion, and suggests the observed cell stiffening is due to an attendant increase in actin stress fibers. This work was supported by the National Cancer Institute under the grant U54 CA143862.

  17. Timely Endocytosis of Cytokinetic Enzymes Prevents Premature Spindle Breakage during Mitotic Exit

    PubMed Central

    Onishi, Masayuki; Yeong, Foong May

    2016-01-01

    Cytokinesis requires the spatio-temporal coordination of membrane deposition and primary septum (PS) formation at the division site to drive acto-myosin ring (AMR) constriction. It has been demonstrated that AMR constriction invariably occurs only after the mitotic spindle disassembly. It has also been established that Chitin Synthase II (Chs2p) neck localization precedes mitotic spindle disassembly during mitotic exit. As AMR constriction depends upon PS formation, the question arises as to how chitin deposition is regulated so as to prevent premature AMR constriction and mitotic spindle breakage. In this study, we propose that cells regulate the coordination between spindle disassembly and AMR constriction via timely endocytosis of cytokinetic enzymes, Chs2p, Chs3p, and Fks1p. Inhibition of endocytosis leads to over accumulation of cytokinetic enzymes during mitotic exit, which accelerates the constriction of the AMR, and causes spindle breakage that eventually could contribute to monopolar spindle formation in the subsequent round of cell division. Intriguingly, the mitotic spindle breakage observed in endocytosis mutants can be rescued either by deleting or inhibiting the activities of, CHS2, CHS3 and FKS1, which are involved in septum formation. The findings from our study highlight the importance of timely endocytosis of cytokinetic enzymes at the division site in safeguarding mitotic spindle integrity during mitotic exit. PMID:27447488

  18. Traction force dynamics predict gap formation in activated endothelium.

    PubMed

    Valent, Erik T; van Nieuw Amerongen, Geerten P; van Hinsbergh, Victor W M; Hordijk, Peter L

    2016-09-10

    In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneous distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps. PMID:27498166

  19. Impact of familial hypertrophic cardiomyopathy-linked mutations in the NH2 terminus of the RLC on β-myosin cross-bridge mechanics.

    PubMed

    Farman, Gerrie P; Muthu, Priya; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2014-12-15

    Familial hypertrophic cardiomyopathy (HCM) is associated with mutations in sarcomeric proteins, including the myosin regulatory light chain (RLC). Here we studied the impact of three HCM mutations located in the NH2 terminus of the RLC on the molecular mechanism of β-myosin heavy chain (MHC) cross-bridge mechanics using the in vitro motility assay. To generate mutant β-myosin, native RLC was depleted from porcine cardiac MHC and reconstituted with mutant (A13T, F18L, and E22K) or wild-type (WT) human cardiac RLC. We characterized the mutant myosin force and motion generation capability in the presence of a frictional load. Compared with WT, all three mutants exhibited reductions in maximal actin filament velocity when tested under low or no frictional load. The actin-activated ATPase showed no significant difference between WT and HCM-mutant-reconstituted myosins. The decrease in velocity has been attributed to a significantly increased duty cycle, as was measured by the dependence of actin sliding velocity on myosin surface density, for all three mutant myosins. These results demonstrate a mutation-induced alteration in acto-myosin interactions that may contribute to the pathogenesis of HCM. PMID:25324513

  20. Microvesicles released from tumor cells disrupt epithelial cell morphology and contractility.

    PubMed

    Bordeleau, Francois; Chan, Bryan; Antonyak, Marc A; Lampi, Marsha C; Cerione, Richard A; Reinhart-King, Cynthia A

    2016-05-24

    During tumor progression, cancer cells interact and communicate with non-malignant cells within their local microenvironment. Microvesicles (MV) derived from human cancer cells play an important role in mediating this communication. Another critical aspect of cancer progression involves widespread ECM remodeling, which occur both at the primary and metastatic sites. ECM remodeling and reorganization within the tumor microenvironment is generally attributed to fibroblasts. Here, using MCF10a cells, a well-characterized breast epithelial cell line that exhibits a non-malignant epithelial phenotype, and MVs shed by aggressive MDA-MB-231 carcinoma cells, we show that non-malignant epithelial cells can participate in ECM reorganization of 3D collagen matrices following their treatment with cancer cell-derived MVs. In addition, MVs trigger several changes in epithelial cells under 3D culture conditions. Furthermore, we show that this ECM reorganization is associated with an increase in cellular traction force following MV treatment, higher acto-myosin contractility, and higher FAK activity. Overall, our findings suggest that MVs derived from tumor cells can contribute to ECM reorganization occurring within the tumor microenvironment by enhancing the contractility of non-malignant epithelial cells. PMID:26477404

  1. RhoA Regulates Peroxisome Association to Microtubules and the Actin Cytoskeleton

    PubMed Central

    Lay, Dorothee; Wiese, Sebastian; Meyer, Helmut E.; Warscheid, Bettina; Saffrich, Rainer; Peränen, Johan; Gorgas, Karin; Just, Wilhelm W.

    2010-01-01

    The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering. PMID:21079737

  2. Collisions of deformable cells lead to collective migration

    SciTech Connect

    Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

    2015-03-17

    Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility – acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignment of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility.

  3. Impacts of Usher Syndrome Type IB Mutations on Human Myosin VIIa Motor Function†

    PubMed Central

    Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

    2010-01-01

    Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3 fold, but reduced the actin-activated ATPase activity to 50% of the wild type. While all the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from acto-myosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa. PMID:18700726

  4. A cis-Regulatory Mutation in Troponin-I of Drosophila Reveals the Importance of Proper Stoichiometry of Structural Proteins During Muscle Assembly

    PubMed Central

    Firdaus, Hena; Mohan, Jayaram; Naz, Sarwat; Arathi, Prabhashankar; Ramesh, Saraf R.; Nongthomba, Upendra

    2015-01-01

    Rapid and high wing-beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations that have been instrumental in the identification of many proteins and residues that are important for muscle assembly, function, and disease. In this article, we present a molecular-genetic characterization of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). We show that fliH is a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms that are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. Our results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications for the identification of mutations and disease phenotypes in other species, including humans. PMID:25747460

  5. Crossbridge mechanism(s) examined by temperature perturbation studies on muscle.

    PubMed

    Ranatunga, K W; Coupland, M E

    2010-01-01

    An overall view of the contractile process that has emerged from -temperature-studies on active muscle is outlined. In isometric muscle, a small rapid temperature-jump (T-jump) enhances an early, pre-phosphate release, step in the acto-myosin (crossbridge) ATPase cycle and induces a characteristic rise in force indicating that crossbridge force generation is endothermic (force rises when heat is absorbed). Sigmoidal temperature dependence of steady force is largely due to the endothermic nature of force generation. During shortening, when muscle force is decreased, the T-jump force generation is enhanced; conversely, when a muscle is lengthening and its force increased, the T-jump force generation is inhibited. Taking T-jump force generation as a signature of the crossbridge - ATPase cycle, the results suggest that during lengthening the ATPase cycle is truncated before endothermic force generation, whereas during shortening this step and the ATPase cycle, are accelerated; this readily provides a molecular basis for the Fenn effect. PMID:20824530

  6. A comparison of selected vertical wind measurement techniques on basis of the EUCAARI IMPACT observations

    NASA Astrophysics Data System (ADS)

    Arabas, S.; Baehr, C.; Boquet, M.; Dufournet, Y.; Pawlowska, H.; Siebert, H.; Unal, C.

    2009-04-01

    The poster presents a comparison of selected methods for determination of the vertical wind in the boundary layer used during the EUCAARI IMPACT campaign that took place in May 2008 in The Netherlands. The campaign covered a monthlong intensified ground-based and airborne measurements in the vicinity of the CESAR observatory in Cabauw. Ground-based vertical wind remote sensing was carried out using the Leosphere WindCube WLS70 IR Doppler lidar, Vaisala LAP3000 radar wind-profiler and the TUDelft TARA S-band radar. In-situ airborne measurements were performed using an ultrasonic anemometer (on the ACTOS helicopter underhung platform) and a 5-hole pressure probe (on the SAFIRE ATR-42 airplane radome). Several in-situ anemometers were deployed on the 200-meter high tower of the CESAR observatory. A summary of the characteristics and principles of the considered techniques is presented. A comparison of the results obtained from different platforms depicts the capabilities of each technique and highlights the time, space and velocity resolutions.

  7. Collisions of deformable cells lead to collective migration.

    PubMed

    Löber, Jakob; Ziebert, Falko; Aranson, Igor S

    2015-01-01

    Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility - acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignment of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility. PMID:25779619

  8. Emerging roles for microtubules in angiosperm pollen tube growth highlight new research cues

    PubMed Central

    Onelli, Elisabetta; Idilli, Aurora I.; Moscatelli, Alessandra

    2015-01-01

    In plants, actin filaments have an important role in organelle movement and cytoplasmic streaming. Otherwise microtubules (MTs) have a role in restricting organelles to specific areas of the cell and in maintaining organelle morphology. In somatic plant cells, MTs also participate in cell division and morphogenesis, allowing cells to take their definitive shape in order to perform specific functions. In the latter case, MTs influence assembly of the cell wall, controlling the delivery of enzymes involved in cellulose synthesis and of wall modulation material to the proper sites. In angiosperm pollen tubes, organelle movement is generally attributed to the acto-myosin system, the main role of which is in distributing organelles in the cytoplasm and in carrying secretory vesicles to the apex for polarized growth. Recent data on membrane trafficking suggests a role of MTs in fine delivery and repositioning of vesicles to sustain pollen tube growth. This review examines the role of MTs in secretion and endocytosis, highlighting new research cues regarding cell wall construction and pollen tube-pistil crosstalk, that help unravel the role of MTs in polarized growth. PMID:25713579

  9. Toxicological evaluation of subchronic use of pioglitazone in mice

    PubMed Central

    Elshama, Said Said; El-Kenawy, Ayman El-Meghawry; Osman, Hosam-Eldin Hussein

    2016-01-01

    Objective(s): Pioglitazone (Actos) is one of the most controversial recent oral antidiabetic drugs. It was originally authorized in the European Union in 2000, and approved as an oral monotherapy for overweight second type of diabetic patients in 2002. It belongs to the thiazolidinedione group which some of its members have been withdrawn from the market due to the hepatotoxicity or cardiotoxicity effects. This study investigates sub-chronic use of pioglitazone induced toxicity in mice by the assessment of renal and liver function tests, cardiac enzymes, and some hematological indices with histological changes of liver, kidney, heart, and bladder. Materials and Methods: 120 albino mice were divided into four groups; 30 in each. The first group (control) received water, second (diabetic) group received alloxan only, while the third and the fourth groups received alloxan with 200 and 400 mg/kg/day of pioglitazone, respectively for 90 days. Results: Prolonged use of pioglitazone induced significant abnormalities of hepatic, renal, and cardiac biomarkers and some hematological indices associated with histopathological changes in the liver, kidney, heart, and bladder that increased based on administered dose. Conclusion: Subchronic use of pioglitazone leads to hepatic, renal, cardiac, hematological, and bladder affection depending on the applied dose.

  10. Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons

    PubMed Central

    Galic, Milos; Tsai, Feng-Chiao; Collins, Sean R; Matis, Maja; Bandara, Samuel; Meyer, Tobias

    2014-01-01

    In the vertebrate central nervous system, exploratory filopodia transiently form on dendritic branches to sample the neuronal environment and initiate new trans-neuronal contacts. While much is known about the molecules that control filopodia extension and subsequent maturation into functional synapses, the mechanisms that regulate initiation of these dynamic, actin-rich structures have remained elusive. Here, we find that filopodia initiation is suppressed by recruitment of ArhGAP44 to actin-patches that seed filopodia. Recruitment is mediated by binding of a membrane curvature-sensing ArhGAP44 N-BAR domain to plasma membrane sections that were deformed inward by acto-myosin mediated contractile forces. A GAP domain in ArhGAP44 triggers local Rac-GTP hydrolysis, thus reducing actin polymerization required for filopodia formation. Additionally, ArhGAP44 expression increases during neuronal development, concurrent with a decrease in the rate of filopodia formation. Together, our data reveals a local auto-regulatory mechanism that limits initiation of filopodia via protein recruitment to nanoscale membrane deformations. DOI: http://dx.doi.org/10.7554/eLife.03116.001 PMID:25498153

  11. Entangled active matter: from ants to living cells

    NASA Astrophysics Data System (ADS)

    Brochard-Wyart, Francoise

    2015-03-01

    We introduce the field of ``Entangled Active Matter'' where the building blocks are transiently bound. We will point out strong similarities between aggregates of ants and cells! We will use multicellular aggregates, a model system for tissues. We characterize the tissue mechanical properties using pipette aspiration technique. The aggregate exhibits a viscoelastic response. We observe aggregate reinforcement with pressure, which may results in pulsed contractions or ``shivering.'' We interpret this reinforcement as a mechano-sensitive active response of the acto-myosin cortex. We describe the spreading of aggregates on rigid and soft substrates, varying both intercellular and substrate adhesion. We find both partial and complete wetting, with a precursor film forming a cellular monolayer in a liquid or gas phase. We model the dynamics of spreading from a balance between active cellular driving forces and permeation of cells to enter into the film. Finally we study the motility of aggregates induced by chemical or rigidity gradients, or spontaneous: on soft substrate, the precursor film is unstable, leading to a symmetry breaking and a global motion of the aggregate. We describe the shapes of migrating aggregates, the flow and the force field responsible of the motion. We monitored the center of mass motion and we characterize the stick-slip motions. LABEX CelTisPhyBio, Institut Curie, France

  12. Collisions of deformable cells lead to collective migration

    DOE PAGESBeta

    Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

    2015-03-17

    Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility – acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignmentmore » of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility.« less

  13. Topography induces differential sensitivity on cancer cell proliferation via Rho-ROCK-Myosin contractility

    PubMed Central

    Chaudhuri, Parthiv Kant; Pan, Catherine Qiurong; Low, Boon Chuan; Lim, Chwee Teck

    2016-01-01

    Although the role of stiffness on proliferative response of cancer cells has been well studied, little is known about the effect of topographic cues in guiding cancer cell proliferation. Here, we examined the effect of topographic cues on cancer cell proliferation using micron scale topographic features and observed that anisotropic features like microgratings at specific dimension could reduce proliferation of non-cancer breast epithelial cells (MCF-10A) but not that for malignant breast cancer cells (MDA-MB-231 and MCF-7). However, isotropic features such as micropillars did not affect proliferation of MCF-10A, indicating that the anisotropic environmental cues are essential for this process. Interestingly, acto-myosin contraction inhibitory drugs, Y-27632 and blebbistatin prevented micrograting-mediated inhibition on proliferation. Here, we propose the concept of Mechanically-Induced Dormancy (MID) where topographic cues could activate Rho-ROCK-Myosin signaling to suppress non-cancerous cells proliferation whereas malignant cells are resistant to this inhibitory barrier and therefore continue uncontrolled proliferation. PMID:26795068

  14. Toxicity data informatics: supporting a new paradigm for toxicity prediction.

    PubMed

    Richard, Ann M; Yang, Chihae; Judson, Richard S

    2008-01-01

    ABSTRACT Chemical toxicity data at all levels of description, from treatment-level dose response data to a high-level summarized toxicity "endpoint," effectively circumscribe, enable, and limit predictive toxicology approaches and capabilities. Several new and evolving public data initiatives focused on the world of chemical toxicity information-as represented here by ToxML (Toxicology XML standard), DSSTox (Distributed Structure-Searchable Toxicity Database Network), and ACToR (Aggregated Computational Toxicology Resource)-are contributing to the creation of a more unified, mineable, and modelable landscape of public toxicity data. These projects address different layers in the spectrum of toxicological data representation and detail and, additionally, span diverse domains of toxicology and chemistry in relation to industry and environmental regulatory concerns. For each of the three projects, data standards are the key to enabling "read-across" in relation to toxicity data and chemical-indexed information. In turn, "read-across" capability enables flexible data mining, as well as meaningful aggregation of lower levels of toxicity information to summarized, modelable endpoints spanning sufficient areas of chemical space for building predictive models. By means of shared data standards and transparent and flexible rules for data aggregation, these and related public data initiatives are effectively spanning the divides among experimental toxicologists, computational modelers, and the world of chemically indexed, publicly available toxicity information. PMID:20020908

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

    PubMed Central

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

    2013-01-01

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

  16. Cytoskeleton and apoptosis.

    PubMed

    Ndozangue-Touriguine, Olivia; Hamelin, Jocelyne; Bréard, Jacqueline

    2008-07-01

    Apoptosis is a genetically programmed and physiological mode of cell death that leads to the removal of unwanted or abnormal cells. Cysteine-proteases called caspases are responsible for the apoptotic execution phase which is characterized by specific biochemical events as well as morphological changes. These changes, which lead to the orderly dismantling of the apoptotic cell, include cell contraction, dynamic membrane blebbing, chromatin condensation, nuclear disintegration, cell fragmentation followed by phagocytosis of the dying cell. They involve major modifications of the cytoskeleton which are largely mediated by cleavage of several of its components by caspases. For example, dynamic membrane blebbing is due to the increased contractility of the acto-myosin system following myosin light chain (MLC) phosphorylation. MLC phosphorylation is a consequence of the cleavage of a Rho GTPase effector, the kinase ROCK I, by caspase-3. This cleavage induces a constitutive kinase activity by removal of an inhibitory domain. Chromatin condensation is facilitated by the processing of lamins by caspases. Collapse of the cytokeratin network is mediated by cleavage of keratin 18. On another hand, the actin cytoskeleton rearrangement needed in the phagocyte for engulfment of the dying cell is due to the activation of the small GTPase Rac, a GTPase of the Rho family that induces actin polymerisation and formation of lamellipodia. In addition to mediating the morphological modifications of the apoptotic cell, several proteins of the cytoskeleton such as actin and keratins are also involved in the regulation of apoptotic signaling. PMID:18462707

  17. Collisions of deformable cells lead to collective migration

    PubMed Central

    Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

    2015-01-01

    Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility – acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignment of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility. PMID:25779619

  18. Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells

    NASA Astrophysics Data System (ADS)

    Dvir, Liron; Nissim, Ronen; Alvarez-Elizondo, Martha B.; Weihs, Daphne

    2015-04-01

    Metastasis formation is a major cause of mortality in cancer patients and includes tumor cell relocation to distant organs. A metastatic cell invades through other cells and extracellular matrix by biochemical attachment and mechanical force application. Force is used to move on or through a 2- or 3-dimensional (3D) environment, respectively, or to penetrate a 2D substrate. We have previously shown that even when a gel substrate is impenetrable, metastatic breast cancer cells can still indent it by applying force. Cells typically apply force through the acto-myosin network, which is mechanically connected to the nucleus. We develop a 3D image-analysis to reveal relative locations of the cell elements, and show that as cells apply force to the gel, a coordinated process occurs that involves cytoskeletal remodeling and repositioning of the nucleus. Our approach shows that the actin and microtubules reorganize in the cell, bringing the actin to the leading edge of the cell. In parallel, the nucleus is transported behind the actin, likely by the cytoskeleton, into the indentation dimple formed in the gel. The nucleus volume below the gel surface correlates with indentation depth, when metastatic breast cancer cells indent gels deeply. However, the nucleus always remains above the gel in benign cells, even when small indentations are observed. Determining mechanical processes during metastatic cell invasion can reveal how cells disseminate in the body and can uncover targets for diagnosis and treatment.

  19. Modeling cell-matrix traction forces in Keratinocyte colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2013-03-01

    Crosstalk between cell-cell and cell-matrix adhesions plays an essential role in the mechanical function of tissues. The traction forces exerted by cohesive keratinocyte colonies with strong cell-cell adhesions are mostly concentrated at the colony periphery. In contrast, for weak cadherin-based intercellular adhesions, individual cells in a colony interact with their matrix independently, with a disorganized distribution of traction forces extending throughout the colony. In this talk I will present a minimal physical model of the colony as contractile elastic media linked by springs and coupled to an elastic substrate. The model captures the spatial distribution of traction forces seen in experiments. For cell colonies with strong cell-cell adhesions, the total traction force of the colony measured in experiments is found to scale with the colony's geometrical size. This scaling suggests the emergence of an effective surface tension of magnitude comparable to that measured for non-adherent, three-dimensional cell aggregates. The physical model supports the scaling and indicates that the surface tension may be controlled by acto-myosin contractility. Supported by the NSF through grant DMR-1004789. This work was done in collaboration with Aaron F. Mertz, Eric R. Dufresne and Valerie Horsley (Yale University) and M. Cristina Marchetti (Syracuse University).

  20. Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis

    PubMed Central

    Vedula, Pavan; Cruz, Lissette A.; Gutierrez, Natasha; Davis, Justin; Ayee, Brian; Abramczyk, Rachel; Rodriguez, Alexis J.

    2016-01-01

    Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation. PMID:27357130

  1. The Filament Sensor for Near Real-Time Detection of Cytoskeletal Fiber Structures

    PubMed Central

    Eltzner, Benjamin; Wollnik, Carina; Gottschlich, Carsten; Huckemann, Stephan; Rehfeldt, Florian

    2015-01-01

    A reliable extraction of filament data from microscopic images is of high interest in the analysis of acto-myosin structures as early morphological markers in mechanically guided differentiation of human mesenchymal stem cells and the understanding of the underlying fiber arrangement processes. In this paper, we propose the filament sensor (FS), a fast and robust processing sequence which detects and records location, orientation, length, and width for each single filament of an image, and thus allows for the above described analysis. The extraction of these features has previously not been possible with existing methods. We evaluate the performance of the proposed FS in terms of accuracy and speed in comparison to three existing methods with respect to their limited output. Further, we provide a benchmark dataset of real cell images along with filaments manually marked by a human expert as well as simulated benchmark images. The FS clearly outperforms existing methods in terms of computational runtime and filament extraction accuracy. The implementation of the FS and the benchmark database are available as open source. PMID:25996921

  2. Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells

    PubMed Central

    Heo, Su-Jin; Thorpe, Stephen D.; Driscoll, Tristan P.; Duncan, Randall L.; Lee, David A.; Mauck, Robert L.

    2015-01-01

    Mechanical cues direct the lineage commitment of mesenchymal stem cells (MSCs). In this study, we identified the operative molecular mechanisms through which dynamic tensile loading (DL) regulates changes in chromatin organization and nuclear mechanics in MSCs. Our data show that, in the absence of exogenous differentiation factors, short term DL elicits a rapid increase in chromatin condensation, mediated by acto-myosin based cellular contractility and the activity of the histone-lysine N-methyltransferase EZH2. The resulting change in chromatin condensation stiffened the MSC nucleus, making it less deformable when stretch was applied to the cell. We also identified stretch induced ATP release and purinergic calcium signaling as a central mediator of this chromatin condensation process. Further, we showed that DL, through differential stabilization of the condensed chromatin state, established a ‘mechanical memory’ in these cells. That is, increasing strain levels and number of loading events led to a greater degree of chromatin condensation that persisted for longer periods of time after the cessation of loading. These data indicate that, with mechanical perturbation, MSCs develop a mechanical memory encoded in structural changes in the nucleus which may sensitize them to future mechanical loading events and define the trajectory and persistence of their lineage specification. PMID:26592929

  3. Asymmetry between pushing and pulling for crawling cells

    NASA Astrophysics Data System (ADS)

    Recho, Pierre; Truskinovsky, Lev

    2013-02-01

    Eukaryotic cells possess motility mechanisms allowing them not only to self-propel but also to exert forces on obstacles (to push) and to carry cargoes (to pull). To study the inherent asymmetry between active pushing and pulling we model a crawling acto-myosin cell extract as a one-dimensional layer of active gel subjected to external forces. We show that pushing is controlled by protrusion and that the macroscopic signature of the protrusion dominated motility mechanism is concavity of the force-velocity relation. In contrast, pulling is driven by protrusion only at small values of the pulling force and it is replaced by contraction when the pulling force is sufficiently large. This leads to more complex convex-concave structure of the force-velocity relation; in particular, competition between protrusion and contraction can produce negative mobility in a biologically relevant range. The model illustrates active readjustment of the force generating machinery in response to changes in the dipole structure of external forces. The possibility of switching between complementary active mechanisms implies that if necessary “pushers” can replace “pullers” and vice versa.

  4. P21-activated kinase 4 (PAK4) is required for metaphase spindle positioning and anchoring.

    PubMed

    Bompard, G; Rabeharivelo, G; Cau, J; Abrieu, A; Delsert, C; Morin, N

    2013-02-14

    The oncogenic kinase PAK4 was recently found to be involved in the regulation of the G1 phase and the G2/M transition of the cell cycle. We have also identified that PAK4 regulates Ran GTPase activity during mitosis. Here, we show that after entering mitosis, PAK4-depleted cells maintain a prolonged metaphase-like state. In these cells, chromosome congression to the metaphase plate occurs with normal kinetics but is followed by an extended period during which membrane blebbing and spindle rotation are observed. These bipolar PAK4-depleted metaphase-like spindles have a defective astral microtubule (MT) network and are not centered in the cell but are in close contact with the cell cortex. As the metaphase-like state persists, centrosome fragmentation occurs, chromosomes scatter from the metaphase plate and move toward the spindle poles with an active spindle assembly checkpoint, a phenotype that is reminiscent of cohesion fatigue. PAK4 also regulates the acto-myosin cytoskeleton and we report that PAK4 depletion results in the induction of cortical membrane blebbing during prometaphase arrest. However, we show that membrane blebs, which are strongly enriched in phospho-cofilin, are not responsible for the poor anchoring of the spindle. As PAK4 depletion interferes with the localization of components of the dynein/dynactin complexes at the kinetochores and on the astral MTs, we propose that loss of PAK4 could induce a change in the activities of motor proteins. PMID:22450748

  5. Dynamics of elastic interactions in soft and biological matter

    NASA Astrophysics Data System (ADS)

    Yuval, Janni; Safran, Samuel A.

    2013-04-01

    Cells probe their mechanical environment and can change the organization of their cytoskeletons when the elastic and viscous properties of their environment are modified. We use a model in which the forces exerted by small, contractile acto-myosin filaments (e.g., nascent stress fibers in stem cells) on the extracellular matrix are modeled as local force dipoles. In some cases, the strain field caused by these force dipoles propagates quickly enough so that only static elastic interactions need be considered. On the other hand, in the case of significant energy dissipation, strain propagation is slower and may be eliminated completely by the relaxation of the cellular cytoskeleton (e.g., by cross-link dissociation). Here, we consider several dissipative mechanisms that affect the propagation of the strain field in adhered cells and consider these effects on the interaction between force dipoles and their resulting mutual orientations. This is a first step in understanding the development of orientational (nematic) or layering (smectic) order in the cytoskeleton. We use the theory to estimate the propagation time of the strain fields over a cellular distance for different mechanisms and find that in some cases it can be of the order of seconds, thus competing with the cytoskeletal relaxation time. Furthermore, for a simple system of two force dipoles, we predict that in some cases the orientation of force dipoles might change significantly with time, e.g., for short times the dipoles exhibit parallel alignment while for later times they align perpendicularly.

  6. ADF and Cofilin1 Control Actin Stress Fibers, Nuclear Integrity, and Cell Survival

    PubMed Central

    Kanellos, Georgios; Zhou, Jing; Patel, Hitesh; Ridgway, Rachel A.; Huels, David; Gurniak, Christine B.; Sandilands, Emma; Carragher, Neil O.; Sansom, Owen J.; Witke, Walter; Brunton, Valerie G.; Frame, Margaret C.

    2015-01-01

    Summary Genetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis. PMID:26655907

  7. Peroxisome proliferator-activated receptor gamma agonists as insulin sensitizers: from the discovery to recent progress.

    PubMed

    Cho, Nobuo; Momose, Yu

    2008-01-01

    An epidemic of metabolic diseases including type 2 diabetes and obesity is undermining the health of people living in industrialized societies. There is an urgent need to develop innovative therapeutics. The peroxisome proliferator-activated receptor gamma (PPARgamma) is one of the ligand-activated transcription factors in the nuclear hormone receptor superfamily and a pivotal regulator of glucose and lipid homeostasis. The discovery of PPARgamma as a target of multimodal insulin sensitizers, represented by thiazolidinediones (TZDs), has attracted remarkable scientific interest and had a great impact on the pharmaceutical industry. With the clinical success of the PPARgamma agonists, pioglitazone (Actos) and rosiglitazone (Avandia), development of novel and potent insulin-sensitizing agents with diverse clinical profiles has been accelerated. Currently, a number of PPARgamma agonists from different chemical classes and with varying pharmacological profiles are being developed. Despite quite a few obstacles to the development of PPAR-related drugs, PPARgamma-targeted agents still hold promise. There are new concepts and encouraging evidence emerging that suggest this class can yield improved anti-diabetic agents. This review covers the discovery of TZDs, provides an overview of PPARgamma including the significance of PPARgamma as a drug target, describes the current status of a wide variety of novel PPARgamma ligands including PPAR dual and pan agonists and selective PPARgamma modulators (SPPARgammaMs), and highlights new approaches for identifying agents targeting PPARgamma in the treatment of type 2 diabetes. PMID:19075761

  8. Axi-symmetric patterns of active polar filaments on spherical and composite surfaces

    NASA Astrophysics Data System (ADS)

    Srivastava, Pragya; Rao, Madan

    2014-03-01

    Experiments performed on Fission Yeast cells of cylindrical and spherical shapes, rod-shaped bacteria and reconstituted cylindrical liposomes suggest the influence of cell geometry on patterning of cortical actin. A theoretical model based on active hydrodynamic description of cortical actin that includes curvature-orientation coupling predicts spontaneous formation of acto-myosin rings, cables and nodes on cylindrical and spherical geometries [P. Srivastava et al, PRL 110, 168104(2013)]. Stability and dynamics of these patterns is also affected by the cellular shape and has been observed in experiments performed on Fission Yeast cells of spherical shape. Motivated by this, we study the stability and dynamics of axi-symmetric patterns of active polar filaments on the surfaces of spherical, saddle shaped and conical geometry and classify the stable steady state patterns on these surfaces. Based on the analysis of the fluorescence images of Myosin-II during ring slippage we propose a simple mechanical model for ring-sliding based on force balance and make quantitative comparison with the experiments performed on Fission Yeast cells. NSF Grant DMR-1004789 and Syracuse Soft Matter Program.

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

    PubMed Central

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

    2014-01-01

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

  10. Sobrevivendo a un tsunami: lecciones de Chile, Hawai y Japon

    USGS Publications Warehouse

    Compilado por Atwater, Brian F.; Cisternas V., Marco; Bourgeois, Joanne; Dudley, Walter C.; Hendley, James W., II; Stauffer, Peter H.

    1999-01-01

    Este folleto contiene historias veridicas que ilustran como sobrevivir, y como no sobrevivir, a un tsunami. Esta publicacion esta dirigida a las personas que viven, trabajan o, simplemente, se divierten a lo largo de las costas que pueden ser afectadas por un tsunami. Tales costas rodean la mayor parte del Oceano Pacifico pero tambien incluyen algunas areas costeras de los Oceanos Atlantico e Indico. Aunque mucha gente llama a los tsunamis 'olas de marea', estos no estan relacionados a las mareas, sino son una serie de olas, o 'tren de olas', generalmente causadas por cambios en el nivel del fondo marino durante los terremotos. Los tsunamis tambien pueden ser generados por la erupcion de volcanes costeros, islas volconicas, deslizamientos submarinos e impactos de grandes meteoritos en el mar. Como sucedio en Sumatra en el 2004, los tsunamis pueden alcanzar alturas de 15 metros, no tan solo en la costa sino tambien kilometros tierra adentro. Los relatos presentados en este folleto fueron seleccionados de entrevistas realizadas a personas que sobrevivieron al tsunami del Oceano Pacifico de 1960. Muchas de estas personas, incluyendo a la enfermera de la foto, se enfrento a las olas generadas a poca distancia, en la costa chilena. En cambio, otros debieron hacer frente al tsunami muchas horas despues, en Hawai y Japon. La mayoria de las entrevistas fueron realizadas a fines de los anos ochenta y en los noventa. Las historias ofrecen una mezcla de lecciones de supervivencia a un tsunami. En algunos casos se presentan las acciones que confiablemente salvaron vidas: poner atencion a los avisos de la naturaleza, abandonar los bienes, dirigirse rapidamente a un sector alto y permanecer alli hasta que el tsunami realmente haya terminado. Otras historias describen como se encontro refugio al subir a construcciones y arboles o flotar sobre desechos, tacticas que tuvieron diferentes resultados y que pueden ser recomendadas solo como actos desesperados de personas atrapadas en

  11. Cardiovascular risk and cardiometabolic protection: role of glitazones.

    PubMed

    Petrazzi, Luisa; Grassi, Davide; Polidoro, Lorella; D'Aurelio, Azzurra; Croce, Giuseppe; Properzi, Giuliana; Tiberti, Sergio; Desideri, Giovambattista; Ferri, Claudio

    2008-01-01

    Thiazolidinediones (TZDs) are widely used in the type 2 diabetes mellitus (DMT2) treatment but have also been tested in cardiovascular prevention. DMT2 is associated with a marked increment in cardiovascular risk, and its prevention represents a main target in cardiometabolic protection. Both Troglitazone (Troglitazone in Prevention of Diabetes study) and Rosiglitazone (Diabetes Reduction Assessment with Ramipril and Rosiglitazone Medication study) significantly reduced new-onset diabetes. A similar topic will be investigated with pioglitazone (Actos Now for Prevention of Diabetes). In the Prospective Pioglitazone Clinical Trial in Macrovascular events the primary end point (all-cause mortality, nonfatal myocardial infarction, stroke, acute coronary syndromes, endovascular or surgical intervention in the coronary/leg arteries and amputation above ankles) was unaffected, whereas the secondary one (all-cause mortality, nonfatal myocardial infarction and stroke) was reduced by pioglitazone (-16%, p=0.027) compared to placebo in 5,238 patients with DMT2 and macrovascular disease. In contrast, a meta-analysis (Nissen and Wolski, N Engl J Med. 2007;356:2457-2471) reported that rosiglitazone treatment is associated with a significant increase in myocardial infarction risk (p=0.03) and a borderline significant increase in the risk of death from cardiovascular causes (p=0.06). Nevertheless, the possibility that rosiglitazone might affect cardiovascular events should be evaluated by the ongoing trial Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of Glycemia in Diabetes (RECORD). Interim findings early from RECORD did not show significant differences between the rosiglitazone and the control group regarding myocardial infarction and death from cardiovascular and any cause. Additional large-scale trials are awaited to clarify the of role TZDs in cardiovascular outcomes. PMID:19034866

  12. Strengthening Adaptation to Extreme Climate Events in Southwestern Amazonia: an Example from the Trinational Acre River Basin in the Madre de Dios/Peru - Acre/Brazil - Pando/Bolivia (MAP) Region.

    NASA Astrophysics Data System (ADS)

    Brown, I. F.

    2015-12-01

    Southwestern Amazonia, where Bolivia, Brazil and Peru meet, faces numerous challenges to the sustainable utilization of land and water resources as the region experiences rapid population and economic growth, expanding agriculture, transportation and energy sectors, along with frequent flooding and droughts. It is also predicted to be one of the most susceptible areas for climate change in the coming decade. The Acre River Basin, one of the few trinational basins in Amazonia, lies at the center of the Madre de Dios Region (Peru), Acre State (Brazil) and Pando Department (Bolivia) or MAP Region. It covers approximately 7,500 km2 and its inhabitants range from indigenous groups avoiding contact with industrial society to more than 60,000 dwellers of a binational urban center. The basin incorporates most the challenges facing the region and this paper discusses steps underway to address the basin's vulnerability to climate-related threats. A trinational group of professionals used GIS databases and local knowledge to classify these threats and possible societal responses. To prioritize threats and to propose responses, this group adapted a method proposed by the Queensland Climate Change Centre of Excellence of Australia to develop climate risk matrices for assessing impacts, adaptation, risk and vulnerability. The three priority climate variables were prolonged and more frequent droughts, more intense flooding, and more days with temperatures > 35oC. The final matrix proposed two areas of concentration - 1) Reduce the vulnerability of communities to hydro-meteorological extreme events and 2) Protect and restore ecosystems that maintain critical water-related resources with actions in public policy, capacity-building, and immediate activities. These results are being incorporated into the Amazon Project of the Global Environment Fund of the United Nations Environment Program, administered by the Amazon Cooperation Treaty Organization (ACTO).

  13. Myosin XI-I is Mechanically and Enzymatically Unique Among Class-XI Myosins in Arabidopsis.

    PubMed

    Haraguchi, Takeshi; Tominaga, Motoki; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2016-08-01

    Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator. PMID:27273580

  14. Velocities of unloaded muscle filaments are not limited by drag forces imposed by myosin cross-bridges

    PubMed Central

    Brizendine, Richard K.; Alcala, Diego B.; Carter, Michael S.; Haldeman, Brian D.; Facemyer, Kevin C.; Baker, Josh E.; Cremo, Christine R.

    2015-01-01

    It is not known which kinetic step in the acto-myosin ATPase cycle limits contraction speed in unloaded muscles (V0). Huxley’s 1957 model [Huxley AF (1957) Prog Biophys Biophys Chem 7:255–318] predicts that V0 is limited by the rate that myosin detaches from actin. However, this does not explain why, as observed by Bárány [Bárány M (1967) J Gen Physiol 50(6, Suppl):197–218], V0 is linearly correlated with the maximal actin-activated ATPase rate (vmax), which is limited by the rate that myosin attaches strongly to actin. We have observed smooth muscle myosin filaments of different length and head number (N) moving over surface-attached F-actin in vitro. Fitting filament velocities (V) vs. N to a detachment-limited model using the myosin step size d = 8 nm gave an ADP release rate 8.5-fold faster and ton (myosin’s attached time) and r (duty ratio) ∼10-fold lower than previously reported. In contrast, these data were accurately fit to an attachment-limited model, V = N·v·d, over the range of N found in all muscle types. At nonphysiologically high N, V = L/ton rather than d/ton, where L is related to the length of myosin’s subfragment 2. The attachment-limited model also fit well to the [ATP] dependence of V for myosin-rod cofilaments at three fixed N. Previously published V0 vs. vmax values for 24 different muscles were accurately fit to the attachment-limited model using widely accepted values for r and N, giving d = 11.1 nm. Therefore, in contrast with Huxley’s model, we conclude that V0 is limited by the actin–myosin attachment rate. PMID:26294254

  15. Tensional Homeostasis in Single Fibroblasts

    PubMed Central

    Webster, Kevin D.; Ng, Win Pin; Fletcher, Daniel A.

    2014-01-01

    Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury. PMID:24988349

  16. IFT88 influences chondrocyte actin organization and biomechanics

    PubMed Central

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

    2016-01-01

    Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. Results IFT88orpk cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88orpk cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88orpk cells. Following membrane blebbing, IFT88orpk cells exhibited slower reformation of the actin cortex. IFT88orpk cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. Conclusions This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. PMID:26493329

  17. Mouse Myosin-19 Is a Plus-end-directed, High-duty Ratio Molecular Motor*

    PubMed Central

    Lu, Zekuan; Ma, Xiao-Nan; Zhang, Hai-Man; Ji, Huan-Hong; Ding, Hao; Zhang, Jie; Luo, Dan; Sun, Yujie; Li, Xiang-dong

    2014-01-01

    Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament. PMID:24825904

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

    PubMed

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

    2014-07-25

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

  19. Relationship between expression of serendipity alpha and cellularisation of the Drosophila embryo as revealed by interspecific transformation.

    PubMed

    Ibnsouda, S; Schweisguth, F; de Billy, G; Vincent, A

    1993-10-01

    A dramatic reorganization of the cytoskeleton underlies the cellularisation of the syncytial Drosophila embryo. Formation of a regular network of acto-myosin filaments, providing a structural framework, and possibly a contractile force as well, appears essential for the synchronous invagination of the plasma membrane between adjacent nuclei. The serendipity alpha (sry alpha) gene is required for this complete reorganization of the microfilaments at the onset of membrane invagination. We compare here the structure and expression of sry alpha between D. pseudoobscura, D. subobscura and D. melanogaster. Interspersion of evolutionarily highly conserved and divergent regions is observed in the protein. One such highly conserved region shows sequence similarities to a motif found in proteins of the ezrin-radixin-moesin (ERM) family. Four 7-13 bp motifs are conserved in the 5' promoter region; two of these are also found, and at the same position relative to the TATA box, in nullo, another zygotic gene recently shown to be involved in cellularisation. The compared patterns of expression of D. melanogaster sry alpha and nullo, and D. pseudoobscura sry alpha reveal a complex regulation of the spatiotemporal accumulation of their transcripts. The D. pseudoobscura sry alpha gene is able to rescue the cellularisation defects associated with a complete loss of sry alpha function in D. melanogaster embryos, even though species-specific aspects of its expression are maintained. Despite their functional homologies, the D. melanogaster and D. pseudoobscura sry alpha RNAs have different subcellular localisations, suggesting that this specific localization has no conserved role in targeting the sry alpha protein to the apical membranes. PMID:8287797

  20. Myosins XI-K, XI-1, and XI-2 are required for development of pavement cells, trichomes, and stigmatic papillae in Arabidopsis

    PubMed Central

    2012-01-01

    Background The positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport. Results Simultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils. Conclusions We conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes. PMID:22672737

  1. An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

    PubMed Central

    Elangovan, R; Capitanio, M; Melli, L; Pavone, F S; Lombardi, V; Piazzesi, G

    2012-01-01

    A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ. With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4–30°C) and substrate concentration. VF was 8.88 ± 0.26 μm s−1 at 30.6°C and decreased to 1.60 ± 0.09 μm s−1 at 4.5°C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40–50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5°C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads. PMID:22199170

  2. Substrate & Cell Compliance Effects on Cell Spreading and Differentiation

    NASA Astrophysics Data System (ADS)

    Discher, Dennis; Sheehan, Maureen; Engler, Adam

    2004-03-01

    The stiffness of the substrate that a cell adheres to is emerging as a critically important physical factor in the response of many cell types. The effects are seen with cells on gels as well as cells on cells - highlighting implications for organism development. The basis for the effects lies in the fact that cells literally 'feel' their substrate. Like other anchorage dependent cells, muscle cells feel their substrate and are found in our studies to spread more and organize their cytoskeleton and focal adhesions much more so on rigid glass and stiff substrates than on soft gels. Such spreading is not necessarily physiological or conducive to biological function. Collagen density certainly factors into cell on gel adhesive spreading, with minimal spreading on very low collagen and a weak maximum in cell spreading on intermediate collagen densities. Bell-shaped curves are readily modeled to highlight the coupling between ligand density and substrate stiffness. Most surprising, however, spreading on soft gels is found to be almost independent of adhesive ligand density: even with high collagen densities, the minimal spreading of cells cannot be over-ridden. Remarkably, muscle cells show the strongest tendency to differentiate and striate their acto-myosin on gels that have a stiffness similar to relaxed muscle. Cells gown on top of other cells also show a very strong tendency to striate, even if the underlying cells do not striate; elasticity measurements appear to unify all of the effects. The implications for organismal development as well as cell biological studies can be very important.

  3. Type 2 diabetes can be prevented with early pharmacological intervention.

    PubMed

    DeFronzo, Ralph A; Abdul-Ghani, Muhammad

    2011-05-01

    In the U.S., ∼ 21 × 10(6) individuals have type 2 diabetes, and twice as many have impaired glucose tolerance (IGT). Approximately 40-50% of individuals with IGT will progress to type 2 diabetes over their lifetime. Therefore, treatment of high-risk individuals with IGT to prevent type 2 diabetes has important medical, economic, social, and human implications. Weight loss, although effective in reducing the conversion of IGT to type 2 diabetes, is difficult to achieve and maintain. Moreover, 40-50% of IGT subjects progress to type 2 diabetes despite successful weight reduction. In contrast, pharmacological treatment of IGT with oral antidiabetic agents that improve insulin sensitivity and preserve β-cell function--the characteristic pathophysiological abnormalities present in IGT and type 2 diabetes--uniformly have been shown to prevent progression of IGT to type 2 diabetes. The most consistent results have been observed with the thiazolidinediones (Troglitazone in the Prevention of Diabetes [TRIPOD], Pioglitazone in the Prevention of Diabetes [PIPOD], Diabetes Reduction Assessment with Ramipril and Rosiglitazone Medication [DREAM], and Actos Now for the Prevention of Diabetes [ACT NOW]), with a 50-70% reduction in IGT conversion to diabetes. Metformin in the U.S. Diabetes Prevention Program (DPP) reduced the development of type 2 diabetes by 31% and has been recommended by the American Diabetes Association (ADA) for treating high-risk individuals with IGT. The glucagon-like peptide-1 analogs, which augment insulin secretion, preserve β-cell function, and promote weight loss, also would be expected to be efficacious in preventing the progression of IGT to type 2 diabetes. Because individuals in the upper tertile of IGT are maximally/near-maximally insulin resistant, have lost 70-80% of their β-cell function, and have an ∼ 10% incidence of diabetic retinopathy, pharmacological intervention, in combination with diet plus exercise, should be instituted. PMID

  4. Active mechanical coupling between the nucleus, cytoskeleton and the extracellular matrix, and the implications for perinuclear actomyosin organization.

    PubMed

    Zemel, Assaf

    2015-03-28

    Experimental and theoretical studies have demonstrated that the polarization of actomyosin forces in the cytoskeleton of adherent cells is governed by local elastic stresses. Based on this phenomenon, and the established observation that the nucleus is mechanically connected to the extracellular matrix (ECM) via the cytoskeleton, we theoretically analyze here the active mechanical coupling between the nucleus, cytoskeleton and the ECM. The cell is modeled as an active spherical inclusion, containing a round nucleus at its center, and embedded in a 3D elastic matrix. We investigate three sources of cellular stress: spreading-induced stress, actomyosin contractility and chromatin entropic forces. Formulating the coupling of actomyosin contractility to the local stress we predict the consequences that the nucleus, cytoskeleton and ECM mechanical properties may have on the overall force-balance in the cell and the perinuclear acto-myosin polarization. We demonstrate that the presence of the nucleus induces symmetry breaking of the elastic stress that, we predict, elastically tends to orient actomyosin alignment tangentially around the nucleus; the softer the nucleus or the matrix, the stronger is the preference for tangential alignment. Spreading induced stresses may induce radial actomyosin alignment near stiff nuclei. In addition, we show that in regions of high actomyosin density myosin motors have an elastic tendency to orient tangentially as often occurs near the cell periphery. These conclusions highlight the role of the nucleus in the regulation of cytoskeleton organization and may provide new insight into the mechanics of stem cell differentiation involving few fold increase in nucleus stiffness. PMID:25652010

  5. Structural changes in isometrically contracting insect flight muscle trapped following a mechanical perturbation.

    PubMed

    Wu, Shenping; Liu, Jun; Reedy, Mary C; Perz-Edwards, Robert J; Tregear, Richard T; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E; Reedy, Michael K; Taylor, Kenneth A

    2012-01-01

    The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.5% changes in length (either a stretch or a release) within 2 ms to isometrically contracting insect flight muscle (IFM) fibers followed after 5-6 ms by rapid freezing against a liquid helium cooled copper mirror. ET of freeze-substituted fibers, embedded and thin-sectioned, provides 3-D cross-bridge images, sorted by multivariate data analysis into ~40 classes, distinct in average structure, population size and lattice distribution. Individual actin subunits are resolved facilitating quasi-atomic modeling of each class average to determine its binding strength (weak or strong) to actin. ~98% of strong-binding acto-myosin attachments present after a length perturbation are confined to "target zones" of only two actin subunits located exactly midway between successive troponin complexes along each long-pitch helical repeat of actin. Significant changes in the types, distribution and structure of actin-myosin attachments occurred in a manner consistent with the mechanical transients. Most dramatic is near disappearance, after either length perturbation, of a class of weak-binding cross-bridges, attached within the target zone, that are highly likely to be precursors of strong-binding cross-bridges. These weak-binding cross-bridges were originally observed in isometrically contracting IFM. Their disappearance following a quick stretch or release can be explained by a recent kinetic model for muscle contraction, as behaviour consistent with their identification as precursors of strong-binding cross-bridges. The results provide a detailed model for

  6. Reliable positioning in a sparse GPS network, eastern Ontario

    NASA Astrophysics Data System (ADS)

    Samadi Alinia, H.; Tiampo, K.; Atkinson, G. M.

    2013-12-01

    GPS raw data. These are based on precise GPS orbit and clock data products with centimeter accuracy computed beforehand. Here, the analysis of 1Hz GPS data is conducted in order to find the most reliable regional network from eight stations (STCO, TYNO, ACTO, INUQ, IVKQ, KLBO, MATQ and ALGO) that cover the study area in eastern Ontario. In this way, the estimated parameters are the total number of ambiguities and resolved ambiguities, posteriori rms of each baseline and the coordinates for each station and their differences with the known coordinates. The positioning accuracy, the corrections and the accuracy of interpolated corrections, and the initialization time required for precise positioning are presented for the various applications.

  7. Computational toxicology as implemented by the U.S. EPA: providing high throughput decision support tools for screening and assessing chemical exposure, hazard and risk.

    PubMed

    Kavlock, Robert; Dix, David

    2010-02-01

    available through the Aggregated Computational Toxicology Resource (ACToR), the Distributed Structure-Searchable Toxicity (DSSTox) Database Network, and other U.S. EPA websites. While initially focused on improving the hazard identification process, the CTRP is placing increasing emphasis on using high-throughput bioactivity profiling data in systems modeling to support quantitative risk assessments, and in developing complementary higher throughput exposure models. This integrated approach will enable analysis of life-stage susceptibility, and understanding of the exposures, pathways, and key events by which chemicals exert their toxicity in developing systems (e.g., endocrine-related pathways). The CTRP will be a critical component in next-generation risk assessments utilizing quantitative high-throughput data and providing a much higher capacity for assessing chemical toxicity than is currently available. PMID:20574897

  8. Spreading and spontaneous motility of multicellular aggregates on soft substrates

    NASA Astrophysics Data System (ADS)

    Brochard-Wyart, Françoise

    2013-03-01

    We first describe the biomechanics of multicellular aggregates, a model system for tissues and tumors. We first characterize the tissue mechanical properties (surface tension, elasticity, viscosity) by a new pipette aspiration technique. The aggregate exhibits a viscoelastic response but, unlike an inert fluid, we observe aggregate reinforcement with pressure, which for a narrow range of pressures results in pulsed contractions or shivering. We interpret this reinforcement as a mechanosensitive active response of the acto-myosin cortex. Such an active behavior has previously been found to cause tissue pulsation during dorsal closure of Drosophila embryo. We then describe the spreading of aggregates on rigid glass substrates, varying both intercellular and substrate adhesion. We find both partial and complete wetting regimes. For the dynamics, we find a universal spreading law at short time, analogous to that of a viscoelastic drop. At long time, we observe, for strong substrate adhesion, a precursor film spreading around the aggregate. Depending on aggregate cohesion, this precursor film can be a dense cellular monolayer (liquid state) or consist of individual cells escaping from the aggregate body (gas state). The transition from liquid to gas state appears also to be present in the progression of a tumor from noninvasive to metastatic, known as the epithelial-mesenchymal transition. Finally, we describe the effect of the substrate rigidity on the phase diagram of wetting. On soft gels decorated with fibronectin and strongly cohesive aggregates, we have observed a wetting transition induced by the substrate rigidity: on ultra soft gels, below an elastic modulus Ec the aggregates do not spread, whereas above Ec we observe a precursor film expending with a diffusive law. The diffusion coefficient D(E) present a maximum for E =Em. A maximum of mobility versus the substrate rigidity had also been observed for single cells. Near Em, we observe a new phenomenon: a cell

  9. Effects of Long-Term Pioglitazone Treatment on Peripheral and Central Markers of Aging

    PubMed Central

    Pancani, Tristano; Searcy, James L.; Anderson, Katie L.; Gant, John C.; Popovic, Jelena; Avdiushko, Margarita G.; Cohen, Don A.; Chen, Kuey-Chu; Porter, Nada M.; Thibault, Olivier

    2010-01-01

    Background Thiazolidinediones (TZDs) activate peroxisome proliferator-activated receptor gamma (PPARγ) and are used clinically to help restore peripheral insulin sensitivity in Type 2 diabetes (T2DM). Interestingly, long-term treatment of mouse models of Alzheimer's disease (AD) with TZDs also has been shown to reduce several well-established brain biomarkers of AD including inflammation, oxidative stress and Aβ accumulation. While TZD's actions in AD models help to elucidate the mechanisms underlying their potentially beneficial effects in AD patients, little is known about the functional consequences of TZDs in animal models of normal aging. Because aging is a common risk factor for both AD and T2DM, we investigated whether the TZD, pioglitazone could alter brain aging under non-pathological conditions. Methods and Findings We used the F344 rat model of aging, and monitored behavioral, electrophysiological, and molecular variables to assess the effects of pioglitazone (PIO-Actos® a TZD) on several peripheral (blood and liver) and central (hippocampal) biomarkers of aging. Starting at 3 months or 17 months of age, male rats were treated for 4–5 months with either a control or a PIO-containing diet (final dose approximately 2.3 mg/kg body weight/day). A significant reduction in the Ca2+-dependent afterhyperpolarization was seen in the aged animals, with no significant change in long-term potentiation maintenance or learning and memory performance. Blood insulin levels were unchanged with age, but significantly reduced by PIO. Finally, a combination of microarray analyses on hippocampal tissue and serum-based multiplex cytokine assays revealed that age-dependent inflammatory increases were not reversed by PIO. Conclusions While current research efforts continue to identify the underlying processes responsible for the progressive decline in cognitive function seen during normal aging, available medical treatments are still very limited. Because TZDs have been

  10. Structural Changes in Isometrically Contracting Insect Flight Muscle Trapped following a Mechanical Perturbation

    PubMed Central

    Wu, Shenping; Liu, Jun; Perz-Edwards, Robert J.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A.

    2012-01-01

    The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.5% changes in length (either a stretch or a release) within 2 ms to isometrically contracting insect flight muscle (IFM) fibers followed after 5–6 ms by rapid freezing against a liquid helium cooled copper mirror. ET of freeze-substituted fibers, embedded and thin-sectioned, provides 3-D cross-bridge images, sorted by multivariate data analysis into ∼40 classes, distinct in average structure, population size and lattice distribution. Individual actin subunits are resolved facilitating quasi-atomic modeling of each class average to determine its binding strength (weak or strong) to actin. ∼98% of strong-binding acto-myosin attachments present after a length perturbation are confined to “target zones” of only two actin subunits located exactly midway between successive troponin complexes along each long-pitch helical repeat of actin. Significant changes in the types, distribution and structure of actin-myosin attachments occurred in a manner consistent with the mechanical transients. Most dramatic is near disappearance, after either length perturbation, of a class of weak-binding cross-bridges, attached within the target zone, that are highly likely to be precursors of strong-binding cross-bridges. These weak-binding cross-bridges were originally observed in isometrically contracting IFM. Their disappearance following a quick stretch or release can be explained by a recent kinetic model for muscle contraction, as behaviour consistent with their identification as precursors of strong-binding cross-bridges. The results provide a detailed model

  11. Force generation examined by laser temperature-jumps in shortening and lengthening mammalian (rabbit psoas) muscle fibres

    PubMed Central

    Ranatunga, K W; Coupland, M E; Pinniger, G J; Roots, H; Offer, G W

    2007-01-01

    phase 2b (endothermic force generation) in isometric muscle. Results are discussed in relation to the previous findings in isometric muscle fibres which showed that a T-jump promotes an early step in the crossbridge–ATPase cycle that generates force. In general, the finding that the T-jump effect on active muscle tension is pronounced during shortening, but is depressed/inhibited during lengthening, is consistent with the expectations from the Fenn effect that energy liberation (and acto-myosin ATPase rate) in muscle are increased during shortening and depressed/inhibited during lengthening. PMID:17916609

  12. Education Through Aerospace Components. (Spanish Title: Educación Através de Elementos Aeroespaciales.) Educação Através de Elementos Aeroespaciais

    NASA Astrophysics Data System (ADS)

    Barbosa Loureda, Oswaldo; Sobral de Araújo, Jéssyca B.

    2008-12-01

    Education is a field that needs development. For such purposes, there are various methods and tools that suggest ideas in favor of the improvement of the Brazilian people in the pedagogical, psychological and cultural aspects. Teaching is an act that demands a lot of care and responsibility; the behavior and performance of an individual in the society is the result of way that people was educated. However, the area of hard sciences demands a special attention, because the acquired knowledge is essential for the personal development of the individual and the technological future of the country. As an alternative or complementary tool for education it is suggested the use of aerospace element, since they show a vast amount of subjects qualitatively dealing with abilities of great importance for the future professional life of the students. A new Race happens, however this time the goal is not the Moon, but knowledge. El área educacional es un campo que necesita desarrollo. Para esto se dispone de diversos métodos y medios que pueden implantar ideas en pro del avance del pueblo brasilero en los aspectos pedagógicos, psicológicos y culturales. Alfabetizar es un acto que exige mucho cuidado y responsabilidad; el comportamento y desempeño de un individuo en la sociedad es el resultado de la manera en que fue educado. En particular, el área de ciencias exactas exige especial atención, pues los conocimientos adquiridos son imprescindibles para el desarrollo personal del individuo y también para el futuro tecnológico del País. Como medio alternativo o complementar de enseñanza se sugiere el uso de elementos aeroespaciales, debido a que compreende una vasta cantidad de disciplinas cualitativamente involucradas en la adquisición de habilidades de gran importancia para su vida profesional futura. Una nueva Carrera está em marcha, sin embargo esta vez la meta no es la Luna, sino el conocimiento. A área educacional é um campo que necessita de desenvolvimento. Para

  13. Los manuales de quimica en Espana (1788--1845): Protagonistas, terminologia, clasificaciones y orden pedagogico

    NASA Astrophysics Data System (ADS)

    Munoz Bello, Maria Rosa

    La presente tesis doctoral es una investigacion sobre los manuales de quimica utilizados en Espana de 1788 a 1845. Este trabajo proporciona una perspectiva general de un tema relevante en las ultimas decadas en Historia de la Ciencia, el estudio de los libros de texto. De acuerdo con las ultimas investigaciones realizadas en este terreno, el acto pedagogico es considerado como un proceso creativo, como espacio de encuentro de actores e intereses muy diversos, matizando las ideas defendidas por Thomas S. Kuhn. Recordemos que segun Kuhn, los libros de texto ofrecen una vision consensuada y normalizada del estado de la ciencia de su epoca, por lo que sus autores eliminan deliberadamente toda controversia y presentan asi una imagen distorsionada de la actividad cientifica. En cambio, se ha mostrado, por ejemplo, que en la ensenanza participan no solamente profesores y alumnos sino tambien otros muchos actores y todos ellos no unicamente con intereses puramente pedagogicos sino tambien con diversos intereses politicos y economicos que pueden conocerse a traves del estudio de los manuales. En esta tesis se pretende analizar los manuales de quimica en Espana desde 1788 hasta 1845. Para poder llevar a cabo la investigacion ha sido necesario precisar el objeto de estudio (libro de texto de quimica) durante el periodo estudiado (1788-1845) ya que no es adecuado adoptar la imagen actual de una disciplina que sufrio sustanciales cambios durante la epoca estudiada. Esta investigacion se centra en un momento especialmente importante para la quimica y que algunos historiadores han llegado a considerar "revolucionario". Durante estos anos se produjo un cambio importante en las teorias quimicas sobre la combustion y el concepto de elemento, asi como una reforma terminologica que originaron la aparicion de importantes controversias. Ademas, debido a la relacion de la quimica con otras disciplinas como la historia natural o la fisica ha sido necesario restringir el objeto de estudio

  14. Force generation examined by laser temperature-jumps in shortening and lengthening mammalian (rabbit psoas) muscle fibres.

    PubMed

    Ranatunga, K W; Coupland, M E; Pinniger, G J; Roots, H; Offer, G W

    2007-11-15

    rate of T-jump tension rise increased with warming (Q10 approximately 2.7), similar to phase 2b (endothermic force generation) in isometric muscle. Results are discussed in relation to the previous findings in isometric muscle fibres which showed that a T-jump promotes an early step in the crossbridge-ATPase cycle that generates force. In general, the finding that the T-jump effect on active muscle tension is pronounced during shortening, but is depressed/inhibited during lengthening, is consistent with the expectations from the Fenn effect that energy liberation (and acto-myosin ATPase rate) in muscle are increased during shortening and depressed/inhibited during lengthening. PMID:17916609

  15. Los manuales de quimica en Espana (1788--1845): Protagonistas, terminologia, clasificaciones y orden pedagogico

    NASA Astrophysics Data System (ADS)

    Munoz Bello, Maria Rosa

    La presente tesis doctoral es una investigacion sobre los manuales de quimica utilizados en Espana de 1788 a 1845. Este trabajo proporciona una perspectiva general de un tema relevante en las ultimas decadas en Historia de la Ciencia, el estudio de los libros de texto. De acuerdo con las ultimas investigaciones realizadas en este terreno, el acto pedagogico es considerado como un proceso creativo, como espacio de encuentro de actores e intereses muy diversos, matizando las ideas defendidas por Thomas S. Kuhn. Recordemos que segun Kuhn, los libros de texto ofrecen una vision consensuada y normalizada del estado de la ciencia de su epoca, por lo que sus autores eliminan deliberadamente toda controversia y presentan asi una imagen distorsionada de la actividad cientifica. En cambio, se ha mostrado, por ejemplo, que en la ensenanza participan no solamente profesores y alumnos sino tambien otros muchos actores y todos ellos no unicamente con intereses puramente pedagogicos sino tambien con diversos intereses politicos y economicos que pueden conocerse a traves del estudio de los manuales. En esta tesis se pretende analizar los manuales de quimica en Espana desde 1788 hasta 1845. Para poder llevar a cabo la investigacion ha sido necesario precisar el objeto de estudio (libro de texto de quimica) durante el periodo estudiado (1788-1845) ya que no es adecuado adoptar la imagen actual de una disciplina que sufrio sustanciales cambios durante la epoca estudiada. Esta investigacion se centra en un momento especialmente importante para la quimica y que algunos historiadores han llegado a considerar "revolucionario". Durante estos anos se produjo un cambio importante en las teorias quimicas sobre la combustion y el concepto de elemento, asi como una reforma terminologica que originaron la aparicion de importantes controversias. Ademas, debido a la relacion de la quimica con otras disciplinas como la historia natural o la fisica ha sido necesario restringir el objeto de estudio

  16. Invasive cancer cells and metastasis

    NASA Astrophysics Data System (ADS)

    Mierke, Claudia Tanja

    2013-12-01

    , vinculin and FAK synergize their functions to regulate the mechanical properties of cells such as stiffness and contractile forces. Finally, the knowledge of the mechanical properties of invasive and non-invasive cells could provide a source for future drug developments to inhibit formation of metastases. This special section also includes two papers from the group of Martin Herrmann, a research paper and a review paper. The research paper by Janko et al deals with the cooperative binding of Annexin A5 to phosphatidylserines on apoptotic cell membranes [6]. This could not alone serve as an 'eat me' signal for macrophages as healthy cells also express Annexin A5 on their cell surface. The authors suggest that the cooperative binding is altered and subsequently the fluidity of Annexin A5 on the membrane. Together this may serve as a signal for phagocytic cells to eat apoptotic cells and leave healthy ones untouched. The paper by Biermann et al reviews the role of biophysical signals in the clearance of apoptotic cells [7]. In addition to the acto-myosin cytoskeleton, the keratin network seems to play a role in cancer research. The paper from the Beil and the Marti group demonstrates that microrheology is a valuable tool to determine the viscoelastic properties of polymer networks such as the keratin network in cells and an arbitrary in vitro network [8]. They describe how the topology of the keratin network affects the overall mechanical behavior of cells. It seems that the field of physical oncology will continue to grow in the future and more research will address the mechanical properties of cancer cells and whole tissues. Biophysical methods will need to be further improved and adapted to the needs of cancer research. References [1] Coughlin M F and Fredberg J J 2013 Phys. Biol. 10 065001 [2] Krause M, te Riet J and Wolf K 2013 Phys. Biol. 10 065002 [3] Munn L L 2013 Phys. Biol. 10 065003 [4] Bordeleau F, Tang L N and Reinhart-King C A 2013 Phys. Biol. 10 065004 [5