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Sample records for allosteric actin contact

  1. Two distinct mechanisms for actin capping protein regulation--steric and allosteric inhibition.

    Science.gov (United States)

    Takeda, Shuichi; Minakata, Shiho; Koike, Ryotaro; Kawahata, Ichiro; Narita, Akihiro; Kitazawa, Masashi; Ota, Motonori; Yamakuni, Tohru; Maéda, Yuichiro; Nitanai, Yasushi

    2010-07-06

    The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the alpha-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the

  2. Optimal allosteric stabilization sites using contact stabilization analysis.

    Science.gov (United States)

    Dickson, Alex; Bailey, Christopher T; Karanicolas, John

    2017-06-05

    Proteins can be destabilized by a number of environmental factors such as temperature, pH, and mutation. The ability to subsequently restore function under these conditions by adding small molecule stabilizers, or by introducing disulfide bonds, would be a very powerful tool, but the physical principles that drive this stabilization are not well understood. The first problem lies is in choosing an appropriate binding site or disulfide bond location to best confer stability to the active site and restore function. Here, we present a general framework for predicting which allosteric binding sites correlate with stability in the active site. Using the Karanicolas-Brooks Gō-like model, we examine the dynamics of the enzyme β-glucuronidase using an Umbrella Sampling method to thoroughly sample the conformational landscape. Each intramolecular contact is assigned a score termed a "stabilization factor" that measures its correlation with structural changes in the active site. We have carried out this analysis for three different scaling strengths for the intramolecular contacts, and we examine how the calculated stabilization factors depend on the ensemble of destabilized conformations. We further examine a locally destabilized mutant of β-glucuronidase that has been characterized experimentally, and show that this brings about local changes in the stabilization factors. We find that the proximity to the active site is not sufficient to determine which contacts can confer active site stability. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein*

    Science.gov (United States)

    Townsend, Philip D.; Rodgers, Thomas L.; Glover, Laura C.; Korhonen, Heidi J.; Richards, Shane A.; Colwell, Lucy J.; Pohl, Ehmke; Wilson, Mark R.; Hodgson, David R. W.; McLeish, Tom C. B.; Cann, Martin J.

    2015-01-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. PMID:26187469

  4. The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein.

    Science.gov (United States)

    Townsend, Philip D; Rodgers, Thomas L; Glover, Laura C; Korhonen, Heidi J; Richards, Shane A; Colwell, Lucy J; Pohl, Ehmke; Wilson, Mark R; Hodgson, David R W; McLeish, Tom C B; Cann, Martin J

    2015-09-04

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Engineering amount of cell-cell contact demonstrates biphasic proliferative regulation through RhoA and the actin cytoskeleton

    International Nuclear Information System (INIS)

    Gray, Darren S.; Liu, Wendy F.; Shen, Colette J.; Bhadriraju, Kiran; Nelson, Celeste M.; Chen, Christopher S.

    2008-01-01

    Endothelial cell-cell contact via VE-cadherin plays an important role in regulating numerous cell functions, including proliferation. However, using different experimental approaches to manipulate cell-cell contact, investigators have observed both inhibition and stimulation of proliferation depending on the adhesive context. In this study, we used micropatterned wells combined with active positioning of cells by dielectrophoresis in order to investigate whether the number of contacting neighbors affected the proliferative response. Varying cell-cell contact resulted in a biphasic effect on proliferation; one contacting neighbor increased proliferation, while two or more neighboring cells partially inhibited this increase. We also observed that cell-cell contact increased the formation of actin stress fibers, and that expression of dominant negative RhoA (RhoN19) blocked the contact-mediated increase in stress fibers and proliferation. Furthermore, examination of heterotypic pairs of untreated cells in contact with RhoN19-expressing cells revealed that intracellular, but not intercellular, tension is required for the contact-mediated stimulation of proliferation. Moreover, engagement of VE-cadherin with cadherin-coated beads was sufficient to stimulate proliferation in the absence of actual cell-cell contact. In all, these results demonstrate that cell-cell contact signals through VE-cadherin, RhoA, and intracellular tension in the actin cytoskeleton to regulate proliferation

  6. Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry*

    Science.gov (United States)

    Stokasimov, Ema; Rubenstein, Peter A.

    2009-01-01

    Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G- and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts. PMID:19605362

  7. Conformational changes in actin induced by its interaction with gelsolin.

    Science.gov (United States)

    Khaitlina, S; Hinssen, H

    1997-08-01

    Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound. We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin. ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex. Despite the nonpolymerizability of ECP-(Ca)actin, this complex can act as a nucleus for the polymerization of intact actin, thus indicating that upon interaction with gelsolin, ECP-(Ca)actin undergoes a conformational change that enables its interaction with another actin monomer. By gel filtration and fluorometry it was shown that the binding of at least one of the ECP-cleaved actins to gelsolin is considerably weaker than of intact actin, suggesting that conformational changes in subdomain 2 of actin monomer may directly or allosterically affect actin-gelsolin interactions. On the other hand, interaction with gelsolin changes the conformation of actin within the DNase I-binding loop, as indicated by inhibition of limited proteolysis of actin by ECP and subtilisin. Cross-linking experiments with gelsolin-nucleated actin filaments using N,N-phenylene-bismaleimide (which cross-links adjacent actin monomers between Cys374 and Lys191) reveal that gelsolin causes a significant increase in the yield of the 115-kDa cross-linking product, confirming the evidence that gelsolin stabilizes or changes the conformation of the C-terminal region of the actin molecule, and these changes are propagated from the capped end along the filament. These results allow us to conclude that nucleation of actin polymerization by gelsolin is promoted by conformational changes within subdomain 2 and at the C-terminus of the actin monomer.

  8. Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation

    Directory of Open Access Journals (Sweden)

    Thompson Erik W

    2009-07-01

    Full Text Available Abstract Background A feature of epithelial to mesenchymal transition (EMT relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. Methods PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1 and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. Results When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4 and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4. Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse

  9. Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation

    International Nuclear Information System (INIS)

    Hugo, Honor J; Wafai, Razan; Blick, Tony; Thompson, Erik W; Newgreen, Donald F

    2009-01-01

    A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive

  10. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    For example, the structural changes that allowed for allosteric regulation of haemoglobin were re- vealed through structural elucidation of the protein in free and oxygen-bound forms by X-ray crystallography. Following this,. X-ray crystallography has been utilized to study a variety of al- losteric proteins including ATCase. 2.

  11. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    triguingly, the substrate or the product of the inhibited enzyme can be structurally different from the inhibitor. ... ulation of proteins in this fashion as 'allosteric' in the year 1961. [9]. The word allostery originated from the ..... flux occurs via the conformational selec- tion pathway at low concentrations of the ligand, while the trend.

  12. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    ... Lecture Workshops · Refresher Courses · Symposia · Live Streaming. Home; Journals; Resonance – Journal of Science Education; Volume 22; Issue 1. Allosteric Regulation of Proteins: A Historical Perspective on the Development of Concepts and Techniques. General Article Volume 22 Issue 1 January 2017 pp 37-50 ...

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

    DEFF Research Database (Denmark)

    Fritzsche, Marco; Fernandes, Ricardo A.; Chang, Veronica T.

    2017-01-01

    optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse....... This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament...... as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions....

  14. Small-molecule allosteric activators of sirtuins.

    Science.gov (United States)

    Sinclair, David A; Guarente, Leonard

    2014-01-01

    The mammalian sirtuins (SIRT1-7) are NAD(+)-dependent lysine deacylases that play central roles in cell survival, inflammation, energy metabolism, and aging. Members of this family of enzymes are considered promising pharmaceutical targets for the treatment of age-related diseases including cancer, type 2 diabetes, inflammatory disorders, and Alzheimer's disease. SIRT1-activating compounds (STACs), which have been identified from a variety of chemical classes, provide health benefits in animal disease models. Recent data point to a common mechanism of allosteric activation by natural and synthetic STACs that involves the binding of STACs to a conserved N-terminal domain in SIRT1. Compared with polyphenols such as resveratrol, the synthetic STACs show greater potency, solubility, and target selectivity. Although considerable progress has been made regarding SIRT1 allosteric activation, key questions remain, including how the molecular contacts facilitate SIRT1 activation, whether other sirtuin family members will be amenable to activation, and whether STACs will ultimately prove safe and efficacious in humans.

  15. Symmetrical retrograde actin flow in the actin fusion structure is involved in osteoclast fusion

    Directory of Open Access Journals (Sweden)

    Jiro Takito

    2017-07-01

    Full Text Available The aim of this study was to elucidate the role of the zipper-like structure (ZLS, a podosome-related structure that transiently appears at the cell contact zone, in osteoclast fusion. Live-cell imaging of osteoclasts derived from RAW264.7 cells transfected with EGFP-actin revealed consistent symmetrical retrograde actin flow in the ZLS, but not in the podosome cluster, the podosome ring or the podosome belt. Confocal imaging showed that the distributions of F-actin, vinculin, paxillin and zyxin in the ZLS were different from those in the podosome belt. Thick actin filament bundles running outside the ZLS appeared to recruit non-muscle myosin IIA. The F-actin-rich domain of the ZLS contained actin-related protein 2/3 complex (Arp2/3. Inhibition of Arp2/3 activity disorganized the ZLS, disrupted actin flow, deteriorated cell-cell adhesion and inhibited osteoclast hypermultinucleation. In contrast, ML-7, an inhibitor of myosin light chain kinase, had little effect on the structure of ZLS and promoted osteoclast hypermultinucleation. These results reveal a link between actin flow in the ZLS and osteoclast fusion. Osteoclast fusion was promoted by branched actin elongation and negatively regulated by actomyosin contraction.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C.; Navaza, Jorge; Dominguez, Roberto (IBS); (BBRI); (UPENN-MED)

    2013-11-20

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

  17. Treatment of Actinic Purpura

    Science.gov (United States)

    2017-01-01

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

  18. Allosteric transition: a comparison of two models

    DEFF Research Database (Denmark)

    Bindslev, Niels

    2013-01-01

    Introduction Two recent models are in use for analysis of allosteric drug action at receptor sites remote from orthosteric binding sites. One is an allosteric two-state mechanical model derived in 2000 by David Hall. The other is an extended operational model developed in 2007 by Arthur Christopo...

  19. The Carboxy-Terminal Third Of Dystrophin Enhances Actin Binding Activity

    Science.gov (United States)

    Henderson, Davin M.; Lin, Ava Yun; Thomas, David D.; Ervasti, James M.

    2012-01-01

    Dystrophin is an actin-binding protein thought to stabilize cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene-actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule (from N-terminal ABD1 through ABD2) bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays compared to full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction in terms of restricting actin’s rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin. PMID:22226838

  20. Histones bundle F-actin filaments and affect actin structure.

    Directory of Open Access Journals (Sweden)

    Edna Blotnick

    Full Text Available Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

  1. Histones bundle F-actin filaments and affect actin structure.

    Science.gov (United States)

    Blotnick, Edna; Sol, Asaf; Muhlrad, Andras

    2017-01-01

    Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

  2. A Novel Alpha Cardiac Actin (ACTC1) Mutation Mapping to a Domain in Close Contact with Myosin Heavy Chain Leads to a Variety of Congenital Heart Defects, Arrhythmia and Possibly Midline Defects

    Science.gov (United States)

    Augière, Céline; Mégy, Simon; El Malti, Rajae; Boland, Anne; El Zein, Loubna; Verrier, Bernard; Mégarbané, André; Deleuze, Jean-François; Bouvagnet, Patrice

    2015-01-01

    Background A Lebanese Maronite family presented with 13 relatives affected by various congenital heart defects (mainly atrial septal defects), conduction tissue anomalies and midline defects. No mutations were found in GATA4 and NKX2-5. Methods and Results A set of 399 poly(AC) markers was used to perform a linkage analysis which peaked at a 2.98 lod score on the long arm of chromosome 15. The haplotype analysis delineated a 7.7 meganucleotides genomic interval which included the alpha-cardiac actin gene (ACTC1) among 36 other protein coding genes. A heterozygous missense mutation was found (c.251T>C, p.(Met84Thr)) in the ACTC1 gene which changed a methionine residue conserved up to yeast. This mutation was absent from 1000 genomes and exome variant server database but segregated perfectly in this family with the affection status. This mutation and 2 other ACTC1 mutations (p.(Glu101Lys) and p.(Met125Val)) which result also in congenital heart defects are located in a region in close apposition to a myosin heavy chain head region by contrast to 3 other alpha-cardiac actin mutations (p.(Ala297Ser),p.(Asp313His) and p.(Arg314His)) which result in diverse cardiomyopathies and are located in a totally different interaction surface. Conclusions Alpha-cardiac actin mutations lead to congenital heart defects, cardiomyopathies and eventually midline defects. The consequence of an ACTC1 mutation may in part be dependent on the interaction surface between actin and myosin. PMID:26061005

  3. A Novel Alpha Cardiac Actin (ACTC1 Mutation Mapping to a Domain in Close Contact with Myosin Heavy Chain Leads to a Variety of Congenital Heart Defects, Arrhythmia and Possibly Midline Defects.

    Directory of Open Access Journals (Sweden)

    Céline Augière

    Full Text Available A Lebanese Maronite family presented with 13 relatives affected by various congenital heart defects (mainly atrial septal defects, conduction tissue anomalies and midline defects. No mutations were found in GATA4 and NKX2-5.A set of 399 poly(AC markers was used to perform a linkage analysis which peaked at a 2.98 lod score on the long arm of chromosome 15. The haplotype analysis delineated a 7.7 meganucleotides genomic interval which included the alpha-cardiac actin gene (ACTC1 among 36 other protein coding genes. A heterozygous missense mutation was found (c.251T>C, p.(Met84Thr in the ACTC1 gene which changed a methionine residue conserved up to yeast. This mutation was absent from 1000 genomes and exome variant server database but segregated perfectly in this family with the affection status. This mutation and 2 other ACTC1 mutations (p.(Glu101Lys and p.(Met125Val which result also in congenital heart defects are located in a region in close apposition to a myosin heavy chain head region by contrast to 3 other alpha-cardiac actin mutations (p.(Ala297Ser,p.(Asp313His and p.(Arg314His which result in diverse cardiomyopathies and are located in a totally different interaction surface.Alpha-cardiac actin mutations lead to congenital heart defects, cardiomyopathies and eventually midline defects. The consequence of an ACTC1 mutation may in part be dependent on the interaction surface between actin and myosin.

  4. ACTIN-DIRECTED TOXIN. ACD toxin-produced actin oligomers poison formin-controlled actin polymerization.

    Science.gov (United States)

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

    2015-07-31

    The actin cross-linking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin cross-linking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here, we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently "poisoned" the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by using actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses. Copyright © 2015, American Association for the Advancement of Science.

  5. Axonal Actin Transport Driven By Metastable Actin Filaments

    Science.gov (United States)

    Chakrabarty, Nilaj; Ganguly, Archan; Roy, Subhojit; Jung, Peter

    Actin is one of the key constituents of the neuronal cytoskeleton and is responsible for driving important cellular processes like axon elongation. Axonal actin is synthesized in the cell body and transported at rates of 0.25 - 3 mm/day, as shown by in-vivo pulse-chase radiolabelling studies. However, the underlying transport mechanisms are unknown. Recent experiments in cultured neurons have revealed a dynamic network of metastable actin filaments (actin trails). Actin trails seem to originate from focal actin hotspots which colocalize with stationary endosomes. Interestingly, the number of actin trails extending anterogradely is higher than the ones extending retrogradely. We hypothesize that the bulk axonal transport of actin originates from this directional asymmetry of the number of actin trails. To test this, we constructed a computational model of actin trail growth and simulated the pulse-chase experiment. In our model, local, metastable trails, which grow with their barbed ends anchored to the hotspots, drive the bulk anterograde transport. Our results indicate that the observed bias of the nucleation probabilities and the elongation rate of actin trails are sufficient to drive the bulk transport of actin at rates that agree with in-vivo pulse chase experiments.

  6. Chemogenomics of allosteric binding sites in GPCRs

    DEFF Research Database (Denmark)

    Gloriam, David E.

    2013-01-01

    profiling. This review describes recent developments structured into ligand-, target- and combined chemogenomic techniques and applications to allosteric GPCR ligands. It also outlines relative strengths and limitations of these techniques and the impact of the increasing crystallographic data....

  7. Effects of solution crowding on actin polymerization reveal the energetic basis for nucleotide-dependent filament stability.

    Science.gov (United States)

    Frederick, Kendra B; Sept, David; De La Cruz, Enrique M

    2008-05-02

    Actin polymerization is a fundamental cellular process involved in cell structure maintenance, force generation, and motility. Phosphate release from filament subunits following ATP hydrolysis destabilizes the filament lattice and increases the critical concentration (C(c)) for assembly. The structural differences between ATP- and ADP-actin are still debated, as well as the energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded intracellular conditions. Here, we investigate the effect of crowding agents on ATP- and ADP-actin polymerization and find that ATP-actin polymerization is largely unaffected by solution crowding, while crowding agents lower the C(c) of ADP-actin in a concentration-dependent manner. The stabilities of ATP- and ADP-actin filaments are comparable in the presence of physiological amounts (approximately 30% w/v) and types (sorbitol) of low molecular weight crowding agents. Crowding agents act to stabilize ADP-F-actin by slowing subunit dissociation. These observations suggest that nucleotide hydrolysis and phosphate release per se do not introduce intrinsic differences in the in vivo filament stability. Rather, the preferential disassembly of ADP-actin filaments in cells is driven through interactions with regulatory proteins. Interpretation of the experimental data according to osmotic stress theory implicates water as an allosteric regulator of actin activity and hydration as the molecular basis for nucleotide-dependent filament stability.

  8. Distinct actin oligomers modulate differently the activity of actin nucleators.

    Science.gov (United States)

    Qu, Zheng; Silvan, Unai; Jockusch, Brigitte M; Aebi, Ueli; Schoenenberger, Cora-Ann; Mannherz, Hans Georg

    2015-10-01

    Polymerization of actin monomers into filaments requires the initial formation of nuclei composed of a few actin subunits; however, their instability has hindered their detailed study. Therefore we used chemically crosslinked actin oligomers to analyse their effect on actin polymerization. Actin dimer (upper dimer, UD), trimer and tetramer intermolecularly crosslinked by phenylene-bismaleimide along the genetic helix (between Lys199 and Cys374) were isolated by gel filtration and found to increasingly stimulate actin polymerization as shown by the pyrene assay and total internal reflection fluorescence microscopy. In contrast, the so-called lower actin dimer (LD) characterized by a Cys374-Cys374 crosslink stimulated actin polymerization only at low but inhibited it at high concentrations. UD and trimer stimulated the repolymerization of actin from complexes with thymosin β4 (Tβ4) or profilin, whereas the LD stimulated repolymerization only from the profilin : actin but not the actin : Tβ4 complex. In vivo, actin polymerization is stimulated by nucleation factors. Therefore the interaction and effects of purified LD, UD and trimer on the actin-nucleating activity of gelsolin, mouse diaphanous related (mDia) formin and the actin-related protein 2/3 (Arp2/3) complex were analysed. Native gel electrophoresis demonstrated binding of LD, UD and trimer to gelsolin and its fragment G1-3, to the FH2 domains of the formins mDia1 and mDia3, and to Arp2/3 complex. UD and trimer increased the nucleating activity of gelsolin and G1-3, but not of the mDia-FH2 domain nor of the Arp2/3 complex. In contrast, LD at equimolar concentration to Arp2/3 complex stimulated its nucleating activity, but inhibited that of mDia-FH2 domains, gelsolin and G1-3, demonstrating differential regulation of their nucleating activity by dimers containing differently oriented actin subunits. © 2015 FEBS.

  9. Cross-reacting material 197 (CRM197) affects actin cytoskeleton of endothelial cells.

    Science.gov (United States)

    Özerman Edis, Bilge; Varol, Başak; Hacıosmanoğlu, Ebru; Ünlü, Ayhan; Bektaş, Muhammet

    2017-10-01

    CRM197, cross-reacting material 197, is a mutant of diphtheria toxin (DTx). CRM197 is used in pharmacology as a carrier protein. It has been recently shown that CRM197 causes breakdown in actin filaments. In order to show intracellular localization of CRM197 and visualize cell structure via actin cytoskeleton, endothelial cells were cultured and subjected to CRM197 in vitro. To address the interaction between CRM197 and actin both experimental and theoretical studies were carried out. Colocalization of CRM197 with actin filaments was determined by immunofluorescence microscopy. Following 24-hour incubation, the loss of cell-cell contact between cells was prominent. CRM197 was shown to bind to G-actin by gel filtration chromatography, and this binding was confirmed by Western blot analysis of eluted samples obtained following chromatography. Based on crystal structure, docked model of CRM197-actin complex was generated. Molecular dynamics simulation revealed that Lys42, Cys218, Cys233 of CRM197 interacts with Gly197, Arg62 and Ser60 of G-actin, respectively. CRM197 binding to G-actin, colocalization of CRM197 with actin filament, and actin cytoskeleton rearrangement resulting in the loss of cell-cell contact show that actin comes into sight as target molecule for CRM197.

  10. The different ways through which specificity works in orthosteric and allosteric drugs.

    Science.gov (United States)

    Nussinov, Ruth; Tsai, Chung-Jung

    2012-01-01

    Currently, there are two types of drugs on the market: orthosteric, which bind at the active site; and allosteric, which bind elsewhere on the protein surface, and allosterically change the conformation of the protein binding site. In this perspective we argue that the different mechanisms through which the two drug types affect protein activity and their potential pitfalls call for different considerations in drug design. The key problem facing orthosteric drugs is side effects which can occur by drug binding to homologous proteins sharing a similar binding site. Hence, orthosteric drugs should have very high affinity to the target; this would allow a low dosage to selectively achieve the goal of target-only binding. By contrast, allosteric drugs work by shifting the free energy landscape. Their binding to the protein surface perturbs the protein surface atoms, and the perturbation propagates like waves, finally reaching the binding site. Effective drugs should have atoms in good contact with the 'right' protein atoms; that is, the contacts should elicit propagation waves optimally reaching the protein binding site target. While affinity is important, the design should consider the protein conformational ensemble and the preferred propagation states. We provide examples from functional in vivo scenarios for both types of cases, and suggest how high potency can be achieved in allosteric drug development.

  11. Actinic reticuloid. Diagnostics

    Directory of Open Access Journals (Sweden)

    E. V. Sokolovskiy

    2016-01-01

    Full Text Available This article is about the case of actinic reticuloid - the rare dermatosis which clinical presentation is similar to atopic dermatitis, T-cell lymphoma. Good treatment effect was obtained by long cycles (2 cycles for 3 months of hydroxychloroquine and sun protective therapy included sunscreens SPF 50, nicotinic acid, sun-safe clothes which blocked ultraviolet radiation without any glucocorticosteroid drugs and cytostatic treatment.

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

    Science.gov (United States)

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

    2015-01-06

    Formins catalyze nucleation and growth of actin filaments. Here, we study the structure and interactions of actin with the FH2 domain of budding yeast formin Bni1p. We built an all-atom model of the formin dimer on an Oda actin filament 7-mer and studied structural relaxation and interprotein interactions by molecular dynamics simulations. These simulations produced a refined model for the FH2 dimer associated with the barbed end of the filament and showed electrostatic interactions between the formin knob and actin target-binding cleft. Mutations of two formin residues contributing to these interactions (R1423N, K1467L, or both) reduced the interaction energies between the proteins, and in coarse-grained simulations, the formin lost more interprotein contacts with an actin dimer than with an actin 7-mer. Biochemical experiments confirmed a strong influence of these mutations on Bni1p-mediated actin filament nucleation, but not elongation, suggesting that different interactions contribute to these two functions of formins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Allosteric regulation of epigenetic modifying enzymes.

    Science.gov (United States)

    Zucconi, Beth E; Cole, Philip A

    2017-08-01

    Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Actin acting at the Golgi.

    Science.gov (United States)

    Egea, Gustavo; Serra-Peinado, Carla; Salcedo-Sicilia, Laia; Gutiérrez-Martínez, Enric

    2013-09-01

    The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in trafficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers.

  15. An allosteric model of the molecular interactions of excitation- contraction coupling in skeletal muscle

    OpenAIRE

    1993-01-01

    A contact interaction is proposed to exist between the voltage sensor of the transverse tubular membrane of skeletal muscle and the calcium release channel of the sarcoplasmic reticulum. This interaction is given a quantitative formulation inspired in the Monod, Wyman, and Changeux model of allosteric transitions in hemoglobin (Monod, J., J. Wyman, and J.-P. Changeux. 1965. Journal of Molecular Biology. 12:88- 118), and analogous to one proposed by Marks and Jones for voltage- dependent Ca ch...

  16. Actin Polymerization and ATP Hydrolysis

    Science.gov (United States)

    Korn, Edward D.; Carlier, Marie-France; Pantaloni, Dominique

    1987-10-01

    F-actin is the major component of muscle thin filaments and, more generally, of the microfilaments of the dynamic, multifunctional cytoskeletal systems of nonmuscle eukaryotic cells. Polymeric F-actin is formed by reversible noncovalent self-association of monomeric G-actin. To understand the dynamics of microfilament systems in cells, the dynamics of polymerization of pure actin must be understood. The following model has emerged from recent work. During the polymerization process, adenosine 5'-triphosphate (ATP) that is bound to G-actin is hydrolyzed to adenosine 5'-diphosphate (ADP) that is bound to F-actin. The hydrolysis reaction occurs on the F-actin subsequent to the polymerization reaction in two steps: cleavage of ATP followed by the slower release of inorganic phosphate (Pi). As a result, at high rates of filament growth a transient cap of ATP-actin subunits exists at the ends of elongating filaments, and at steady state a stabilizing cap of ADP \\cdot Pi-actin subunits exists at the barbed ends of filaments. Cleavage of ATP results in a highly stable filament with bound ADP \\cdot Pi, and release of Pi destabilizes the filament. Thus these two steps of the hydrolytic reaction provide potential mechanisms for regulating the monomer-polymer transition.

  17. ALLO: A tool to discriminate and prioritize allosteric pockets.

    Science.gov (United States)

    Akbar, Rahmad; Helms, Volkhard

    2018-04-01

    Allosteric proteins make up a substantial proportion of human drug targets. Thus, rational design of small molecule binders that target these proteins requires the identification of putative allosteric pockets and an understanding of their potential activity. Here, we characterized allosteric pockets using a set of physicochemical descriptors and compared them to pockets that are found on the surface of a protein. Further, we trained predictive models capable of discriminating allosteric pockets from orthosteric pockets and models capable of prioritizing allosteric pockets in a set of pockets found on a given protein. Such models might be useful for identifying novel allosteric sites and in turn, potentially new allosteric drug targets. Datasets along with a Python program encapsulating the predictive models are available at http://github.com/fibonaccirabbits/allo. © 2017 John Wiley & Sons A/S.

  18. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

    Wu, Peng; Clausen, Mads Hartvig; Nielsen, Thomas E.

    2015-01-01

    current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance. In spite of the small number of identified allosteric inhibitors in comparison with that of inhibitors targeting the ATP pocket, encouraging results, such as the FDA-approval of the first small...

  19. Agonism/antagonism switching in allosteric ensembles.

    Science.gov (United States)

    Motlagh, Hesam N; Hilser, Vincent J

    2012-03-13

    Ligands for several transcription factors can act as agonists under some conditions and antagonists under others. The structural and molecular bases of such effects are unknown. Previously, we demonstrated how the folding of intrinsically disordered (ID) protein sequences, in particular, and population shifts, in general, could be used to mediate allosteric coupling between different functional domains, a model that has subsequently been validated in several systems. Here it is shown that population redistribution within allosteric systems can be used as a mechanism to tune protein ensembles such that a given ligand can act as both an agonist and an antagonist. Importantly, this mechanism can be robustly encoded in the ensemble, and does not require that the interactions between the ligand and the protein differ when it is acting either as an agonist or an antagonist. Instead, the effect is due to the relative probabilities of states prior to the addition of the ligand. The ensemble view of allostery that is illuminated by these studies suggests that rather than being seen as switches with fixed responses to allosteric activation, ensembles can evolve to be "functionally pluripotent," with the capacity to up or down regulate activity in response to a stimulus. This result not only helps to explain the prevalence of intrinsic disorder in transcription factors and other cell signaling proteins, it provides important insights about the energetic ground rules governing site-to-site communication in all allosteric systems.

  20. Metalloregulatory proteins: metal selectivity and allosteric switching.

    Science.gov (United States)

    Reyes-Caballero, Hermes; Campanello, Gregory C; Giedroc, David P

    2011-07-01

    Prokaryotic organisms have evolved the capacity to quickly adapt to a changing and challenging microenvironment in which the availability of both biologically required and non-essential transition metal ions can vary dramatically. In all bacteria, a panel of metalloregulatory proteins controls the expression of genes encoding membrane transporters and metal trafficking proteins that collectively manage metal homeostasis and resistance. These "metal sensors" are specialized allosteric proteins, in which the direct binding of a specific or small number of "cognate" metal ion(s) drives a conformational change in the regulator that allosterically activates or inhibits operator DNA binding, or alternatively, distorts the promoter structure thereby converting a poor promoter to a strong one. In this review, we discuss our current understanding of the features that control metal specificity of the allosteric response in these systems, and the role that structure, thermodynamics and conformational dynamics play in mediating allosteric activation or inhibition of DNA binding. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; El-Fakahany, E. E.

    2010-01-01

    Roč. 3, č. 9 (2010), s. 2838-2860 ISSN 1424-8247 R&D Projects: GA ČR GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic acetylcholine receptors * allosteric modulation * Alzheimer ´s disease Subject RIV: CE - Biochemistry

  2. Actin binding proteins and spermiogenesis

    Science.gov (United States)

    Mruk, Dolores D

    2011-01-01

    Drebrin E, an actin-binding protein lacking intrinsic activity in the regulation of actin dynamics (e.g., polymerization, capping, nucleation, branching, cross-linking, bundling and severing), is known to recruit actin regulatory proteins to a specific cellular site. Herein, we critically evaluate recent findings in the field which illustrate that drebrin E works together with two other actin-binding proteins, namely Arp3 (actin-related protein 3, a component of the Arp2/3 complex that simultaneously controls actin nucleation for polymerization and branching of actin filaments) and Eps8 (epidermal growth factor receptor pathway substrate 8 that controls capping of the barbed ends of actin filaments, as well as actin filament bundling) to regulate the homeostasis of F-actin filament bundles at the ectoplasmic specialization (ES), a testis-specific atypical adherens junction (AJ) in the seminiferous epithelium. This is mediated by the strict temporal and spatial expression of these three actin-binding proteins at the apical and basal ES at the Sertoli cell-spermatid (step 8–19) and Sertoli-Sertoli cell interface, respectively, during the seminiferous epithelial cycle of spermatogenesis. In this Commentary, we put forth a possible model by which drebrin E may be acting as a platform upon which proteins (e.g., Arp3) that are needed to alter the conformation of actin filament bundles at the ES can be recruited to the site, thus facilitating changes in cell shape and cell position in the epithelium during spermiogenesis and spermiation. In short, drebrin E may be acting as a “logistic” distribution center to manage different regulatory proteins at the apical ES, thereby regulating the dynamics of actin filament bundles and modulating the plasticity of the apical ES. This would allow adhesion to be altered continuously throughout the epithelial cycle to accommodate spermatid movement in the seminiferous epithelium during spermiogenesis and spermiation. We also

  3. Hydrogen-deuterium exchange study of an allosteric energy cycle.

    Science.gov (United States)

    Beckett, Dorothy

    2012-01-01

    Elucidation of mechanisms of energy transduction through macromolecules in allosteric systems requires application of a broad range of techniques and approaches. High-resolution structures of the end states in an allosteric system provide invaluable clues about allosteric mechanism. Thermodynamic and kinetic studies reveal the rules that govern the transitions between states in the system. Acquisition of detailed molecular level information about allosteric mechanism requires interrogation of the structural and dynamic properties of both intermediates and end states in the allosteric cycle. Many experimental and computational tools have been developed to probe allostery. Among these are hydrogen-deuterium exchange detected by either NMR spectroscopy or mass spectrometry. This article provides a detailed description of application of hydrogen exchange detected by mass spectrometry (HDX-MS) to investigate an allosteric system.

  4. Small Molecule-Induced Allosteric Activation of the Vibrio Cholerae RTX Cysteine Protease Domain

    Energy Technology Data Exchange (ETDEWEB)

    Lupardus, P.J.; Shen, A.; Bogyo, M.; Garcia, K.C.

    2009-05-19

    Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP{sub 6}), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP{sub 6}. InsP{sub 6} binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP{sub 6} binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

  5. Agonism/antagonism switching in allosteric ensembles

    OpenAIRE

    Motlagh, Hesam N.; Hilser, Vincent J.

    2012-01-01

    Ligands for several transcription factors can act as agonists under some conditions and antagonists under others. The structural and molecular bases of such effects are unknown. Previously, we demonstrated how the folding of intrinsically disordered (ID) protein sequences, in particular, and population shifts, in general, could be used to mediate allosteric coupling between different functional domains, a model that has subsequently been validated in several systems. Here it is shown that pop...

  6. Rapid and dynamic arginylation of the leading edge β-actin is required for cell migration.

    Science.gov (United States)

    Pavlyk, Iuliia; Leu, Nicolae A; Vedula, Pavan; Kurosaka, Satoshi; Kashina, Anna

    2018-04-01

    β-actin plays key roles in cell migration. Our previous work demonstrated that β-actin in migratory non-muscle cells is N-terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of β-actin arginylation in cell migration. We found that arginylated β-actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact-inhibited cells in confluent cultures, suggesting that arginylated β-actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact-inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of β-actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Exploiting protein flexibility to predict the location of allosteric sites

    Directory of Open Access Journals (Sweden)

    Panjkovich Alejandro

    2012-10-01

    Full Text Available Abstract Background Allostery is one of the most powerful and common ways of regulation of protein activity. However, for most allosteric proteins identified to date the mechanistic details of allosteric modulation are not yet well understood. Uncovering common mechanistic patterns underlying allostery would allow not only a better academic understanding of the phenomena, but it would also streamline the design of novel therapeutic solutions. This relatively unexplored therapeutic potential and the putative advantages of allosteric drugs over classical active-site inhibitors fuel the attention allosteric-drug research is receiving at present. A first step to harness the regulatory potential and versatility of allosteric sites, in the context of drug-discovery and design, would be to detect or predict their presence and location. In this article, we describe a simple computational approach, based on the effect allosteric ligands exert on protein flexibility upon binding, to predict the existence and position of allosteric sites on a given protein structure. Results By querying the literature and a recently available database of allosteric sites, we gathered 213 allosteric proteins with structural information that we further filtered into a non-redundant set of 91 proteins. We performed normal-mode analysis and observed significant changes in protein flexibility upon allosteric-ligand binding in 70% of the cases. These results agree with the current view that allosteric mechanisms are in many cases governed by changes in protein dynamics caused by ligand binding. Furthermore, we implemented an approach that achieves 65% positive predictive value in identifying allosteric sites within the set of predicted cavities of a protein (stricter parameters set, 0.22 sensitivity, by combining the current analysis on dynamics with previous results on structural conservation of allosteric sites. We also analyzed four biological examples in detail, revealing

  8. The future of type 1 cannabinoid receptor allosteric ligands.

    Science.gov (United States)

    Alaverdashvili, Mariam; Laprairie, Robert B

    2018-02-01

    Allosteric modulation of the type 1 cannabinoid receptor (CB1R) holds great therapeutic potential. This is because allosteric modulators do not possess intrinsic efficacy, but instead augment (positive allosteric modulation) or diminish (negative allosteric modulation) the receptor's response to endogenous ligand. Consequently, CB1R allosteric modulators have an effect ceiling which allows for the tempering of CB1R signaling without the desensitization, tolerance, dependence, and psychoactivity associated with orthosteric compounds. Pain, movement disorders, epilepsy, obesity are all potential therapeutic targets for CB1R allosteric modulation. Several challenges exist for the development of CB1R allosteric modulators, such as receptor subtype specificity, translation to in vivo systems, and mixed allosteric/agonist/inverse agonist activity. Despite these challenges, elucidation of crystal structures of CB1R and compound design based on structure-activity relationships will advance the field. In this review, we will cover recent progress for CB1R allosteric modulators and discuss the future promise of this research.

  9. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

    Science.gov (United States)

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-01-01

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division. DOI: http://dx.doi.org/10.7554/eLife.08828.001 PMID:26305500

  10. Boolean gates on actin filaments

    Energy Technology Data Exchange (ETDEWEB)

    Siccardi, Stefano, E-mail: ssiccardi@2ssas.it [The Unconventional Computing Centre, University of the West of England, Bristol (United Kingdom); Tuszynski, Jack A., E-mail: jackt@ualberta.ca [Department of Oncology, University of Alberta, Edmonton, Alberta (Canada); Adamatzky, Andrew, E-mail: andrew.adamatzky@uwe.ac.uk [The Unconventional Computing Centre, University of the West of England, Bristol (United Kingdom)

    2016-01-08

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

  11. Synthesis of Novel Allosteric Agonists and Allosteric Modulators for Nicotinic Acetylcholine Receptors

    OpenAIRE

    Dhankher, P.

    2013-01-01

    In healthy individuals, the α7 and α4β2 nAChRs are concentrated in regions of the brain involved with learning, cognition and memory, which are relevant to diseases such as Alzheimer’s disease. Hence, these receptors have become significant from a pharmacological and drug discovery perspective. The tetrahydroquinoline compound 4BP-TQS has been reported to act as a potent allosteric agonist on the α7 nAChR. The natural product desformylflustrabromine is able to act as a positive allosteric mod...

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

    Science.gov (United States)

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

    2016-01-01

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

  13. Actin capping protein and its inhibitor CARMIL: how intrinsically disordered regions function

    International Nuclear Information System (INIS)

    Takeda, Shuichi; Maéda, Yuichiro; Koike, Ryotaro; Ota, Motonori; Nitanai, Yasushi; Minakata, Shiho

    2011-01-01

    The actin capping protein (CP) tightly binds to the barbed end of actin filaments to block further elongation. The β-tentacle in CP is an important region that ensures stable interaction with actin filaments. CARMIL inhibits the interaction of CP with actin filaments via the C-terminal portion containing the CP-binding motif, located in an intrinsically disordered region. We have proposed an allosteric inhibition model in which CARMIL suppresses CP by the population shift mechanism. Here, we solved a crystal structure of CP in complex with a CARMIL-derived peptide, CA32. The new structure clearly represents the α-helical form of the β-tentacle that was invisible in other CP/CARMIL peptide complex structures. In addition, we exhaustively performed a normal mode analysis with the elastic network model on all available crystal structures of the CP/CARMIL peptide complexes, including the new structure. We concluded that the CP-binding motif is necessary and sufficient for altering the fluctuation of CP, which is essential for attenuating the barbed-end-capping activity along the population shift mechanism. The roles and functions of the β-tentacle and the CP-binding motif are discussed in terms of their intrinsically disordered nature

  14. Actin capping protein and its inhibitor CARMIL: how intrinsically disordered regions function

    Science.gov (United States)

    Takeda, Shuichi; Koike, Ryotaro; Nitanai, Yasushi; Minakata, Shiho; Maéda, Yuichiro; Ota, Motonori

    2011-06-01

    The actin capping protein (CP) tightly binds to the barbed end of actin filaments to block further elongation. The β-tentacle in CP is an important region that ensures stable interaction with actin filaments. CARMIL inhibits the interaction of CP with actin filaments via the C-terminal portion containing the CP-binding motif, located in an intrinsically disordered region. We have proposed an allosteric inhibition model in which CARMIL suppresses CP by the population shift mechanism. Here, we solved a crystal structure of CP in complex with a CARMIL-derived peptide, CA32. The new structure clearly represents the α-helical form of the β-tentacle that was invisible in other CP/CARMIL peptide complex structures. In addition, we exhaustively performed a normal mode analysis with the elastic network model on all available crystal structures of the CP/CARMIL peptide complexes, including the new structure. We concluded that the CP-binding motif is necessary and sufficient for altering the fluctuation of CP, which is essential for attenuating the barbed-end-capping activity along the population shift mechanism. The roles and functions of the β-tentacle and the CP-binding motif are discussed in terms of their intrinsically disordered nature.

  15. Boolean gates on actin filaments

    Science.gov (United States)

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

    2016-01-01

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

  16. Allosteric modulation of G-protein coupled receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, Tracy A

    2004-01-01

    are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric sites....... In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines...... the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands....

  17. Orthosteric and Allosteric Regulation in Trypsin-Like Peptidases

    DEFF Research Database (Denmark)

    Kromann-Tofting, Tobias

    peptides and Camelid derived antibody fragments, so-called nanobodies. Allosteric regulation of activity in trypsin-like serine peptidases is in general poorly understood, as the propagation of the allosteric signal from the ligand binding site to the active site in some cases is subtle and based...... exclusively on a change in side chain and backbone dynamics along the allosteric trajectory. This thesis describes the characterisation of two allosteric monoclonal antibodies and the development and characterisation of an allosteric nanobody against murine uPA. Insights into their binding mechanisms, using X...... approach for molecular intervention with the function of trypsin-like serine peptidases. In the thesis, I also describe the development of nanobodies that specifically target zymogen activation of uPA, by preventing its proteolytic cleavage by plasmin....

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

    Science.gov (United States)

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

    2000-01-01

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

  19. ETA-receptor antagonists or allosteric modulators?

    DEFF Research Database (Denmark)

    De Mey, Jo G R; Compeer, Matthijs G; Lemkens, Pieter

    2011-01-01

    . In resistance arteries, the long-lasting contractile effects can only be partly and reversibly relaxed by low-molecular-weight ET(A) antagonists (ERAs). However, the neuropeptide calcitonin-gene-related peptide selectively terminates binding of ET1 to ET(A). We propose that ET1 binds polyvalently to ET......(A) and that ERAs and the physiological antagonist allosterically reduce ET(A) functions. Combining the two-state model and the two-domain model of GPCR function and considering receptor activation beyond agonist binding might lead to better anti-endothelinergic drugs. Future studies could lead to compounds...

  20. Arabidopsis actin-depolymerizing factor7 severs actin filaments and regulates actin cable turnover to promote normal pollen tube growth.

    Science.gov (United States)

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-09-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana actin-depolymerizing factor7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7-enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes.

  1. Actin microfilament dynamics in locomoting cells

    Science.gov (United States)

    Theriot, Julie A.; Mitchison, Timothy J.

    1991-07-01

    The dynamic behaviour of actin filaments has been directly observed in living, motile cells using fluorescence photoactivation. In goldfish epithelial keratocytes, the actin microfilaments in the lamellipodium remain approximately fixed relative to the substrate as the cell moves over them, regardless of cell speed. The rate of turnover of actin subunits in the lamellipodium is remarkably rapid. Cell movement is directly and tightly coupled to the formation of new actin filaments at the leading edge.

  2. Actin' as a Death Signal

    NARCIS (Netherlands)

    Geijtenbeek, Teunis B. H.

    2012-01-01

    Cell death needs to be detected by immune cells. In this issue of Immunity, Ahrens et al. (2012) and Zhang et al. (2012) show that actin filaments become exposed on necrotic cells and act as ligands for the C-type lectin receptor Clec9a

  3. Emerging Computational Methods for the Rational Discovery of Allosteric Drugs.

    Science.gov (United States)

    Wagner, Jeffrey R; Lee, Christopher T; Durrant, Jacob D; Malmstrom, Robert D; Feher, Victoria A; Amaro, Rommie E

    2016-06-08

    Allosteric drug development holds promise for delivering medicines that are more selective and less toxic than those that target orthosteric sites. To date, the discovery of allosteric binding sites and lead compounds has been mostly serendipitous, achieved through high-throughput screening. Over the past decade, structural data has become more readily available for larger protein systems and more membrane protein classes (e.g., GPCRs and ion channels), which are common allosteric drug targets. In parallel, improved simulation methods now provide better atomistic understanding of the protein dynamics and cooperative motions that are critical to allosteric mechanisms. As a result of these advances, the field of predictive allosteric drug development is now on the cusp of a new era of rational structure-based computational methods. Here, we review algorithms that predict allosteric sites based on sequence data and molecular dynamics simulations, describe tools that assess the druggability of these pockets, and discuss how Markov state models and topology analyses provide insight into the relationship between protein dynamics and allosteric drug binding. In each section, we first provide an overview of the various method classes before describing relevant algorithms and software packages.

  4. An allosteric model of the molecular interactions of excitation-contraction coupling in skeletal muscle.

    Science.gov (United States)

    Ríos, E; Karhanek, M; Ma, J; González, A

    1993-09-01

    A contact interaction is proposed to exist between the voltage sensor of the transverse tubular membrane of skeletal muscle and the calcium release channel of the sarcoplasmic reticulum. This interaction is given a quantitative formulation inspired in the Monod, Wyman, and Changeux model of allosteric transitions in hemoglobin (Monod, J., J. Wyman, and J.-P. Changeux. 1965. Journal of Molecular Biology. 12:88-118), and analogous to one proposed by Marks and Jones for voltage-dependent Ca channels (Marks, T. N., and S. W. Jones. 1992. Journal of General Physiology. 99:367-390). The allosteric protein is the calcium release channel, a homotetramer, with two accessible states, closed and open. The kinetics and equilibrium of this transition are modulated by voltage sensors (dihydropyridine receptors) pictured as four units per release channel, each undergoing independent voltage-driven transitions between two states (resting and activating). For each voltage sensor that moves to the activating state, the tendency of the channel to open increases by an equal (large) factor. The equilibrium and kinetic equations of the model are solved and shown to reproduce well a number of experimentally measured relationships including: charge movement (Q) vs. voltage, open probability of the release channel (Po) vs. voltage, the transfer function relationship Po vs. Q, and the kinetics of charge movement, release activation, and deactivation. The main consequence of the assumption of allosteric coupling is that primary effects on the release channel are transmitted backward to the voltage sensor and give secondary effects. Thus, the model reproduces well the effects of perchlorate, described in the two previous articles, under the assumption that the primary effect is to increase the intrinsic tendency of the release channel to open, with no direct effects on the voltage sensor. This modification of the open-closed equilibrium of the release channel causes a shift in the equilibrium

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

    DEFF Research Database (Denmark)

    Rasmussen, Izabela; Pedersen, Line Hjortshøj; Byg, Luise

    2010-01-01

    Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin d...... dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton....

  6. Allosteric Equilibria in the Binding of Fibrinogen to Platelets

    Science.gov (United States)

    de Cristofaro, Raimondo; Landolfi, Raffaele; de Candia, Erica; Castagnola, Massimo; di Cera, Enrico; Wyman, Jeffries

    1988-11-01

    The binding of fibrinogen to platelets occurs according to the law of mass action. The platelet receptor binds reversibly a single fibrinogen molecule and undergoes a conformational transition between two allosteric states, T and R, that differ in their affinity for fibrinogen. The equilibrium between the two forms is shifted by ADP toward the R (high-affinity) state, thus promoting the aggregation process. This model opens the way to consideration of allosteric modulation of the binding of fibrinogen to its platelet receptor.

  7. The actin multigene family of Paramecium tetraurelia

    Directory of Open Access Journals (Sweden)

    Wagner Erika

    2007-03-01

    Full Text Available Abstract Background A Paramecium tetraurelia pilot genome project, the subsequent sequencing of a Megabase chromosome as well as the Paramecium genome project aimed at gaining insight into the genome of Paramecium. These cells display a most elaborate membrane trafficking system, with distinct, predictable pathways in which actin could participate. Previously we had localized actin in Paramecium; however, none of the efforts so far could proof the occurrence of actin in the cleavage furrow of a dividing cell, despite the fact that actin is unequivocally involved in cell division. This gave a first hint that Paramecium may possess actin isoforms with unusual characteristics. The genome project gave us the chance to search the whole Paramecium genome, and, thus, to identify and characterize probably all actin isoforms in Paramecium. Results The ciliated protozoan, P. tetraurelia, contains an actin multigene family with at least 30 members encoding actin, actin-related and actin-like proteins. They group into twelve subfamilies; a large subfamily with 10 genes, seven pairs and one trio with > 82% amino acid identity, as well as three single genes. The different subfamilies are very distinct from each other. In comparison to actins in other organisms, P. tetraurelia actins are highly divergent, with identities topping 80% and falling to 30%. We analyzed their structure on nucleotide level regarding the number and position of introns. On amino acid level, we scanned the sequences for the presence of actin consensus regions, for amino acids of the intermonomer interface in filaments, for residues contributing to ATP binding, and for known binding sites for myosin and actin-specific drugs. Several of those characteristics are lacking in several subfamilies. The divergence of P. tetraurelia actins and actin-related proteins between different P. tetraurelia subfamilies as well as with sequences of other organisms is well represented in a phylogenetic

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

    Science.gov (United States)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

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

  9. Actin dynamics in mouse fibroblasts in microgravity

    Science.gov (United States)

    Moes, Maarten J. A.; Bijvelt, Jose J.; Boonstra, Johannes

    2007-09-01

    After stimulating with the growth factor PDGF, cells exhibit abundant membrane ruffling and other morphological changes under normal gravity conditions. These morphological changes are largely determined by the actin microfilament system. Now these actin dynamics were studied under microgravity conditions in mouse fibroblasts during the DELTA mission. The aim of the present study was to describe the actin morphology in detail, to establish the effect of PDGF on actin morphology and to study the role of several actin-interacting proteins involved in introduced actin dynamics in microgravity. Identical experiments were conducted at 1G on earth as a reference. No results in microgravity were obtained due to a combination of malfunctioning hardware and unfulfilled temperature requirements.

  10. Actin organization and dynamics in filamentous fungi.

    Science.gov (United States)

    Berepiki, Adokiye; Lichius, Alexander; Read, Nick D

    2011-11-02

    Growth and morphogenesis of filamentous fungi is underpinned by dynamic reorganization and polarization of the actin cytoskeleton. Actin has crucial roles in exocytosis, endocytosis, organelle movement and cytokinesis in fungi, and these processes are coupled to the production of distinct higher-order structures (actin patches, cables and rings) that generate forces or serve as tracks for intracellular transport. New approaches for imaging actin in living cells are revealing important similarities and differences in actin architecture and organization within the fungal kingdom, and have yielded key insights into cell polarity, tip growth and long-distance intracellular transport. In this Review, we discuss the contribution that recent live-cell imaging and mutational studies have made to our understanding of the dynamics and regulation of actin in filamentous fungi.

  11. Conformational and dynamic differences between actin filaments polymerized from ATP- or ADP-actin monomers.

    Science.gov (United States)

    Nyitrai, M; Hild, G; Hartvig, N; Belágyi, J; Somogyi, B

    2000-12-29

    Conformational and dynamic properties of actin filaments polymerized from ATP- or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys(374) residue of actin was smaller in filaments from ADP-actin than in filaments from ATP-actin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys(374) from the longitudinal filament axis. Temperature-dependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys(374) of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.

  12. GPCRs and actin-cytoskeleton dynamics.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; González-Espinosa, Claudia; Espinosa-Riquer, Zyanya P; Macías-Silva, Marina

    2016-01-01

    A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Co-transcriptional nuclear actin dynamics.

    Science.gov (United States)

    Percipalle, Piergiorgio

    2013-01-01

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

  14. Bioinformatics study of the mangrove actin genes

    Science.gov (United States)

    Basyuni, M.; Wasilah, M.; Sumardi

    2017-01-01

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

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

    OpenAIRE

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

    2014-01-01

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, an...

  16. Pharmacological treatment of actinic keratosis

    Directory of Open Access Journals (Sweden)

    Ewa Zwierzyńska

    2016-09-01

    Full Text Available Actinic keratosis (AK is a disease characterized by hyperkeratotic lesions on skin damaged by ultraviolet. radiation. These lesions may progress to squamous cell or basal cell carcinoma. Currently pharmacotherapy and different surgical procedures are used in AK therapy. The most common treatment options are 5-fluorouracil, imiquimod, diclofenac, ingenol mebutate, and first and third generation retinoids (retinol, adapalene, tazarotene. Furthermore, research is being carried out in order to test new medications including nicotinamide, resiquimod, piroxicam, potassium dobesilate and oleogel based on a triterpene extract (betulin, betulinic acid. Recently, the preventive effect of acetylsalicylic acid and celecoxib has also been investigated.

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

    Science.gov (United States)

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

    2014-09-15

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

  18. Mechanical hysteresis in actin networks.

    Science.gov (United States)

    Majumdar, Sayantan; Foucard, Louis C; Levine, Alex J; Gardel, Margaret L

    2018-03-14

    Understanding the response of complex materials to external force is central to fields ranging from materials science to biology. Here, we describe a novel type of mechanical adaptation in cross-linked networks of F-actin, a ubiquitous protein found in eukaryotic cells. We show that shear stress changes the network's nonlinear mechanical response even long after that stress is removed. The duration, magnitude and direction of forcing history all change this mechanical response. While the mechanical hysteresis is long-lived, it can be simply erased by force application in the opposite direction. We further show that the observed mechanical adaptation is consistent with stress-dependent changes in the nematic order of the constituent filaments. Thus, this mechanical hysteresis arises from the changes in non-linear response that originates from stress-induced changes to filament orientation. This demonstrates that F-actin networks can exhibit analog read-write mechanical hysteretic properties, which can be used for adaptation to mechanical stimuli.

  19. Novel inhibitors complexed with glutamate dehydrogenase: allosteric regulation by control of protein dynamics.

    Science.gov (United States)

    Li, Ming; Smith, Christopher J; Walker, Matthew T; Smith, Thomas J

    2009-08-21

    Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)(+) as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

  20. Novel Inhibitors Complexed with Glutamate Dehydrogenase: ALLOSTERIC REGULATION BY CONTROL OF PROTEIN DYNAMICS

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ming; Smith, Christopher J.; Walker, Matthew T.; Smith, Thomas J.; (Danforth)

    2009-12-01

    Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P){sup +} as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

  1. In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

    Science.gov (United States)

    Mitchell, J J; Woodcock-Mitchell, J L; Perry, L; Zhao, J; Low, R B; Baldor, L; Absher, P M

    1993-07-01

    alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Unconventional actin conformations localize on intermediate filaments in mitosis

    International Nuclear Information System (INIS)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-01

    Research highlights: → Unconventional actin conformations colocalize with vimentin on a cage-like structure in metaphase HEK 293T cells. → These conformations are detected with the anti-actin antibodies 1C7 ('lower dimer') and 2G2 ('nuclear actin'), but not C4 (monomeric actin). → Mitotic unconventional actin cables are independent of filamentous actin or microtubules. → Unconventional actin colocalizes with vimentin on a nocodazole-induced perinuclear dense mass of cables. -- Abstract: Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel 'lower dimer' actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

  3. Immunology: Is Actin at the Lytic Synapse a Friend or a Foe?

    Science.gov (United States)

    Hammer, John A

    2018-02-19

    Cytotoxic T cells and natural killer cells defend us against disease by secreting lytic granules. Whether actin facilitates or thwarts lytic granule secretion has been an open question. Recent results now indicate that the answer depends on the maturation stage of the immune cell-target cell contact. Published by Elsevier Ltd.

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

    Science.gov (United States)

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

    2011-10-18

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

  5. Allosterically tunable, DNA-based switches triggered by heavy metals.

    Science.gov (United States)

    Porchetta, Alessandro; Vallée-Bélisle, Alexis; Plaxco, Kevin W; Ricci, Francesco

    2013-09-11

    Here we demonstrate the rational design of allosterically controllable, metal-ion-triggered molecular switches. Specifically, we designed DNA sequences that adopt two low energy conformations, one of which does not bind to the target ion and the other of which contains mismatch sites serving as specific recognition elements for mercury(II) or silver(I) ions. Both switches contain multiple metal binding sites and thus exhibit homotropic allosteric (cooperative) responses. As heterotropic allosteric effectors we employ single-stranded DNA sequences that either stabilize or destabilize the nonbinding state, enabling dynamic range tuning over several orders of magnitude. The ability to rationally introduce these effects into target-responsive switches could be of value in improving the functionality of DNA-based nanomachines.

  6. Dynamic Regulation of Sarcomeric Actin Filaments in Striated Muscle

    OpenAIRE

    Ono, Shoichiro

    2010-01-01

    In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin sub...

  7. Specification of Architecture and Function of Actin Structures by Actin Nucleation Factors.

    Science.gov (United States)

    Skau, Colleen T; Waterman, Clare M

    2015-01-01

    The actin cytoskeleton is essential for diverse processes in mammalian cells; these processes range from establishing cell polarity to powering cell migration to driving cytokinesis to positioning intracellular organelles. How these many functions are carried out in a spatiotemporally regulated manner in a single cytoplasm has been the subject of much study in the cytoskeleton field. Recent work has identified a host of actin nucleation factors that can build architecturally diverse actin structures. The biochemical properties of these factors, coupled with their cellular location, likely define the functional properties of actin structures. In this article, we describe how recent advances in cell biology and biochemistry have begun to elucidate the role of individual actin nucleation factors in generating distinct cellular structures. We also consider how the localization and orientation of actin nucleation factors, in addition to their kinetic properties, are critical to their ability to build a functional actin cytoskeleton.

  8. Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

    Science.gov (United States)

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

    2010-09-09

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

  9. Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

    Directory of Open Access Journals (Sweden)

    Shenping Wu

    2010-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Shenping; Liu, Jun; Reedy, Mary C.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A. (UPENN); (Duke); (MRCLMB); (FSU); (Jikei-Med)

    2010-10-22

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

  11. Modulation of microfilament protein composition by transfected cytoskeletal actin genes

    Energy Technology Data Exchange (ETDEWEB)

    Ng, S.Y.; Erba, H.; Latter, G.; Kedes, L.; Leavitt, J.

    1988-04-01

    HuT-14T is a highly tumorigenic fibroblast cell line which exhibits a reduced steady-state level of ..beta..-actin due to coding mutations in one of two ..beta..-actin alleles. The normal rate of total actin synthesis could be restored in some clones of cells following transfection of the functional ..beta..-actin gene but not following transfection of the functional ..gamma..-actin gene. In ..gamma..-actin gene-transfected substrains that have increased rates of ..gamma..-actin synthesis, ..beta..-actin synthesis is further reduced in a manner consistent with an autoregulatory mechanism, resulting in abnormal ratios of actin isoforms. Thus, both ..beta..- and ..gamma..-actin proteins can apparently regulate the synthesis of their coexpressed isoforms. In addition, decreased synthesis of normal ..beta..-actin seems to correlate with a concomitant down-regulation of tropomyosin isoforms.

  12. Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Aguado, Carmen; Mato, Eugenia; Sánchez-Ruíz, Yován; Esteban, Inmaculada; Alberch, Jordi; Knecht, Erwin; Egea, Gustavo

    2008-05-01

    In this study, we report the formation of several cytoplasmic inclusion bodies composed of filamentous actin (F-actin) and generated by experimental treatments using depolymerizing or stabilizing actin toxins in neuronal and non-neuronal mammalian cell lines. The actin-stabilizing toxin jasplakinolide (Jpk) induced, in a microtubule-dependent manner, a single, large F-actin aggregate, which contained beta- and gamma-actin, ADF/cofilin, cortactin, and the actin nucleator Arp2/3. This aggregate was tightly associated with the Golgi complex and mitochondria, and was surrounded by vimentin intermediate filaments, microtubules and MAP4. Therefore, the Jpk-induced single, large F-actin aggregate fits the established criteria for being considered an aggresome. Lysosomes and/or autophagic vacuoles, proteasomes and microtubules were found to directly participate in the dissolution of this F-actin aggresome. Finally, the model reported here is simple, highly reproducible and reversible, and it provides an opportunity to test pharmacological agents that interfere with the formation, maintenance and/or disappearance of F-actin-enriched pathological inclusion bodies.

  13. Nuclear Actin and Myosins in Adenovirus Infection

    Science.gov (United States)

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

    2015-01-01

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

  14. Metal ion coupled protein folding and allosteric motions

    Science.gov (United States)

    Wang, Wei

    2014-03-01

    Many proteins need the help of cofactors for their successful folding and functioning. Metal ions, i.e., Zn2+, Ca2+, and Mg2+ etc., are typical biological cofactors. Binding of metal ions can reshape the energy landscapes of proteins, thereby modifying the folding and allosteric motions. For example, such binding may make the intrinsically disordered proteins have funneled energy landscapes, consequently, ensures their spontaneous folding. In addition, the binding may activate certain biological processes by inducing related conformational changes of regulation proteins. However, how the local interactions involving the metal ion binding can induce the global conformational motions of proteins remains elusive. Investigating such question requires multiple models with different details, including quantum mechanics, atomistic models, and coarse grained models. In our recent work, we have been developing such multiscale methods which can reasonably model the metal ion binding induced charge transfer, protonation/deprotonation, and large conformational motions of proteins. With such multiscale model, we elucidated the zinc-binding induced folding mechanism of classical zinc finger and the calcium-binding induced dynamic symmetry breaking in the allosteric motions of calmodulin. In addition, we studied the coupling of folding, calcium binding and allosteric motions of calmodulin domains. In this talk, I will introduce the above progresses on the metal ion coupled protein folding and allosteric motions. We thank the finacial support from NSFC and the 973 project.

  15. The structure and allosteric regulation of glutamate dehydrogenase.

    Science.gov (United States)

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2011-09-01

    Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a 'housekeeping' enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an 'antenna' like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. The allosteric communication pathways in KIX domain of CBP

    Science.gov (United States)

    Palazzesi, Ferruccio; Barducci, Alessandro; Tollinger, Martin; Parrinello, Michele

    2013-01-01

    Allosteric regulation plays an important role in a myriad of biomacromolecular processes. Specifically, in a protein, the process of allostery refers to the transmission of a local perturbation, such as ligand binding, to a distant site. Decades after the discovery of this phenomenon, models built on static images of proteins are being reconsidered with the knowledge that protein dynamics plays an important role in its function. Molecular dynamics simulations are a valuable tool for studying complex biomolecular systems, providing an atomistic description of their structure and dynamics. Unfortunately, their predictive power has been limited by the complexity of the biomolecule free-energy surface and by the length of the allosteric timescale (in the order of milliseconds). In this work, we are able to probe the origins of the allosteric changes that transcription factor mixed lineage leukemia (MLL) causes to the interactions of KIX domain of CREB-binding protein (CBP) with phosphorylated kinase inducible domain (pKID), by combing all-atom molecular dynamics with enhanced sampling methods recently developed in our group. We discuss our results in relation to previous NMR studies. We also develop a general simulations protocol to study allosteric phenomena and many other biological processes that occur in the micro/milliseconds timescale. PMID:23940332

  17. Actinic Granuloma with Focal Segmental Glomerulosclerosis

    Directory of Open Access Journals (Sweden)

    Ruedee Phasukthaworn

    2016-02-01

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

  18. Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments.

    Science.gov (United States)

    Bubb, M R; Spector, I; Bershadsky, A D; Korn, E D

    1995-02-24

    Swinholide A, isolated from the marien sponge Theonella swinhoei, is a 44-carbon ring dimeric dilactone macrolide with a 2-fold axis of symmetry. Recent studies have elucidated its unusual structure and shown that it has potent cytotoxic activity. We now report that swinholide A disrupts the actin cytoskeleton of cells grown in culture, sequesters actin dimers in vitro in both polymerizing and non-polymerizing buffers with a binding stoichiometry of one swinholide A molecule per actin dimer, and rapidly severs F-actin in vitro with high cooperativity. These unique properties are sufficient to explain the cytotoxicity of swinholide A. They also suggest that swinholide A might be a model for studies of the mechanism of action of F-actin severing proteins and be therapeutically useful in conditions where filamentous actin contributes to pathologically high viscosities.

  19. Structural differences explain diverse functions of Plasmodium actins.

    Directory of Open Access Journals (Sweden)

    Juha Vahokoski

    2014-04-01

    Full Text Available Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.

  20. Functional Properties and Mechanism of Action of PPTQ, an Allosteric Agonist and Low Nanomolar Positive Allosteric Modulator at GABAA Receptors

    DEFF Research Database (Denmark)

    Madjroh, Nawid; Olander, Emma Rie; Bundgaard, Christoffer

    2018-01-01

    The former sedative-hypnotic and recreational drug methaqualone (Quaalude) is a moderately potent, non-selective positive allosteric modulator (PAM) at GABAA receptors (GABAARs) (Hammer et al., 2015). In the present study, we have identified a novel methaqualone analog, 2-phenyl-3-(p...

  1. Extracellular loop 2 of the free Fatty Acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric modulator

    DEFF Research Database (Denmark)

    Smith, Nicola J; Ward, Richard J; Stoddart, Leigh A

    2011-01-01

    Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molec...

  2. Mechanics model for actin-based motility.

    Science.gov (United States)

    Lin, Yuan

    2009-02-01

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

  3. Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

    Science.gov (United States)

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

    2013-01-01

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

  4. Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea

    Directory of Open Access Journals (Sweden)

    Ligia Cristina Kalb Souza

    2013-08-01

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

  5. Branching and capping determine the force–velocity relationships of branching actin networks

    International Nuclear Information System (INIS)

    Smith, Daniel B; Liu, Jian

    2013-01-01

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

  6. Distortion of the Actin A-Triad Results in Contractile Disinhibition and Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Meera C. Viswanathan

    2017-09-01

    Full Text Available Striated muscle contraction is regulated by the movement of tropomyosin over the thin filament surface, which blocks or exposes myosin binding sites on actin. Findings suggest that electrostatic contacts, particularly those between K326, K328, and R147 on actin and tropomyosin, establish an energetically favorable F-actin-tropomyosin configuration, with tropomyosin positioned in a location that impedes actomyosin associations and promotes relaxation. Here, we provide data that directly support a vital role for these actin residues, termed the A-triad, in tropomyosin positioning in intact functioning muscle. By examining the effects of an A295S α-cardiac actin hypertrophic cardiomyopathy-causing mutation, over a range of increasingly complex in silico, in vitro, and in vivo Drosophila muscle models, we propose that subtle A-triad-tropomyosin perturbation can destabilize thin filament regulation, which leads to hypercontractility and triggers disease. Our efforts increase understanding of basic thin filament biology and help unravel the mechanistic basis of a complex cardiac disorder.

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

    Science.gov (United States)

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

    2016-01-15

    The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails--dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens. © 2016 Borinskaya et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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

    Science.gov (United States)

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

    1990-05-01

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

  9. Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton.

    Science.gov (United States)

    Marat, Andrea L; Ioannou, Maria S; McPherson, Peter S

    2012-01-01

    The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)-domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1-3/DENND1A-C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

  10. Separation of actin-dependent and actin-independent lipid rafts

    NARCIS (Netherlands)

    Klappe, Karin; Hummel, Ina; Kok, Jan Willem

    2013-01-01

    Lipid rafts have been isolated on the basis of their resistance to various detergents and more recently by using detergent-free procedures. The actin cytoskeleton is now recognized as a dynamic regulator of lipid raft stability. We carefully analyzed the effects of the cortical actin-disrupting

  11. Light-activated DNA binding in a designed allosteric protein

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R. (UC)

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  12. Macrolide antibiotics allosterically predispose the ribosome for translation arrest.

    Science.gov (United States)

    Sothiselvam, Shanmugapriya; Liu, Bo; Han, Wei; Ramu, Haripriya; Klepacki, Dorota; Atkinson, Gemma Catherine; Brauer, Age; Remm, Maido; Tenson, Tanel; Schulten, Klaus; Vázquez-Laslop, Nora; Mankin, Alexander S

    2014-07-08

    Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.

  13. Green fluorescent protein-mtalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro

    NARCIS (Netherlands)

    Ketelaar, T.; Anthony, R.G.; Hussey, P.J.

    2004-01-01

    Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that

  14. Chronic actinic dermatitis - A study of clinical features

    Directory of Open Access Journals (Sweden)

    Somani Vijay

    2005-01-01

    Full Text Available Background: Chronic actinic dermatitis (CAD, one of the immune mediated photo-dermatoses, comprises a spectrum of conditions including persistent light reactivity, photosensitive eczema and actinic reticuloid. Diagnostic criteria were laid down about 20 years back, but clinical features are the mainstay in diagnosis. In addition to extreme sensitivity to UVB, UVA and/or visible light, about three quarters of patients exhibit contact sensitivity to several allergens, which may contribute to the etiopathogenesis of CAD. This study was undertaken to examine the clinical features of CAD in India and to evaluate the relevance of patch testing and photo-aggravation testing in the diagnosis of CAD. Methods: The clinical data of nine patients with CAD were analyzed. Histopathology, patch testing and photo-aggravation testing were also performed. Results: All the patients were males. The average age of onset was 57 years. The first episode was usually noticed in the beginning of summer. Later the disease gradually tended to be perennial, without any seasonal variations. The areas affected were mainly the photo-exposed areas in all patients, and the back in three patients. Erythroderma was the presenting feature in two patients. The palms and soles were involved in five patients. Patch testing was positive in seven of nine patients. Conclusions: The diagnosis of CAD mainly depended upon the history and clinical features. The incidence of erythroderma and palmoplantar eczema was high in our series. Occupation seems to play a role in the etiopathogenesis of CAD.

  15. Glutamyl phosphate is an activated intermediate in actin crosslinking by actin crosslinking domain (ACD toxin.

    Directory of Open Access Journals (Sweden)

    Elena Kudryashova

    Full Text Available Actin Crosslinking Domain (ACD is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K(0.5 = 30 µM reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg(2+-GTP to support crosslinking, but the kinetic parameters (K(M = 8 µM and 50 µM for ATP and GTP, respectively suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0-9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions.

  16. Glutamyl phosphate is an activated intermediate in actin crosslinking by actin crosslinking domain (ACD) toxin.

    Science.gov (United States)

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

    2012-01-01

    Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K(0.5) = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg(2+)-GTP to support crosslinking, but the kinetic parameters (K(M) = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0-9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions.

  17. Evolution of allosteric regulation in chorismate mutases from early plants

    Energy Technology Data Exchange (ETDEWEB)

    Kroll, Kourtney; Holland, Cynthia K.; Starks, Courtney M.; Jez, Joseph M.

    2017-09-28

    Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 Å X-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.

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

    Directory of Open Access Journals (Sweden)

    Paves Heiti

    2004-04-01

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

  19. Antibodies to actin in autoimmune haemolytic anaemia

    Directory of Open Access Journals (Sweden)

    Ritzmann Mathias

    2010-03-01

    Full Text Available Abstract Background In autoimmune haemolytic anaemia (AIHA, autoreactive antibodies directed against red blood cells are up-regulated, leading to erythrocyte death. Mycoplasma suis infections in pigs induce AIHA of both the warm and cold types. The aim of this study was to identify the target autoantigens of warm autoreactive IgG antibodies. Sera from experimentally M. suis-infected pigs were screened for autoreactivity. Results Actin-reactive antibodies were found in the sera of 95% of all animals tested. The reactivity was species-specific, i.e. reactivity with porcine actin was significantly higher than with rabbit actin. Sera of animals previously immunised with the M. suis adhesion protein MSG1 showed reactivity with actin prior to infection with M. suis indicating that molecular mimicry is involved in the specific autoreactive mechanism. A potentially cross-reactive epitope was detected. Conclusions This is the first report of autoreactive anti-actin antibodies involved in the pathogenesis of autoimmune haemolytic anaemia.

  20. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    Science.gov (United States)

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  1. The structure and allosteric regulation of mammalian glutamate dehydrogenase.

    Science.gov (United States)

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2012-03-15

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Glutamate dehydrogenase: structure, allosteric regulation, and role in insulin homeostasis.

    Science.gov (United States)

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2014-01-01

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine and inhibitors include GTP, palmitoyl CoA, and ATP. Spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds blocked the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  3. Trade-offs and constraints in allosteric sensing.

    Science.gov (United States)

    Martins, Bruno M C; Swain, Peter S

    2011-11-01

    Sensing extracellular changes initiates signal transduction and is the first stage of cellular decision-making. Yet relatively little is known about why one form of sensing biochemistry has been selected over another. To gain insight into this question, we studied the sensing characteristics of one of the biochemically simplest of sensors: the allosteric transcription factor. Such proteins, common in microbes, directly transduce the detection of a sensed molecule to changes in gene regulation. Using the Monod-Wyman-Changeux model, we determined six sensing characteristics--the dynamic range, the Hill number, the intrinsic noise, the information transfer capacity, the static gain, and the mean response time--as a function of the biochemical parameters of individual sensors and of the number of sensors. We found that specifying one characteristic strongly constrains others. For example, a high dynamic range implies a high Hill number and a high capacity, and vice versa. Perhaps surprisingly, these constraints are so strong that most of the space of characteristics is inaccessible given biophysically plausible ranges of parameter values. Within our approximations, we can calculate the probability distribution of the numbers of input molecules that maximizes information transfer and show that a population of one hundred allosteric transcription factors can in principle distinguish between more than four bands of input concentrations. Our results imply that allosteric sensors are unlikely to have been selected for high performance in one sensing characteristic but for a compromise in the performance of many.

  4. Identification of the Allosteric Regulatory Site of Insulysin

    Energy Technology Data Exchange (ETDEWEB)

    Noinaj, Nicholas; Bhasin, Sonia K.; Song, Eun Suk; Scoggin, Kirsten E.; Juliano, Maria A.; Juliano, Luiz; Hersh, Louis B.; Rodgers, David W. (U. Sao Paulo); (Kentucky)

    2012-05-25

    Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the A{beta} peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP. The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits. Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.

  5. Identification of the Allosteric Regulatory Site of Insulysin

    Energy Technology Data Exchange (ETDEWEB)

    Noinaj, Nicholas; Bhasin, Sonia K.; Song, Eun Suk; Scoggin, Kirsten E.; Juliano, Maria A.; Juliano, Luiz; Hersh, Louis B.; Rodgers, David W.; Gerrard, Juliet Ann

    2011-06-24

    Background Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the Aβ peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP. Principal Findings The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits. Conclusions/Significance Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.

  6. Physical guidance of the actin cytoskeleton and cell migration dynamics in epithelial cells

    Science.gov (United States)

    Lee, Rachel; Schmidt, B. U. Sebastian; Campanello, Leonard; Hourwitz, Matt J.; Fourkas, John T.; Losert, Wolfgang

    Many cell types have been shown to exhibit contact guidance, in which cells sense and follow the texture of their environment. Contact guidance can lead to persistent directional migration that does not require the coordinated spatial and temporal cues required for guidance cues such as chemical concentration (i.e. chemotaxis). Actin polymerization has been shown to be guided by topographical features (esotaxis) in Dictyostelium discoideum cells, leading to guided cell migration. In this work, we show that actin dynamics are also guided by nanotopography in epithelial MCF10A cells despite large differences in the normal migration behavior of these two cell types. The existence of esotaxis and guided migration across phyla suggests that cytoskeletal dynamics play an important role in texture sensing and directional cell migration.

  7. Nuclear Actin and Lamins in Viral Infections

    Science.gov (United States)

    Cibulka, Jakub; Fraiberk, Martin; Forstova, Jitka

    2012-01-01

    Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections. PMID:22590674

  8. HIV infection of T cells: actin-in and actin-out.

    Science.gov (United States)

    Liu, Yin; Belkina, Natalya V; Shaw, Stephen

    2009-04-14

    Three studies shed light on the decade-old observation that the actin cytoskeleton is hijacked to facilitate entry of HIV into its target cells. Polymerization of actin is required to assemble high concentrations of CD4 and CXCR4 at the plasma membrane, which promote viral binding and entry in both the simple model of infection by free virus and the more physiologically relevant route of infection through the virological synapse. Three types of actin-interacting proteins-filamin, ezrin/radixin/moesin (ERM), and cofilin-are now shown to play critical roles in this process. Filamin binds to both CD4 and CXCR4 in a manner promoted by signaling of the HIV gp120 glycoprotein. ERM proteins attach actin filaments to the membrane and may promote polymerization of actin. Early in the process of viral entry, cofilin is inactivated, which is proposed to facilitate the early assembly of actin filaments, but cofilin is reported to be activated soon thereafter to facilitate postentry events. This complex role of cofilin may help to reconcile the paradox that actin polymerization promotes initial binding and fusion steps but inhibits some subsequent early postentry events.

  9. Human muscle LIM protein dimerizes along the actin cytoskeleton and cross-links actin filaments.

    Science.gov (United States)

    Hoffmann, Céline; Moreau, Flora; Moes, Michèle; Luthold, Carole; Dieterle, Monika; Goretti, Emeline; Neumann, Katrin; Steinmetz, André; Thomas, Clément

    2014-08-01

    The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. Innovation: Contact

    African Journals Online (AJOL)

    Principal Contact. Ruth Hoskins Editor University of KwaZulu-Natal, Information Studies Programme Email: hoskinsr@ukzn.ac.za. Support Contact. Gita Ramdass Email: ramdass@ukzn.ac.za. ISSN: 1025-8892. AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's · More ...

  11. Non-Straub type actin from molluscan catch muscle

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-05-27

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

  12. The role of actin and myosin during spermatogenesis.

    Science.gov (United States)

    Sun, Xiao; Kovacs, Tamas; Hu, Yan-Jun; Yang, Wan-Xi

    2011-08-01

    Spermatogenesis is a transitionary process in which the diploid spermatogonia transform into haploid mature spermatozoa. Actin and myosin have been implicated in various aspects during spermatogenesis. Actin is present in the form of monomer, oligomer and polymer within cells, the latter is called microfilament. There are five actin-containing structures during spermatogenesis, i.e., ectoplasmic specialization, acroplaxome, manchette in mammals, actin cones in Drosophila and acroframosome in Caridean shrimp. They are involved in the shaping and differentiating of spermatids. Along with spermatogenesis, the actin cytoskeletons show active remodeling in this process. Some actin binding or actin regulated proteins have been demonstrated to regulate dynamic changes of the actin-containing structures. Myosin, actin-dependent molecular motor, plays an important role during spermatogenesis, such as involving in acrosome biogenesis, vesicle transport, gene transcription and nuclear shaping. The actin cytoskeleton and actin binding/regulated proteins cooperate to facilitate spermatogenesis. In this review, we summarize the existing knowledge about the cytoskeletal structures consisting of actin, actin binding/regulated proteins and myosin during spermatogenesis.

  13. Nucleotide exchange and rheometric studies with F-actin prepared from ATP- or ADP-monomeric actin

    OpenAIRE

    Newman, J.; Zaner, K.S.; Schick, K.L.; Gershman, L.C.; Selden, L.A.; Kinosian, H.J.; Travis, J.L.; Estes, J.E.

    1993-01-01

    It has recently been reported that polymer actin made from monomer containing ATP (ATP-actin) differed in EM appearance and rheological characteristics from polymer made from ADP-containing monomers (ADP-actin). Further, it was postulated that the ATP-actin polymer was more rigid due to storage of the energy released by ATP hydrolysis during polymerization (Janmey et al. 1990. Nature 347:95-99). Electron micrographs of our preparations of ADP-actin and ATP-actin polymers show no major differe...

  14. Virtual Screening for Potential Allosteric Inhibitors of Cyclin-Dependent Kinase 2 from Traditional Chinese Medicine

    Directory of Open Access Journals (Sweden)

    Fang Lu

    2016-09-01

    Full Text Available Cyclin-dependent kinase 2 (CDK2, a member of Cyclin-dependent kinases (CDKs, plays an important role in cell division and DNA replication. It is regarded as a desired target to treat cancer and tumor by interrupting aberrant cell proliferation. Compared to lower subtype selectivity of CDK2 ATP-competitive inhibitors, CDK2 allosteric inhibitor with higher subtype selectivity has been used to treat CDK2-related diseases. Recently, the first crystal structure of CDK2 with allosteric inhibitor has been reported, which provides new opportunities to design pure allosteric inhibitors of CDK2. The binding site of the ATP-competition inhibitors and the allosteric inhibitors are partially overlapped in space position, so the same compound might interact with the two binding sites. Thus a novel screening strategy was essential for the discovery of pure CDK2 allosteric inhibitors. In this study, pharmacophore and molecular docking were used to screen potential CDK2 allosteric inhibitors and ATP-competition inhibitors from Traditional Chinese Medicine (TCM. In the docking result of the allosteric site, the compounds which can act with the CDK2 ATP site were discarded, and the remaining compounds were regarded as the potential pure allosteric inhibitors. Among the results, prostaglandin E1 and nordihydroguaiaretic acid (NDGA were available and their growth inhibitory effect on human HepG2 cell lines was determined by MTT assay. The two compounds could substantially inhibit the growth of HepG2 cell lines with an estimated IC50 of 41.223 μmol/L and 45.646 μmol/L. This study provides virtual screening strategy of allosteric compounds and a reliable method to discover potential pure CDK2 allosteric inhibitors from TCM. Prostaglandin E1 and NDGA could be regarded as promising candidates for CDK2 allosteric inhibitors.

  15. Actin dynamics and the elasticity of cytoskeletal networks

    Directory of Open Access Journals (Sweden)

    2009-09-01

    Full Text Available The structural integrity of a cell depends on its cytoskeleton, which includes an actin network. This network is transient and depends upon the continual polymerization and depolymerization of actin. The degradation of an actin network, and a corresponding reduction in cell stiffness, can indicate the presence of disease. Numerical simulations will be invaluable for understanding the physics of these systems and the correlation between actin dynamics and elasticity. Here we develop a model that is capable of generating actin network structures. In particular, we develop a model of actin dynamics which considers the polymerization, depolymerization, nucleation, severing, and capping of actin filaments. The structures obtained are then fed directly into a mechanical model. This allows us to qualitatively assess the effects of changing various parameters associated with actin dynamics on the elasticity of the material.

  16. STRUCTURE AND FUNCTION OF PALLADIN’S ACTIN BINDING DOMAIN

    Science.gov (United States)

    Beck, Moriah R.; Dixon, Richard D.S.; Goicoechea, Silvia M.; Murphy, Grant S.; Brungardt, Joseph G.; Beam, Matthew T.; Srinath, Pavan; Patel, Julie; Mohiuddin, Jahan; Otey, Carol A.; Campbell, Sharon L.

    2013-01-01

    Here we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct F-actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and crosslinking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin crosslinking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo. PMID:23806659

  17. Non equivalence of the chains in the allosteric interaction of the hemoglobin

    International Nuclear Information System (INIS)

    Jacchieri, S.G.

    1983-01-01

    The importance, for the temperature dependence of the cooperative behaviour of hemoglobin, of the functional non equivalence of the polypeptide chains from which the hemoglobin molecule is built is studied. With such purpose thermodynamic allosteric parameters are introduced called 'mean allosteric parameters' which relate the last two oxygen bindings to the firsttwo ones. It is shown that the mean allosteric free energy is strongly correlated to the Hill parameter which is a classic measure of cooperativity; hence, the mean allosteric free energy measures the hemoglobin cooperativity. Recent experimental data show that the mean allosteric free energy decreasses with temperature; this is due to the mean allosteric enthalphy and entropy being positive quantities. To analise such behaviour in terms of thermodynamic's arguments equations are derived for the thermodynamic parameters of oxygen binding to hemoglobin in terms of those of its chains. Since the obtained equations have a great number of terms the same treatment is applied to a hypothetic dimer from which simpler relations are derived. From both cases it is concluded that the positive character of the mean allosteric enthalpy and entropy is due to the presence of cooperative and anticooperative terms. Since the last terms are absent in the equations of allosteric homoproteins, the characteristic temperature-dependence of hemoglobin's cooperativity depends on the presence of non-equivalent chains. (Author) [pt

  18. The therapeutic potential of allosteric ligands for free fatty acid sensitive GPCRs

    DEFF Research Database (Denmark)

    Hudson, Brian D; Ulven, Trond; Milligan, Graeme

    2013-01-01

    of identifying allosteric leads and their often flat or confusing SAR. The present review will consider the advantages and challenges associated with allosteric GPCR ligands, and examine how the particular properties of these ligands may be exploited to uncover the therapeutic potential for free fatty acid...

  19. Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis

    Science.gov (United States)

    Spracklen, Andrew J.; Kelpsch, Daniel J.; Chen, Xiang; Spracklen, Cassandra N.; Tootle, Tina L.

    2014-01-01

    Prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton—temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling. PMID:24284900

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

    Science.gov (United States)

    Brugerolle, G; Bricheux, G; Coffe, G

    1996-01-01

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

  1. Towards the Structure Determination of a Modulated Protein Crystal: The Semicrystalline State of Profilin:Actin

    Science.gov (United States)

    Borgstahl, G.; Lovelace, J.; Snell, E. H.; Bellamy, H.

    2003-01-01

    One of the remaining challenges to structural biology is the solution of modulated structures. While small molecule crystallographers have championed this type of structure, to date, no modulated macromolecular structures have been determined. Modulation of the molecular structures within the crystal can produce satellite reflections or a superlattice of reflections in reciprocal space. We have developed the data collection methods and strategies that are needed to collect and analyze these data. If the macromolecule's crystal lattice is composed of physiologically relevant packing contacts, structural changes induced under physiological conditions can cause distortion relevant to the function and biophysical processes of the molecule making up the crystal. By careful measurement of the distortion, and the corresponding three-dimensional structure of the distorted molecule, we will visualize the motion and mechanism of the biological macromolecule(s). We have measured the modulated diffraction pattern produced by the semicrystalline state of profilin:actin crystals using highly parallel and highly monochromatic synchrotron radiation coupled with fine phi slicing (0.001-0.010 degrees) for structure determination. These crystals present these crystals present a unique opportunity to address an important question in structural biology. The modulation is believed to be due to the formation of actin helical filaments from the actin beta ribbon upon the pH-induced dissociation of profilin. To date, the filamentous state of actin has resisted crystallization and no detailed structures are available. The semicrystalline state profilin:actin crystals provides a unique opportunity to understand the many conformational states of actin. This knowledge is essential for understanding the dynamics underlying shape changes and motility of eukaryotic cells. Many essential processes, such as cytokinesis, phagocytosis, and cellular migration depend upon the capacity of the actin

  2. Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro.

    Directory of Open Access Journals (Sweden)

    Yaron Ideses

    Full Text Available During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]. Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing

  3. ACTIN BINDING PROTEIN 29 from Lilium pollen plays an important role in dynamic actin remodeling.

    Science.gov (United States)

    Xiang, Yun; Huang, Xi; Wang, Ting; Zhang, Yan; Liu, Qinwen; Hussey, Patrick J; Ren, Haiyun

    2007-06-01

    Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca(2+)- and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.

  4. Actin, actin-related proteins and profilin in diatoms: a comparative genomic analysis.

    Science.gov (United States)

    Aumeier, Charlotte; Polinski, Ellen; Menzel, Diedrik

    2015-10-01

    Diatoms are heterokont unicellular algae with a widespread distribution throughout all aquatic habitats. Research on diatoms has advanced significantly over the last decade due to available genetic transformation methods and publicly available genome databases. Yet up to now, proteins involved in the regulation of the cytoskeleton in diatoms are largely unknown. Consequently, this work focuses on actin and actin-related proteins (ARPs) encoded in the diatom genomes of Thalassiosira pseudonana, Thalassiosira oceanica, Phaeodactylum tricornutum, Fragilariopsis cylindrus and Pseudo-nitzschia multiseries. Our comparative genomic study revealed that most diatoms possess only a single conventional actin and a small set of ARPs. Among these are the highly conserved cytoplasmic Arp1 protein and the nuclear Arp4 as well as Arp6. Diatom genomes contain genes coding for two structurally different homologues of Arp4 that might serve specific functions. All diatom species examined here lack ARP2 and ARP3 proteins, suggesting that diatoms are not capable of forming the Arp2/3 complex, which is essential in most eukaryotes for actin filament branching and plus-end dynamics. Interestingly, none of the sequenced representatives of the Bacillariophyta phylum code for profilin. Profilin is an essential actin-binding protein regulating the monomer actin pool and is involved in filament plus-end dynamics. This is the first report of organisms not containing profilin. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.

    Directory of Open Access Journals (Sweden)

    Rohini Shrivastava

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

  6. Bioinformatic scaling of allosteric interactions in biomedical isozymes

    Science.gov (United States)

    Phillips, J. C.

    2016-09-01

    Allosteric (long-range) interactions can be surprisingly strong in proteins of biomedical interest. Here we use bioinformatic scaling to connect prior results on nonsteroidal anti-inflammatory drugs to promising new drugs that inhibit cancer cell metabolism. Many parallel features are apparent, which explain how even one amino acid mutation, remote from active sites, can alter medical results. The enzyme twins involved are cyclooxygenase (aspirin) and isocitrate dehydrogenase (IDH). The IDH results are accurate to 1% and are overdetermined by adjusting a single bioinformatic scaling parameter. It appears that the final stage in optimizing protein functionality may involve leveling of the hydrophobic limits of the arms of conformational hydrophilic hinges.

  7. The origin and evolution of green algal and plant actins.

    Science.gov (United States)

    An, S S; Möpps, B; Weber, K; Bhattacharya, D

    1999-02-01

    The Viridiplantae are subdivided into two groups: the Chlorophyta, which includes the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Prasinophyceae; and the Streptophyta, which includes the Charophyceae and all land plants. Within the Streptophyta, the actin genes of the angiosperms diverge nearly simultaneously from each other before the separation of monocots and dicots. Previous evolutionary analyses have provided limited insights into the gene duplications that have produced these complex gene families. We address the origin and diversification of land plant actin genes by studying the phylogeny of actins within the green algae, ferns, and fern allies. Partial genomic sequences or cDNAs encoding actin were characterized from Cosmarium botrytis (Zygnematales), Selaginella apoda (Selaginellales), Anemia phyllitidis (Polypodiales), and Psilotum triquetrum (Psilotales). Selaginella contains at least two actin genes. One sequence (Ac2) diverges within a group of fern sequences that also includes the Psilotum Ac1 actin gene and one gymnosperm sequence (Cycas revoluta Cyc3). This clade is positioned outside of the angiosperm actin gene radiation. The second Selaginella sequence (Ac1) is the sister to all remaining land plant actin sequences, although the internal branches in this portion of the tree are very short. Use of complete actin-coding regions in phylogenetic analyses provides support for the separation of angiosperm actins into two classes. N-terminal "signature" sequence analyses support these groupings. One class (VEG) includes actin genes that are often expressed in vegetative structures. The second class (REP) includes actin genes that trace their ancestry within the vegetative actins and contains members that are largely expressed in reproductive structures. Analysis of intron positions within actin genes shows that sequences from both Selaginella and Cosmarium contain the conserved 20-3, 152-1, and 356-3 introns found in many members of the

  8. Allosteric modulators of the hERG K{sup +} channel

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Zhiyi, E-mail: z.yu@lacdr.leidenuniv.nl; Klaasse, Elisabeth, E-mail: elisabethklaasse@hotmail.com; Heitman, Laura H., E-mail: l.h.heitman@lacdr.leidenuniv.nl; IJzerman, Adriaan P., E-mail: ijzerman@lacdr.leidenuniv.nl

    2014-01-01

    Drugs that block the cardiac K{sup +} channel encoded by the human ether-à-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K{sup +} channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [{sup 3}H]astemizole and [{sup 3}H]dofetilide to the hERG K{sup +} channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC{sub 50} values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC{sub 50} values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K{sup +} channel, which is discussed in the light of findings on other ion channels. - Highlights: • Allosteric modulators on the hERG K{sup +} channel were evaluated in binding assays. • LUF6200 was identified as a potent allosteric inhibitor. • Potassium ions were found to behave as allosteric enhancers. • Positive cooperativity and distinct allosteric sites for them were proposed.

  9. The Calponin Regulatory Region Is Intrinsically Unstructured: Novel Insight into Actin-Calponin and Calmodulin-Calponin Interfaces Using NMR Spectroscopy

    Science.gov (United States)

    Pfuhl, Mark; Al-Sarayreh, Sameeh; El-Mezgueldi, Mohammed

    2011-01-01

    Calponin is an actin- and calmodulin-binding protein believed to regulate the function of actin. Low-resolution studies based on proteolysis established that the recombinant calponin fragment 131–228 contained actin and calmodulin recognition sites but failed to precisely identify the actin-binding determinants. In this study, we used NMR spectroscopy to investigate the structure of this functionally important region of calponin and map its interaction with actin and calmodulin at amino-acid resolution. Our data indicates that the free calponin peptide is largely unstructured in solution, although four short amino-acid stretches corresponding to residues 140–146, 159–165, 189–195, and 199–205 display the propensity to form α-helices. The presence of four sequential transient helices probably provides the conformational malleability needed for the promiscuous nature of this region of calponin. We identified all amino acids involved in actin binding and demonstrated for the first time, to our knowledge, that the N-terminal flanking region of Lys137-Tyr144 is an integral part of the actin-binding site. We have also delineated the second actin-binding site to amino acids Thr180-Asp190. Ca2+-calmodulin binding extends beyond the previously identified minimal sequence of 153–163 and includes most amino acids within the stretch 143–165. In addition, we found that calmodulin induces chemical shift perturbations of amino acids 188–190 demonstrating for the first time, to our knowledge, an effect of Ca2+-calmodulin on this region. The spatial relationship of the actin and calmodulin contacts as well as the transient α-helical structures within the regulatory region of calponin provides a structural framework for understanding the Ca2+-dependent regulation of the actin-calponin interaction by calmodulin. PMID:21463585

  10. ALKBH4-dependent demethylation of actin regulates actomyosin dynamics

    DEFF Research Database (Denmark)

    Li, M.-M.; Shi, Y.; Niu, Y.

    2013-01-01

    -type but not catalytically inactive ALKBH4. Similar to actin and myosin knock-out mice, homozygous Alkbh4 mutant mice display early embryonic lethality. These findings imply that ALKBH4-dependent actin demethylation regulates actomyosin function by promoting actin-non-muscle myosin II interaction.......-dependent processes such as cytokinesis and cell migration. ALKBH4-deficient cells display elevated K84me1 levels. Non-muscle myosin II only interacts with unmethylated actin and its proper recruitment to and interaction with actin depend on ALKBH4. ALKBH4 co-localizes with the actomyosin-based contractile ring...

  11. The allosteric transition of GroEL induced by metal fluoride-ADP complexes.

    Science.gov (United States)

    Inobe, Tomonao; Kikushima, Kenji; Makio, Tadashi; Arai, Munehito; Kuwajima, Kunihiro

    2003-05-23

    To understand the mechanism of a functionally important ATP-induced allosteric transition of GroEL, we have studied the effect of a series of metal fluoride-ADP complexes and vanadate-ADP on GroEL by kinetic fluorescence measurement of pyrene-labeled GroEL and by small-angle X-ray scattering measurement of wild-type GroEL. The metal fluorides and vanadate, complexed with ADP, are known to mimic the gamma-phosphate group of ATP, but they differ in geometry and size; it is expected that these compounds will be useful for investigating the strikingly high specificity of GroEL for ATP that enables the induction of the allosteric transition. The kinetic fluorescence measurement revealed that aluminium, beryllium, and gallium ions, when complexed with the fluoride ion and ADP, induced a biphasic fluorescence change of pyrenyl GroEL, while scandium and vanadate ions did not induce any kinetically observed change in fluorescence. The burst phase and the first phase of the fluorescence kinetics were reversible, while the second phase and subsequent changes were irreversible. The dependence of the burst-phase and the first-phase fluorescence changes on the ADP concentration indicated that the burst phase represents non-cooperative nucleotide binding to GroEL, and that the first phase represents the allosteric transition of GroEL. Both the amplitude and the rate constant of the first phase of the fluorescence kinetics were well understood in terms of a kinetic allosteric model, which is a combination of transition state theory and the Monod-Wyman-Changeux allosteric model. From the kinetic allosteric model analysis, the relative free energy of the transition state in the metal fluoride-ADP-induced allosteric transition of GroEL was found to be larger than the corresponding free energy of the ATP-induced allosteric transition by more than 5.5kcal/mol. However, the X-ray scattering measurements indicated that the allosteric state induced by these metal fluoride-ADP complexes is

  12. Skeletal muscle myopathy mutations at the actin tropomyosin interface that cause gain- or loss-of-function.

    Science.gov (United States)

    Memo, Massimiliano; Marston, Steven

    2013-08-01

    It is well known that the regulation of muscle contraction relies on the ability of tropomyosin to switch between different positions on the actin filament, but it is still not well understood which amino acids are directly involved in the different states of the interaction. Recently the structure of the actin-tropomyosin interface has been determined both in the absence and presence of myosin heads. Interestingly, a number of mutations in tropomyosin that are associated with skeletal muscle myopathy are located within this interface. We first give an overview of the functional effect of mutations on amino acids that are involved in the contact with actin asp25, which represent a pattern repeated seven times along tropomyosin. It is explained how some of these amino acids (R167 and R244) which are thought to be involved in a salt bridge contact with actin in the closed state can produce a loss-of-function when mutated, while other positively charged tropomyosin amino acids positioned on the downstream side of the contact (K7, K49, R91, K168) can produce a gain-of-function when mutated. We then consider mutations of amino acids involved in another salt bridge contact between the two proteins in the closed state, actin K326N (which binds on five different points of tropomyosin) and tropomyosin ∆E139 and E181K, and we report how all of these mutations produce a gain-of-function. These observations can be important to validate the proposed structures and to understand more deeply how mutations affect the function of these proteins and to enable prediction of their outcomes.

  13. The allosteric switching mechanism in bacteriophage MS2

    Science.gov (United States)

    Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F.

    2016-07-01

    We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.

  14. Studies on allosteric phenomena in glycogen phosphorylase b.

    Science.gov (United States)

    Madsen, N B; Avramovic-Zikic, O; Lue, P F; Honikel, K O

    1976-03-26

    This article attempts to trace, from a personal point of view, the history of discoveries of allosteric phenomena in phosphorylase b and the later development of systematic attempts to fit the data into comprehensive theoretical models. Work from our own laboratory is emphasized, but we try to integrate this into the results from other investigators and show their contributions to our ideas and experiments. Finally, some recent unpublished data is presented together with some conclusions and predictions from a new hypothesis. The discoveries by Carl and Gerty Cori of the activation of phosphorylase by AMP, the inhibition of glucose and the enzymatic interconversion of two forms fo the enzyme with different control properties helped lay the foundations of our present understanding of allosteric mechanisms. The later discovery of the oligomeric nature of phosphorylase and its relationship to AMP binding served as a basis for many years of research into the structure-function relationships of phosphorylase and other enzymes. Data showing that AMP lowers the entropy of activation is discussed with respect to the role of the nucleotide and its binding close to the active site. The discovery of the control of phosphorylase b by common metabolites and the impetus this gave to the intensive kinetic studies of the last ten years, wherein fitting to theoretical models has been a common feature, is reviewed.

  15. Allosteric Inhibition of Macrophage Migration Inhibitory Factor Revealed by Ibudilast

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Y.; Crichlow, G; Vermeire, J; Leng, L; Du, X; Hodsdon, M; Bucala, R; Cappello, M; Gross, M; et al.

    2010-01-01

    AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

  16. The allosteric switching mechanism in bacteriophage MS2

    Energy Technology Data Exchange (ETDEWEB)

    Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F., E-mail: hagan@brandeis.edu [Martin Fisher School of Physics, Brandeis University, Waltham, Massachusetts 02474 (United States)

    2016-07-21

    We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.

  17. Allosteric inhibitors of Coxsackie virus A24 RNA polymerase.

    Science.gov (United States)

    Schein, Catherine H; Rowold, Diane; Choi, Kyung H

    2016-02-15

    Coxsackie virus A24 (CVA24), a causative agent of acute hemorrhagic conjunctivitis, is a prototype of enterovirus (EV) species C. The RNA polymerase (3D(pol)) of CVA24 can uridylylate the viral peptide linked to the genome (VPg) from distantly related EV and is thus, a good model for studying this reaction. Once UMP is bound, VPgpU primes RNA elongation. Structural and mutation data have identified a conserved binding surface for VPg on the RNA polymerase (3D(pol)), located about 20Å from the active site. Here, computational docking of over 60,000 small compounds was used to select those with the lowest (best) specific binding energies (BE) for this allosteric site. Compounds with varying structures and low BE were assayed for their effect on formation of VPgU by CVA24-3D(pol). Two compounds with the lowest specific BE for the site inhibited both uridylylation and formation of VPgpolyU at 10-20μM. These small molecules can be used to probe the role of this allosteric site in polymerase function, and may be the basis for novel antiviral compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

    Directory of Open Access Journals (Sweden)

    Mirco Müller

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

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

    Science.gov (United States)

    Hong, Nan Hyung; Qi, Aidong

    2015-01-01

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

  20. Profilin connects actin assembly with microtubule dynamics

    Czech Academy of Sciences Publication Activity Database

    Nejedla, M.; Sadi, S.; Sulimenko, Vadym; de Almeida, F.N.; Blom, H.; Dráber, Pavel; Aspenstrom, P.; Karlsson, R.

    2016-01-01

    Roč. 27, č. 15 (2016), s. 2381-2393 ISSN 1059-1524 R&D Projects: GA ČR GA16-25159S Institutional support: RVO:68378050 Keywords : cross-linked profilin * arp2/3 complex * f-actin * microfilament system * migrating cell s * focal adhesions * cultured- cell s * messenger-rna * living cell s * protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.685, year: 2016

  1. Late complications of rxtherapy: actinic sarcoma

    Energy Technology Data Exchange (ETDEWEB)

    Buffat, J.D.

    1975-09-09

    Relation of two cases of benign tumors: a vertebral osteoblastoma and a cerebellar medulloblastoma which, after operation, have had radiotherapy. 20 years later for one case and 14 years for the other one actinic sarcomas will appear, and, in spite of usual therapy, the death is coming rapidly. We are certainly in presence of two exceptional cases, but each physician must be conscious, before to attempt a treatment, that very distant complication can eventually occur.

  2. In Vivo Investigation of Escitalopram’s Allosteric Site on the Serotonin Transporter

    Science.gov (United States)

    Murray, Karen E.; Ressler, Kerry J.; Owens, Michael J.

    2015-01-01

    Escitalopram is a commonly prescribed antidepressant of the selective serotonin reuptake inhibitor class. Clinical evidence and mapping of the serotonin transporter (SERT) identified that escitalopram, in addition to its binding to a primary uptake-blocking site, is capable of binding to the SERT via an allosteric site that is hypothesized to alter escitalopram’s kinetics at the SERT. The studies reported here examined the in vivo role of the SERT allosteric site in escitalopram action. A knockin mouse model that possesses an allosteric-null SERT was developed. Autoradiographic studies indicated that the knockin protein was expressed at a lower density than endogenous mouse SERT (approximately 10–30% of endogenous mouse SERT), but the knockin mice are a viable tool to study the allosteric site. Microdialysis studies in the ventral hippocampus found no measurable decrease in extracellular serotonin response after local escitalopram challenge in mice without the allosteric site compared to mice with the site (p = 0.297). In marble burying assays there was a modest effect of the absence of the allosteric site, with a larger systemic dose of escitalopram (10-fold) necessary for the same effect as in mice with intact SERT (p = 0.023). However, there was no effect of the allosteric site in the tail suspension test. Together these data suggest that there may be a regional specificity in the role of the allosteric site. The lack of a robust effect overall suggests that the role of the allosteric site for escitalopram on the SERT may not produce meaningful in vivo effects. PMID:26621784

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

    Science.gov (United States)

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

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition. © 2015. Published by The Company of Biologists Ltd.

  4. Contact hysteroscopy.

    Science.gov (United States)

    Baggish, M S; Barbot, J

    1983-06-01

    In 1907 innovations in optics and illumination made by Maximilian Nitze were applied to hysteroscopy by Charles David, who wrote a treatise of hysteroscopy. David improved illumination by placing an electric incandescent bulb at the intrauterine end of his endoscope and also sealed the distal end of the tube with a piece of glass. The history of the contact endoscope that the authors personally used is connected to the invention by Vulmiere (1952) of a revolutionary illumination process in endoscopy--the "cold light" process. The components of cold light consist of a powerful external light source that is transmitted via a special optical guide into the endometrial cavity. The 1st application of his principle (1963) was an optical trochar contained in a metallic sheath. This simple endoscope was perfected, and in 1973 Barbot and Parent, in France, began to use it to examine the uterine cavity. Discussion focuses on methods, instrumentation, method for examination (grasping the instrument, setup, light source, anesthesia, dilatation, technique, and normal endometrium); cervical neoplasia; nonneoplastic lesions of the endometrium (endometrial polyp, submucous myoma, endometrial hyperplasia); intrauterine device localization; neoplastic lesions of the endometrium; precursors (adenocarcinoma); hysteroscopy in pregnancy (embryoscopy, hydatidiform mole, postpartum hemorrhage, incomplete abortion, spontaneous abortion, induced abortions, and amnioscopy); and examinations of children and infants. The contact endoscope must make light contact with the structure to be viewed. The principles of contact endoscopy depend on an interpretation of color, contour, vascular pattern, and a sense of touch. These are computed together and a diagnosis is made on the basis of previously learned clinical pathologic correlations. The contact endoscope is composed of 3 parts: an optical guide; a cylindric chamber that collects and traps ambient light; and a magnifying eyepiece. The phase of

  5. Spreading and contraction in phagocytosis: The role of actin organization and curvature

    Science.gov (United States)

    Curtis, Jennifer E.

    Phagocytosis is the process used by immune cells to engulf and remove foreign objects from the body. The engulfment is realized by the formation of an actin-driven `phagocytic cup' of the cell membrane, which quickly crawls up and then surrounds the object via constriction. In this study, we resolve the paradox of how actin-driven protrusion of the plasma membrane can co-exist with a contractile actin belt proposed to mechanically-drive the closure of the phagocytic cup. To do this we quantitatively assessed macrophage phagocytic behavior in a planar geometry, a process known as frustrated phagocytosis. Our results reveal that phagocytosis occurs in a binary manner, such that once it is initiated, frustrated phagocytosis proceeds at a prescribed rate, resulting in peak contact areas that correspond to a roughly 225% increase in apparent cell surface area. Upon reaching their maximum area, the majority of macrophages enter a period of late-stage contraction. During the contraction phase, cells exert significant stress on the underlying substrate. Contraction also corresponds with dramatic reorganization of the F-actin cytoskeleton, in particular the formation of a bundled contractile belt around the cell perimeter. In contrast to other studies of phagocytosis, our work definitively illustrates that whatever signals trigger late-stage phagocytic contraction must be independent of particle size and curvature. Mounting evidence suggests that membrane tension is involved in late-stage signaling. The idea that tension is linked to late-stage contraction is reinforced by our finding that the peak-contact area roughly corresponds to the area threshold that results in increased cortical tension, as measured by Lam et al., and that reducing tension through hypertonic buffer shock enables the cells to spread further before the onset of contraction. Supported by NSF Grants #PHYS-0848797 and SRN-POLS 1205878.

  6. Allosteric regulation of metabolism in cancer: endogenous mechanisms and considerations for drug design.

    Science.gov (United States)

    Macpherson, Jamie A; Anastasiou, Dimitrios

    2017-12-01

    Alterations in metabolic processes have been linked to various diseases, including cancer. Although gene expression can dictate long-term metabolic adaptation, many metabolic changes found in cancer are associated with altered allosteric properties of the underlying enzymes. Small molecule-protein interactions and intracellular signalling converge to orchestrate these allosteric mechanisms, which, emerging evidence suggests, constitute a promising therapeutic avenue. In this review we focus on glucose and energy metabolism to illustrate the role of allostery in cancer physiology and we discuss approaches to streamline the process of targeting aberrant allosteric pathways with small molecules. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  8. The Nance-Horan syndrome protein encodes a functional WAVE homology domain (WHD) and is important for co-ordinating actin remodelling and maintaining cell morphology.

    Science.gov (United States)

    Brooks, Simon P; Coccia, Margherita; Tang, Hao R; Kanuga, Naheed; Machesky, Laura M; Bailly, Maryse; Cheetham, Michael E; Hardcastle, Alison J

    2010-06-15

    Nance-Horan syndrome (NHS) is an X-linked developmental disorder, characterized by bilateral congenital cataracts, dental anomalies, facial dysmorphism and mental retardation. Null mutations in a novel gene, NHS, cause the syndrome. The NHS gene appears to have multiple isoforms as a result of alternative transcription, but a cellular function for the NHS protein has yet to be defined. We describe NHS as a founder member of a new protein family (NHS, NHSL1 and NHSL2). Here, we demonstrate that NHS is a novel regulator of actin remodelling and cell morphology. NHS localizes to sites of cell-cell contact, the leading edge of lamellipodia and focal adhesions. The N-terminus of isoforms NHS-A and NHS-1A, implicated in the pathogenesis of NHS, have a functional WAVE homology domain that interacts with the Abi protein family, haematopoietic stem/progenitor cell protein 300 (HSPC300), Nap1 and Sra1. NHS knockdown resulted in the disruption of the actin cytoskeleton. We show that NHS controls cell morphology by maintaining the integrity of the circumferential actin ring and controlling lamellipod formation. NHS knockdown led to a striking increase in cell spreading. Conversely, ectopic overexpression of NHS inhibited lamellipod formation. Remodelling of the actin cytoskeleton and localized actin polymerization into branched actin filaments at the plasma membrane are essential for mediating changes in cell shape, migration and cell contact. Our data identify NHS as a new regulator of actin remodelling. We suggest that NHS orchestrates actin regulatory protein function in response to signalling events during development.

  9. Stress generation by myosin minifilaments in actin bundles.

    Science.gov (United States)

    Dasanayake, Nilushi L; Carlsson, Anders E

    2013-06-01

    Forces and stresses generated by the action of myosin minifilaments are analyzed in idealized computer-generated actin bundles, and compared to results for isotropic actin networks. The bundles are generated as random collections of actin filaments in two dimensions with constrained orientations, crosslinked and attached to two fixed walls. Myosin minifilaments are placed on actin filament pairs and allowed to move and deform the network so that it exerts forces on the walls. The vast majority of simulation runs end with contractile minifilament stress, because minifilaments rotate into energetically stable contractile configurations. This process is aided by the bending and stretching of actin filaments, which accomodate minifilament rotation. Stresses for bundles are greater than those for isotropic networks, and antiparallel filaments generate more tension than parallel filaments. The forces transmitted by the actin network to the walls of the simulation cell often exceed the tension in the minifilament itself.

  10. Covalent interactions of acetaldehyde with the actin/microfilament system.

    Science.gov (United States)

    Xu, D S; Jennett, R B; Smith, S L; Sorrell, M F; Tuma, D J

    1989-01-01

    The covalent binding of [14C]acetaldehyde to purified rabbit skeletal muscle actin was characterized. As we have found for other cytoskeletal proteins, actin formed stable covalent adducts under reductive and non-reductive conditions. Under non-reductive conditions, individual and competition binding studies versus albumin both showed that the G-form of actin is more reactive toward acetaldehyde than the F-form. When proteins were compared on an 'equi-lysine' basis under non-reducing conditions, G-actin was found to preferentially compete with albumin for binding to acetaldehyde. Time-course dialysis studies indicated that acetaldehyde-actin adducts become more stable with prolonged incubation at 37 degrees C. These data raise the possibility that actin could be a preferential target for adduct formation in cellular systems and will serve as the basis for ongoing studies aimed at defining the role of acetaldehyde-protein adducts in ethanol-induced cell injury.

  11. Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Wei; Chun, Eugene; Thompson, Aaron A.; Chubukov, Pavel; Xu, Fei; Katritch, Vsevolod; Han, Gye Won; Roth, Christopher B.; Heitman, Laura H.; IJzerman, Adriaan P.; Cherezov, Vadim; Stevens, Raymond C. (Scripps); (Leiden/Amsterdam); (Receptos)

    2012-08-31

    Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A{sub 2A} adenosine receptor by replacing its third intracellular loop with apocytochrome b{sub 562}RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp{sup 2.50}. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.

  12. Enhancing NMDA Receptor Function: Recent Progress on Allosteric Modulators

    Directory of Open Access Journals (Sweden)

    Lulu Yao

    2017-01-01

    Full Text Available The N-methyl-D-aspartate receptors (NMDARs are subtype glutamate receptors that play important roles in excitatory neurotransmission and synaptic plasticity. Their hypo- or hyperactivation are proposed to contribute to the genesis or progression of various brain diseases, including stroke, schizophrenia, depression, and Alzheimer’s disease. Past efforts in targeting NMDARs for therapeutic intervention have largely been on inhibitors of NMDARs. In light of the discovery of NMDAR hypofunction in psychiatric disorders and perhaps Alzheimer’s disease, efforts in boosting NMDAR activity/functions have surged in recent years. In this review, we will focus on enhancing NMDAR functions, especially on the recent progress in the generation of subunit-selective, allosteric positive modulators (PAMs of NMDARs. We shall also discuss the usefulness of these newly developed NMDAR-PAMs.

  13. The therapeutic potential of allosteric ligands for free fatty acid sensitive GPCRs

    DEFF Research Database (Denmark)

    Hudson, Brian D; Ulven, Trond; Milligan, Graeme

    2013-01-01

    G protein coupled receptors (GPCRs) are the most historically successful therapeutic targets. Despite this success there are many important aspects of GPCR pharmacology and function that have yet to be exploited to their full therapeutic potential. One in particular that has been gaining attention...... in recent times is that of GPCR ligands that bind to allosteric sites on the receptor distinct from the orthosteric site of the endogenous ligand. As therapeutics, allosteric ligands possess many theoretical advantages over their orthosteric counterparts, including more complex modes of action, improved...... of identifying allosteric leads and their often flat or confusing SAR. The present review will consider the advantages and challenges associated with allosteric GPCR ligands, and examine how the particular properties of these ligands may be exploited to uncover the therapeutic potential for free fatty acid...

  14. Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Steen, Anne; Jensen, Pia C

    2011-01-01

    molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5...... chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago......-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5...

  15. Ca2+ bound to the high affinity divalent cation-binding site of actin enhances actophorin-induced depolymerization of muscle F-actin but inhibits actophorin-induced depolymerization of Acanthamoeba F-actin.

    Science.gov (United States)

    Mossakowska, M; Korn, E D

    1996-08-01

    The cation tightly bound to actin, Mg2+ or Ca2+, affects the ability of actophorin to accelerate depolymerization of filaments and bind to monomers of actin prepared from rabbit skeletal muscle and Acanthamoeba castellanii. Actophorin interacted similarly with muscle and Acanthamoeba Mg2(+)-F-actin but depolymerized muscle Mg2(+)-F-actin more efficiently. Muscle Ca2(+)-F-actin depolymerized about 5 times more rapidly than Mg2(+)-F-actin in the presence of actophorin but Acanthamoeba Ca2(+)-F-actin was highly resistant to actophorin. Muscle actin subunits dissociated more rapidly than Acanthamoeba actin subunits from copolymers of muscle and Acanthamoeba Ca2(+)-actin upon addition of actophorin although Acanthamoeba actin dissociated much more rapidly from copolymers than from its homopolymer. The Kd of the 1:1 complex between actophorin and monomeric actin was somewhat lower for muscle Mg2(+)-ATP-G-actin than for both Acanthamoeba Mg2(+)-ATP-G-actin and muscle Ca2(+)-ATP-G-actin. The data for the interactions of actophorin with Acanthamoeba Ca2(+)-ATP-G-actin or muscle and amoeba Mg2(+)- and Ca2(+)-ADP-G-actin were incompatible with the formation of 1:1 actin: actophorin complexes and, thus, Kd values could not be calculated. While it may not be surprising that actophorin would interact differently with Mg2(+)- and Ca2(+)-actin, it is unexpected that the nature of the tightly bound cation would have such dramatically opposite effects on the ability of actophorin to depolymerize muscle and Acanthamoeba F-actin. Differential severing by actophorin, with Acanthamoeba Ca2(+)-actin being almost totally resistant, is sufficient to explain the results but other possibilities cannot be ruled out.

  16. A Robust Actin Filaments Image Analysis Framework.

    Directory of Open Access Journals (Sweden)

    Mitchel Alioscha-Perez

    2016-08-01

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

  17. Myosin Vs organize actin cables in fission yeast.

    Science.gov (United States)

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

    2012-12-01

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

  18. Probing actin polymerization by intermolecular cross-linking

    OpenAIRE

    1988-01-01

    We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an app...

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

    Science.gov (United States)

    1991-01-01

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

  20. Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*

    Science.gov (United States)

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

    2013-01-01

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

  1. Electrostatics control actin filament nucleation and elongation kinetics.

    Science.gov (United States)

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

    2013-04-26

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

  2. Daylight photodynamic therapy for actinic keratosis

    DEFF Research Database (Denmark)

    Wiegell, Stine; Wulf, H C; Szeimies, R-M

    2011-01-01

    Photodynamic therapy (PDT) is an attractive therapy for non-melanoma skin cancers including actinic keratoses (AKs) because it allows treatment of large areas; it has a high response rate and results in an excellent cosmesis. However, conventional PDT for AKs is associated with inconveniently long...... clinic visits and discomfort during therapy. In this article, we critically review daylight-mediated PDT, which is a simpler and more tolerable treatment procedure for PDT. We review the effective light dose, efficacy and safety, the need for prior application of sunscreen, and potential clinical scope...

  3. Dynamic buckling of actin within filopodia

    DEFF Research Database (Denmark)

    Leijnse, Natascha; Oddershede, Lene B; Bendix, Pól Martin

    2015-01-01

    Filopodia are active tubular structures protruding from the cell surface which allow the cell to sense and interact with the surrounding environment through repetitive elongation-retraction cycles. The mechanical behavior of filopodia has been studied by measuring the traction forces exerted...... on external substrates.(1) These studies have revealed that internal actin flow can transduce a force across the cell surface through transmembrane linkers like integrins. In addition to the elongation-retraction behavior filopodia also exhibit a buckling and rotational behavior. Filopodial buckling...

  4. Change in Allosteric Network Affects Binding Affinities of PDZ Domains: Analysis through Perturbation Response Scanning

    Science.gov (United States)

    Gerek, Z. Nevin; Ozkan, S. Banu

    2011-01-01

    The allosteric mechanism plays a key role in cellular functions of several PDZ domain proteins (PDZs) and is directly linked to pharmaceutical applications; however, it is a challenge to elaborate the nature and extent of these allosteric interactions. One solution to this problem is to explore the dynamics of PDZs, which may provide insights about how intramolecular communication occurs within a single domain. Here, we develop an advancement of perturbation response scanning (PRS) that couples elastic network models with linear response theory (LRT) to predict key residues in allosteric transitions of the two most studied PDZs (PSD-95 PDZ3 domain and hPTP1E PDZ2 domain). With PRS, we first identify the residues that give the highest mean square fluctuation response upon perturbing the binding sites. Strikingly, we observe that the residues with the highest mean square fluctuation response agree with experimentally determined residues involved in allosteric transitions. Second, we construct the allosteric pathways by linking the residues giving the same directional response upon perturbation of the binding sites. The predicted intramolecular communication pathways reveal that PSD-95 and hPTP1E have different pathways through the dynamic coupling of different residue pairs. Moreover, our analysis provides a molecular understanding of experimentally observed hidden allostery of PSD-95. We show that removing the distal third alpha helix from the binding site alters the allosteric pathway and decreases the binding affinity. Overall, these results indicate that (i) dynamics plays a key role in allosteric regulations of PDZs, (ii) the local changes in the residue interactions can lead to significant changes in the dynamics of allosteric regulations, and (iii) this might be the mechanism that each PDZ uses to tailor their binding specificities regulation. PMID:21998559

  5. Fumarate analogs act as allosteric inhibitors of the human mitochondrial NAD(P)+-dependent malic enzyme.

    Science.gov (United States)

    Hsieh, Ju-Yi; Liu, Jyung-Hurng; Yang, Pai-Chun; Lin, Chi-Li; Liu, Guang-Yaw; Hung, Hui-Chih

    2014-01-01

    Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P)-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P)-ME_R67A/R91A and m-NAD(P)-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P)-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P)-ME enzyme activity.

  6. Fumarate analogs act as allosteric inhibitors of the human mitochondrial NAD(P+-dependent malic enzyme.

    Directory of Open Access Journals (Sweden)

    Ju-Yi Hsieh

    Full Text Available Human mitochondrial NAD(P+-dependent malic enzyme (m-NAD(P-ME is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P-ME_R67A/R91A and m-NAD(P-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P-ME enzyme activity.

  7. Prioritized Contact Transport Stream

    Science.gov (United States)

    Hunt, Walter Lee, Jr. (Inventor)

    2015-01-01

    A detection process, contact recognition process, classification process, and identification process are applied to raw sensor data to produce an identified contact record set containing one or more identified contact records. A prioritization process is applied to the identified contact record set to assign a contact priority to each contact record in the identified contact record set. Data are removed from the contact records in the identified contact record set based on the contact priorities assigned to those contact records. A first contact stream is produced from the resulting contact records. The first contact stream is streamed in a contact transport stream. The contact transport stream may include and stream additional contact streams. The contact transport stream may be varied dynamically over time based on parameters such as available bandwidth, contact priority, presence/absence of contacts, system state, and configuration parameters.

  8. 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

    Directory of Open Access Journals (Sweden)

    Holweg Carola L

    2008-07-01

    organelles such as peroxisomes. Potential roles of MYA2 may also exist in the cell nucleus. Whether the low quality of the F-actin-labeling by MYA2-head6IQ compared to other F-actin-binding proteins (ABPs signifies a weak association of the myosin with actin filaments remains to be proven by other means than in vivo. Clues for the mode of contact between the myosin molecules and F-actin so far cannot be drawn from sequence-related data.

  9. Plasmin enzymatic activity in the presence of actin

    Directory of Open Access Journals (Sweden)

    Yusova E. I.

    2015-10-01

    Full Text Available Aim. To study the changes in the plasmin activity towards substrates with high and low molecular mass in the presence of actin. Methods. The proteins used for this investigation were obtained by affinity chromatography and gel-filtration. The plasmin enzymatic activity was determined by a turbidimetric assay and a chromogenic substrate-based assay. The enzyme linked immunosorbent assay and biotin-avidin-phosphatase system were used to study the interaction of plasminogen and its fragments with actin. Results. It was shown that G-actin causes 1.5-fold decrease in the rate of polymeric fibrin hydrolysis by plasmin and Glu-plasminogen activated by the tissue plasminogen activator. However, actin did not impede plasmin autolysis and had no influence on its amidase activity. We have studied an interaction of biotinylated Glu-plasminogen and its fragments (kringle 1-3, kringle 4 and mini-plasminogen with immobilized G-actin. Glu-plasminogen and kringle 4 had a high affinity towards actin (C50 is 113 and 117 nM correspondingly. Mini-plasminogen and kringe 4 did not bind to actin. A similar affinity of Glu-plasminogen and kringle 1-3 towards actin proves the involvement of the kringle 1-3 lysine-binding sites of the native plasminogen form in the actin interaction. Conclusions. Actin can modulate plasmin specificity towards high molecular mass substrates through its interaction with lysine-binding sites of the enzyme kringle domains. Actin inhibition of the fibrinolytic activity of plasmin is due to its competition with fibrin for thelysine binding sites of plasminogen/plasmin.

  10. Force Transmission in the Actin Cytoskeleton

    Science.gov (United States)

    Gardel, Margaret

    2012-02-01

    The ability of cells to sense and generate mechanical forces is essential to numerous aspects of their physiology, including adhesion, migration, division and differentiation. To a large degree, cellular tension is regulated by the transmission of myosin II-generated forces through the filamentous actin (F-actin) cytoskeleton. While transmission of myosin-generated stresses from the molecular to cellular length scale is well understood in the context of highly organized sarcomeres found in striated muscle, non-muscle and smooth muscle cells contain a wide variety of bundles and networks lacking sarcomeric organization. I will describe the in vitro and in vivo approaches we use to study force transmission in such disordered actomyosin assemblies. Our in vivo results are showing that highly organized stress fibers contribute surprisingly little to the overall extent of cellular tension as compared to disordered actomyosin meshworks. Our in vitro results are demonstrating the mechanisms of symmetry breaking in disordered actomyosin bundles that facilitate the formation of contractile bundles with well-defined ``contractile elements.'' These results provide insight into the self-organization of actomyosin cytoskeleton in non-muscle cells that regulate and maintain cellular tension.

  11. Actin binding proteins, spermatid transport and spermiation*

    Science.gov (United States)

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  12. An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome.

    Science.gov (United States)

    Gil-Krzewska, Aleksandra; Saeed, Mezida B; Oszmiana, Anna; Fischer, Elizabeth R; Lagrue, Kathryn; Gahl, William A; Introne, Wendy J; Coligan, John E; Davis, Daniel M; Krzewski, Konrad

    2017-12-11

    Chediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells. We sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS. We generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells. Loss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells. The cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel

  13. The IpaC carboxyterminal effector domain mediates Src-dependent actin polymerization during Shigella invasion of epithelial cells.

    Directory of Open Access Journals (Sweden)

    Joëlle Mounier

    2009-01-01

    Full Text Available Shigella, the causative agent of bacillary dysentery, invades epithelial cells by locally reorganizing the actin cytoskeleton. Shigella invasion requires actin polymerization dependent on the Src tyrosine kinase and a functional bacterial type III secretion (T3S apparatus. Using dynamic as well as immunofluorescence microscopy, we show that the T3S translocon component IpaC allows the recruitment of the Src kinase required for actin polymerization at bacterial entry sites during the initial stages of Shigella entry. Src recruitment occurred at bacterial-cell contact sites independent of actin polymerization at the onset of the invasive process and was still observed in Shigella strains mutated for translocated T3S effectors of invasion. A Shigella strain with a polar mutation that expressed low levels of the translocator components IpaB and IpaC was fully proficient for Src recruitment and bacterial invasion. In contrast, a Shigella strain mutated in the IpaC carboxyterminal effector domain that was proficient for T3S effector translocation did not induce Src recruitment. Consistent with a direct role for IpaC in Src activation, cell incubation with the IpaC last 72 carboxyterminal residues fused to the Iota toxin Ia (IaC component that translocates into the cell cytosol upon binding to the Ib component led to Src-dependent ruffle formation. Strikingly, IaC also induced actin structures resembling bacterial entry foci that were enriched in activated Src and were inhibited by the Src inhibitor PP2. These results indicate that the IpaC effector domain determines Src-dependent actin polymerization and ruffle formation during bacterial invasion.

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

    Science.gov (United States)

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

    2015-06-01

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

  15. Tropomyosin isoforms bias actin track selection by vertebrate myosin Va

    Science.gov (United States)

    Sckolnick, Maria; Krementsova, Elena B.; Warshaw, David M.; Trybus, Kathleen M.

    2016-01-01

    Tropomyosin (Tpm) isoforms decorate actin with distinct spatial and temporal localization patterns in cells and thus may function to sort actomyosin processes by modifying the actin track affinity for specific myosin isoforms. We examined the effect of three Tpm isoforms on the ability of myosin Va (myoVa) to engage with actin in vitro in the absence or presence of the cargo adapter melanophilin (Mlph), which links myoVa to Rab27a-melanosomes for in vivo transport. We show that both the myosin motor domain and the cargo adapter Mlph, which has an actin-binding domain that acts as a tether, are sensitive to the Tpm isoform. Actin–Tpm3.1 and actin–Tpm1.8 were equal or better tracks compared to bare actin for myoVa-HMM based on event frequency, run length, and speed. The full-length myoVa-Mlph complex showed high-frequency engagement with actin-Tpm3.1 but not with actin-Tpm1.8. Actin–Tpm4.2 excluded both myoVa-HMM and full-length myoVa-Mlph from productive interactions. Of importance, Tpm3.1 is enriched in the dendritic protrusions and cortical actin of melanocytes, where myoVa-Mlph engages in melanosome transport. These results support the hypothesis that Tpm isoforms constitute an “actin–Tpm code” that allows for spatial and temporal sorting of actomyosin function in the cell. PMID:27535431

  16. Deafness and espin-actin self-organization in stereocilia

    Science.gov (United States)

    Wong, Gerard C. L.

    2009-03-01

    Espins are F-actin-bundling proteins associated with large parallel actin bundles found in hair cell stereocilia in the ear, as well as brush border microvilli and Sertoli cell junctions. We examine actin bundle structures formed by different wild-type espin isoforms, fragments, and naturally-occurring human espin mutants linked to deafness and/or vestibular dysfunction. The espin-actin bundle structure consisted of a hexagonal arrangement of parallel actin filaments in a non-native twist state. We delineate the structural consequences caused by mutations in espin's actin-bundling module. For espin mutation with a severely damaged actin-bundling module, which are implicated in deafness in mice and humans, oriented nematic-like actin filament structures, which strongly impinges on bundle mechanical stiffness. Finally, we examine what makes espin different, via a comparative study of bundles formed by espin and those formed by fascin, a prototypical bundling protein found in functionally different regions of the cell, such as filopodia.

  17. Actin-mediated cytoplasmic organization of plant cells

    NARCIS (Netherlands)

    Honing, van der H.S.

    2011-01-01

    In this thesis, I present results that give insight in the role of the actin cytoskeleton in the production of an organized cytoplasm in plant cells, which is, for instance, required for proper cell morphogenesis.

    Chapter 1 is a review in which we discuss the possible role of actin-based

  18. Mapping allostery through computational glycine scanning and correlation analysis of residue-residue contacts.

    Science.gov (United States)

    Johnson, Quentin R; Lindsay, Richard J; Nellas, Ricky B; Fernandez, Elias J; Shen, Tongye

    2015-02-24

    Understanding allosteric mechanisms is essential for the physical control of molecular switches and downstream cellular responses. However, it is difficult to decode essential allosteric motions in a high-throughput scheme. A general two-pronged approach to performing automatic data reduction of simulation trajectories is presented here. The first step involves coarse-graining and identifying the most dynamic residue-residue contacts. The second step is performing principal component analysis of these contacts and extracting the large-scale collective motions expressed via these residue-residue contacts. We demonstrated the method using a protein complex of nuclear receptors. Using atomistic modeling and simulation, we examined the protein complex and a set of 18 glycine point mutations of residues that constitute the binding pocket of the ligand effector. The important motions that are responsible for the allostery are reported. In contrast to conventional induced-fit and lock-and-key binding mechanisms, a novel "frustrated-fit" binding mechanism of RXR for allosteric control was revealed.

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

    Science.gov (United States)

    Wang, Xinxin

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

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

    Science.gov (United States)

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

    2014-01-01

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

  1. Structural modeling and molecular dynamics simulation of the actin filament.

    Science.gov (United States)

    Splettstoesser, Thomas; Holmes, Kenneth C; Noé, Frank; Smith, Jeremy C

    2011-07-01

    Actin is a major structural protein of the eukaryotic cytoskeleton and enables cell motility. Here, we present a model of the actin filament (F-actin) that not only incorporates the global structure of the recently published model by Oda et al. but also conserves internal stereochemistry. A comparison is made using molecular dynamics simulation of the model with other recent F-actin models. A number of structural determents such as the protomer propeller angle, the number of hydrogen bonds, and the structural variation among the protomers are analyzed. The MD comparison is found to reflect the evolution in quality of actin models over the last 6 years. In addition, simulations of the model are carried out in states with both ADP or ATP bound and local hydrogen-bonding differences characterized. Copyright © 2011 Wiley-Liss, Inc.

  2. Actin polymerization contributes to neutrophil chemotactic dysfunction following thermal injury.

    Science.gov (United States)

    Hasslen, S R; Ahrenholz, D H; Solem, L D; Nelson, R D

    1992-11-01

    The agent(s) and mechanism(s) responsible for suppression of neutrophil chemotaxis in association with major thermal injury have not been identified. We have proposed that the reduced random motility characterizing patients' cells may contribute to their generalized chemotactic dysfunction. Here we report that actin polymerization may be responsible for the loss of neutrophil motility associated with major thermal injury. Using a fluorescent ligand specific for polymerized or filamentous actin (NBD-phallacidin) in conjunction with flow cytometry, we have discovered that peripheral blood and exudate neutrophils from patients with major thermal injury contain increased levels of actin in a stably polymerized form. Because cyclic polymerization and depolymerization of actin is essential to cell motility, we suggest that actin polymerization may contribute in a major way to the attenuation of neutrophil random and chemotactic functions induced by major thermal injury.

  3. Measuring Actin Flow in 3D Cell Protrusions

    Science.gov (United States)

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

    2013-01-01

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

  4. Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae

    Directory of Open Access Journals (Sweden)

    Benjamin B. A. Raymond

    2018-02-01

    Full Text Available Mycoplasma hyopneumoniae, an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15 using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM, and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

  5. Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Raymond, Benjamin B A; Madhkoor, Ranya; Schleicher, Ina; Uphoff, Cord C; Turnbull, Lynne; Whitchurch, Cynthia B; Rohde, Manfred; Padula, Matthew P; Djordjevic, Steven P

    2018-01-01

    Mycoplasma hyopneumoniae , an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15) using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM), and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii) microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii) more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv) biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

  6. Cyclic hardening in bundled actin networks.

    Science.gov (United States)

    Schmoller, K M; Fernández, P; Arevalo, R C; Blair, D L; Bausch, A R

    2010-01-01

    Nonlinear deformations can irreversibly alter the mechanical properties of materials. Most soft materials, such as rubber and living tissues, display pronounced softening when cyclically deformed. Here we show that, in contrast, reconstituted networks of crosslinked, bundled actin filaments harden when subject to cyclical shear. As a consequence, they exhibit a mechano-memory where a significant stress barrier is generated at the maximum of the cyclic shear strain. This unique response is crucially determined by the network architecture: at lower crosslinker concentrations networks do not harden, but soften showing the classic Mullins effect known from rubber-like materials. By simultaneously performing macrorheology and confocal microscopy, we show that cyclic shearing results in structural reorganization of the network constituents such that the maximum applied strain is encoded into the network architecture.

  7. Ultrastructural localization of actin and actin-binding proteins in the nucleus

    Czech Academy of Sciences Publication Activity Database

    Dingová, Hana; Fukalová, Jana; Maninová, Miloslava; Philimonenko, Vlada; Hozák, Pavel

    2009-01-01

    Roč. 131, č. 3 (2009), s. 425-434 ISSN 0948-6143 R&D Projects: GA MŠk LC545 Grant - others:MŠk(CZ) LC06063 Program:LC Institutional research plan: CEZ:AV0Z50520514 Keywords : nuclear actin * ultrastructure * actin–binding proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.021, year: 2009

  8. Titin-Actin Interaction: PEVK-Actin-Based Viscosity in a Large Animal

    Directory of Open Access Journals (Sweden)

    Charles S. Chung

    2011-01-01

    Full Text Available Titin exhibits an interaction between its PEVK segment and the actin filament resulting in viscosity, a speed dependent resistive force, which significantly influences diastolic filling in mice. While diastolic disease is clinically pervasive, humans express a more compliant titin (N2BA:N2B ratio ~0.5–1.0 than mice (N2BA:N2B ratio ~0.2. To examine PEVK-actin based viscosity in compliant titin-tissues, we used pig cardiac tissue that expresses titin isoforms similar to that in humans. Stretch-hold experiments were performed at speeds from 0.1 to 10 lengths/s from slack sarcomere lengths (SL to SL of 2.15 μm. Viscosity was calculated from the slope of stress-relaxation vs stretch speed. Recombinant PEVK was added to compete off native interactions and this found to reduce the slope by 35%, suggesting that PEVK-actin interactions are a strong contributor of viscosity. Frequency sweeps were performed at frequencies of 0.1–400 Hz and recombinant protein reduced viscous moduli by 40% at 2.15 μm and by 50% at 2.25 μm, suggesting a SL-dependent nature of viscosity that might prevent SL ``overshoot’’ at long diastolic SLs. This study is the first to show that viscosity is present at physiologic speeds in the pig and supports the physiologic relevance of PEVK-actin interactions in humans in both health and disease.

  9. Coarse-grained molecular simulations of allosteric cooperativity

    Energy Technology Data Exchange (ETDEWEB)

    Nandigrami, Prithviraj; Portman, John J. [Department of Physics, Kent State University, Kent, Ohio 44242 (United States)

    2016-03-14

    Interactions between a protein and a ligand are often accompanied by a redistribution of the population of thermally accessible conformations. This dynamic response of the protein’s functional energy landscape enables a protein to modulate binding affinities and control binding sensitivity to ligand concentration. In this paper, we investigate the structural origins of binding affinity and allosteric cooperativity of binding two Ca{sup 2+} ions to each domain of Calmodulin (CaM) through simulations of a simple coarse-grained model. In this model, the protein’s conformational transitions between open and closed conformational ensembles are simulated explicitly and ligand binding and unbinding are treated implicitly within the grand canonical ensemble. Ligand binding is cooperative because the binding sites are coupled through a shift in the dominant conformational ensemble upon binding. The classic Monod-Wyman-Changeux model of allostery with appropriate binding free energies to the open and closed ensembles accurately describes the simulated binding thermodynamics. The simulations predict that the two domains of CaM have distinct binding affinity and cooperativity. In particular, the C-terminal domain binds Ca{sup 2+} with higher affinity and greater cooperativity than the N-terminal domain. From a structural point of view, the affinity of an individual binding loop depends sensitively on the loop’s structural compatibility with the ligand in the bound ensemble, as well as the conformational flexibility of the binding site in the unbound ensemble.

  10. Allosteric potentiation of quisqualate receptors by a nootropic drug aniracetam.

    Science.gov (United States)

    Ito, I; Tanabe, S; Kohda, A; Sugiyama, H

    1990-05-01

    1. Allosteric potentiation of the ionotropic quisqualate (iQA) receptor by a nootropic drug aniracetam (1-p-anisoyl-2-pyrrolidinone) was investigated using Xenopus oocytes injected with rat brain mRNA and rat hippocampal slices. 2. Aniracetam potentiates the iQA responses induced in Xenopus oocytes by rat brain mRNA in a reversible manner. This effect was observed above the concentrations of 0.1 mM. Kainate. N-methyl-D-aspartate and gamma-aminobutyric acid responses induced in the same oocytes were not affected. 3. The specific potentiation of iQA responses was accompanied by an increase in the conductance change of iQA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) responses, but the affinity of receptors for agonist and the ion-selectivity of the channels (reversal potentials) were not changed. 4. Aniracetam reversibly potentiated the iQA responses recorded intracellularly from the pyramidal cells in the CA1 region of rat hippocampal slices. The excitatory postsynaptic potentials (EPSPs) in Schaffer collateral-commissural-CA1 synapses were also potentiated by aniracetam. 5. Population EPSPs recorded in the mossy fibre-CA3 synapses as well as Schaffer-commissural synapses were also potentiated by aniracetam. The amplitudes of the potentiation were not changed by the formation of long-term potentiation.

  11. Molecular investigations into the mechanics of actin in different nucleotide states.

    Science.gov (United States)

    Lee, Ji Y; Iverson, Tyler M; Dima, Ruxandra I

    2011-01-13

    Actin plays crucial roles in the mechanical response of cells to applied forces. For example, during cell adhesion, under the action of forces transmitted through integrins, actin filaments (F-actin) induce intracellular mechanical movements leading to changes in the cell shape. Muscle contraction results from the interaction of F-actin with the molecular motor myosin. Thus, understanding the origin of actin's mechanical flexibility is required to understand the basis of fundamental cellular processes. F-actin results from the polymerization of globular actin (G-actin), which contains one tightly bound nucleotide (ATP or ADP). Experiments revealed that G-actin is more flexible than F-actin, but no molecular-level understanding of this differential behavior exists. To probe the basis of the mechanical behavior of actin, we study the force response of G-actin bound with ATP (G-ATP) or ADP (G-ADP). We investigate the global unfolding of G-actin under forces applied at its ends and its mechanical resistance along the actin-actin and actin-myosin bonds in F-actin. Our study reveals that the nucleotide plays an important role in the global unfolding of actin, leading to multiple unfolding scenarios which emphasize the differences between the G-ATP and G-ADP states. Furthermore, our simulations show that G-ATP is more flexible than G-ADP and that the actin-myosin interaction surface responds faster to force than the actin-actin interaction surface. The deformation of G-actin under tension revealed in our simulations correlates very well with experimental data on G-actin domain flexibility.

  12. Triggering actin comets versus membrane ruffles: distinctive effects of phosphoinositides on actin reorganization.

    Science.gov (United States)

    Ueno, Tasuku; Falkenburger, Björn H; Pohlmeyer, Christopher; Inoue, Takanari

    2011-12-13

    A limited set of phosphoinositide membrane lipids regulate diverse cellular functions including proliferation, differentiation, and migration. We developed two techniques based on rapamycin-induced protein dimerization to rapidly change the concentration of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. First, using a membrane-recruitable form of PI(4)P 5-kinase, we increased PI(4,5)P(2) synthesis from phosphatidylinositol 4-phosphate [PI(4)P] and found that COS-7, HeLa, and human embryonic kidney 293 cells formed bundles of motile actin filaments known as actin comets. In contrast, a second technique that increased the concentration of PI(4,5)P(2) without consuming PI(4)P induced membrane ruffles. These distinct phenotypes were mediated by dynamin-mediated vesicular trafficking and mutually inhibitory crosstalk between the small guanosine triphosphatases Rac and RhoA. Our results indicate that the effect of PI(4,5)P(2) on actin reorganization depends on the abundance of other phosphoinositides, such as PI(4)P. Thus, combinatorial regulation of phosphoinositide concentrations may contribute to the diversity of phosphoinositide functions.

  13. EDITORIAL: Close contact Close contact

    Science.gov (United States)

    Demming, Anna

    2010-07-01

    The development of scanning probe techniques, such as scanning tunnelling microscopy [1], has often been touted as the catalyst for the surge in activity and progress in nanoscale science and technology. Images of nanoscale structural detail have served as an invaluable investigative resource and continue to fascinate with the fantastical reality of an intricate nether world existing all around us, but hidden from view of the naked eye by a disparity in scale. As is so often the case, the invention of the scanning tunnelling microscope heralded far more than just a useful new apparatus, it demonstrated the scope for exploiting the subtleties of electronic contact. The shrinking of electronic devices has been a driving force for research into molecular electronics, in which an understanding of the nature of electronic contact at junctions is crucial. In response, the number of experimental techniques in molecular electronics has increased rapidly in recent years. Scanning tunnelling microscopes have been used to study electron transfer through molecular films on a conducting substrate, and the need to monitor the contact force of scanning tunnelling electrodes led to the use of atomic force microscopy probes coated in a conducting layer as studied by Cui and colleagues in Arizona [2]. In this issue a collaboration of researchers at Delft University and Leiden University in the Netherlands report a new device architecture for the independent mechanical and electrostatic tuning of nanoscale charge transport, which will enable thorough studies of molecular transport in the future [3]. Scanning probes can also be used to pattern surfaces, such as through spatially-localized Suzuki and Heck reactions in chemical scanning probe lithography. Mechanistic aspects of spatially confined Suzuki and Heck chemistry are also reported in this issue by researchers in Oxford [4]. All these developments in molecular electronics fabrication and characterization provide alternative

  14. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    Science.gov (United States)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  15. Cannabidiol is a negative allosteric modulator of the cannabinoid CB1 receptor.

    Science.gov (United States)

    Laprairie, R B; Bagher, A M; Kelly, M E M; Denovan-Wright, E M

    2015-10-01

    Cannabidiol has been reported to act as an antagonist at cannabinoid CB1 receptors. We hypothesized that cannabidiol would inhibit cannabinoid agonist activity through negative allosteric modulation of CB1 receptors. Internalization of CB1 receptors, arrestin2 recruitment, and PLCβ3 and ERK1/2 phosphorylation, were quantified in HEK 293A cells heterologously expressing CB1 receptors and in the STHdh(Q7/Q7) cell model of striatal neurons endogenously expressing CB1 receptors. Cells were treated with 2-arachidonylglycerol or Δ(9)-tetrahydrocannabinol alone and in combination with different concentrations of cannabidiol. Cannabidiol reduced the efficacy and potency of 2-arachidonylglycerol and Δ(9)-tetrahydrocannabinol on PLCβ3- and ERK1/2-dependent signalling in cells heterologously (HEK 293A) or endogenously (STHdh(Q7/Q7)) expressing CB1 receptors. By reducing arrestin2 recruitment to CB1 receptors, cannabidiol treatment prevented internalization of these receptors. The allosteric activity of cannabidiol depended upon polar residues being present at positions 98 and 107 in the extracellular amino terminus of the CB1 receptor. Cannabidiol behaved as a non-competitive negative allosteric modulator of CB1 receptors. Allosteric modulation, in conjunction with effects not mediated by CB1 receptors, may explain the in vivo effects of cannabidiol. Allosteric modulators of CB1 receptors have the potential to treat CNS and peripheral disorders while avoiding the adverse effects associated with orthosteric agonism or antagonism of these receptors. © 2015 The British Pharmacological Society.

  16. Causality, transfer entropy, and allosteric communication landscapes in proteins with harmonic interactions.

    Science.gov (United States)

    Hacisuleyman, Aysima; Erman, Burak

    2017-06-01

    A fast and approximate method of generating allosteric communication landscapes in proteins is presented by using Schreiber's entropy transfer concept in combination with the Gaussian Network Model of proteins. Predictions of the model and the allosteric communication landscapes generated show that information transfer in proteins does not necessarily take place along a single path, but an ensemble of pathways is possible. The model emphasizes that knowledge of entropy only is not sufficient for determining allosteric communication and additional information based on time delayed correlations should be introduced, which leads to the presence of causality in proteins. The model provides a simple tool for mapping entropy sink-source relations into pairs of residues. By this approach, residues that should be manipulated to control protein activity may be determined. This should be of great importance for allosteric drug design and for understanding the effects of mutations on function. The model is applied to determine allosteric communication in three proteins, Ubiquitin, Pyruvate Kinase, and the PDZ domain. Predictions are in agreement with molecular dynamics simulations and experimental evidence. Proteins 2017; 85:1056-1064. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Binding and discerning interactions of PTP1B allosteric inhibitors: novel insights from molecular dynamics simulations.

    Science.gov (United States)

    Shinde, Ranajit Nivrutti; Sobhia, M Elizabeth

    2013-09-01

    The α7 helix is either disordered or missing in the three co-crystal structures of allosteric inhibitors with protein tyrosine phosphatase 1B (PTP1B). It was modeled in each complex using the open form of PTP1B structure and studied using molecular dynamics (MD) simulations for 25 ns. B-factor analysis of the residues sheds light on its disordered nature in the co-crystal structures. Further, the ability of inhibitors to act as allosteric inhibitor was studied and established using novel hydrogen bond criteria. The MD simulations were utilized to determine the relative importance of electrostatic and hydrophobic component in to the binding of inhibitors. It was revealed that the hydrophobic interactions predominantly drive the molecular recognition of these inhibitors. Per residue energy decomposition analysis attributed dissimilar affinities of three inhibitors to the several hydrogen bonds and non-bonded interactions. Among the secondary structure elements that surround the allosteric site, helices α6, α7 and loop α6-α7 were notorious in providing variable affinities to the inhibitors. A novel hydrophobic pocket lined by the α7 helix residues Val287, Asn289 and Trp291 was identified in the allosteric site. This study provides useful insights for the rational design of high affinity PTP1B allosteric inhibitors. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Crosstalk between Rac1-mediated actin regulation and ROS production.

    Science.gov (United States)

    Acevedo, Alejandro; González-Billault, Christian

    2018-02-20

    The small RhoGTPase Rac1 is implicated in a variety of events related to actin cytoskeleton rearrangement. Remarkably, another event that is completely different from those related to actin regulation has the same relevance; the Rac1-mediated production of reactive oxygen species (ROS) through NADPH oxidases (NOX). Each outcome involves different Rac1 downstream effectors; on one hand, events related to the actin cytoskeleton require Rac1 to bind to WAVEs proteins and PAKs that ultimately promote actin branching and turnover, on the other, NOX-derived ROS production demands active Rac1 to be bound to a cytosolic activator of NOX. How Rac1-mediated signaling ends up promoting actin-related events, NOX-derived ROS, or both is poorly understood. Rac1 regulators, including scaffold proteins, are known to exert tight control over its functions. Hence, evidence of Rac1 regulatory events leading to both actin remodeling and NOX-mediated ROS generation are discussed. Moreover, cellular functions linked to physiological and pathological conditions that exhibit crosstalk between Rac1 outcomes are analyzed, while plausible roles in neuronal functions (and dysfunctions) are highlighted. Together, discussed evidence shed light on cellular mechanisms which requires Rac1 to direct either actin- and/or ROS-related events, helping to understand crucial roles of Rac1 dual functionality. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Treadmilling of actin filaments via Brownian dynamics simulations

    DEFF Research Database (Denmark)

    Guo, Kunkun; Shillcock, Julian C.; Lipowsky, Reinhard

    2010-01-01

    Actin polymerization is coupled to the hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (Pi). Therefore, each protomer within an actin filament can attain three different nucleotide states corresponding to bound ATP, ADP / Pi, and ADP....... These protomer states form spatial patterns on the growing (or shrinking) filaments. Using Brownian dynamics simulations, the growth behavior of long filaments is studied, together with the associated protomer patterns, as a function of ATP-actin monomer concentration, CT, within the surrounding solution...

  20. Highly Dynamic Host Actin Reorganization around Developing Plasmodium Inside Hepatocytes

    Science.gov (United States)

    Gomes-Santos, Carina S. S.; Itoe, Maurice A.; Afonso, Cristina; Henriques, Ricardo; Gardner, Rui; Sepúlveda, Nuno; Simões, Pedro D.; Raquel, Helena; Almeida, António Paulo; Moita, Luis F.; Frischknecht, Friedrich; Mota, Maria M.

    2012-01-01

    Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver. PMID:22238609

  1. Actin purification from a gel of rat brain extracts.

    Science.gov (United States)

    Levilliers, N; Peron-Renner, M; Coffe, G; Pudles, J

    1984-01-01

    Actin, 99% pure, has been recovered from rat brain with a high yield (greater than 15 mg/100 g brain). We have shown that: 1. a low ionic strength extract from rat brain tissue is capable of giving rise to a gel; 2. actin is the main gel component and its proportion is one order of magnitude higher than in the original extract; 3. actin can be isolated from this extract by a three-step procedure involving gelation, dissociation of the gel in 0.6 M KCl, followed by one or two depolymerization-polymerization cycles.

  2. Design of Elastic Networks with Evolutionary Optimized Long-Range Communication as Mechanical Models of Allosteric Proteins.

    Science.gov (United States)

    Flechsig, Holger

    2017-08-08

    Allosteric effects often underlie the activity of proteins, and elucidating generic design aspects and functional principles unique to allosteric phenomena represent a major challenge. Here an approach consisting of the in silico design of synthetic structures, which, as the principal element of allostery, encode dynamical long-range coupling among two sites, is presented. The structures are represented by elastic networks, similar to coarse-grained models of real proteins. A strategy of evolutionary optimization was implemented to iteratively improve allosteric coupling. In the designed structures, allosteric interactions were analyzed in terms of strain propagation, and simple pathways that emerged during evolution were identified as signatures through which long-range communication was established. Moreover, robustness of allosteric performance with respect to mutations was demonstrated. As it turned out, the designed prototype structures reveal dynamical properties resembling those found in real allosteric proteins. Hence, they may serve as toy models of complex allosteric systems, such as proteins. Application of the developed modeling scheme to the allosteric transition in the myosin V molecular motor was also demonstrated. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    Science.gov (United States)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  4. Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins.

    Science.gov (United States)

    Yao, Xin-Qiu; Malik, Rabia U; Griggs, Nicholas W; Skjærven, Lars; Traynor, John R; Sivaramakrishnan, Sivaraj; Grant, Barry J

    2016-02-26

    G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  6. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

    Science.gov (United States)

    Foda, Zachariah H.; Shan, Yibing; Kim, Eric T.; Shaw, David E.; Seeliger, Markus A.

    2015-01-01

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

  7. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase.

    Science.gov (United States)

    Foda, Zachariah H; Shan, Yibing; Kim, Eric T; Shaw, David E; Seeliger, Markus A

    2015-01-20

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

  8. Discovery and Characterization of Biased Allosteric Agonists of the Chemokine Receptor CXCR3

    DEFF Research Database (Denmark)

    Milanos, Lampros; Brox, Regine; Frank, Theresa

    2016-01-01

    In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces...... weak chemotaxis and leads to receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial...... agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or β-arrestin 2 recruitment. Concomitant structure-activity relationship studies indicated very steep structure-activity relationships, which steer the ligand bias between the β-arrestin 2 and G...

  9. Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation

    Directory of Open Access Journals (Sweden)

    George P. Lisi

    2018-02-01

    Full Text Available The enzyme imidazole glycerol phosphate synthase (IGPS is a model for studies of long-range allosteric regulation in enzymes. Binding of the allosteric effector ligand N'-[5'-phosphoribulosylformimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR stimulates millisecond (ms timescale motions in IGPS that enhance its catalytic function. We studied the effect of temperature on these critical conformational motions and the catalytic mechanism of IGPS from the hyperthermophile Thermatoga maritima in an effort to understand temperature-dependent allostery. Enzyme kinetic and NMR dynamics measurements show that apo and PRFAR-activated IGPS respond differently to changes in temperature. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG relaxation dispersion experiments performed at 303, 323, and 343 K (30, 50, and 70°C reveal that millisecond flexibility is enhanced to a higher degree in apo IGPS than in the PRFAR-bound enzyme as the sample temperature is raised. We find that the flexibility of the apo enzyme is nearly identical to that of its PRFAR activated state at 343 K, whereas conformational motions are considerably different between these two forms of the enzyme at room temperature. Arrhenius analyses of these flexible sites show a varied range of activation energies that loosely correlate to allosteric communities identified by computational methods and reflect local changes in dynamics that may facilitate conformational sampling of the active conformation. In addition, kinetic assays indicate that allosteric activation by PRFAR decreases to 65-fold at 343 K, compared to 4,200-fold at 303 K, which mirrors the decreased effect of PRFAR on ms motions relative to the unactivated enzyme. These studies indicate that at the growth temperature of T. maritima, PFRAR is a weaker allosteric activator than it is at room temperature and illustrate that the allosteric mechanism of IGPS is temperature dependent.

  10. Organism-adapted specificity of the allosteric regulation of pyruvate kinase in lactic acid bacteria.

    Directory of Open Access Journals (Sweden)

    Nadine Veith

    Full Text Available Pyruvate kinase (PYK is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric

  11. Microfilament dynamics during cell movement and chemotaxis monitored using a GFP-actin fusion protein.

    Science.gov (United States)

    Westphal, M; Jungbluth, A; Heidecker, M; Mühlbauer, B; Heizer, C; Schwartz, J M; Marriott, G; Gerisch, G

    1997-03-01

    The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, both spontaneously and in response to external signals. In particular, actin polymerization induced by a gradient of chemoattractant leads to local accumulation of filamentous actin and protrusion of a 'leading edge' of the cell in the direction of the gradient. In order to study the dynamics of actin in these processes, actin was tagged at its amino terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of D. discoideum. Purified GFP-actin was capable of copolymerizing with actin. In the transfected cells of D. discoideum studied, GFP-actin made up 10-20% of the total actin. Microfilaments containing GFP-actin were capable of generating force with myosin in an in vitro assay. Observations of single living cells using fluorescence microscopy showed that the fusion protein was enriched in cell projections, including filopodia and leading edges, and that the fusion protein reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated, or aggregated. When confocal sections of fixed cells containing GFP-actin were labeled with fluorescent phalloidin, which binds only to filamentous actin, there was a correlation between the areas of GFP-actin and phalloidin fluorescence, but there were distinct sites in which GFP-actin was more prominent. Double labeling with GFP-actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP-actin are networks or bundles of filamentous actin. Other clusters of GFP-actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins.

  12. Thymosin beta4 sequesters actin in cystic fibrosis sputum and decreases sputum cohesivity in vitro

    NARCIS (Netherlands)

    Rubin, Bruce K.; Kater, Arnon P.; Goldstein, Allan L.

    2006-01-01

    Filamentous actin (F-actin) forms polymers that contribute to the abnormal biophysical properties of sputum. Thymosin beta4 (Tbeta4) is the major monomeric actin-sequestering peptide in cells and can depolymerize F-actin. Tbeta4 could potentially decrease sputum viscoelasticity and adhesivity and

  13. Nanosecond electric pulses trigger actin responses in plant cells

    International Nuclear Information System (INIS)

    Berghoefer, Thomas; Eing, Christian; Flickinger, Bianca; Hohenberger, Petra; Wegner, Lars H.; Frey, Wolfgang; Nick, Peter

    2009-01-01

    We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

  14. First steps in the direction of synthetic, allosteric, direct inhibitors of thrombin and factor Xa.

    Science.gov (United States)

    Verghese, Jenson; Liang, Aiye; Sidhu, Preet Pal Singh; Hindle, Michael; Zhou, Qibing; Desai, Umesh R

    2009-08-01

    Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that (i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; (ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and (iii) the mechanism of inhibition is allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes.

  15. First Steps in the Direction of Synthetic, Allosteric, Direct Inhibitors of Thrombin and Factor Xa

    Science.gov (United States)

    Verghese, Jenson; Liang, Aiye; Sidhu, Preet Pal Singh; Hindle, Michael; Zhou, Qibing; Desai, Umesh R.

    2009-01-01

    Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and iii) the mechanism of inhibition is allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes. PMID:19540113

  16. Old drug new tricks: Chlorhexidine acts as a potential allosteric inhibitor toward PAK1.

    Science.gov (United States)

    Huang, Han-Wei; Zhang, Xiang-Yu; Song, Pei-Lu; Jiang, Hai-Lun; Li, Wei; Wang, Peng-Liang; Wang, Jian; Liu, Fu-Nan

    2018-01-01

    This paper describes the identification of chlorhexidine, an agent commonly used in clinical as a novel potential allosteric inhibitor of PAK1. In cellular assays, chlorhexidine showed a good inhibitory profile, and its inhibitory profile was even better than IPA-3, a well-known allosteric inhibitor. In pharmacology experiments, chlorhexidine successfully inhibited the relief of PAK1 dimer and inhibited the activation of PAK1. Our findings offer an insight for the new drug development of PAK1 inhibitor. We also provide a possible explanation for the phenomenon that the application of the chlorhexidine in peritoneal lavage inhibited the development of tumor. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1

    DEFF Research Database (Denmark)

    Hindie, Valerie; Stroba, Adriana; Zhang, Hua

    2009-01-01

    Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation...... and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present...

  18. Identification and Structure-Function Study of Positive Allosteric Modulators of Kainate Receptors

    DEFF Research Database (Denmark)

    Larsen, Anja Probst; Fièvre, Sabine; Frydenvang, Karla

    2017-01-01

    as the AMPA receptor subunit GluA1i (5-fold). X-ray structures of the three modulators in the GluK1 ligand-binding domain were determined, locating two modulator-binding sites at the GluK1 dimer interface. In conclusion, this study may enable the design of new positive allosteric modulators selective for KARs......Kainate receptors (KARs) consist of a class of ionotropic glutamate receptors, which exert diverse pre- and postsynaptic functions through complex signaling regulating the activity of neural circuits. Whereas numerous small-molecule positive allosteric modulators of the ligand-binding domain of (S...

  19. The role of actin networks in cellular mechanosensing

    Science.gov (United States)

    Azatov, Mikheil

    Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

  20. The role of antihistamines in chronic actinic dermatitis treatment

    Directory of Open Access Journals (Sweden)

    E. V. Orlov

    2016-01-01

    Full Text Available Inveterate actinic dermatitis is an immunologically mediated photodermatosis characterized by itchy eczematous dermhelminthiasis exposed to sunlight. The disease proceeds in the same way as the atopic eczema or atopic dermatitis. The treatment of patients with inveterate actinic dermatitis is similar to the treatment of patients with atopic dermatitis and eczema. Administration of the modern antihistaminic preparation desloratadine (Aerius in the treatment has a positive effect on the skin process relief and on some cellular and humoral immunity factors.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gomibuchi, Yuki [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Uyeda, Taro Q.P. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan)

    2013-11-29

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

  2. Amphidinolide H, a novel type of actin-stabilizing agent isolated from dinoflagellate

    International Nuclear Information System (INIS)

    Saito, Shin-ya; Feng Jue; Kira, Atsushi; Kobayashi, Jun'ichi; Ohizumi, Yasushi

    2004-01-01

    The effect of novel cytotoxic marine macrolide, amphidinolide H (Amp-H), on actin dynamics was investigated in vitro. Amp-H attenuated actin depolymerization induced by diluting F-actin. This effect remained after washing out of unbound Amp-H by filtration. In the presence of either Amp-H or phalloidin, lag phase, which is the rate-limiting step of actin polymerization, was shortened. Phalloidin decreased the polymerization-rate whereas Amp-H did not. Meanwhile, the effects of both compounds were the same when barbed end of actin was capped by cytochalasin D. Quartz crystal microbalance system revealed interaction of Amp-H with G-actin and F-actin. Amp-H also enhanced the binding of phalloidin to F-actin. We concluded that Amp-H stabilizes actin in a different manner from that of phalloidin and serves as a novel pharmacological tool for analyzing actin-mediated cell function

  3. Actin polymerization drives polar growth in Arabidopsis root hair cells.

    Science.gov (United States)

    Vazquez, Luis Alfredo Bañuelos; Sanchez, Rosana; Hernandez-Barrera, Alejandra; Zepeda-Jazo, Isaac; Sánchez, Federico; Quinto, Carmen; Torres, Luis Cárdenas

    2014-01-01

    In plants, the actin cytoskeleton is a prime regulator of cell polarity, growth, and cytoplasmic streaming. Tip growth, as observed in root hairs, caulonema, and pollen tubes, is governed by many factors, including calcium gradients, exocytosis and endocytosis, reactive oxygen species, and the cytoskeleton. Several studies indicate that the polymerization of G-actin into F-actin also contributes to tip growth. The structure and function of F-actin within the apical dome is variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in the elongating apices of tip-growing cells suggests that this cytoskeletal array is tightly regulated. We recently reported that sublethal concentrations of fluorescently labeled cytochalasin could be used to visualize the distribution of microfilament plus ends using fluorescence microscopy, and found that the tip region of the growing root hair cells of a legume plant exhibits a clear response to the nodulation factors secreted by Rhizobium. (1) In this current work, we expanded our analysis using confocal microscopy and demonstrated the existence of highly dynamic fluorescent foci along Arabidopsis root hair cells. Furthermore, we show that the strongest fluorescence signal accumulates in the tip dome of the growing root hair and seems to be in close proximity to the apical plasma membrane. Based on these findings, we propose that actin polymerization within the dome of growing root hair cells regulates polar growth.

  4. Rheology of Membrane-Attached Minimal Actin Cortices.

    Science.gov (United States)

    Noeding, Helen; Schoen, Markus; Kramer, Corinna; Doerrer, Nils; Kuerschner, Aileen; Geil, Burkhard; Mey, Ingo P; Heussinger, Claus; Janshoff, Andreas; Steinem, Claudia

    2018-03-28

    The actin cortex is a thin cross-linked network attached to the plasma membrane, being responsible for the cell's shape during migration, division and growth. In a reductionist approach, we created a minimal actin cortex (MAC) attached to a lipid membrane to correlate the filamentous actin architecture with its viscoelastic properties. The system is composed of a supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer doped with the receptor lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2) to which a constitutively active mutant of ezrin, being a direct membrane-cytoskeleton linker, is bound. The formation of the MAC on the supported lipid bilayer is analyzed as a function of increasing PtdIns(4,5)P2/ezrin pinning points revealing an increase in the intersections between actin filaments, i.e., the node density of the MAC. Bead tracking microrheology on the membrane attached actin network provides information about its viscoelastic properties. The results show that ezrin serves as a dynamic cross-linker for the actin cortex attached to the lipid bilayer and that the stiffness of the network is influenced by the pinning point density, relating the plateau storage modulus G0 to the node density of the MAC.

  5. Liquid droplets of cross-linked actin filaments

    Science.gov (United States)

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

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

  6. Nucleotide effects on the structure and dynamics of actin.

    Science.gov (United States)

    Zheng, Xiange; Diraviyam, Karthikeyan; Sept, David

    2007-08-15

    Adenosine 5'-triphosphate or ATP is the primary energy source within the cell, releasing its energy via hydrolysis into adenosine 5'-diphosphate or ADP. Actin is an important ATPase involved in many aspects of cellular function, and the binding and hydrolysis of ATP regulates its polymerization into actin filaments as well as its interaction with a host of actin-associated proteins. Here we study the dynamics of monomeric actin in ATP, ADP-Pi, and ADP states via molecular dynamics simulations. As observed in some crystal structures we see that the DNase-I loop is an alpha-helix in the ADP state but forms an unstructured coil domain in the ADP-Pi and ATP states. We also find that this secondary structure change is reversible, and by mimicking nucleotide exchange we can observe the transition between the helical and coil states. Apart from the DNase-I loop, we also see several key structural differences in the nucleotide binding cleft as well as in the hydrophobic cleft between subdomains 1 and 3 where WH2-containing proteins have been shown to interact. These differences provide a structural basis for understanding the observed differences between the various nucleotide states of actin and provide some insight into how ATP regulates the interaction of actin with itself and other proteins.

  7. Functional characterisation of filamentous actin probe expression in neuronal cells.

    Directory of Open Access Journals (Sweden)

    Shrujna Patel

    Full Text Available Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.

  8. Nuclear actin filaments recruit cofilin and actin-related protein 3, and their formation is connected with a mitotic block

    Czech Academy of Sciences Publication Activity Database

    Kalendová, Alžběta; Kalasová, Ilona; Yamazaki, S.; Uličná, Lívia; Harata, M.; Hozák, Pavel

    2014-01-01

    Roč. 142, č. 2 (2014), s. 139-152 ISSN 0948-6143 R&D Projects: GA ČR GAP305/11/2232; GA MŠk LD12063; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : nuclear actin * transcription * mitosis * actin-related protein 3 * cofilin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.927, year: 2013

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  10. ACTIN BINDING PROTEIN29 from Lilium Pollen Plays an Important Role in Dynamic Actin Remodeling[W][OA

    Science.gov (United States)

    Xiang, Yun; Huang, Xi; Wang, Ting; Zhang, Yan; Liu, Qinwen; Hussey, Patrick J.; Ren, Haiyun

    2007-01-01

    Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263–amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca2+- and/or phosphatidylinositol 4,5-bisphosphate–regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth. PMID:17586658

  11. A Coincidence Detection Mechanism Controls PX-BAR Domain-Mediated Endocytic Membrane Remodeling via an Allosteric Structural Switch.

    Science.gov (United States)

    Lo, Wen-Ting; Vujičić Žagar, Andreja; Gerth, Fabian; Lehmann, Martin; Puchkov, Dymtro; Krylova, Oxana; Freund, Christian; Scapozza, Leonardo; Vadas, Oscar; Haucke, Volker

    2017-11-20

    Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P 2 ) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P 2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists.

    Science.gov (United States)

    Hoyer, Inna; Haas, Ann-Karin; Kreuchwig, Annika; Schülein, Ralf; Krause, Gerd

    2013-02-01

    The TSHR (thyrotropin receptor) is activated endogenously by the large hormone thyrotropin and activated pathologically by auto-antibodies. Both activate and bind at the extracellular domain. Recently, SMLs (small-molecule ligands) have been identified, which bind in an allosteric binding pocket within the transmembrane domain. Modelling driven site-directed mutagenesis of amino acids lining this pocket led to the delineation of activation and inactivation sensitive residues. Modified residues showing CAMs (constitutively activating mutations) indicate signalling-sensitive positions and mark potential trigger points for agonists. Silencing mutations lead to an impairment of basal activity and mark contact points for antagonists. Mapping these residues on to a structural model of TSHR indicates locations where an SML may switch the receptor to an inactive or active conformation. In the present article, we report the effects of SMLs on these signalling-sensitive amino acids at the TSHR. Surprisingly, the antagonistic effect of SML compound 52 was reversed to an agonistic effect, when tested at the CAM Y667A. Switching agonism to antagonism and the reverse by changing either SMLs or residues covering the binding pocket provides detailed knowledge about discriminative pharmacophores. It prepares the basis for rational optimization of new high-affinity antagonists to interfere with the pathogenic activation of the TSHR.

  13. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... One Use Facts About Colored Contacts and Halloween Safety Colored Contact Lens Facts Over-the-Counter Costume ... Costume Contact Lenses Can Ruin Vision Eye Makeup Safety In fact, it is illegal to sell colored ...

  14. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... with Colored Contact Lenses Julian: Teenager Blinded In One Eye By Non-Prescription Contact Lens Laura: Vision ... Robyn: Blurry Vision and Daily Eye Drops After One Use Facts About Colored Contacts and Halloween Safety ...

  15. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... One Use Facts About Colored Contacts and Halloween Safety Colored Contact Lens Facts Over-the-Counter Costume ... use of colored contact lenses , from the U.S. Food and Drug Administration (FDA). Are the colored lenses ...

  16. Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium-exchange to characterize allosteric regulation†

    Science.gov (United States)

    Prasannan, Charulata B.; Artigues, Antonio; Fenton, Aron W.

    2011-01-01

    There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g. active site and allosteric site). Both NMR, by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both H2O and D2O. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in D2O. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in D2O, but the effector continues to elicit an allosteric response. PMID:21701851

  17. Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium exchange to characterize allosteric regulation.

    Science.gov (United States)

    Prasannan, Charulata B; Artigues, Antonio; Fenton, Aron W

    2011-08-01

    There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g., active and allosteric sites). Both nuclear magnetic resonance (NMR), by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both H(2)O and D(2)O. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in D(2)O. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in D(2)O, but the effector continues to elicit an allosteric response.

  18. Molecular mechanism of allosteric modulation at GPCRs: insight from a binding kinetics study at the human A1 adenosine receptor.

    Science.gov (United States)

    Guo, Dong; Venhorst, Suzanne N; Massink, Arnault; van Veldhoven, Jacobus P D; Vauquelin, Georges; IJzerman, Adriaan P; Heitman, Laura H

    2014-12-01

    Many GPCRs can be allosterically modulated by small-molecule ligands. This modulation is best understood in terms of the kinetics of the ligand-receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands. Here, we describe the application of a so-called competition association assay at the adenosine A1 receptor for this purpose. We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A1 receptor in the absence or presence of two allosteric modulators. We also tested three bitopic ligands, in which an orthosteric and an allosteric pharmacophore were covalently linked with different spacer lengths. The relevance of the competition association assay for the binding kinetics of the bitopic ligands was also explored by analysing simulated data. The binding kinetics of an unlabelled orthosteric ligand were affected by the addition of an allosteric modulator and such effects were probe- and concentration-dependent. Covalently linking the orthosteric and allosteric pharmacophores into one bitopic molecule had a substantial effect on the overall on- or off-rate. The competition association assay is a useful tool for exploring the allosteric modulation of the human adenosine A1 receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well. © 2014 The British Pharmacological Society.

  19. Selective Negative Allosteric Modulation Of Metabotropic Glutamate Receptors - A Structural Perspective of Ligands and Mutants

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Isberg, Vignir; Tehan, Benjamin G

    2015-01-01

    The metabotropic glutamate receptors have a wide range of modulatory functions in the central nervous system. They are among the most highly pursued drug targets, with relevance for several neurological diseases, and a number of allosteric modulators have entered clinical trials. However, so far ...

  20. 2013 Philip S. Portoghese Medicinal Chemistry Lectureship: Drug Discovery Targeting Allosteric Sites†

    Science.gov (United States)

    2015-01-01

    The identification of sites on receptors topographically distinct from the orthosteric sites, so-called allosteric sites, has heralded novel approaches and modes of pharmacology for target modulation. Over the past 20 years, our understanding of allosteric modulation has grown significantly, and numerous advantages, as well as caveats (e.g., flat structure–activity relationships, species differences, “molecular switches”), have been identified. For multiple receptors and proteins, numerous examples have been described where unprecedented levels of selectivity are achieved along with improved physiochemical properties. While not a panacea, these novel approaches represent exciting opportunities for tool compound development to probe the pharmacology and therapeutic potential of discrete molecular targets, as well as new medicines. In this Perspective, in commemoration of the 2013 Philip S. Portoghese Medicinal Chemistry Lectureship (LindsleyC. W.Adventures in allosteric drug discovery. Presented at the 246th National Meeting of the American Chemical Society, Indianapolis, IN, September 10, 2013; The 2013 Portoghese Lectureship), several vignettes of drug discovery campaigns targeting novel allosteric mechanisms will be recounted, along with lessons learned and guidelines that have emerged for successful lead optimization. PMID:25180768

  1. "Molecular Switches" on mGluR Allosteric Ligands That Modulate Modes of Pharmacology

    Science.gov (United States)

    Wood, Michael R.; Hopkins, Corey R.; Brogan, John T.; Conn, P. Jeffrey; Lindsley, Craig W.

    2013-01-01

    G-Protein-coupled receptors (GPCRs) represent the largest class of drug targets, accounting for more than 40% of marketed drugs; however, discovery efforts for many GPCRs have failed to provide viable drug candidates. Historically, drug discovery efforts have focused on developing ligands that act at the orthosteric site of the endogenous agonist. Recently, efforts have focused on functional assay paradigms and the discovery of ligands that act at allosteric sites to modulate receptor function in either a positive, negative, or neutral manner. Allosteric modulators have numerous advantages over orthosteric ligands, including high subtype selectivity; the ability to mimic physiological conditions; the lack of densensitization, downregulation, and internalization; and reduced side effects. Despite these virtues, challenging issues have now arisen for allosteric modulators of metabotropic glutamate receptors (mGluRs): shallow SAR, ligand-directed trafficking, and the identification of subtle “molecular switches” that modulate the modes of pharmacology. Here, we will discuss the impact of modest structural changes to multiple mGluR allosteric ligands scaffolds that unexpectedly modulate pharmacology and raise concerns over metabolism and the pharmacology of metabolites. PMID:21341760

  2. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α–β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  3. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB a–b-subunit interface and affects multiple steps in the enzyme’s overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  4. Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; Randáková, Alena; El-Fakahany, E. E.; Doležal, Vladimír

    2009-01-01

    Roč. 9, č. 15 (2009), s. 1-20 ISSN 1471-2210 R&D Projects: GA ČR GA305/09/0681; GA MŠk(CZ) LC554; GA AV ČR(CZ) IAA500110703 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * allosteric modulation * rapacuronium Subject RIV: ED - Physiology

  5. Allosteric Regulation of the Rotational Speed in a Light-Driven Molecular Motor

    NARCIS (Netherlands)

    Faulkner, Adele; van Leeuwen, Thomas; Feringa, Ben L; Wezenberg, Sander J

    2016-01-01

    The rotational speed of an overcrowded alkene-based molecular rotary motor, having an integrated 4,5-diazafluorenyl coordination motif, can be regulated allosterically via the binding of metal ions. DFT calculations have been used to predict the relative speed of rotation of three different (i.e.

  6. Entropy Transfer between Residue Pairs and Allostery in Proteins: Quantifying Allosteric Communication in Ubiquitin.

    Science.gov (United States)

    Hacisuleyman, Aysima; Erman, Burak

    2017-01-01

    It has recently been proposed by Gunasakaran et al. that allostery may be an intrinsic property of all proteins. Here, we develop a computational method that can determine and quantify allosteric activity in any given protein. Based on Schreiber's transfer entropy formulation, our approach leads to an information transfer landscape for the protein that shows the presence of entropy sinks and sources and explains how pairs of residues communicate with each other using entropy transfer. The model can identify the residues that drive the fluctuations of others. We apply the model to Ubiquitin, whose allosteric activity has not been emphasized until recently, and show that there are indeed systematic pathways of entropy and information transfer between residues that correlate well with the activities of the protein. We use 600 nanosecond molecular dynamics trajectories for Ubiquitin and its complex with human polymerase iota and evaluate entropy transfer between all pairs of residues of Ubiquitin and quantify the binding susceptibility changes upon complex formation. We explain the complex formation propensities of Ubiquitin in terms of entropy transfer. Important residues taking part in allosteric communication in Ubiquitin predicted by our approach are in agreement with results of NMR relaxation dispersion experiments. Finally, we show that time delayed correlation of fluctuations of two interacting residues possesses an intrinsic causality that tells which residue controls the interaction and which one is controlled. Our work shows that time delayed correlations, entropy transfer and causality are the required new concepts for explaining allosteric communication in proteins.

  7. Nootropic α7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators

    Science.gov (United States)

    Ng, Herman J.; Whittemore, Edward R.; Tran, Minhtam B.; Hogenkamp, Derk J.; Broide, Ron S.; Johnstone, Timothy B.; Zheng, Lijun; Stevens, Karen E.; Gee, Kelvin W.

    2007-01-01

    Activation of brain α7 nicotinic acetylcholine receptors (α7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of α7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective α7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-α-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at α7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of α7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction. PMID:17470817

  8. Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators.

    Science.gov (United States)

    Ng, Herman J; Whittemore, Edward R; Tran, Minhtam B; Hogenkamp, Derk J; Broide, Ron S; Johnstone, Timothy B; Zheng, Lijun; Stevens, Karen E; Gee, Kelvin W

    2007-05-08

    Activation of brain alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of alpha7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective alpha7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-alpha-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at alpha7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of alpha7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.

  9. Molecular mechanism of allosteric communication in Hsp70 revealed by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Federica Chiappori

    Full Text Available Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD modulate substrate recognition at the Substrate Binding Domain (SBD. Herein, a comparative analysis of an allosteric (Hsp70-DnaK and a non-allosteric structural homolog (Hsp110-Sse1 of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.

  10. Allosteric Modulation of SULT2A1 by Celecoxib and Nimesulide: Computational Analyses

    OpenAIRE

    Yalcin, Emine Bihter; Struzik, Scott M.; King, Roberta S.

    2008-01-01

    We used protein-ligand docking and minimization to identify celecoxib as an allosteric modulator of SULT2A1-catalyzed estradiol sulfonation. Subsequent to celecoxib docking and complex minimization, conformational changes in SULT2A1 allowed estradiol docking to an alternative binding region with predicted preference for 17β-OH-E2 sulfonation over 3-OH-E2 sulfonation.

  11. Participation of actin on Giardia lamblia growth and encystation.

    Directory of Open Access Journals (Sweden)

    Araceli Castillo-Romero

    Full Text Available BACKGROUND: Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia. METHODOLOGY AND PRINCIPAL FINDINGS: By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45% and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of fluorescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%. CONCLUSIONS AND SIGNIFICANCE: All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression.

  12. Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton

    Science.gov (United States)

    Hou, Guichuan; Mohamalawari, Deepti R.; Blancaflor, Elison B.

    2003-01-01

    The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.

  13. Enzyme-substrate complexes of allosteric citrate synthase: evidence for a novel intermediate in substrate binding.

    Science.gov (United States)

    Duckworth, Harry W; Nguyen, Nham T; Gao, Yin; Donald, Lynda J; Maurus, Robert; Ayed, Ayeda; Bruneau, Brigitte; Brayer, Gary D

    2013-12-01

    The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding. © 2013.

  14. A Strong Contractile Actin Fence and Large Adhesions Direct Human Pluripotent Colony Morphology and Adhesion

    Directory of Open Access Journals (Sweden)

    Elisa Närvä

    2017-07-01

    Full Text Available Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM. Human pluripotent stem cells (hPSCs have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure.

  15. Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

    Energy Technology Data Exchange (ETDEWEB)

    Cowieson, Nathan P.; King, Gordon; Cookson, David; Ross, Ian; Huber, Thomas; Hume, David A.; Kobe, Bostjan; Martin, Jennifer L. (Queensland); (Aust. Synch.)

    2008-08-21

    Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

  16. Oral acetylsalicylic acid and prevalence of actinic keratosis

    Directory of Open Access Journals (Sweden)

    Juliano Schmitt

    2014-01-01

    Full Text Available Objective: To investigate the influence of a regular oral use of acetylsalicylic acid in the prevalence of actinic keratosis. Methods: A case-control study with dermatologic outpatients above 50 years of age assessed between 2009 and 2011. Cases were defined as those who had been under regular use of oral acetylsalicylic acid for more than six consecutive months. The assessment focused on: age, sex, skin-type, tobacco smoking, use of medication, occurrence of individual or family skin cancer, and sunscreen and sun exposure habits. Actinic keratoses were counted in the medial region of the face and upper limbs. Counts were adjusted by co-variables based on a generalized linear model. Results: A total of 74 cases and 216 controls were assessed. The median time of acetylsalicylic acid use was 36 months. Cases differed from controls as to the highest age, highest prevalence of use of angiotensin-converting enzyme inhibitors and fewer keratosis on the face and on the upper limbs (p<0.05. The multivariate model showed that the use of acetylsalicylic acid was associated to lower counts of face actinic keratosis and upper-limb erythematous actinic keratosis (p<0.05, regardless of other risk factors. Conclusion: The regular use of oral acetylsalicylic acid for more than six months was associated to a lower prevalence of actinic keratosis, especially facial and erythematous ones.

  17. Microscale Mechanics of Actin Networks During Dynamic Assembly and Dissociation

    Science.gov (United States)

    Gurmessa, Bekele; Robertson-Anderson, Rae; Ross, Jennifer; Nguyen, Dan; Saleh, Omar

    Actin is one of the key components of the cytoskeleton, enabling cells to move and divide while maintaining shape by dynamic polymerization, dissociation and crosslinking. Actin polymerization and network formation is driven by ATP hydrolysis and varies depending on the concentrations of actin monomers and crosslinking proteins. The viscoelastic properties of steady-state actin networks have been well-characterized, yet the mechanical properties of these non-equilibrium systems during dynamic assembly and disassembly remain to be understood. We use semipermeable microfluidic devices to induce in situ dissolution and re-polymerization of entangled and crosslinked actin networks, by varying ATP concentrations in real-time, while measuring the mechanical properties during disassembly and re-assembly. We use optical tweezers to sinusoidally oscillate embedded microspheres and measure the resulting force at set time-intervals and in different regions of the network during cyclic assembly/disassembly. We determine the time-dependent viscoelastic properties of non-equilibrium network intermediates and the reproducibility and homogeneity of network formation and dissolution. Results inform the role that cytoskeleton reorganization plays in the dynamic multifunctional mechanics of cells. NSF CAREER Award (DMR-1255446) and a Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

  18. All-Round Manipulation of the Actin Cytoskeleton by HIV.

    Science.gov (United States)

    Ospina Stella, Alberto; Turville, Stuart

    2018-02-05

    While significant progress has been made in terms of human immunodeficiency virus (HIV) therapy, treatment does not represent a cure and remains inaccessible to many people living with HIV. Continued mechanistic research into the viral life cycle and its intersection with many aspects of cellular biology are not only fundamental in the continued fight against HIV, but also provide many key observations of the workings of our immune system. Decades of HIV research have testified to the integral role of the actin cytoskeleton in both establishing and spreading the infection. Here, we review how the virus uses different strategies to manipulate cellular actin networks and increase the efficiency of various stages of its life cycle. While some HIV proteins seem able to bind to actin filaments directly, subversion of the cytoskeleton occurs indirectly by exploiting the power of actin regulatory proteins, which are corrupted at multiple levels. Furthermore, this manipulation is not restricted to a discrete class of proteins, but rather extends throughout all layers of the cytoskeleton. We discuss prominent examples of actin regulators that are exploited, neutralized or hijacked by the virus, and address how their coordinated deregulation can lead to changes in cellular behavior that promote viral spreading.

  19. Incorporation of β-actin loading control into zymography.

    Science.gov (United States)

    Govindasamy, Natasha; Yan, MengJie; Jurasz, Paul

    2016-11-01

    Gelatin zymography and immunoblot are widely used gel electrophoresis techniques to study matrix metalloproteinases-2 and -9. Each method has its advantages and disadvantages. Zymography is exquisitely sensitive but offers no loading control to ensure equal sample loading. Immunoblot is a 100-1000-fold less sensitive, but allows for the probing of a sample loading control such as β-actin to ensure accurate protein loading. In this report, we describe two simple protocols that combine gelatin zymography to study MMP-2 and -9 levels with an in-gel β-actin immunoblot loading control, thus combining sensitivity and accuracy in a single assay. The protocols incorporate the loading of molecular weight markers to demarcate MMP-2/-9 from the β-actin. The first protocol utilizes the overlay of a 10% zymography gel over a 5% Tris-Glycine separating gel from which the β-actin is transferred. The second protocol involves the direct transfer of the β-actin from a single 10% zymography gel.

  20. Localization of actin in pollen tubes of Ornithogalum virens L.

    Directory of Open Access Journals (Sweden)

    Małgorzata Stępka

    2014-01-01

    Full Text Available The germinating pollen grain (in vivo on the stigma or in vitro in germination medium forms a pollen tube which transports the vegetative nucleus and generative cell/two sperm cells participating in the process of double fertilization. The growth of the tube and the transport of organelles and the cells occur due to two major motor systems existing in the pollen tubes of higher plants: the tubuline-dynein/kinesin and the actin-myosin system. In pollen tubes of Ornithogalum virens the actin filaments were labelled with TRITC-phalloidin (2 µg/ml in the PIPES buffer and the 10% sucrose, without the fixative and DMSO. Omission of the fixative and permeabilizing agent (DMSO allowed better preservation of the structure, and the "fluorescence" of actin was observed in living pollen tubes. Observations in CLSM (confocal laser scanning microscope showed that actin is distributed in the vicinity of the cell membrane. This could support the view that actin filaments and the plasmalemma form the pollen tube cortex along which the cytoplasmic movement of organelles, and cell transport occurs.

  1. Novel Actin-like Filament Structure from Clostridium tetani*

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2012-06-15

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

  3. Addition of electrophilic lipids to actin alters filament structure

    International Nuclear Information System (INIS)

    Gayarre, Javier; Sanchez, David; Sanchez-Gomez, Francisco J.; Terron, Maria C.; Llorca, Oscar; Perez-Sala, Dolores

    2006-01-01

    Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Δ 12,14 -PGJ 2 (15d-PGJ 2 ) and PGA 1 in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA 1 and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ 2 or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ 2 at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles

  4. Oral acetylsalicylic acid and prevalence of actinic keratosis.

    Science.gov (United States)

    Schmitt, Juliano; Miot, Hélio

    2014-01-01

    To investigate the influence of a regular oral use of acetylsalicylic acid in the prevalence of actinic keratosis. A case-control study with dermatologic outpatients above 50 years of age assessed between 2009 and 2011. Cases were defined as those who had been under regular use of oral acetylsalicylic acid for more than six consecutive months. The assessment focused on: age, sex, skin-type, tobacco smoking, use of medication, occurrence of individual or family skin cancer, and sunscreen and sun exposure habits. Actinic keratoses were counted in the medial region of the face and upper limbs. Counts were adjusted by co-variables based on a generalized linear model. A total of 74 cases and 216 controls were assessed. The median time of acetylsalicylic acid use was 36 months. Cases differed from controls as to the highest age, highest prevalence of use of angiotensin-converting enzyme inhibitors and fewer keratosis on the face and on the upper limbs (pacetylsalicylic acid was associated to lower counts of face actinic keratosis and upper-limb erythematous actinic keratosis (pacetylsalicylic acid for more than six months was associated to a lower prevalence of actinic keratosis, especially facial and erythematous ones.

  5. Multiple roles for the actin cytoskeleton during regulated exocytosis

    Science.gov (United States)

    Porat-Shliom, Natalie; Milberg, Oleg; Masedunskas, Andrius; Weigert, Roberto

    2014-01-01

    Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules. PMID:22986507

  6. Regulation of myotube formation by the actin-binding factor drebrin

    Directory of Open Access Journals (Sweden)

    Mancini Annalisa

    2011-12-01

    Full Text Available Abstract Background Myogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified. Methods RNA interference (RNAi, chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells. Results In a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated. Conclusions Our findings reveal that Dbn1 expression is

  7. Spatially restricted actin-regulatory signaling contributes to synapse morphology

    Science.gov (United States)

    Nicholson, Daniel A.; Cahill, Michael E.; Tulisiak, Christopher T.; Geinisman, Yuri; Penzes, Peter

    2012-01-01

    The actin cytoskeleton in dendritic spines is organized into microdomains, but how signaling molecules that regulate actin are spatially governed is incompletely understood. Here we examine how the localization of the RacGEF kalirin-7, a well-characterized regulator of actin in spines, varies as a function of postsynaptic density (PSD) area and spine volume. Using serial section electron microscopy (EM), we find that extrasynaptic, but not synaptic, expression of kalirin-7 varies directly with synapse size and spine volume. Moreover, we find that overall expression levels of kalirin-7 differ in spines bearing perforated and non-perforated synapses, due primarily to extrasynaptic pools of kalirin-7 expression in the former. Overall, our findings indicate that kalirin-7 is differentially compartmentalized in spines as a function of both synapse morphology and spine size. PMID:22458534

  8. Spiral actin-polymerization waves can generate amoeboidal cell crawling

    Energy Technology Data Exchange (ETDEWEB)

    Dreher, A.; Aranson, I. S.; Kruse, K.

    2014-05-01

    Amoeboidal cell crawling on solid substrates is characterized by protrusions that seemingly appear randomly along the cell periphery and drive the cell forward. For many cell types, it is known that the protrusions result from polymerization of the actin cytoskeleton. However, little is known about how the formation of protrusions is triggered and whether the appearance of subsequent protrusions is coordinated. Recently, the spontaneous formation of actin-polymerization waves was observed. These waves have been proposed to orchestrate the cytoskeletal dynamics during cell crawling. Here, we study the impact of cytoskeletal polymerization waves on cell migration using a phase-field approach. In addition to directionally moving cells, we find states reminiscent of amoeboidal cell crawling. In this framework, new protrusions are seen to emerge from a nucleation process, generating spiral actin waves in the cell interior. Nucleation of new spirals does not require noise, but occurs in a state that is apparently displaying spatio-temporal chaos.

  9. Condensation of F-Actin by Dimensional Reduction

    Science.gov (United States)

    Bruinsma, Robijn; Christian, Cyron; Mueller, Kei; Bausch, Andreas; Wall, Wolfgang

    2012-02-01

    We present a Brownian Dynamics simulation of the equilibrium condensation of F-actin in the presence of linker molecules. The filaments are modeled as worm-like chains, using finite element analysis. At low linker concentrations, the systems forms a gel whose physical properties do not depend on the linker molecules. If the linker concentration is increased then for isotropic linkers only a single mode of condensation is encountered: bundle formation. If the linker molecules impose a preferential angle between F-actin filaments, then condensation takes place either into a either a hexatic or squaratic two-dimensional liquid crystal phase or into a heterogeneous cluster. Condensation is driven by competition between linker and filament entropy, which imposes dimensional reduction on the F-actin aggregate.

  10. Formation of actin networks in microfluidic concentration gradients

    Directory of Open Access Journals (Sweden)

    Natalja eStrelnikova

    2016-05-01

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

  11. [Correct contact lens hygiene].

    Science.gov (United States)

    Blümle, S; Kaercher, T; Khaireddin, R

    2013-06-01

    Although contact lenses have long been established in ophthalmology, practical aspects of handling contact lenses is becoming increasingly less important in the clinical training as specialist for ophthalmology. Simultaneously, for many reasons injuries due to wearing contact lenses are increasing. In order to correct this discrepancy, information on contact lenses and practical experience with them must be substantially increased from a medical perspective. This review article deals with the most important aspects for prevention of complications, i.e. contact lens hygiene.

  12. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  13. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking.

    Directory of Open Access Journals (Sweden)

    Christopher Arnette

    Full Text Available The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.

  14. Modelling phagosomal lipid networks that regulate actin assembly

    Directory of Open Access Journals (Sweden)

    Schwarz Roland

    2008-12-01

    Full Text Available Abstract Background When purified phagosomes are incubated in the presence of actin under appropriate conditions, microfilaments start growing from the membrane in a process that is affected by ATP and the lipid composition of the membrane. Isolated phagosomes are metabolically active organelles that contain enzymes and metabolites necessary for lipid interconversion. Hence, addition of ATP, lipids, and actin to the system alter the steady-state composition of the phagosomal membrane at the same time that the actin nucleation is initiated. Our aim was to model all these processes in parallel. Results We compiled detailed experimental data on the effects of different lipids and ATP on actin nucleation and we investigated experimentally lipid interconversion and ATP metabolism in phagosomes by using suitable radioactive compounds. In a first step, a complex lipid network interconnected by chemical reactions catalyzed by known enzymes was modelled in COPASI (Complex Pathway Simulator. However, several lines of experimental evidence indicated that only the phosphatidylinositol branch of the network was active, an observation that dramatically reduced the number of parameters in the model. The results also indicated that a lipid network-independent ATP-consuming activity should be included in the model. When this activity was introduced, the set of differential equations satisfactorily reproduced the experimental data. On the other hand, a molecular mechanism connecting membrane lipids, ATP, and the actin nucleation process is still missing. We therefore adopted a phenomenological (black-box approach to represent the empirical observations. We proposed that lipids and ATP influence the dynamic interconversion between active and inactive actin nucleation sites. With this simple model, all the experimental data were satisfactorily fitted with a single positive parameter per lipid and ATP. Conclusion By establishing an active 'dialogue' between an

  15. Health related quality of life in patients with actinic keratosis

    DEFF Research Database (Denmark)

    Tennvall, Gunnel Ragnarson; Norlin, J M; Malmberg, I

    2015-01-01

    BACKGROUND: Actinic keratosis (AK) is a common skin condition that may progress to non-melanoma skin cancer (NMSC). The disease may influence Health Related Quality of Life (HRQoL), but studies of HRQoL in patients with AK are limited. The purpose of the study was to analyze HRQoL in patients......-center setting. Dermatologists assessed AK severity and patients completed: Actinic Keratosis Quality of Life Questionnaire (AKQoL), Dermatology Life Quality Index (DLQI), and EQ-5D-5 L including EQ-VAS. Differences between categorical subgroups were tested with Wilcoxon rank-sum test. The relationship between...

  16. Actin and Arp2/3 localize at the centrosome of interphase cells

    Energy Technology Data Exchange (ETDEWEB)

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan, E-mail: jan.gettemans@vib-ugent.be

    2011-01-07

    Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

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

    Directory of Open Access Journals (Sweden)

    Sundaravadivel Balasubramanian

    2010-07-01

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

  18. Actin in Mung Bean Mitochondria and Implications for Its Function[W][OA

    Science.gov (United States)

    Lo, Yih-Shan; Cheng, Ning; Hsiao, Lin-June; Annamalai, Arunachalam; Jauh, Guang-Yuh; Wen, Tuan-Nan; Dai, Hwa; Chiang, Kwen-Sheng

    2011-01-01

    Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin–green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography–tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed. PMID:21984697

  19. Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim.

    Science.gov (United States)

    Shao, Xiaowei; Li, Qingsen; Mogilner, Alex; Bershadsky, Alexander D; Shivashankar, G V

    2015-05-19

    Cells constantly sense and respond to mechanical signals by reorganizing their actin cytoskeleton. Although a number of studies have explored the effects of mechanical stimuli on actin dynamics, the immediate response of actin after force application has not been studied. We designed a method to monitor the spatiotemporal reorganization of actin after cell stimulation by local force application. We found that force could induce transient actin accumulation in the perinuclear region within ∼ 2 min. This actin reorganization was triggered by an intracellular Ca(2+) burst induced by force application. Treatment with the calcium ionophore A23187 recapitulated the force-induced perinuclear actin remodeling. Blocking of actin polymerization abolished this process. Overexpression of Klarsicht, ANC-1, Syne Homology (KASH) domain to displace nesprins from the nuclear envelope did not abolish Ca(2+)-dependent perinuclear actin assembly. However, the endoplasmic reticulum- and nuclear membrane-associated inverted formin-2 (INF2), a potent actin polymerization activator (mutations of which are associated with several genetic diseases), was found to be important for perinuclear actin assembly. The perinuclear actin rim structure colocalized with INF2 on stimulation, and INF2 depletion resulted in attenuation of the rim formation. Our study suggests that cells can respond rapidly to external force by remodeling perinuclear actin in a unique Ca(2+)- and INF2-dependent manner.

  20. Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

    Directory of Open Access Journals (Sweden)

    Eric A. Vitriol

    2015-04-01

    Full Text Available Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin regulated by the ordered assembly from and disassembly into actin monomers (G-actin. Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer-binding protein thymosin β4 (Tβ4 for optimal leading-edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it does not interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions.

  1. Benzodiazepine modulation of partial agonist efficacy and spontaneously active GABAA receptors supports an allosteric model of modulation

    OpenAIRE

    Downing, Scott S; Lee, Yan T; Farb, David H; Gibbs, Terrell T

    2005-01-01

    Benzodiazepines (BZDs) have been used extensively for more than 40 years because of their high therapeutic index and low toxicity. Although BZDs are understood to act primarily as allosteric modulators of GABAA receptors, the mechanism of modulation is not well understood.The applicability of an allosteric model with two binding sites for γ-aminobutyric acid (GABA) and one for a BZD-like modulator was investigated.This model predicts that BZDs should enhance the efficacy of partial agonists.C...

  2. Delineation of the functional properties and the mechanism of action of AA29504, an allosteric agonist and positive allosteric modulator of GABAAreceptors.

    Science.gov (United States)

    Olander, Emma Rie; Madjroh, Nawid; Bunch, Lennart; Söderhielm, Pella Cecilia; Jensen, Anders A

    2018-04-01

    The retigabine analog 2-amino-4-[(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504) is a positive allosteric modulator (PAM) of γ-aminobutyric acid A receptors (GABA A Rs), and the modulator has been used in ex vivo/in vivo studies to probe the physiological roles of native δ-containing GABA A Rs. In this study, the functional properties and mode of action of AA29504 were investigated at human GABA A Rs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. AA29504 was found to be an allosteric GABA A R agonist displaying low intrinsic activities at 3-30 μM. AA29504 was essentially equipotent as a PAM at the 13 GABA A R subtypes tested (EC 50 : 0.45-5.2 μM), however GABA EC 5 -evoked currents through αβδ subtypes were modulated to substantially higher levels than those through αβγ 2S subtypes (relative to GABA I max ). While the δ/γ 2S -difference clearly was key for this differential GABA efficacy modulation, studies of the AA29504-mediated modulation of different α 4,5,6 -containing αβ, αβγ 2S and αβδ GABA A Rs revealed the α-subunit identity to be another important determinant. Based on its functional properties at numerous mutant GABA A Rs and on in silico analysis of its low-energy conformations, AA29504 is proposed to act through an allosteric site in the transmembrane β (+) /α (-) interface in the GABA A R also targeted by etomidate and several other modulators. In contrast to these modulators, however, AA29504 did not display substantial β 2 /β 3 -over-β 1 GABA A R preference, which challenges the notion of ligands targeting this site always possessing this subtype-selectivity profile. Hence, the detailed pharmacological profiling of AA29504 both highlights the complexity of allosteric GABA A R modulation and provides valuable information about this modulator as a pharmacological tool. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. The actin cytoskeleton in root hairs: all is fine at the tip

    NARCIS (Netherlands)

    Ketelaar, T.

    2013-01-01

    Filamentous actin forms characteristic bundles in plant cells that facilitate cytoplasmic streaming. In contrast, networks of actin exhibiting fast turnover are found especially near sites of rapid cell expansion. These networks may serve various functions including delivering and retaining vesicles

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

    Directory of Open Access Journals (Sweden)

    Almudena García-Ortiz

    2017-04-01

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

  5. The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding.

    Science.gov (United States)

    Wilson, C J; Zhan, H; Swint-Kruse, L; Matthews, K S

    2007-01-01

    In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli. This proposal was followed by an explication of allosteric behavior by Monod and colleagues. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function.

  6. Signaling within Allosteric Machines: Signal Transmission Pathways Inside G Protein-Coupled Receptors.

    Science.gov (United States)

    Bartuzi, Damian; Kaczor, Agnieszka A; Matosiuk, Dariusz

    2017-07-15

    In recent years, our understanding of function of G protein-coupled receptors (GPCRs) has changed from a picture of simple signal relays, transmitting only a particular signal to a particular G protein heterotrimer, to versatile machines, capable of various responses to different stimuli and being modulated by various factors. Some recent reports provide not only the data on ligands/modulators and resultant signals induced by them, but also deeper insights into exact pathways of signal migration and mechanisms of signal transmission through receptor structure. Combination of these computational and experimental data sheds more light on underlying mechanisms of signal transmission and signaling bias in GPCRs. In this review we focus on available clues on allosteric pathways responsible for complex signal processing within GPCRs structures, with particular emphasis on linking compatible in silico- and in vitro-derived data on the most probable allosteric connections.

  7. ATP-competitive inhibitors of the mitotic kinesin KSP that function via an allosteric mechanism.

    Science.gov (United States)

    Luo, Lusong; Parrish, Cynthia A; Nevins, Neysa; McNulty, Dean E; Chaudhari, Amita M; Carson, Jeffery D; Sudakin, Valery; Shaw, Antony N; Lehr, Ruth; Zhao, Huizhen; Sweitzer, Sharon; Lad, Latesh; Wood, Kenneth W; Sakowicz, Roman; Annan, Roland S; Huang, Pearl S; Jackson, Jeffrey R; Dhanak, Dashyant; Copeland, Robert A; Auger, Kurt R

    2007-11-01

    The mitotic kinesin KSP (kinesin spindle protein, or Eg5) has an essential role in centrosome separation and formation of the bipolar mitotic spindle. Its exclusive involvement in the mitotic spindle of proliferating cells presents an opportunity for developing new anticancer agents with reduced side effects relative to antimitotics that target tubulin. Ispinesib is an allosteric small-molecule KSP inhibitor in phase 2 clinical trials. Mutations that attenuate ispinesib binding to KSP have been identified, which highlights the need for inhibitors that target different binding sites. We describe a new class of selective KSP inhibitors that are active against ispinesib-resistant forms of KSP. These ATP-competitive KSP inhibitors do not bind in the nucleotide binding pocket. Cumulative data from generation of resistant cells, site-directed mutagenesis and photo-affinity labeling suggest that they compete with ATP binding via a novel allosteric mechanism.

  8. The role of mechanics in actin stress fiber kinetics.

    Science.gov (United States)

    Elson, E L; Genin, G M

    2013-10-01

    The dynamic responses of actin stress fibers within a cell's cytoskeleton are central to the development and maintenance of healthy tissues and organs. Disturbances to these underlie a broad range of pathologies. Because of the importance of these responses, extensive experiments have been conducted in vitro to characterize actin cytoskeleton dynamics of cells cultured upon two-dimensional substrata, and the first experiments have been conducted for cells within three-dimensional tissue models. Three mathematical models exist for predicting the dynamic behaviors observed. Surprisingly, despite differing viewpoints on how actin stress fibers are stabilized or destabilized, all of these models are predictive of a broad range of available experimental data. Coarsely, the models of Kaunas and co-workers adopt a strategy whereby mechanical stretch can hasten the depolymerization actin stress fibers that turn over constantly, while the models of Desphande and co-workers adopt a strategy whereby mechanical stress is required to activate the formation of stress fibers and subsequently stabilize them. In three-dimensional culture, elements of both approaches appear necessary to predict observed phenomena, as embodied by the model of Lee et al. After providing a critical review of existing models, we propose lines of experimentation that might be able to test the different principles underlying their kinetic laws. © 2013 Elsevier Inc. All rights reserved.

  9. Fragmentation of Human Erythrocyte Actin following Exposure to Hypoxia

    Czech Academy of Sciences Publication Activity Database

    Risso, A.; Santamaria, B.; Pistarino, E.; Cosulich, M. E.; Pompach, Petr; Bezouška, Karel; Antonutto, G.

    2010-01-01

    Roč. 123, č. 1 (2010), s. 6-13 ISSN 0001-5792 Institutional research plan: CEZ:AV0Z50200510 Keywords : beta-Actin * Erythrocytes * Hypoxia Subject RIV: EE - Microbiology, Virology Impact factor: 1.316, year: 2010

  10. T cell antigen receptor activation and actin cytoskeleton remodeling

    Science.gov (United States)

    Kumari, Sudha; Curado, Silvia; Mayya, Viveka

    2013-01-01

    T cells constitute a crucial arm of the adaptive immune system and their optimal function is required for a healthy immune response. After the initial step of T cell-receptor (TCR) triggering by antigenic peptide complexes on antigen presenting cell (APC), the T cell exhibits extensive cytoskeletal remodeling. This cytoskeletal remodeling leads to formation of an “immunological synapse” [1] characterized by regulated clustering, segregation and movement of receptors at the interface. Synapse formation regulates T cell activation and response to antigenic peptides and proceeds via feedback between actin cytoskeleton and TCR signaling. Actin polymerization participates in various events during the synapse formation, maturation, and eventually its disassembly. There is increasing knowledge about the actin effectors that couple TCR activation to actin rearrangements [2, 3], and how defects in these effectors translate into impairment of T cell activation. In this review we aim to summarize and integrate parts of what is currently known about this feedback process. In addition, in light of recent advancements in our understanding of TCR triggering and translocation at the synapse, we speculate on the organizational and functional diversity of microfilament architecture in the T cell. PMID:23680625

  11. Interconnection between actin cytoskeleton and plant defense signaling

    Czech Academy of Sciences Publication Activity Database

    Janda, Martin; Matoušková, J.; Burketová, Lenka; Valentová, O.

    2014-01-01

    Roč. 9, č. 11 (2014) ISSN 1559-2316 R&D Projects: GA ČR(CZ) GAP501/11/1654 Institutional support: RVO:61389030 Keywords : Actin * Cytoskeleton * Pathogen Subject RIV: ED - Physiology http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Alerting&SrcApp=Alerting&DestApp=MEDLINE&DestLinkType=FullRecord&UT=25482795

  12. Control of the actin cytoskeleton in plant cell growth

    NARCIS (Netherlands)

    Hussey, P.J.; Ketelaar, M.J.; Deeks, M.J.

    2006-01-01

    Plant cells grow through increases in volume and cell wall surface area. The mature morphology of a plant cell is a product of the differential rates of expansion between neighboring zones of the cell wall during this process. Filamentous actin arrays are associated with plant cell growth, and the

  13. The actin Cytoskeleton in Root Hairs: a cell elongation device

    NARCIS (Netherlands)

    Ketelaar, T.; Emons, A.M.C.

    2009-01-01

    The actin cytoskeleton plays an important role in root hair development. It is involved in both the delivery of growth materials to the expanding tip of root hairs and the regulation of the area of tip growth. This review starts with a discussion of the techniques that are available to visualize the

  14. Force Exertion and Transmission in Cross-Linked Actin Networks

    Science.gov (United States)

    Stam, Samantha

    Cells are responsive to external cues in their environment telling them to proliferate or migrate within their surrounding tissue. Sensing of cues that are mechanical in nature, such stiffness of a tissue or forces transmitted from other cells, is believed to involve the cytoskeleton of a cell. The cytoskeleton is a complex network of proteins consisting of polymers that provide structural support, motor proteins that remodel these structures, and many others. We do not yet have a complete understanding of how cytoskeletal components respond to either internal or external mechanical force and stiffness. Such an understanding should involve mechanisms by which constituent molecules, such as motor proteins, are responsive to mechanics. Additionally, physical models of how forces are transmitted through biopolymer networks are necessary. My research has focused on networks formed by the cytoskeletal filament actin and the molecular motor protein myosin II. Actin filaments form networks and bundles that form a structural framework of the cell, and myosin II slides actin filaments. In this thesis, we show that stiffness of an elastic load that opposes myosin-generated actin sliding has a very sharp effect on the myosin force output in simulations. Secondly, we show that the stiffness and connectivity of cytoskeletal filaments regulates the contractility and anisotropy of network deformations that transmit force on material length scales. Together, these results have implications for predicting and interpreting the deformations and forces in biopolymeric active materials.

  15. Onchocercal DNA amplification using beta actin gene primers ...

    African Journals Online (AJOL)

    Onchocercal DNA amplification using beta actin gene primers compared with first internal transcribed spacer sequences for monitoring onchocerciasis eradication strategy. ... Out of the 12 amplicons in agarose gel, there were 6 sharp and 6 faint bands of 100bp molecular weight as documented. The sharp bands included 3 ...

  16. The roles of the actin cytoskeleton in fear memory formation

    Directory of Open Access Journals (Sweden)

    Raphael eLamprecht

    2011-07-01

    Full Text Available The formation and storage of fear memory is needed to adapt behavior and avoid danger during subsequent fearful events. However, fear memory may also play a significant role in stress and anxiety disorders. When fear becomes disproportionate to that necessary to cope with a given stimulus, or begins to occur in inappropriate situations, a fear or anxiety disorder exists. Thus, the study of cellular and molecular mechanisms underpinning fear memory may shed light on the formation of memory and on anxiety and stress related disorders. Evidence indicates that fear learning leads to changes in neuronal synaptic transmission and morphology in brain areas underlying fear memory formation including the amygdala and hippocampus. The actin cytoskeleton has been shown to participate in these key neuronal processes. Recent findings show that the actin cytoskeleton is needed for fear memory formation and extinction. Moreover, the actin cytoskeleton is involved in synaptic plasticity and in neuronal morphogenesis in brain areas that mediate fear memory. The actin cytoskeleton may therefore mediate between synaptic transmission during fear learning and long-term cellular alterations mandatory for fear memory formation.

  17. Actin and myosin contribute to mammalian mitochondrial DNA maintenance

    Science.gov (United States)

    Reyes, A.; He, J.; Mao, C. C.; Bailey, L. J.; Di Re, M.; Sembongi, H.; Kazak, L.; Dzionek, K.; Holmes, J. B.; Cluett, T. J.; Harbour, M. E.; Fearnley, I. M.; Crouch, R. J.; Conti, M. A.; Adelstein, R. S.; Walker, J. E.; Holt, I. J.

    2011-01-01

    Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance. PMID:21398640

  18. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

    NARCIS (Netherlands)

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

    2013-01-01

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies.

  19. Real-world approach to actinic keratosis management

    DEFF Research Database (Denmark)

    Dirschka, Thomas; Gupta, Girish; Micali, Giuseppe

    2017-01-01

    Actinic keratosis (AK) is a chronic skin disease in which multiple clinical and subclinical lesions co-exist across large areas of sun-exposed skin, resulting in field cancerisation. Lesions require treatment because of their potential to transform into invasive squamous cell carcinoma. This arti...

  20. Decidable and undecidable arithmetic functions in actin filament networks

    Science.gov (United States)

    Schumann, Andrew

    2018-01-01

    The plasmodium of Physarum polycephalum is very sensitive to its environment, and reacts to stimuli with appropriate motions. Both the sensory and motor stages of these reactions are explained by hydrodynamic processes, based on fluid dynamics, with the participation of actin filament networks. This paper is devoted to actin filament networks as a computational medium. The point is that actin filaments, with contributions from many other proteins like myosin, are sensitive to extracellular stimuli (attractants as well as repellents), and appear and disappear at different places in the cell to change aspects of the cell structure—e.g. its shape. By assembling and disassembling actin filaments, some unicellular organisms, like Amoeba proteus, can move in response to various stimuli. As a result, these organisms can be considered a simple reversible logic gate—extracellular signals being its inputs and motions its outputs. In this way, we can implement various logic gates on amoeboid behaviours. These networks can embody arithmetic functions within p-adic valued logic. Furthermore, within these networks we can define the so-called diagonalization for deducing undecidable arithmetic functions.

  1. Role of cell-matrix contacts in cell migration and epithelial-mesenchymal transformation.

    Science.gov (United States)

    Hay, E D

    1990-12-02

    Epithelial cells make contact with extracellular matrix via receptors on the basal surface that interact with the basal actin cortex. In 3D matrix, the mesenchymal cell makes contact with matrix all around its circumference via similar receptors. When moving, the fibroblasts is constantly constructing a new front end. We postulate in a 'fixed cortex' theory of cell motility that the circumferential actin cortex is firmly attached to matrix and that the myosin-rich endoplasm slides past it into the continually forming new front end. During epithelial-mesenchymal transformation, the presumptive mesenchymal cell seems to turn on the new front end mechanism as a way of emigrating from the epithelium into the underlying matrix with which it makes 'fixed' contacts. Master genes may exist that regulate the expression of epithelial genes on the one hand, and mesenchymal genes on the other.

  2. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... One Use Facts About Colored Contacts and Halloween Safety Colored Contact Lens Facts Over-the-Counter Costume ... new application of artificial intelligence shows whether a patient’s eyes point to high blood pressure or risk ...

  3. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... sell contacts without a prescription are breaking the law, and may be fined $11,000 per violation. " ... wear any kind of contact lens. In Butler's case, the lenses caused an infection and left her ...

  4. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... One Use Facts About Colored Contacts and Halloween Safety Colored Contact Lens Facts Over-the-Counter Costume ... Academy Jobs at the Academy Financial Relationships with Industry Medical Disclaimer Privacy Policy Terms of Service For ...

  5. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... in a pair of colored contact lenses, Laura Butler of Parkersburg, W.Va., had "extreme pain in ... to wear any kind of contact lens. In Butler's case, the lenses caused an infection and left ...

  6. Contact Angle Goniometer

    Data.gov (United States)

    Federal Laboratory Consortium — Description:The FTA32 goniometer provides video-based contact angle and surface tension measurement. Contact angles are measured by fitting a mathematical expression...

  7. Multiple Josephson contact interferometer

    International Nuclear Information System (INIS)

    Zappe, H.H.

    1978-01-01

    The interferometer (quantum interference between two parallel contacts) displays a mid connector and contacts of the same size, or contacts at which the middle one is twice the size as the other two, or a double connector and three contacts by which the middle contact carries twice the current as the other two. Also there can be provided interferometers with three and four contacts as well as with symmetrical double current connectors and the same largest Josephson current through all contacts. Because all contacts display the same phase state in the voltage free switching state, the amplification property can be increased and current dissipation can be decreased in a way that logic circuits with high integration degree and high switching velocities can be designed. (DG) [de

  8. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... Cleveland. "This is far from the truth." Real People, Real Problems with Colored Contact Lenses Julian: Teenager ... the lenses. Never share contact lenses with another person. Get follow up exams with your eye care ...

  9. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... popping touch. But colored contact lenses are popular year-round, not just at Halloween. But few know the ... also available in Spanish . Follow The Academy Professionals: Education Guidelines News Multimedia Public & Patients: Contact Us About ...

  10. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... Contact Lens Facts Over-the-Counter Costume Contacts May Contain Chemicals Harmful to Eyes Four Ways Over- ... without a prescription are breaking the law, and may be fined $11,000 per violation. "Many of ...

  11. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... Costume Contacts May Contain Chemicals Harmful to Eyes Four Ways Over-the-Counter Costume Contact Lenses Can ... was in severe pain and on medication for four weeks, and couldn't see well enough to ...

  12. Corporate Consumer Contact API

    Data.gov (United States)

    General Services Administration — The data in the Corporate Consumer Contact API is based on the content you can find in the Corporate Consumer Contact listing in the Consumer Action Handbook (PDF)....

  13. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... not require the same level of care or consideration as a standard contact lens because they can ... sell contacts without a prescription are breaking the law, and may be fined $11,000 per violation. " ...

  14. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... lens because they can be purchased over-the-counter or on the Internet," says Thomas Steinemann, MD, ... Halloween Safety Colored Contact Lens Facts Over-the-Counter Costume Contacts May Contain Chemicals Harmful to Eyes ...

  15. Contact Us about Asbestos

    Science.gov (United States)

    How to contact EPA for more information on asbestos, including state and regional contacts, EPA’s Asbestos Abatement/Management Ombudsman and the Toxic Substances Control Act (TSCA) Assistance Information Service (TSCA Hotline).

  16. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... like a suction cup." Halloween is a popular time for people to use colored contact lenses to ... wear costume contact lenses for Halloween or any time of year, follow these guidelines: Get an eye ...

  17. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... One Use Facts About Colored Contacts and Halloween Safety Colored Contact Lens Facts Over-the-Counter Costume ... an ophthalmologist — an eye medical doctor — who will measure each eye and talk to you about proper ...

  18. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... contact lenses , from the U.S. Food and Drug Administration (FDA). Are the colored lenses you are considering ... Follow The Academy Professionals: Education Guidelines News Multimedia Public & Patients: Contact Us About the Academy Jobs at ...

  19. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... prescription. Follow the contact lens care directions for cleaning, disinfecting, and wearing the lenses. Never share contact ... with Industry Medical Disclaimer Privacy Policy Terms of Service For Advertisers For Media Ophthalmology Job Center © American ...

  20. Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists.

    Science.gov (United States)

    Ajram, Laura; Begg, Malcolm; Slack, Robert; Cryan, Jenni; Hall, David; Hodgson, Simon; Ford, Alison; Barnes, Ashley; Swieboda, Dawid; Mousnier, Aurelie; Solari, Roberto

    2014-04-15

    The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Quantifying Allosteric Communication via Both Concerted Structural Changes and Conformational Disorder with CARDS.

    Science.gov (United States)

    Singh, Sukrit; Bowman, Gregory R

    2017-04-11

    Allosteric (i.e., long-range) communication within proteins is crucial for many biological processes, such as the activation of signaling cascades in response to specific stimuli. However, the physical basis for this communication remains unclear. Existing computational methods for identifying allostery focus on the role of concerted structural changes, but recent experimental work demonstrates that disorder is also an important factor. Here, we introduce the Correlation of All Rotameric and Dynamical States (CARDS) framework for quantifying correlations between both the structure and disorder of different regions of a protein. To quantify disorder, we draw inspiration from methods for quantifying "dynamic heterogeneity" from chemical physics to classify segments of a dihedral's time evolution as being in either ordered or disordered regimes. To demonstrate the utility of this approach, we apply CARDS to the Catabolite Activator Protein (CAP), a transcriptional activator that is regulated by Cyclic Adenosine MonoPhosphate (cAMP) binding. We find that CARDS captures allosteric communication between the two cAMP-Binding Domains (CBDs). Importantly, CARDS reveals that this coupling is dominated by disorder-mediated correlations, consistent with NMR experiments that establish allosteric coupling between the CBDs occurs without a concerted structural change. CARDS also recapitulates an enhanced role for disorder in the communication between the DNA-Binding Domains (DBDs) and CBDs in the S62F variant of CAP. Finally, we demonstrate that using CARDS to find communication hotspots identifies regions of CAP that are in allosteric communication without foreknowledge of their identities. Therefore, we expect CARDS to be of great utility for both understanding and predicting allostery.

  2. Sparse networks of directly coupled, polymorphic, and functional side chains in allosteric proteins.

    Science.gov (United States)

    Soltan Ghoraie, Laleh; Burkowski, Forbes; Zhu, Mu

    2015-03-01

    Recent studies have highlighted the role of coupled side-chain fluctuations alone in the allosteric behavior of proteins. Moreover, examination of X-ray crystallography data has recently revealed new information about the prevalence of alternate side-chain conformations (conformational polymorphism), and attempts have been made to uncover the hidden alternate conformations from X-ray data. Hence, new computational approaches are required that consider the polymorphic nature of the side chains, and incorporate the effects of this phenomenon in the study of information transmission and functional interactions of residues in a molecule. These studies can provide a more accurate understanding of the allosteric behavior. In this article, we first present a novel approach to generate an ensemble of conformations and an efficient computational method to extract direct couplings of side chains in allosteric proteins, and provide sparse network representations of the couplings. We take the side-chain conformational polymorphism into account, and show that by studying the intrinsic dynamics of an inactive structure, we are able to construct a network of functionally crucial residues. Second, we show that the proposed method is capable of providing a magnified view of the coupled and conformationally polymorphic residues. This model reveals couplings between the alternate conformations of a coupled residue pair. To the best of our knowledge, this is the first computational method for extracting networks of side chains' alternate conformations. Such networks help in providing a detailed image of side-chain dynamics in functionally important and conformationally polymorphic sites, such as binding and/or allosteric sites. © 2014 Wiley Periodicals, Inc.

  3. mGluR5 Positive Allosteric Modulation Enhances Extinction Learning Following Cocaine Self-Administration

    OpenAIRE

    Cleva, Richard M.; Hicks, Megan P.; Gass, Justin T.; Wischerath, Kelly C.; Plasters, Elizabeth T.; Widholm, John J.; Olive, M. Foster

    2011-01-01

    Extinction of classically and instrumentally conditioned behaviors, such as conditioned fear and drug-seeking behavior, is a process of active learning, and recent studies indicate that potentiation of glutamatergic transmission facilitates extinction learning. In this study we investigated the effects of the type 5 metabotropic glutamate receptors (mGluR5) positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) on the extinction of cocaine-seeking behavior in ...

  4. Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

    Science.gov (United States)

    Gill, JasKiran K.; Savolainen, Mari; Young, Gareth T.; Zwart, Ruud; Sher, Emanuele; Millar, Neil S.

    2011-01-01

    Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular “orthosteric” binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site. PMID:21436053

  5. Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase.

    Science.gov (United States)

    Tomita, Takeo; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2011-10-28

    Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is unique in that the enzyme is subject to allosteric activation by leucine. To elucidate the structural basis for leucine-induced allosteric activation of GDH, we determined the crystal structures of the GdhB-Glu and GdhA-GdhB-Leu complexes at 2.1 and 2.6 Å resolution, respectively. The GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits. The GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex. Consistent with the structure, replacement of the amino acid residues of T. thermophilus GDH responsible for leucine binding made T. thermophilus GDH insensitive to leucine. Equivalent amino acid replacement caused a similar loss of sensitivity to leucine in human GDH2, suggesting that human GDH2 also uses the same allosteric site for regulation by leucine.

  6. Molecular sites for the positive allosteric modulation of glycine receptors by endocannabinoids.

    Directory of Open Access Journals (Sweden)

    Gonzalo E Yévenes

    Full Text Available Glycine receptors (GlyRs are transmitter-gated anion channels of the Cys-loop superfamily which mediate synaptic inhibition at spinal and selected supraspinal sites. Although they serve pivotal functions in motor control and sensory processing, they have yet to be exploited as drug targets partly because of hitherto limited possibilities for allosteric control. Endocannabinoids (ECs have recently been characterized as direct allosteric GlyR modulators, but the underlying molecular sites have remained unknown. Here, we show that chemically neutral ECs (e.g. anandamide, AEA are positive modulators of α(1, α(2 and α(3 GlyRs, whereas acidic ECs (e.g. N-arachidonoyl-glycine; NA-Gly potentiate α(1 GlyRs but inhibit α(2 and α(3. This subunit-specificity allowed us to identify the underlying molecular sites through analysis of chimeric and mutant receptors. We found that alanine 52 in extracellular loop 2, glycine 254 in transmembrane (TM region 2 and intracellular lysine 385 determine the positive modulation of α(1 GlyRs by NA-Gly. Successive substitution of non-conserved extracellular and TM residues in α(2 converted NA-Gly-mediated inhibition into potentiation. Conversely, mutation of the conserved lysine within the intracellular loop between TM3 and TM4 attenuated NA-Gly-mediated potentiation of α(1 GlyRs, without affecting inhibition of α(2 and α(3. Notably, this mutation reduced modulation by AEA of all three GlyRs. These results define molecular sites for allosteric control of GlyRs by ECs and reveal an unrecognized function for the TM3-4 intracellular loop in the allosteric modulation of Cys-loop ion channels. The identification of these sites may help to understand the physiological role of this modulation and facilitate the development of novel therapeutic approaches to diseases such as spasticity, startle disease and possibly chronic pain.

  7. Allosteric mechanisms within the adenosine A2A-dopamine D2 receptor heterotetramer

    Science.gov (United States)

    Ferré, Sergi; Bonaventura, Jordi; Tomasi, Dardo; Navarro, Gemma; Moreno, Estefanía; Cortés, Antonio; Lluís, Carme; Casadó, Vicent; Volkow, Nora D.

    2017-01-01

    The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provides a main functional unit and oligomeric entities can be viewed as multiples of dimers. For GPCR heteromers, experimental evidence supports a tetrameric structure, comprised of two different homodimers, each able to signal with its preferred G protein. GPCR homomers and heteromers can act as the conduit of allosteric interactions between orthosteric ligands. The well-known agonist/agonist allosteric interaction in the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer, by which A2AR agonists decrease the affinity of D2R agonists, gave the first rationale for the use of A2AR antagonists in Parkinson’s disease. We review new pharmacological findings that can be explained in the frame of a tetrameric structure of the A2AR-D2R heteromer: first, ligand-independent allosteric modulations by the D2R that result in changes of the binding properties of A2AR ligands; second, differential modulation of the intrinsic efficacy of D2R ligands for G protein-dependent and independent signaling; third, the canonical antagonistic Gs-Gi interaction within the frame of the heteromer; and fourth, the ability of A2AR antagonists, including caffeine, to also exert the same allosteric modulations of D2R ligands than A2AR agonists, while A2AR agonists and antagonists counteract each other’s effects. These findings can have important clinical implications when evaluating the use of A2AR antagonists. They also call for the need of monitoring caffeine intake when evaluating the effect of D2R ligands, when used as therapeutic agents in neuropsychiatric disorders or as probes in imaging studies. PMID:26051403

  8. Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.

    Science.gov (United States)

    Olsen, Richard W

    2015-01-01

    GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation. © 2015 Elsevier Inc. All rights reserved.

  9. Discovery of a novel allosteric modulator of 5-HT3 receptor

    DEFF Research Database (Denmark)

    Trattnig, Sarah M; Harpsøe, Kasper; Thygesen, Sarah B

    2012-01-01

    class of negative allosteric modulators of the 5HT3 receptors (5HT3Rs). PU02 (6[(1naphthylmethyl)thio]9Hpurine) is a potent and selective antagonist displaying IC50 values ~1 µM at 5-HT3Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5HT3A...

  10. Substituted benzoxazinones as potent positive allosteric AMPA receptor modulators: part II.

    Science.gov (United States)

    Mueller, Rudolf; Rachwal, Stanislaw; Tedder, Martina E; Li, Yong-Xin; Zhong, Sheng; Hampson, Aidan; Ulas, Jolanta; Varney, Mark; Nielsson, Lena; Rogers, Gary

    2011-07-01

    AMPA receptors (AMPARs) are an important therapeutic target in the CNS. A series of substituted benzoxazinone derivatives with good to very good in vitro activity as positive allosteric AMPAR modulators was synthesized and evaluated. The appropriate substituent choice on the benzoxazinone fragment improved the affinity towards the AMPA receptor significantly in comparison to our lead molecule CX614. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Benzotriazinone and benzopyrimidinone derivatives as potent positive allosteric AMPA receptor modulators.

    Science.gov (United States)

    Mueller, Rudolf; Rachwal, Stanislaw; Lee, Steven; Zhong, Sheng; Li, Yong-Xin; Haroldsen, Peter; Herbst, Todd; Tanimura, Susan; Varney, Mark; Johnson, Steven; Rogers, Gary; Street, Leslie J

    2011-10-15

    AMPA receptors (AMPARs) have been demonstrated to be an important therapeutic CNS target. A series of substituted benzotriazinone and benzopyrimidinone derivatives were prepared with the aim to improve in vivo activity over the previously reported bis-benzoxazinone based AMPAKINE series from our laboratory. These compounds were shown to be potent, positive allosteric AMPAR modulators that have better in vivo activity and improved metabolic stability over the analogous benzoxazinone derivatives. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. High–resolution crystal structure of deoxy hemoglobin complexed with a potent allosteric effector

    OpenAIRE

    Safo, Martin K.; Moure, Carmen M.; Burnett, James C.; Joshi, Gajanan S.; Abraham, Donald J.

    2001-01-01

    The crystal structure of human deoxy hemoglobin (Hb) complexed with a potent allosteric effector (2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid) = RSR-13) is reported at 1.85 Å resolution. Analysis of the hemoglobin:effector complex indicates that two of these molecules bind to the central water cavity of deoxy Hb in a symmetrical fashion, and that each constrains the protein by engaging in hydrogen bonding and hydrophobic interactions with three of its four subu...

  13. Roles of cortical actin microfilament patterning in division plane orientation in plants.

    Science.gov (United States)

    Kojo, Kei H; Higaki, Takumi; Kutsuna, Natsumaro; Yoshida, Yuya; Yasuhara, Hiroki; Hasezawa, Seiichiro

    2013-09-01

    In land plant cells, division planes are precisely predicted by the microtubule preprophase band and cortical actin microfilament pattern called the actin-depleted zone or actin microfilament twin peaks. However, the function of cortical actin microfilament patterning is not clear. In this study, we report that treatment with the inhibitor 2,3,5-triiodobenzonic acid (TIBA) or jasplakinolide increased the amount of thick actin microfilaments in tobacco BY-2 cells at interphase. However, during the division of BY-2 cells, these inhibitors did not induce visible alteration of actin microfilament thickness but altered cortical actin microfilament patterning without significant disorganization of the microtubule preprophase band. TIBA treatment induced a single intensity peak of actin microfilament distribution around the cell center, whereas jasplakinolide caused the appearance of triple peaks relative to the distribution of actin microfilament around the cell center, in approximately one-third of the cells at metaphase. Dual observations of microtubules and actin microfilaments revealed that abnormal cortical actin microfilament patterning with single or triple peaks is correlated with oblique mitotic spindles in BY-2 cells. In addition, oblique cell plates were frequently observed in BY-2 cells and Arabidopsis thaliana root cells treated with TIBA or jasplakinolide. These results provide evidence for the critical roles of cortical actin microfilament patterning in spindle and cell plate orientation.

  14. DeActs : genetically encoded tools for perturbing the actin cytoskeleton in single cells

    NARCIS (Netherlands)

    Harterink, Martin; Santos Esteves da Silva, Marta; Will, Lena; Turan, Julia; Ibrahim, Adiljan; Lang, Alexander E; Van Battum, Eljo Y; Pasterkamp, R Jeroen; Kapitein, Lukas C; Kudryashov, Dmitri; Barres, Ben A; Hoogenraad, Casper C; Zuchero, J Bradley

    2017-01-01

    The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively

  15. Multidrug Resistance-Related Protein 1 (MRP1) Function and Localization Depend on Cortical Actin

    NARCIS (Netherlands)

    Hummel, Ina; Klappe, Karin; Ercan, Cigdem; Kok, Jan Willem

    MRP1 (ABCC1) is known to be localized in lipid rafts. Here we show in two different cell lines that localization of Mrp1/MRP1 (Abcc1/ABCC1) in lipid rafts and its function as an efflux pump are dependent on cortical actin. Latrunculin B disrupts both cortical actin and actin stress fibers. This

  16. Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers

    NARCIS (Netherlands)

    Ketelaar, T.; Honing, van der H.S.; Emons, A.M.C.

    2010-01-01

    In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be

  17. Contact lens in keratoconus

    OpenAIRE

    Rathi, Varsha M; Mandathara, Preeji S; Dumpati, Srikanth

    2013-01-01

    Contact lenses are required for the visual improvement in patients with keratoconus. Various contact lens options, such as rigid gas permeable (RGP) lenses, soft and soft toric lenses, piggy back contact lenses (PBCL), hybrid lenses and scleral lenses are availble. This article discusses about selection of a lens depending on the type of keratoconus and the fitting philosophies of various contact lenses including the starting trial lens. A Medline search was carried out for articles in the En...

  18. Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A. (Novartis)

    2017-03-01

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

  19. Tuning Transcriptional Regulation through Signaling: A Predictive Theory of Allosteric Induction.

    Science.gov (United States)

    Razo-Mejia, Manuel; Barnes, Stephanie L; Belliveau, Nathan M; Chure, Griffin; Einav, Tal; Lewis, Mitchell; Phillips, Rob

    2018-04-25

    Allosteric regulation is found across all domains of life, yet we still lack simple, predictive theories that directly link the experimentally tunable parameters of a system to its input-output response. To that end, we present a general theory of allosteric transcriptional regulation using the Monod-Wyman-Changeux model. We rigorously test this model using the ubiquitous simple repression motif in bacteria by first predicting the behavior of strains that span a large range of repressor copy numbers and DNA binding strengths and then constructing and measuring their response. Our model not only accurately captures the induction profiles of these strains, but also enables us to derive analytic expressions for key properties such as the dynamic range and [EC 50 ]. Finally, we derive an expression for the free energy of allosteric repressors that enables us to collapse our experimental data onto a single master curve that captures the diverse phenomenology of the induction profiles. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. The structure of brain glycogen phosphorylase-from allosteric regulation mechanisms to clinical perspectives.

    Science.gov (United States)

    Mathieu, Cécile; Dupret, Jean-Marie; Rodrigues Lima, Fernando

    2017-02-01

    Glycogen phosphorylase (GP) is the key enzyme that regulates glycogen mobilization in cells. GP is a complex allosteric enzyme that comprises a family of three isozymes: muscle GP (mGP), liver GP (lGP), and brain GP (bGP). Although the three isozymes display high similarity and catalyze the same reaction, they differ in their sensitivity to the allosteric activator adenosine monophosphate (AMP). Moreover, inactivating mutations in mGP and lGP have been known to be associated with glycogen storage diseases (McArdle and Hers disease, respectively). The determination, decades ago, of the structure of mGP and lGP have allowed to better understand the allosteric regulation of these two isoforms and the development of specific inhibitors. Despite its important role in brain glycogen metabolism, the structure of the brain GP had remained elusive. Here, we provide an overview of the human brain GP structure and its relationship with the two other members of this key family of the metabolic enzymes. We also summarize how this structure provides valuable information to understand the regulation of bGP and to design specific ligands of potential pharmacological interest. © 2016 Federation of European Biochemical Societies.

  1. Identification of an allosteric binding site for RORγt inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Scheepstra, Marcel; Leysen, Seppe; vanAlmen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc (Merck); (Eindhoven)

    2015-12-07

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

  2. Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands.

    Science.gov (United States)

    Sattin, Sara; Tao, Jiahui; Vettoretti, Gerolamo; Moroni, Elisabetta; Pennati, Marzia; Lopergolo, Alessia; Morelli, Laura; Bugatti, Antonella; Zuehlke, Abbey; Moses, Mike; Prince, Thomas; Kijima, Toshiki; Beebe, Kristin; Rusnati, Marco; Neckers, Len; Zaffaroni, Nadia; Agard, David A; Bernardi, Anna; Colombo, Giorgio

    2015-09-21

    Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 Å from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules' effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A

    Energy Technology Data Exchange (ETDEWEB)

    Quinlan, Casey L.; Kaiser, Stephen E.; Bolaños, Ben; Nowlin, Dawn; Grantner, Rita; Karlicek-Bryant, Shannon; Feng, Jun Li; Jenkinson, Stephen; Freeman-Cook, Kevin; Dann, Stephen G.; Wang, Xiaoli; Wells, Peter A.; Fantin, Valeria R.; Stewart, Al E.; Grant, Stephan K. (Pfizer)

    2017-05-29

    S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.

  4. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... Ophthalmologist Patient Stories Español Eye Health / News Halloween Hazard: The Hidden Dangers of Buying Decorative Contact Lenses ... One Use Facts About Colored Contacts and Halloween Safety Colored Contact Lens Facts Over-the-Counter Costume ...

  5. New Cosmetic Contact Allergens

    Directory of Open Access Journals (Sweden)

    An Goossens

    2015-02-01

    Full Text Available Allergic and photo-allergic contact dermatitis, and immunologic contact urticaria are potential immune-mediated adverse effects from cosmetics. Fragrance components and preservatives are certainly the most frequently observed allergens; however, all ingredients must be considered when investigating for contact allergy.

  6. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... an Ophthalmologist Patient Stories Español Eye Health / News Halloween Hazard: The Hidden Dangers of Buying Decorative Contact ... After One Use Facts About Colored Contacts and Halloween Safety Colored Contact Lens Facts Over-the-Counter ...

  7. Hydrogenation of passivated contacts

    Energy Technology Data Exchange (ETDEWEB)

    Nemeth, William; Yuan, Hao-Chih; LaSalvia, Vincenzo; Stradins, Pauls; Page, Matthew R.

    2018-03-06

    Methods of hydrogenation of passivated contacts using materials having hydrogen impurities are provided. An example method includes applying, to a passivated contact, a layer of a material, the material containing hydrogen impurities. The method further includes subsequently annealing the material and subsequently removing the material from the passivated contact.

  8. Analysis of actin FLAP dynamics in the leading lamella.

    Directory of Open Access Journals (Sweden)

    Igor R Kuznetsov

    2010-04-01

    Full Text Available The transport of labeled G-actin from the mid-lamella region to the leading edge in a highly motile malignant rat fibroblast line has been studied using fluorescence localization after photobleaching or FLAP, and the transit times recorded in these experiments were so fast that simple diffusion was deemed an insufficient explanation (see Zicha et al., Science, v. 300, pp. 142-145 [1].We re-examine the Zicha FLAP experiments using a two-phase reactive interpenetrating flow formalism to model the cytoplasm and the transport dynamics of bleached and unbleached actin. By allowing an improved treatment of effects related to the retrograde flow of the cytoskeleton and of the geometry and finite thickness of the lamella, this new analysis reveals a mechanism that can realistically explain the timing and the amplitude of all the FLAP signals observed in [1] without invoking special transport modalities.We conclude that simple diffusion is sufficient to explain the observed transport rates, and that variations in the transport of labeled actin through the lamella are minor and not likely to be the cause of the observed physiological variations among different segments of the leading edge. We find that such variations in labeling can easily arise from differences and changes in the microscopic actin dynamics inside the edge compartment, and that the key dynamical parameter in this regard is the so-called "dilatation rate" (the velocity of cytoskeletal retrograde flow divided by a characteristic dimension of the edge compartment where rapid polymerization occurs. If our dilatation hypothesis is correct, the transient kinetics of bleached actin relocalization constitute a novel and very sensitive method for probing the cytoskeletal dynamics in leading edge micro-environments which are otherwise very difficult to directly interrogate.

  9. Analysis of actin FLAP dynamics in the leading lamella.

    Science.gov (United States)

    Kuznetsov, Igor R; Herant, Marc; Dembo, Micah

    2010-04-15

    The transport of labeled G-actin from the mid-lamella region to the leading edge in a highly motile malignant rat fibroblast line has been studied using fluorescence localization after photobleaching or FLAP, and the transit times recorded in these experiments were so fast that simple diffusion was deemed an insufficient explanation (see Zicha et al., Science, v. 300, pp. 142-145 [1]). We re-examine the Zicha FLAP experiments using a two-phase reactive interpenetrating flow formalism to model the cytoplasm and the transport dynamics of bleached and unbleached actin. By allowing an improved treatment of effects related to the retrograde flow of the cytoskeleton and of the geometry and finite thickness of the lamella, this new analysis reveals a mechanism that can realistically explain the timing and the amplitude of all the FLAP signals observed in [1] without invoking special transport modalities. We conclude that simple diffusion is sufficient to explain the observed transport rates, and that variations in the transport of labeled actin through the lamella are minor and not likely to be the cause of the observed physiological variations among different segments of the leading edge. We find that such variations in labeling can easily arise from differences and changes in the microscopic actin dynamics inside the edge compartment, and that the key dynamical parameter in this regard is the so-called "dilatation rate" (the velocity of cytoskeletal retrograde flow divided by a characteristic dimension of the edge compartment where rapid polymerization occurs). If our dilatation hypothesis is correct, the transient kinetics of bleached actin relocalization constitute a novel and very sensitive method for probing the cytoskeletal dynamics in leading edge micro-environments which are otherwise very difficult to directly interrogate.

  10. Differential sensitivity to detergents of actin cytoskeleton from nerve endings.

    Science.gov (United States)

    Cubí, Roger; Matas, Lluís A; Pou, Marta; Aguilera, José; Gil, Carles

    2013-11-01

    Detergent-resistant membranes (DRM), an experimental model used to study lipid rafts, are typically extracted from cells by means of detergent treatment and subsequent ultracentrifugation in density gradients, Triton X-100 being the detergent of choice in most of the works. Since lipid rafts are membrane microdomains rich in cholesterol, depletion of this component causes solubilization of DRM with detergent. In previous works from our group, the lack of effect of cholesterol depletion on DRM solubilization with Triton X-100 was detected in isolated rat brain synaptosomes. In consequence, the aim of the present work is to explore reasons for this observation, analyzing the possible role of the actin cytoskeleton, as well as the use of an alternative detergent, Brij 98, to overcome the insensitivity to Triton X-100 of cholesterol-depleted DRM. Brij 98 yields Brij-DRM that are highly dependent on cholesterol, since marker proteins (Flotillin-1 and Thy-1), as well as actin, appear solubilized after MCD treatment. Pretreatment with Latrunculin A results in a significant increase in Flotillin-1, Thy-1 and actin solubilization by Triton X-100 after cholesterol depletion. Studies with transmission electron microscopy show that combined treatment with MCD and Latrunculin A leads to a significant increase in solubilization of DRM with Triton X-100. Thus, Triton-DRM resistance to cholesterol depletion can be explained, at least partially, thanks to the scaffolding action of the actin cytoskeleton, without discarding differential effects of Brij 98 and Triton X-100 on specific membrane components. In conclusion, the detergent of choice is important when events that depend on the actin cytoskeleton are going to be studied. © 2013.

  11. GH32 family activity: a topological approach through protein contact networks.

    Science.gov (United States)

    Cimini, Sara; Di Paola, Luisa; Giuliani, Alessandro; Ridolfi, Alessandra; De Gara, Laura

    2016-11-01

    The application of Protein Contact Networks methodology allowed to highlight a novel response of border region between the two domains to substrate binding. Glycoside hydrolases (GH) are enzymes that mainly hydrolyze the glycosidic bond between two carbohydrates or a carbohydrate and a non-carbohydrate moiety. These enzymes are involved in many fundamental and diverse biological processes in plants. We have focused on the GH32 family, including enzymes very similar in both sequence and structure, each having however clear specificities of substrate preferences and kinetic properties. Structural and topological differences among proteins of the GH32 family have been here identified by means of an emerging approach (Protein Contact network, PCN) based on the formalization of 3D structures as contact networks among amino-acid residues. The PCN approach proved successful in both reconstructing the already known functional domains and in identifying the structural counterpart of the properties of GH32 enzymes, which remain uncertain, like their allosteric character. The main outcome of the study was the discovery of the activation upon binding of the border (cleft) region between the two domains. This reveals the allosteric nature of the enzymatic activity for all the analyzed forms in the GH32 family, a character yet to be highlighted in biochemical studies. Furthermore, we have been able to recognize a topological signature (graph energy) of the different affinity of the enzymes towards small and large substrates.

  12. Roles of the actin cytoskeleton and an actin-binding protein in wheat resistance against Puccinia striiformis f. sp. tritici.

    Science.gov (United States)

    Song, Xiaohe; Ma, Qing; Hao, Xinyuan; Li, Hongli

    2012-01-01

    Elucidating resistance mechanisms of plant cells against pathogens is essential to develop novel strategies of disease control. The actin cytoskeleton was found intimately involved in plant defense. In order to reveal how actin would be involved in the interaction between wheat and the stripe rust Puccinia striiformis f. sp. tritici, prior to fungal inoculation, wheat leaves were treated with cytochalasin A, an inhibitor of actin polymerization. Our results showed reduced incidence of hypersensitive cell death and delayed accumulation of H(2)O(2) in wheat leaves treated with cytochalasin A compared to the control. We also found that the TaPRO profilin gene exhibited significantly different expression levels in host leaves when comparing compatible and incompatible interactions. Real-time PCR analysis revealed that the expression transcript of TaPRO was lower at each time point in incompatible interactions when compared to compatible ones, and the largest difference between the two interactions occurred at 12 h post-inoculation. Both pharmacological and gene expression results collectively support the notion that the compromise of the actin microfilament is linked to the compatible interaction between the stripe rust fungus and the leaves of its wheat host.

  13. Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.

    Directory of Open Access Journals (Sweden)

    Luis Vidali

    2009-05-01

    Full Text Available Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments.In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore.Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells.

  14. Contact lens in keratoconus

    Directory of Open Access Journals (Sweden)

    Varsha M Rathi

    2013-01-01

    Full Text Available Contact lenses are required for the visual improvement in patients with keratoconus. Various contact lens options, such as rigid gas permeable (RGP lenses, soft and soft toric lenses, piggy back contact lenses (PBCL, hybrid lenses and scleral lenses are availble. This article discusses about selection of a lens depending on the type of keratoconus and the fitting philosophies of various contact lenses including the starting trial lens. A Medline search was carried out for articles in the English language with the keywords keratoconus and various contact lenses such as Rose k lens, RGP lens, hybrid lens, scleral lens and PBCL.

  15. Contact lens in keratoconus

    Science.gov (United States)

    Rathi, Varsha M; Mandathara, Preeji S; Dumpati, Srikanth

    2013-01-01

    Contact lenses are required for the visual improvement in patients with keratoconus. Various contact lens options, such as rigid gas permeable (RGP) lenses, soft and soft toric lenses, piggy back contact lenses (PBCL), hybrid lenses and scleral lenses are availble. This article discusses about selection of a lens depending on the type of keratoconus and the fitting philosophies of various contact lenses including the starting trial lens. A Medline search was carried out for articles in the English language with the keywords keratoconus and various contact lenses such as Rose k lens, RGP lens, hybrid lens, scleral lens and PBCL. PMID:23925325

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

    Science.gov (United States)

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

    2016-01-01

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

  17. A function for filamentous alpha-smooth muscle actin: Retardation of motility in human breast fibroblasts

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Petersen, Ole William

    1996-01-01

    Actins are known to comprise six mammalian isoforms of which beta- and gamma-nonmuscle actins are present in all cells, whereas alpha-smooth muscle (alpha-sm) actin is normally restricted to cells of the smooth muscle lineages. alpha-Sm actin has been found also to be expressed transiently in cer...... of less prominent focal adhesions as revealed by immunofluorescence staining against vinculin, talin, and beta1-integrin. We propose that an important function of filamentous alpha-sm actin is to immobilize the cells....

  18. SPARC Interacts with Actin in Skeletal Muscle in Vitro and in Vivo

    DEFF Research Database (Denmark)

    Jørgensen, Louise H; Jepsen, Pia Lørup; Boysen, Anders

    2017-01-01

    to actin. This interaction is present in regenerating myofibers of patients with Duchenne muscular dystrophy, polymyositis, and compartment syndrome. Analysis of the α-, β-, and γ-actin isoforms in SPARC knockout myoblasts reveals a changed expression pattern with dominance of γ-actin. In SPARC knockout...... stimulation protocol, we find a defective force recovery. Therefore, SPARC appears to be an important modulator of the actin cytoskeleton, implicating maintenance of muscular function. This direct interaction with actin suggests a new role of SPARC during tissue remodeling....

  19. Inhibitory effects of pectenotoxins from marine algae on the polymerization of various actin isoforms.

    Science.gov (United States)

    Butler, Suzanne C; Miles, Christopher O; Karim, Amna; Twiner, Michael J

    2012-04-01

    Pectenotoxins (PTXs) are marine toxins produced by dinoflagellates and which accumulate in shellfish. There are at least 14 different analogs of PTX with slight variations in structure leading to different chemical properties and consequently different toxicities. Since preliminary studies have shown that the parent compound PTX1 targets actin, we investigated the effects of two analogs, PTX2 and PTX2 seco acid, on the polymerization and depolymerization of skeletal muscle actin, smooth muscle actin, cardiac muscle actin, and non-muscle actin. Optimized actin assays using fluorescently labeled skeletal muscle actin and SDS-PAGE were jointly used to determine the relative amounts of filamentous and globular actin formed during polymerization and depolymerization experiments. Our findings suggest that PTX2 causes a dose-dependent decrease in both the rate and yield of skeletal muscle actin polymerization (IC50 values of 44 and 177 nM; respectively), with no significant effects on depolymerization. Moreover, the inhibitory effects of PTX2 are conserved towards other actin isoforms (i.e., smooth muscle, cardiac muscle, and non-muscle), as the inhibitory effects on actin polymerization were also observed with similar IC50 values (range: 19-94 nM). No inhibitory effects on polymerization were observed for PTX2 seco acid, suggesting an intact lactone ring is necessary for bioactivity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Dynamics of actin cables in polarized growth of the filamentous fungus Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Anna eBergs

    2016-05-01

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

  1. The conserved Tarp actin binding domain is important for chlamydial invasion.

    Directory of Open Access Journals (Sweden)

    Travis J Jewett

    2010-07-01

    Full Text Available The translocated actin recruiting phosphoprotein (Tarp is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.

  2. Probing the flexibility of tropomyosin and its binding to filamentous actin using molecular dynamics simulations.

    Science.gov (United States)

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

    2013-10-15

    Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Variable actin dynamics requirement for the exit of different cargo from the trans-Golgi network.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Colonna, Cecilia; Cortegano, Miguel; Calvo, María; Martínez, Susana E; Egea, Gustavo

    2007-08-07

    Efficient post-Golgi trafficking depends on microtubules, but actin filaments and actin-associated proteins are also postulated. Here we examined, by inverse fluorescence recovery after photobleaching, the role of actin dynamics in the exit from the TGN of fluorescent-tagged apical or basolateral and raft or non-raft-associated cargoes. Either the actin-stabilizing jasplakinolide or the actin-depolymerising latrunculin B variably but significantly inhibited post-Golgi traffic of non-raft associated apical p75NTR and basolateral VSV-G cargoes. The TGN-exit of the apical-destined VSV-G mutant was impaired only by latrunculin B. Strikingly, the raft-associated GPI-anchor protein was not affected by either actin toxin. Results indicate that actin dynamics participates in the TGN egress of both apical- and basolateral-targeted proteins but is not needed for apical raft-associated cargo.

  4. ADF/cofilin-mediated actin retrograde flow directs neurite formation in the developing brain.

    Science.gov (United States)

    Flynn, Kevin C; Hellal, Farida; Neukirchen, Dorothee; Jacob, Sonja; Tahirovic, Sabina; Dupraz, Sebastian; Stern, Sina; Garvalov, Boyan K; Gurniak, Christine; Shaw, Alisa E; Meyn, Liane; Wedlich-Söldner, Roland; Bamburg, James R; Small, J Victor; Witke, Walter; Bradke, Frank

    2012-12-20

    Neurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Rac1 acts in conjunction with Nedd4 and dishevelled-1 to promote maturation of cell-cell contacts

    NARCIS (Netherlands)

    M. Nethe (Micha); B.J. de Kreuk (Bart-Jan); D.V.F. Tauriello (Daniele); E.C. Anthony (Eloise); B. Snoek (Barbara); T. Stumpel (Thomas); M. Salinas; K. Maurice (Karelle); D. Geerts (Dirk); A.M. Deelder (André); P. Hensbergen (Paul); P.L. Hordijk (Peter )

    2012-01-01

    textabstractThe Rho-GTPase Rac1 promotes actin polymerization and membrane protrusion that mediate initial contact and subsequent maturation of cell-cell junctions. Here we report that Rac1 associates with the ubiquitin-protein ligase neural precursor cell expressed developmentally down-regulated 4

  6. A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and F-actin organization resulting in dwarf, lintless cotton plants.

    Science.gov (United States)

    Thyssen, Gregory N; Fang, David D; Turley, Rickie B; Florane, Christopher B; Li, Ping; Mattison, Christopher P; Naoumkina, Marina

    2017-04-01

    Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single-celled trichomes that comprise cotton lint fibers. Using mapping-by-sequencing, virus-induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li 1 short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F-actin in Li 1 plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F-actin did not uniformly decorate the nuclear envelope. While wild-type lint fiber trichome cells contained long longitudinal actin cables, the short Li 1 fiber cells accumulated disoriented transverse cables. The polymerization-defective Gly65Val allele in Li 1 plants likely disrupts processive elongation of F-actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li 1 mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  7. Sound attenuation of polymerizing actin reflects supramolecular structures: viscoelastic properties of actin gels modified by cytochalasin D, profilin and alpha-actinin.

    Science.gov (United States)

    Wagner, O; Schüler, H; Hofmann, P; Langer, D; Dancker, P; Bereiter-Hahn, J

    2001-05-01

    Polymerization and depolymerization of cytoskeletal elements maintaining cytoplasmic stiffness are key factors in the control of cell crawling. Rheometry is a significant tool in determining the mechanical properties of the single elements in vitro. Viscoelasticity of gels formed by these polymers strongly depends on both the length and the associations of the filaments (e.g. entanglements, annealings and side-by-side associations). Ultrasound attenuation is related to viscosity, sound velocity and supramolecular structures in the sample. In combination with a small glass fibre (2 mm x 50 microm), serving as a viscosity sensor, an acoustic microscope was used to measure the elasticity and acoustic attenuation of actin solutions. Changes in acoustic attenuation of polymerizing actin by far exceed the values expected from calculations based on changes in viscosity and sound velocity. During the lag-phase of actin polymerization, attenuation slightly decreases, depending on actin concentration. After the half-maximum viscosity is accomplished and elasticity turns into steady state, attenuation distinctly rises. Changes in ultrasound attenuation depend on actin concentration, and they are modulated by the addition of alpha-actinin, cytochalasin D and profilin. Thus absorption and scattering of sound on the polymerization of actin is related to the packing density of the actin net, entanglements and the length of the actin filaments. Shortening of actin filaments by cytochalasin D was also confirmed by electron micrographs and falling-ball viscosimetry. In addition to viscosity and elasticity, the attenuation of sound proved to be a valuable parameter in characterizing actin polymerization and the supramolecular associations of F-actin.

  8. HPC Analysis of Multiple Binding Sites Communication and Allosteric Modulations in Drug Design: The HSP Case Study.

    Science.gov (United States)

    Chiappori, Federica; Milanesi, Luciano; Merelli, Ivan

    2016-01-01

    Allostery is a long-range macromolecular mechanism of internal regulation, in which the binding of a ligand in an allosteric site induces distant conformational changes in a distant portion of the protein, modifying its activity. From the drug design point of view, this mechanism can be exploited to achieve important therapeutic effects, since ligands able to bind allosteric sites may be designed to regulate target proteins. Computational tools are a valid support in this sense, since they allow the characterization of allosteric communications within proteins, which are essential to design modulator ligands. While considering long-range interactions in macromolecules, the principal drug design tool available to researcher is molecular dynamics, and related applications, since it allows the evaluation of conformational changes of a protein bound to a ligand. In particular, all-atoms molecular dynamics is suitable to verify the internal mechanisms that orchestrate allosteric communications, in order to identify key residues and internal pathways that modify the protein behaviour. The problem is that these techniques are heavily time-consuming and computationally intensive, thus high performance computing systems, including parallel computing and GPU-accelerated computations, are necessary to achieve results in a reasonable time. In this review, we will discuss how it is possible to exploit in silico approaches to characterize allosteric modulations and long-range interactions within proteins, describing the case study of the Heat Shock Proteins, a class of chaperons regulated by stress conditions, which is particularly important since it is involved in many cancers and neurodegenerative diseases.

  9. Actin microfilaments in presumptive statocytes of root caps and coleoptiles

    Science.gov (United States)

    White, R. G.; Sack, F. D.

    1990-01-01

    Rhodamine-phalloidin was used to determine the distribution of actin microfilament bundles (mfb) in cells thought to be the site of gravity perception (statocytes) in coleoptiles and root caps of Zea mays and Hordeum vulgare. In coleoptile cells, amyloplasts were usually observed in close proximity to thick mfb, which often appeared to divide into finer mfb adjacent to individual amyloplasts. The nucleus in these cells was surrounded by an extensive network of mfb, which were connected to thicker transvacuolar mfb. Columella cells of the root cap contained an extensive reticulum of fine mfb throughout the protoplast, but lacked the much thicker mfb seen in coleoptile cells. The distribution and extent of mfb observed in fixed cells correlates with patterns of streaming and amyloplast movement seen in living cells. A possible role for actin mfb in the perception of gravity is discussed.

  10. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Hu, Huimin; Qiu, Weimin

    2018-01-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined...... differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte...... differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating h...

  11. Osmosensation in vasopressin neurons: changing actin density to optimize function.

    Science.gov (United States)

    Prager-Khoutorsky, Masha; Bourque, Charles W

    2010-02-01

    The proportional relation between circulating vasopressin concentration and plasma osmolality is fundamental for body fluid homeostasis. Although changes in the sensitivity of this relation are associated with pathophysiological conditions, central mechanisms modulating osmoregulatory gain are unknown. Here, we review recent data that sheds important light on this process. The cell autonomous osmosensitivity of vasopressin neurons depends on cation channels comprising a variant of the transient receptor potential vanilloid 1 (TRPV1) channel. Hyperosmotic activation is mediated by a mechanical process where sensitivity increases in proportion with actin filament density. Moreover, angiotensin II amplifies osmotic activation by a rapid stimulation of actin polymerization, suggesting that neurotransmitter-induced changes in cytoskeletal organization in osmosensory neurons can mediate central changes in osmoregulatory gain. (c) 2009 Elsevier Ltd. All rights reserved.

  12. Lamin A/C and polymeric actin in genome organization

    Czech Academy of Sciences Publication Activity Database

    Ondřej, V.; Lukášová, Emilie; Kroupová, Jana; Matula, P.; Kozubek, Stanislav

    2008-01-01

    Roč. 26, č. 4 (2008), s. 356-361 ISSN 1016-8478 R&D Projects: GA AV ČR(CZ) 1QS500040508; GA MŠk(CZ) LC535 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : lamin A/C * polymeric actin * chromosome territories Subject RIV: BO - Biophysics Impact factor: 2.023, year: 2008

  13. Dynamics and Morphology of Microvilli Driven by Actin Polymerization

    Science.gov (United States)

    Gov, Nir S.

    2006-07-01

    Many different cell types have dynamic protrusions, called microvilli, on their surface. We model these structures as arising from the balance between the force of actin polymerization and the restoring force of the membrane. From this simple model we calculate the distribution function of microvilli heights for several cells. We further describe the phase diagram and the resulting morphology of the microvilli aggregates on the cell surface.

  14. Actinic inspection of multilayer defects on EUV masks

    International Nuclear Information System (INIS)

    Barty, A; Liu, Y; Gullikson, E; Taylor, J S; Wood, O

    2005-01-01

    The production of defect-free mask blanks, and the development of techniques for inspecting and qualifying EUV mask blanks, remains a key challenge for EUV lithography. In order to ensure a reliable supply of defect-free mask blanks, it is necessary to develop techniques to reliably and accurately detect defects on un-patterned mask blanks. These inspection tools must be able to accurately detect all critical defects whilst simultaneously having the minimum possible false-positive detection rate. There continues to be improvement in high-speed non-actinic mask blank inspection tools, and it is anticipated that these tools can and will be used by industry to qualify EUV mask blanks. However, the outstanding question remains one of validating that non-actinic inspection techniques are capable of detecting all printable EUV defects. To qualify the performance of non-actinic inspection tools, a unique dual-mode EUV mask inspection system has been installed at the Advanced Light Source (ALS) synchrotron at Lawrence Berkeley National Laboratory. In high-speed inspection mode, whole mask blanks are scanned for defects using 13.5-nm wavelength light to identify and map all locations on the mask that scatter a significant amount of EUV light. In imaging, or defect review mode, a zone plate is placed in the reflected beam path to image a region of interest onto a CCD detector with an effective resolution on the mask of 100-nm or better. Combining the capabilities of the two inspection tools into one system provides the unique capability to determine the coordinates of native defects that can be used to compare actinic defect inspection with visible light defect inspection tools under commercial development, and to provide data for comparing scattering models for EUV mask defects

  15. Mutual regulation of plant phospholipase D and the actin cytoskeleton

    Czech Academy of Sciences Publication Activity Database

    Pleskot, Roman; Potocký, Martin; Pejchar, Přemysl; Linek, J.; Bezvoda, R.; Martinec, Jan; Valentová, O.; Novotná, Z.; Žárský, Viktor

    2010-01-01

    Roč. 62, č. 3 (2010), s. 494-507 ISSN 0960-7412 R&D Projects: GA AV ČR IAA601110916; GA MŠk(CZ) LC06034; GA ČR GA522/05/0340 Institutional research plan: CEZ:AV0Z50380511 Keywords : phospholipase D * actin * signaling Subject RIV: ED - Physiology Impact factor: 6.948, year: 2010

  16. The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling.

    Directory of Open Access Journals (Sweden)

    Jong T Chun

    Full Text Available BACKGROUND: Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca(2+. Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 serves as the precursor of inositol 1,4,5 trisphosphate (InsP(3 and also regulates actin-binding proteins, PIP2 might be involved in these two processes. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca(2+ and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca(2+ wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE. These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca(2+ signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+ waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule

  17. Myosin lever arm directs collective motion on cellular actin network.

    Science.gov (United States)

    Hariadi, Rizal F; Cale, Mario; Sivaramakrishnan, Sivaraj

    2014-03-18

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions.

  18. Memory Dynamics in Cross-linked Actin Networks

    Science.gov (United States)

    Scheff, Danielle; Majumdar, Sayantan; Gardel, Margaret

    Cells demonstrate the remarkable ability to adapt to mechanical stimuli through rearrangement of the actin cytoskeleton, a cross-linked network of actin filaments. In addition to its importance in cell biology, understanding this mechanical response provides strategies for creation of novel materials. A recent study has demonstrated that applied stress can encode mechanical memory in these networks through changes in network geometry, which gives rise to anisotropic shear response. Under later shear, the network is stiffer in the direction of the previously applied stress. However, the dynamics behind the encoding of this memory are unknown. To address this question, we explore the effect of varying either the rigidity of the cross-linkers or the length of actin filament on the time scales required for both memory encoding and over which it later decays. While previous experiments saw only a long-lived memory, initial results suggest another mechanism where memories relax relatively quickly. Overall, our study is crucial for understanding the process by which an external stress can impact network arrangement and thus the dynamics of memory formation.

  19. Treadmilling of actin filaments via Brownian dynamics simulations

    Science.gov (United States)

    Guo, Kunkun; Shillcock, Julian; Lipowsky, Reinhard

    2010-10-01

    Actin polymerization is coupled to the hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (Pi). Therefore, each protomer within an actin filament can attain three different nucleotide states corresponding to bound ATP, ADP/Pi, and ADP. These protomer states form spatial patterns on the growing (or shrinking) filaments. Using Brownian dynamics simulations, the growth behavior of long filaments is studied, together with the associated protomer patterns, as a function of ATP-actin monomer concentration, CT, within the surrounding solution. For concentrations close to the critical concentration CT=CT,cr, the filaments undergo treadmilling, i.e., they grow at the barbed and shrink at the pointed end, which leads to directed translational motion of the whole filament. The corresponding nonequilibrium states are characterized by several global fluxes and by spatial density and flux profiles along the filaments. We focus on a certain set of transition rates as deduced from in vitro experiments and find that the associated treadmilling (or turnover) rate is about 0.08 monomers per second.

  20. How cellular membrane properties are affected by the actin cytoskeleton.

    Science.gov (United States)

    Lemière, J; Valentino, F; Campillo, C; Sykes, C

    2016-11-01

    Lipid membranes define the boundaries of living cells and intracellular compartments. The dynamic remodelling of these membranes by the cytoskeleton, a very dynamic structure made of active biopolymers, is crucial in many biological processes such as motility or division. In this review, we present some aspects of cellular membranes and how they are affected by the presence of the actin cytoskeleton. We show that, in parallel with the direct study of membranes and cytoskeleton in vivo, biomimetic in vitro systems allow reconstitution of biological processes in a controlled environment. In particular, we show that liposomes, or giant unilamellar vesicles, encapsulating a reconstituted actin network polymerizing at their membrane are suitable models of living cells and can be used to decipher the relative contributions of membrane and actin on the mechanical properties of the cellular interface. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. The Evolution of the Actin Binding NET Superfamily

    Directory of Open Access Journals (Sweden)

    Tim eHawkins

    2014-06-01

    Full Text Available The arabidopsis Networked protein superfamily are plant-specific actin binding proteins which specifically label different membrane compartments and identify specialized sites of interaction between actin and membranes unique to plants. There are 13 members of the superfamily in arabidopsis which group into 4 distinct clades or subfamilies. NET homologues are absent from the genomes of metazoa and fungi, furthermore in Plantae NET sequences are also absent from the genome of mosses and more ancient extant plant clades. A single subfamily of the NET proteins are found encoded in the club moss genome; an extant species of the earliest vascular plants. Gymnosperms have examples from subfamilies 4 and 3 with a hybrid form of NET1 and 2 which shows characteristics of both NET1 and NET2. In addition to NET3 and 4 subfamilies, the NET1 and pollen-expressed NET2 subfamilies are only found as independent sequences in angiosperms. This is consistent with the divergence of reproductive actin. The four subfamilies are conserved across monocots and eudicots with the numbers of members of each clade expanding at this point due in part to regions of genome duplication. Since the emergence of the NET superfamily at the dawn of vascular plants they have continued to develop and diversify in a manner which has mirrored the divergence and complexity of plant species through evolution in the ‘March of Progress’.

  2. Antibodies to filamentous actin (F-actin) in type 1 autoimmune hepatitis.

    Science.gov (United States)

    Granito, A; Muratori, L; Muratori, P; Pappas, G; Guidi, M; Cassani, F; Volta, U; Ferri, A; Lenzi, M; Bianchi, F B

    2006-03-01

    To evaluate the diagnostic significance of anti-filamentous actin antibodies (A-FAA) assessed with a commercial ELISA in comparison with immunofluorescence reactivity and patterns of anti-smooth muscle antibodies (SMA); and to correlate A-FAA positivity with clinical, immunogenetic, laboratory, and histological features in patients with autoimmune hepatitis type 1 (AIH-1). We studied 78 consecutive untreated AIH-1 patients and 160 controls: 22 with autoimmune hepatitis type 2 (AIH-2), 51 with hepatitis C, 17 with coeliac disease (CD), 20 with primary biliary cirrhosis (PBC) and 50 blood donors. SMA was evaluated by indirect immunofluorescence (IIF) on frozen sections of rat tissues, and A-FAA with a modified commercial ELISA. SMA was detected by IIF in 61 (78%) of 78 AIH-1 patients, of whom 47 (60%) had the SMA-T/G and 14 (18%) the SMA-V pattern. Of the pathological controls, 32 (20%) had the SMA-V pattern (25 with hepatitis C, 2 with AIH-2, 2 with PBC, 3 with CD). A-FAA were present in 55 AIH-1 patients (70.5%; 46 with SMA-T/G, 7 with SMA-V, and 2 SMA-negative), and in 10 controls (6%), of whom five had hepatitis C, two AIH-2, two PBC and one CD. The association between A-FAA and the SMA-T/G pattern was statistically significant (p<0.0001). A-FAA levels were higher in SMA-T/G positive than SMA-V positive AIH-1 patients and controls (p<0.0001). A-FAA positivity was significantly associated with higher gamma-globulin and IgG levels, but did not correlate with other considered parameters. The modified A-FAA ELISA strictly correlates with the SMA-T/G pattern and is a reliable and operator independent assay for AIH-1. Detection of A-FAA, even if devoid of prognostic relevance, may be useful when interpretative doubts of standard IIF arise.

  3. Antibodies to filamentous actin (F‐actin) in type 1 autoimmune hepatitis

    Science.gov (United States)

    Granito, A; Muratori, L; Muratori, P; Pappas, G; Guidi, M; Cassani, F; Volta, U; Ferri, A; Lenzi, M; Bianchi, F B

    2006-01-01

    Aims To evaluate the diagnostic significance of anti‐filamentous actin antibodies (A‐FAA) assessed with a commercial ELISA in comparison with immunofluorescence reactivity and patterns of anti‐smooth muscle antibodies (SMA); and to correlate A‐FAA positivity with clinical, immunogenetic, laboratory, and histological features in patients with autoimmune hepatitis type 1 (AIH‐1). Methods We studied 78 consecutive untreated AIH‐1 patients and 160 controls: 22 with autoimmune hepatitis type 2 (AIH‐2), 51 with hepatitis C, 17 with coeliac disease (CD), 20 with primary biliary cirrhosis (PBC) and 50 blood donors. SMA was evaluated by indirect immunofluorescence (IIF) on frozen sections of rat tissues, and A‐FAA with a modified commercial ELISA. Results SMA was detected by IIF in 61 (78%) of 78 AIH‐1 patients, of whom 47 (60%) had the SMA‐T/G and 14 (18%) the SMA‐V pattern. Of the pathological controls, 32 (20%) had the SMA‐V pattern (25 with hepatitis C, 2 with AIH‐2, 2 with PBC, 3 with CD). A‐FAA were present in 55 AIH‐1 patients (70.5%; 46 with SMA‐T/G, 7 with SMA‐V, and 2 SMA‐negative), and in 10 controls (6%), of whom five had hepatitis C, two AIH‐2, two PBC and one CD. The association between A‐FAA and the SMA‐T/G pattern was statistically significant (p<0.0001). A‐FAA levels were higher in SMA‐T/G positive than SMA‐V positive AIH‐1 patients and controls (p<0.0001). A‐FAA positivity was significantly associated with higher γ‐globulin and IgG levels, but did not correlate with other considered parameters. Conclusion The modified A‐FAA ELISA strictly correlates with the SMA‐T/G pattern and is a reliable and operator independent assay for AIH‐1. Detection of A‐FAA, even if devoid of prognostic relevance, may be useful when interpretative doubts of standard IIF arise. PMID:16505279

  4. The role of actin in root hair morphogenesis : studies with lipochito-oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug

    NARCIS (Netherlands)

    Miller, D.D.; Ruijter, de N.C.A.; Bisseling, T.; Emons, A.M.C.

    1999-01-01

    Root hairs develop from bulges on root epidermal cells and elongate by tip growth, in which Golgi vesicles are targeted, released and inserted into the plasma membrane on one side of the cell. We studied the role of actin in vesicle delivery and retention by comparing the actin filament

  5. Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter

    DEFF Research Database (Denmark)

    Plenge, Per; Shi, Lei; Beuming, Thijs

    2012-01-01

    The serotonin transporter (SERT) controls synaptic serotonin levels and is the primary target for antidepressants, including selective serotonin reuptake inhibitors (e.g. (S)-citalopram) and tricyclic antidepressants (e.g. clomipramine). In addition to a high affinity binding site, SERT possesses...... a low affinity allosteric site for antidepressants. Binding to the allosteric site impedes dissociation of antidepressants from the high affinity site, which may enhance antidepressant efficacy. Here we employ an induced fit docking/molecular dynamics protocol to identify the residues that may...... effects of Zn(2+) binding in an engineered site and the covalent attachment of benzocaine-methanethiosulfonate to a cysteine introduced in the extracellular vestibule. The data provide a mechanistic explanation for the allosteric action of antidepressants at SERT and suggest that the role of the vestibule...

  6. Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor.

    Science.gov (United States)

    Haas, Ann-Karin; Kleinau, Gunnar; Hoyer, Inna; Neumann, Susanne; Furkert, Jens; Rutz, Claudia; Schülein, Ralf; Gershengorn, Marvin C; Krause, Gerd

    2011-01-01

    The thyrotropin receptor (TSHR) exhibits elevated cAMP signaling in the basal state and becomes fully activated by thyrotropin. Previously we presented evidence that small-molecule ligands act allosterically within the transmembrane region in contrast to the orthosteric extracellular hormone-binding sites. Our goal in this study was to identify positions that surround the allosteric pocket and that are sensitive for inactivation of TSHR. Homology modeling combined with site-directed mutagenesis and functional characterization revealed seven mutants located in the allosteric binding site that led to a decrease of basal cAMP signaling activity. The majority of these silencing mutations, which constrain the TSHR in an inactive conformation, are found in two clusters when mapped onto the 3D structural model. We suggest that the amino acid positions identified herein are indicating locations where small-molecule antagonists, both neutral antagonists and inverse agonists, might interfere with active TSHR conformations.

  7. Actin nemaline myopathy mouse reproduces disease, suggests other actin disease phenotypes and provides cautionary note on muscle transgene expression.

    Directory of Open Access Journals (Sweden)

    Gianina Ravenscroft

    Full Text Available Mutations in the skeletal muscle α-actin gene (ACTA1 cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G of ACTA1 (identified in a severe nemaline myopathy patient fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ~30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations.

  8. Actin Nemaline Myopathy Mouse Reproduces Disease, Suggests Other Actin Disease Phenotypes and Provides Cautionary Note on Muscle Transgene Expression

    Science.gov (United States)

    Ravenscroft, Gianina; Jackaman, Connie; Sewry, Caroline A.; McNamara, Elyshia; Squire, Sarah E.; Potter, Allyson C.; Papadimitriou, John; Griffiths, Lisa M.; Bakker, Anthony J.; Davies, Kay E.; Laing, Nigel G.; Nowak, Kristen J.

    2011-01-01

    Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations. PMID:22174871

  9. Actin cytoskeleton and small heat shock proteins: how do they interact?

    Science.gov (United States)

    Mounier, Nicole; Arrigo, André-Patrick

    2002-01-01

    Actin and small heat shock proteins (sHsps) are ubiquitous and multifaceted proteins that exist in 2 reversible forms, monomers and multimers, ie, the microfilament of the cytoskeleton and oligomers of the sHsps, generally, supposed to be in a spherical and hollow form. Two situations are described in the literature, where the properties of actin are modulated by sHsps; the actin polymerization is inhibited in vitro by some sHsps acting as capping proteins, and the actin cytoskeleton is protected by some sHsps against the disruption induced by various stressful conditions. We propose that a direct actin-sHsp interaction occurs to inhibit actin polymerization and to participate in the in vivo regulation of actin filament dynamics. Protection of the actin cytoskeleton would result from an F-actin–sHsp interaction in which microfilaments would be coated by small oligomers of phosphorylated sHsps. Both proteins share common structural motives suggesting direct binding sites, but they remain to be demonstrated. Some sHsps would behave with the actin cytoskeleton as actin-binding proteins capable of either capping a microfilament when present as a nonphosphorylated monomer or stabilizing and protecting the microfilament when organized in small, phosphorylated oligomers. PMID:12380684

  10. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    Science.gov (United States)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  11. FIMBRIN1 is involved in lily pollen tube growth by stabilizing the actin fringe.

    Science.gov (United States)

    Su, Hui; Zhu, Jinsheng; Cai, Chao; Pei, Weike; Wang, Jiaojiao; Dong, Huaijian; Ren, Haiyun

    2012-11-01

    An actin fringe structure in the subapex plays an important role in pollen tube tip growth. However, the precise mechanism by which the actin fringe is generated and maintained remains largely unknown. Here, we cloned a 2606-bp full-length cDNA encoding a deduced 77-kD fimbrin-like protein from lily (Lilium longiflorum), named FIMBRIN1 (FIM1). Ll-FIM1 was preferentially expressed in pollen and concentrated at actin fringe in the subapical region, as well as in longitudinal actin-filament bundles in the shank of pollen tubes. Microinjection of Ll-FIM1 antibody into lily pollen tubes inhibited tip growth and disrupted the actin fringe. Furthermore, we verified the function of Ll-FIM1 in the fim5 mutant of its closest relative, Arabidopsis thaliana. Pollen tubes of fim5 mutants grew with a larger diameter in early stages but could recover into normal forms in later stages, despite significantly slower growth rates. The actin fringe of the fim5 mutants, however, was impaired during both early and late stages. Impressively, stable expression of fim5pro:GFP:Ll-FIM1 rescued the actin fringe and the growth rate of Arabidopsis fim5 pollen tubes. In vitro biochemical analysis showed that Ll-FIM1 could bundle actin filaments. Thus, our study has identified a fimbrin that may stabilize the actin fringe by cross-linking actin filaments into bundles, which is important for proper tip growth of lily pollen tubes.

  12. Latrunculin B-induced plant dwarfism: Plant cell elongation is F-actin-dependent.

    Science.gov (United States)

    Baluska, F; Jasik, J; Edelmann, H G; Salajová, T; Volkmann, D

    2001-03-01

    Marine macrolides latrunculins are highly specific toxins which effectively depolymerize actin filaments (generally F-actin) in all eukaryotic cells. We show that latrunculin B is effective on diverse cell types in higher plants and describe the use of this drug in probing F-actin-dependent growth and in plant development-related processes. In contrast to other eukaryotic organisms, cell divisions occurs in plant cells devoid of all actin filaments. However, the alignment of the division planes is often distorted. In addition to cell division, postembryonic development and morphogenesis also continue in the absence of F-actin. These experimental data suggest that F-actin is of little importance in the morphogenesis of higher plants, and that plants can develop more or less normally without F-actin. In contrast, F-actin turns out to be essential for cell elongation. When latrunculin B was added during germination, morphologically normal Arabidopsis and rye seedlings developed but, as a result of the absence of cell elongation, these were stunted, resembling either genetic dwarfs or environmental bonsai plants. In conclusion, F-actin is essential for the plant cell elongation, while this F-actin-dependent cell elongation is not an essential feature of plant-specific developmental programs.

  13. Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum

    International Nuclear Information System (INIS)

    Schwartz, M.A.; Luna, E.J.

    1986-01-01

    The binding of native, 125 I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. The authors conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins

  14. Altered cell mechanics from the inside: dispersed single wall carbon nanotubes integrate with and restructure actin.

    Science.gov (United States)

    Holt, Brian D; Shams, Hengameh; Horst, Travis A; Basu, Saurav; Rape, Andrew D; Wang, Yu-Li; Rohde, Gustavo K; Mofrad, Mohammad R K; Islam, Mohammad F; Dahl, Kris Noel

    2012-05-23

    With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs) are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  15. Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

    Directory of Open Access Journals (Sweden)

    Mohammad F. Islam

    2012-05-01

    Full Text Available With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  16. The 5’cap of Tobacco Mosaic Virus (TMV) is required for virion attachment to the actin/ER network during early infection

    DEFF Research Database (Denmark)

    Christensen, Nynne Meyn; Tilsner, Jens; Bell, Karen

    to the motile cortical actin/ER network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actindependent RNA movement. The 5’ methylguanosine TMV cap was shown to be required for vRNA anchoring to the ER. TMV vRNA lacking the 5’cap failed to form granules...... the fluorescent vRNA pool nor co-injected GFP left the injected trichome, indicating that the synthesis of unlabelled progeny viral (v)RNA is required to initiate cell-cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3-vRNA formed granules that became anchored...... on the same ER-bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome....

  17. Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states.

    Science.gov (United States)

    Rould, Mark A; Wan, Qun; Joel, Peteranne B; Lowey, Susan; Trybus, Kathleen M

    2006-10-20

    Actin filament growth and disassembly, as well as affinity for actin-binding proteins, is mediated by the nucleotide-bound state of the component actin monomers. The structural differences between ATP-actin and ADP-actin, however, remain controversial. We expressed a cytoplasmic actin in Sf9 cells, which was rendered non-polymerizable by virtue of two point mutations in subdomain 4 (A204E/P243K). This homogeneous monomer, called AP-actin, was crystallized in the absence of toxins, binding proteins, or chemical modification, with ATP or ADP at the active site. The two surface mutations do not perturb the structure. Significant differences between the two states are confined to the active site region and sensor loop. The active site cleft remains closed in both states. Minor structural shifts propagate from the active site toward subdomain 2, but dissipate before reaching the DNase binding loop (D-loop), which remains disordered in both the ADP and ATP states. This result contrasts with previous structures of actin made monomeric by modification with tetramethylrhodamine, which show formation of an alpha-helix at the distal end of the D-loop in the ADP-bound but not the ATP-bound form (Otterbein, L. R., Graceffa, P., and Dominguez, R. (2001) Science 293, 708-711). Our reanalysis of the TMR-modified actin structures suggests that the nucleotide-dependent formation of the D-loop helix may result from signal propagation through crystal packing interactions. Whereas the observed nucleotide-dependent changes in the structure present significantly different surfaces on the exterior of the actin monomer, current models of the actin filament lack any actin-actin interactions that involve the region of these key structural changes.

  18. A peek into tropomyosin binding and unfolding on the actin filament.

    Directory of Open Access Journals (Sweden)

    Abhishek Singh

    Full Text Available BACKGROUND: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. PRINCIPAL FINDINGS: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering, and chain dissociation (analyzed using circular dichroism. CONCLUSIONS: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest

  19. Animal Research International: Contact

    African Journals Online (AJOL)

    Principal Contact. Dr. J. E. Eyo Dr. Department of Zoology, University of Nig Department of Zoology, POBox 3146, University of Nigeria, Nsukka, Enugu State, Nigeria. Phone: 234 42 308030. Email: joseph.eyo@unn.edu.ng. Support Contact. N. S. Oluah Phone: +234-83732127. Email: ndubusioluah@yahoo.com.

  20. Contact Hamiltonian mechanics

    Energy Technology Data Exchange (ETDEWEB)

    Bravetti, Alessandro, E-mail: alessandro.bravetti@iimas.unam.mx [Instituto de Investigaciones en Matemáticas Aplicadas y en Sistemas, Universidad Nacional Autónoma de México, A. P. 70543, México, DF 04510 (Mexico); Cruz, Hans, E-mail: hans@ciencias.unam.mx [Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, A. P. 70543, México, DF 04510 (Mexico); Tapias, Diego, E-mail: diego.tapias@nucleares.unam.mx [Facultad de Ciencias, Universidad Nacional Autónoma de México, A.P. 70543, México, DF 04510 (Mexico)

    2017-01-15

    In this work we introduce contact Hamiltonian mechanics, an extension of symplectic Hamiltonian mechanics, and show that it is a natural candidate for a geometric description of non-dissipative and dissipative systems. For this purpose we review in detail the major features of standard symplectic Hamiltonian dynamics and show that all of them can be generalized to the contact case.

  1. Discovery and Innovation: Contact

    African Journals Online (AJOL)

    Principal Contact. Prof. Keto Mshigeni Editor-in-Chief Academy Science Publishers. PO Box 14798-00200. Nairobi. Kenya. Phone: 254 (20) 884401-5. Fax: 254 (20) 884406. Email: aas@aasciences.org. Support Contact. Prof. Keto Mshigeni Email: aas@aasciences.org. ISSN: 1015-079X. AJOL African Journals Online.

  2. LBS Management Review: Contact

    African Journals Online (AJOL)

    Principal Contact. Dr Obinna Muogboh Managing Editor Lagos Business School Pan African University 2 Ahmed Onibudo Street, P.O. Box 73688, Victoria Island, Lagos, NIGERIA Email: omuogboh@lbs.edu.ng. Support Contact. Editor Email: omuogboh@lbs.edu.ng. ISSN: 1118-3713. AJOL African Journals Online.

  3. African Health Sciences: Contact

    African Journals Online (AJOL)

    Principal Contact. Dr James Tumwine Editor-in-Chief. Makerere University Medical School P. O. Box 7072 Kampala Uganda. Phone: 256-41-530020/1. Email: kabaleimc@gmail.com. Support Contact. Pauline Salamula Email: paulinesalamula@gmail.com. ISSN: 1680-6905. AJOL African Journals Online. HOW TO USE ...

  4. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... Get follow up exams with your eye care provider. If you notice redness, swelling, excessive discharge, pain or discomfort from wearing contact lenses, remove the lenses and seek immediate medical attention from an ophthalmologist. Related resources: Learn how to properly care for contact lenses . ...

  5. Contact Quality in Participation

    DEFF Research Database (Denmark)

    Simonsen, Jesper; Jensen, Olav Storm

    2016-01-01

    We investigate the concept of participation from the perspective of quality of the contact in the communicative interactions between participants. We argue for the need for an academic-personal competence that qualifies the human contact central in all Participatory Design (PD) activities as a wa...

  6. Electric contact arcing

    International Nuclear Information System (INIS)

    Cuthrell, R.E.

    1976-01-01

    Electrical contacts must function properly in many types of components used in nuclear weapon systems. Design, application, and testing of these components require detailed knowledge of chemical and physical phenomena associated with stockpile storage, stockpile testing, and operation. In the past, investigation of these phenomena has led to significant discoveries on the effects of surface contaminants, friction and wear, and the mechanics of closure on contact performance. A recent investigation of contact arcing phenomena which revealed that, preceding contact closure, arcs may occur at voltages lower than had been previously known is described. This discovery is important, since arcing may damage contacts, and repetitive testing of contacts performed as part of a quality assurance program might produce cumulative damage that would yield misleading life-test data and could prevent proper operation of the contacts at some time in the future. This damage can be avoided by determining the conditions under which arcing occurs, and ensuring that these conditions are avoided in contact testing

  7. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... Eye Health A-Z Symptoms Glasses & Contacts Tips & Prevention News Ask an Ophthalmologist Patient Stories Español Eye ... colored contact lenses , from the U.S. Food and Drug Administration (FDA). Are the colored lenses you are ...

  8. Sciences & Nature: Contact

    African Journals Online (AJOL)

    Principal Contact. Ehouan Etienne Ehile Professor University of Abobo-Adjamé 02 BP 801 Abidjan 02. Phone: (+225) 2030 4201. Fax: (+225) 2030 4203. Email: eh_ehile@yahoo.fr. Support Contact. Irie Zoro Bi Email: banhiakalou@yahoo.fr. ISSN: 1812-0741. AJOL African Journals Online. HOW TO USE AJOL.

  9. Colored Contact Lens Dangers

    Medline Plus

    Full Text Available ... in Cleveland. "This is far from the truth." Real People, Real Problems with Colored Contact Lenses Julian: Teenager Blinded ... use of colored contact lenses , from the U.S. Food and Drug Administration (FDA). Are the colored lenses ...

  10. African Zoology: Contact

    African Journals Online (AJOL)

    Principal Contact. Lester Isaacs Phone: +27466229698. Fax: +2746 622 9550. Email: lester@nisc.co.za. Support Contact. NISC office. Email: info@nisc.co.za. ISSN: 2224-073X. AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's · More about AJOL · AJOL's Partners ...

  11. Factor XII Contact Activation.

    Science.gov (United States)

    Naudin, Clément; Burillo, Elena; Blankenberg, Stefan; Butler, Lynn; Renné, Thomas

    2017-11-01

    Contact activation is the surface-induced conversion of factor XII (FXII) zymogen to the serine protease FXIIa. Blood-circulating FXII binds to negatively charged surfaces and this contact to surfaces triggers a conformational change in the zymogen inducing autoactivation. Several surfaces that have the capacity for initiating FXII contact activation have been identified, including misfolded protein aggregates, collagen, nucleic acids, and platelet and microbial polyphosphate. Activated FXII initiates the proinflammatory kallikrein-kinin system and the intrinsic coagulation pathway, leading to formation of bradykinin and thrombin, respectively. FXII contact activation is well characterized in vitro and provides the mechanistic basis for the diagnostic clotting assay, activated partial thromboplastin time. However, only in the past decade has the critical role of FXII contact activation in pathological thrombosis been appreciated. While defective FXII contact activation provides thromboprotection, excess activation underlies the swelling disorder hereditary angioedema type III. This review provides an overview of the molecular basis of FXII contact activation and FXII contact activation-associated disease states. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  12. Afrika Statistika: Contact

    African Journals Online (AJOL)

    Principal Contact. Prof. Gane Samb Lo Editor Université Gaston Berger BP 234, Université Saint-Louis Sénégal Phone: 221 961 23 40. Fax: 221 961 53 38. Email: ganesamblo@yahoo.com. Support Contact. Mamadou Camara Email: mdoucamara@gmail.com. ISSN: 2316-090X. AJOL African Journals Online. HOW TO ...

  13. Nigerian Food Journal: Contact

    African Journals Online (AJOL)

    Nigerian Food Journal. ... Nigerian Food Journal: Contact. Journal Home > About the Journal > Nigerian Food Journal: Contact. Log in or Register to get access to full text downloads. ... Mailing Address. Department of Food Science and Technology University of Agriculture, Makurdi, Nigeria ...

  14. Lettuce contact allergy

    DEFF Research Database (Denmark)

    Paulsen, Evy; Andersen, Klaus E

    2016-01-01

    degradability of lettuce allergens, it is recommended to patch test with freshly cut lettuce stem and supplement this with Compositae mix. As contact urticaria and protein contact dermatitis may present as dermatitis, it is important to perform prick-prick tests, and possibly scratch patch tests as well. Any...

  15. Targeting the Akt1 allosteric site to identify novel scaffolds through virtual screening.

    Science.gov (United States)

    Yilmaz, Oya Gursoy; Olmez, Elif Ozkirimli; Ulgen, Kutlu O

    2014-02-01

    Preclinical data and tumor specimen studies report that AKT kinases are related to many human cancers. Therefore, identification and development of small molecule inhibitors targeting AKT and its signaling pathway can be therapeutic in treatment of cancer. Numerous studies report inhibitors that target the ATP-binding pocket in the kinase domains, but the similarity of this site, within the kinase family makes selectivity a major problem. The sequence identity amongst PH domains is significantly lower than that in kinase domains and developing more selective inhibitors is possible if PH domain is targeted. This in silico screening study is the first time report toward the identification of potential allosteric inhibitors expected to bind the cavity between kinase and PH domains of Akt1. Structural information of Akt1 was used to develop structure-based pharmacophore models comprising hydrophobic, acceptor, donor and ring features. The 3D structural information of previously identified allosteric Akt inhibitors obtained from literature was employed to develop a ligand-based pharmacophore model. Database was generated with drug like subset of ZINC and screening was performed based on 3D similarity to the selected pharmacophore hypotheses. Binding modes and affinities of the ligands were predicted by Glide software. Top scoring hits were further analyzed considering 2D similarity between the compounds, interactions with Akt1, fitness to pharmacophore models, ADME, druglikeness criteria and Induced-Fit docking. Using virtual screening methodologies, derivatives of 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol were proposed as potential leads for allosteric inhibition of Akt1. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Intra-subunit flexibility underlies activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

    Science.gov (United States)

    Chrisman, Paul A; Podair, Julie I; Jobe, Emily M; Levandoski, Mark M

    2014-04-01

    Allosteric modulation is a general feature of nicotinic acetylcholine receptors, yet the structural components and movements important for conversions among functional states are not well understood. In this study, we examine the communication between the binding sites for agonist and the modulator morantel (Mor) of neuronal α3β2 receptors, measuring evoked currents of receptors expressed in Xenopus oocytes with the two-electrode voltage-clamp method. We hypothesized that movement along an interface of β sheets connecting the agonist and modulator sites is necessary for allosteric modulation. To address this, we created pairs of substituted cysteines that span the cleft formed where the outer β sheet meets the β sheet constituting the (-)-face of the α3 subunit; the three pairs were L158C-A179C, L158C-G181C and L158C-K183C. Employing a disulfide trapping approach in which bonds are formed between neighboring cysteines under oxidation conditions, we found that oxidation treatments decreased the amplitude of currents evoked by either the agonist (ACh) or co-applied agonist and modulator (ACh + Mor), by as much as 51%, consistent with the introduced bond decreasing channel efficacy. Reduction treatment increased evoked currents up to 89%. The magnitude of the oxidation effects depended on whether agonists were present during oxidation and on the cysteine pair. Additionally, the cysteine mutations themselves decreased Mor potentiation, implicating these residues in modulation. Our findings suggest that these β sheets in the α3 subunit move with respect to each other during activation and modulation, and the residues studied highlight the contribution of this intramolecular allosteric pathway to receptor function. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Disruption of integrin-fibronectin complexes by allosteric but not ligand-mimetic inhibitors.

    Science.gov (United States)

    Mould, A Paul; Craig, Susan E; Byron, Sarah K; Humphries, Martin J; Jowitt, Thomas A

    2014-12-15

    Failure of Arg-Gly-Asp (RGD)-based inhibitors to reverse integrin-ligand binding has been reported, but the prevalence of this phenomenon among integrin heterodimers is currently unknown. In the present study we have investigated the interaction of four different RGD-binding integrins (α5β1, αVβ1, αVβ3 and αVβ6) with fibronectin (FN) using surface plasmon resonance. The ability of inhibitors to reverse ligand binding was assessed by their capacity to increase the dissociation rate of pre-formed integrin-FN complexes. For all four receptors we showed that RGD-based inhibitors (such as cilengitide) were completely unable to increase the dissociation rate. Formation of the non-reversible state occurred very rapidly and did not rely on the time-dependent formation of a high-affinity state of the integrin, or the integrin leg regions. In contrast with RGD-based inhibitors, Ca2+ (but not Mg2+) was able to greatly increase the dissociation rate of integrin-FN complexes, with a half-maximal response at ~0.4 mM Ca2+ for αVβ3-FN. The effect of Ca2+ was overcome by co-addition of Mn2+, but not Mg2+. A stimulatory anti-β1 monoclonal antibody (mAb) abrogated the effect of Ca2+ on α5β1-FN complexes; conversely, a function-blocking mAb mimicked the effect of Ca2+. These results imply that Ca2+ acts allosterically, probably through binding to the adjacent metal-ion-dependent adhesion site (ADMIDAS), and that the α1 helix in the β subunit I domain is the key element affected by allosteric modulators. The data suggest an explanation for the limited clinical efficacy of RGD-based integrin antagonists, and we propose that allosteric antagonists could prove to be of greater therapeutic benefit.

  18. Allosteric activation of sodium-calcium exchange by picomolar concentrations of cadmium.

    Science.gov (United States)

    Le, Hoa Dinh; Omelchenko, Alexander; Hryshko, Larry V; Uliyanova, Alexandra; Condrescu, Madalina; Reeves, John P

    2005-02-15

    Chinese hamster ovary cells expressing the bovine cardiac Na+-Ca2+ exchanger (NCX1.1) accumulated Cd2+ after a lag period of several tens of seconds. The lag period reflects the progressive allosteric activation of exchange activity by Cd2+ as it accumulates within the cytosol. The lag period was greatly reduced in cells expressing a mutant exchanger, Delta(241-680), that does not require allosteric activation by Ca2+ for activity. Non-transfected cells did not show Cd2+ uptake under the same conditions. In cells expressing NCX1.1, the lag period was nearly abolished following an elevation of the cytosolic Ca2+ concentration. Cytosolic Cd2+ concentrations estimated at 0.5-2 pm markedly stimulated the subsequent uptake of Ca2+ by Na+-Ca2+ exchange. Outward exchange currents in membrane patches from Xenopus oocytes expressing the canine NCX1.1 were rapidly and reversibly stimulated by 3 pm Cd2+ applied at the cytosolic membrane surface. Exchange currents activated by 3 pm Cd2+ were 40% smaller than currents activated by 1 mum cytosolic Ca2+. Current amplitudes declined by 30% and the rate of current development fell sharply upon repetitive applications of Na+ in the presence of 3 pm Cd2+. Cd2+ mimicked the anomalous inhibitory effects of Ca2+ on outward exchange currents generated by the Drosophila exchanger CALX1.1. We conclude that the regulatory sites responsible for allosteric Ca2+ activation bind Cd2+ with high affinity and that Cd2+ mimics the regulatory effects of Ca2+ at concentrations 5 orders of magnitude lower than Ca2+.

  19. Convergent transmission of RNAi guide-target mismatch information across Argonaute internal allosteric network.

    Directory of Open Access Journals (Sweden)

    Thomas T Joseph

    Full Text Available In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand "seed region" have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the

  20. Contact materials for nanoelectronics

    KAUST Repository

    Alshareef, Husam N.

    2011-02-01

    In this article, we review current research activities in contact material development for electronic and nanoelectronic devices. A fundamental issue in contact materials research is to understand and control interfacial reactions and phenomena that modify the expected device performance. These reactions have become more challenging and more difficult to control as new materials have been introduced and as device sizes have entered the deep nanoscale. To provide an overview of this field of inquiry, this issue of MRS Bulletin includes articles on gate and contact materials for Si-based devices, junction contact materials for Si-based devices, and contact materials for alternate channel substrates (Ge and III-V), nanodevices. © 2011 Materials Research Society.

  1. Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists

    OpenAIRE

    Ajram, Laura; Begg, Malcolm; Slack, Robert; Cryan, Jenni; Hall, David; Hodgson, Simon; Ford, Alison; Barnes, Ashley; Swieboda, Dawid; Mousnier, Aurelie; Solari, Roberto

    2014-01-01

    The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in in...

  2. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    Directory of Open Access Journals (Sweden)

    Klein-Seetharaman Judith

    2008-02-01

    Full Text Available Abstract Metabotropic glutamate receptors (mGluRs are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%, the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%, the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86% and 8/9 (89% for ArgusLab and 10/14 (71% and 7/9 (78% for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by

  3. Muscarinic receptor M4 positive allosteric modulators attenuate central effects of cocaine

    DEFF Research Database (Denmark)

    Dall, Camilla; Weikop, Pia; Dencker, Ditte

    2017-01-01

    allosteric modulators VU0152100 and VU0467154 in a drug discrimination assay and a conditioned place preference assay, including extinction and reinstatement of place preference. Specificity of the cocaine discrimination effect was verified using knockout mice lacking either M1or M4receptors (M1-/-, M4....... As previously shown with VU0152100, VU0467154 almost eliminated cocaine-induced hyperactivity and striatal dopamine efflux. VU0467154 failed to attenuate acquisition of cocaine-conditioned place preference, but facilitated extinction and prevented reinstatement of the conditioned place preference. CONCLUSIONS...

  4. Allosteric ATPase behavior: the onset of laser-sustained enzyme cooperation

    Science.gov (United States)

    Causa, F.; Costato, Michele; Milani, Marziale; Bolognani, Lorenzo

    1995-01-01

    A two level model is considered to describe the dynamics of a biological system undergoing cyclic oscillations from one state to another separated by an energy quantum interval. This is typically met in enzyme activated reactions, involving the ADP-ATP cycle. General results can be obtained analytically, the dynamics of the system being investigated from an energetic point of view. Numerical solutions show how an enzymatic system can be driven across different regimes where cooperation (allostericity) and oscillations appears. The model can be extended to the case of an external energy supply in the form of electromagnetic radiation, providing clues for a physical understanding of nonthermal laser interaction with biosystems.

  5. Allosteric control of internal electron transfer in cytochrome cd1 nitrite reductase

    DEFF Research Database (Denmark)

    Farver, Ole; Kroneck, Peter M H; Zumft, Walter G

    2003-01-01

    Cytochrome cd1 nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water. Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have...... been studied and found to be dominated by pronounced interactions between the c and the d1 hemes. The interactions are expressed both in dramatic changes in the internal electron-transfer rates between these sites and in marked cooperativity in their electron affinity. The results constitute a prime...... example of intraprotein control of the electron-transfer rates by allosteric interactions....

  6. Substituted 3-Benzylcoumarins as Allosteric MEK1 Inhibitors: Design, Synthesis and Biological Evaluation as Antiviral Agents

    Directory of Open Access Journals (Sweden)

    Ping Xu

    2013-05-01

    Full Text Available In order to find novel antiviral agents, a series of allosteric MEK1 inhibitors were designed and synthesized. Based on docking results, multiple optimizations were made on the coumarin scaffold. Some of the derivatives showed excellent MEK1 binding affinity in the appropriate enzymatic assays and displayed obvious inhibitory effects on the ERK pathway in a cellular assay. These compounds also significantly inhibited virus (EV71 replication in HEK293 and RD cells. Several compounds showed potential as agents for the treatment of viral infective diseases, with the most potent compound 18 showing an IC50 value of 54.57 nM in the MEK1 binding assay.

  7. Biased signaling of lipids and allosteric actions of synthetic molecules for GPR119

    DEFF Research Database (Denmark)

    Hassing, Helle A; Fares, Suzan; Larsen, Olav

    2016-01-01

    GPR119 is a Gαs-coupled lipid-sensor in the gut, where it mediates release of incretin hormones from the enteroendocrine cells and in pancreatic α-cells, where it releases insulin. Naturally occurring lipids such as monoacylglycerols (MAGs) and N-acylethanolamines (NAEs), like oleoylethanolamide...... for 2h with the 2-MAG-lipase inhibitor JZL84 doubled the constitutive activity, indicating that endogenous lipids contribute to the apparent constitutive activity. Finally, besides being an agonist, AR231453 acted as a positive allosteric modulator of OEA and increased its potency by 54-fold at 100nM AR...

  8. Structural insight to mutation effects uncover a common allosteric site in class C GPCRs

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Boesgaard, Michael W; Munk, Christian

    2017-01-01

    MOTIVATION: Class C G protein-coupled receptors (GPCRs) regulate important physiological functions and allosteric modulators binding to the transmembrane domain constitute an attractive and, due to a lack of structural insight, a virtually unexplored potential for therapeutics and the food industry....... Combining pharmacological site-directed mutagenesis data with the recent class C GPCR experimental structures will provide a foundation for rational design of new therapeutics. RESULTS: We uncover one common site for both positive and negative modulators with different amino acid layouts that can...

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

    Science.gov (United States)

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

    2014-01-01

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

  10. System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells

    Science.gov (United States)

    Nowak, Jacqueline; Ivakov, Alexander; Somssich, Marc; Persson, Staffan; Nikoloski, Zoran

    2017-01-01

    The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms. PMID:28655850

  11. Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Shi, Kaikai; Frary, Charles

    2015-01-01

    Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene...... expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize...... polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1...

  12. Lithium preserves F-actin from the disarrangement induced by either DNase I or cytochalasin D.

    Science.gov (United States)

    DalleDonne, I; Milzani, A; Fascio, U; Ratti, A; Colombo, R

    1993-01-01

    Light scattering at 546 nm, which is mainly related to the presence of rodlike particles longer than 50 nm, showed that Li+ accelerates the formation of actin filaments. Intermolecular cross-linking with N,N'-1,4-phenylene-bismaleimide proved that the observed enhancement in the light-scattering intensity is caused by the increase in the concentration of actin oligomers, which gradually elongate to form longer filaments. DNase-I-related F-actin disassembly was reduced in the presence of lithium ions, as demonstrated by fluorimetric and viscometric experiments. Li(+)-F-actin showed an apparently similar behaviour when exposed to cytochalasin D. We confirm that Li+ acts on actin polymerization by stabilizing actin nuclei and polymers. The stabilization of cytoskeletal polymers really appears as one of the mechanisms by which lithium ions influence some of the cell activities.

  13. The interaction between the adaptor protein APS and Enigma is involved in actin organisation

    DEFF Research Database (Denmark)

    Barres, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick

    2005-01-01

    and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin...... cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest...... that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation....

  14. Contacts to semiconductors

    International Nuclear Information System (INIS)

    Tove, P.A.

    1975-08-01

    Contacts to semiconductors play an important role in most semiconductor devices. These devices range from microelectronics to power components, from high-sensitivity light or radiation detectors to light-emitting of microwave-generating components. Silicon is the dominating material but compound semiconductors are increasing in importance. The following survey is an attempt to classify contact properties and the physical mechanisms involved, as well as fabrication methods and methods of investigation. The main interest is in metal-semiconductor type contacts where a few basic concepts are dealt with in some detail. (Auth.)

  15. Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    Science.gov (United States)

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments. PMID:25978408

  16. Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Iman Jalilian

    Full Text Available The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm, in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

  17. Cell stress promotes the association of phosphorylated HspB1 with F-actin.

    Directory of Open Access Journals (Sweden)

    Joseph P Clarke

    Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

  18. Actin expression is induced and three isoforms are differentially expressed during germination in Zea mays.

    Science.gov (United States)

    Díaz-Camino, Claudia; Conde, Renaud; Ovsenek, Nick; Villanueva, Marco A

    2005-02-01

    Previous analysis of actin in a dicotyledonous plant, Phaseolus vulgaris (or common bean), showed very low actin levels in cotyledons but they were concentrated in the embryo axis. Upon imbibition, actin expression increased 5-fold and a maximum of four actin isoforms were observed, two of them transient and two major ones were steadily expressed. In this work, analysis of the actin expression in a monocotyledonous plant, Zea mays (or maize), and over a longer period of germination/growth, showed that striking similarities exist. Actin is present in all the seed components, but it is mainly concentrated in the embryo axis. The expression of maize actin was induced during post-imbibition at both the protein and mRNA levels. Sharp increases in actin appeared from 24-48 h and again from 72-96 h. A more modest and steady actin mRNA increase in expression was observed; however, it did not appear as dramatic as in the case of common bean due to the presence of readily detectable amounts of message in the dry maize seed. The isoform distribution in the dry seed showed a pattern of at least three isovariants of pIs approximately 5.0, 5.1, and 5.2, which were differentially expressed at the various post-imbibition times analysed. Two of the actin isoforms at 48 h post-imbibition cross-reacted with a phosphotyrosine-specific antibody and they are the product of three expressed genes as shown by in vitro translation assays. These data indicate that maize actin protein and mRNA expression is induced upon the trigger of germination, and the isoform expression kinetics and patterns resemble those from bean, suggesting that, in both species, actin expression at these early germination/growth stages is a highly regulated event.

  19. Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Fortanet, Jorge Garcia; Chen, Christine Hiu-Tung; Chen, Ying-Nan P.; Chen, Zhouliang; Deng, Zhan; Firestone, Brant; Fekkes, Peter; Fodor, Michelle; Fortin, Pascal D.; Fridrich, Cary; Grunenfelder, Denise; Ho, Samuel; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Keen, Nick; LaBonte, Laura R.; Larrow, Jay; Lenoir, Francois; Liu, Gang; Liu, Shumei; Lombardo, Franco; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Ramsey, Timothy; Sellers, William R.; Shultz, Michael D.; Stams, Travis; Towler, Christopher; Wang, Ping; Williams, Sarah L.; Zhang, Ji-Hu; LaMarche, Matthew J. (Novartis)

    2016-09-08

    SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein–ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

  20. Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation.

    Science.gov (United States)

    Fulton, Melody D; Hanold, Laura E; Ruan, Zheng; Patel, Sneha; Beedle, Aaron M; Kannan, Natarajan; Kennedy, Eileen J

    2018-03-15

    Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide "stapling" to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Identification of novel allosteric modulator binding sites in NMDA receptors: A molecular modeling study.

    Science.gov (United States)

    Kane, Lucas T; Costa, Blaise M

    2015-09-01

    The dysfunction of N-methyl-d-Aspartate receptors (NMDARs), a subtype of glutamate receptors, is correlated with schizophrenia, stroke, and many other neuropathological disorders. However, not all NMDAR subtypes equally contribute towards these disorders. Since NMDARs composed of different GluN2 subunits (GluN2A-D) confer varied physiological properties and have different distributions in the brain, pharmacological agents that target NMDARs with specific GluN2 subunits have significant potential for therapeutic applications. In our previous research, we have identified a family of novel allosteric modulators that differentially potentiate and/or inhibit NMDARs of differing GluN2 subunit composition. To further elucidate their molecular mechanisms, in the present study, we have identified four potential binding sites for novel allosteric modulators by performing molecular modeling, docking, and in silico mutations. The molecular determinants of the modulator binding sites (MBS), analysis of particular MBS electrostatics, and the specific loss or gain of binding after mutations have revealed modulators that have strong potential affinities for specific MBS on given subunits and the role of key amino acids in either promoting or obstructing modulator binding. These findings will help design higher affinity GluN2 subunit-selective pharmaceuticals, which are currently unavailable to treat psychiatric and neurological disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

    Science.gov (United States)

    Manna, Moutusi; Niemelä, Miia; Tynkkynen, Joona; Javanainen, Matti; Kulig, Waldemar; Müller, Daniel J; Rog, Tomasz; Vattulainen, Ilpo

    2016-01-01

    There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human β2-adrenergic receptor (β2AR) – a prototypical G protein-coupled receptor – is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates β2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5–7 of the receptor. The alternative mechanism, where the β2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of β2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions. DOI: http://dx.doi.org/10.7554/eLife.18432.001 PMID:27897972

  3. Discovery of Peptidomimetic Ligands of EED as Allosteric Inhibitors of PRC2

    Energy Technology Data Exchange (ETDEWEB)

    Barnash, Kimberly D.; The, Juliana; Norris-Drouin, Jacqueline L.; Cholensky, Stephanie H.; Worley, Beau M.; Li, Fengling; Stuckey, Jacob I.; Brown, Peter J.; Vedadi, Masoud; Arrowsmith, Cheryl H.; Frye, Stephen V.; James, Lindsey I. (UNC); (Toronto)

    2017-02-06

    The function of EED within polycomb repressive complex 2 (PRC2) is mediated by a complex network of protein–protein interactions. Allosteric activation of PRC2 by binding of methylated proteins to the embryonic ectoderm development (EED) aromatic cage is essential for full catalytic activity, but details of this regulation are not fully understood. EED’s recognition of the product of PRC2 activity, histone H3 lysine 27 trimethylation (H3K27me3), stimulates PRC2 methyltransferase activity at adjacent nucleosomes leading to H3K27me3 propagation and, ultimately, gene repression. By coupling combinatorial chemistry and structure-based design, we optimized a low-affinity methylated jumonji, AT-rich interactive domain 2 (Jarid2) peptide to a smaller, more potent peptidomimetic ligand (Kd = 1.14 ± 0.14 μM) of the aromatic cage of EED. Our strategy illustrates the effectiveness of applying combinatorial chemistry to achieve both ligand potency and property optimization. Furthermore, the resulting ligands, UNC5114 and UNC5115, demonstrate that targeted disruption of EED’s reader function can lead to allosteric inhibition of PRC2 catalytic activity.

  4. Allosteric mechanism of action of the therapeutic anti-IgE antibody omalizumab.

    Science.gov (United States)

    Davies, Anna M; Allan, Elizabeth G; Keeble, Anthony H; Delgado, Jean; Cossins, Benjamin P; Mitropoulou, Alkistis N; Pang, Marie O Y; Ceska, Tom; Beavil, Andrew J; Craggs, Graham; Westwood, Marta; Henry, Alistair J; McDonnell, James M; Sutton, Brian J

    2017-06-16

    Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Allosteric Control of Substrate Specificity of the Escherichia coli ADP-glucose Pyrophosphorylase

    Science.gov (United States)

    Ebrecht, Ana C.; Solamen, Ligin; Hill, Benjamin L.; Iglesias, Alberto A.; Olsen, Kenneth W.; Ballicora, Miguel A.

    2017-06-01

    The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. However, there is growing evidence that many enzymes can catalyze alternative reactions. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. ADP-glucose pyrophosphorylase from Escherichia coli is an example of an enzyme that needs to select the correct substrate from a broad spectrum of alternatives. This selection will guide the flow of carbohydrate metabolism towards the synthesis of reserve polysaccharides. Here, we show that the allosteric activator fructose-1,6-bisphosphate plays a role in such selection by increasing the catalytic efficiency of the enzyme towards the use of ATP rather than other nucleotides. In the presence of fructose-1,6-bisphosphate, the kcat/S0.5 for ATP was near 600-fold higher that other nucleotides, whereas in the absence of activator was only 3-fold higher. We propose that the allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates.

  6. The Structural Basis for Allosteric Inhibition of a Threonine-sensitive Aspartokinase

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xuying; Pavlovsky, Alexander G.; Viola, Ronald E. (Toledo)

    2008-10-08

    The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK. Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation.

  7. Characterization of an allosteric citalopram-binding site at the serotonin transporter

    DEFF Research Database (Denmark)

    Chen, Fenghua; Breum Larsen, Mads; Neubauer, Henrik Amtoft

    2005-01-01

          rate, of [3H]S-citalopram from human SERT, is retarded by the presence of       serotonin, as well as by several antidepressants, when present in the       dissociation buffer. Dissociation of [3H]S-citalopram from SERT is most       potently inhibited by S-citalopram followed by R...... is independent of       temperature, or the presence of Na+ in the dissociation buffer.       Dissociation of [3H]S-citalopram from a complex with the SERT       double-mutant, N208Q/N217Q, which has been suggested to be unable to       self-assemble into oligomeric complexes, is retarded to an extent similar......       to that found with the wild-type, raising the possibility that the       allosteric mechanism is mediated within a single subunit. A       species-scanning mutagenesis study comparing human and bovine SERT       revealed that Met180, Tyr495 and Ser513 are important residues in       mediating the allosteric...

  8. Notes on stochastic (bio)-logic gates: computing with allosteric cooperativity

    Science.gov (United States)

    Agliari, Elena; Altavilla, Matteo; Barra, Adriano; Dello Schiavo, Lorenzo; Katz, Evgeny

    2015-05-01

    Recent experimental breakthroughs have finally allowed to implement in-vitro reaction kinetics (the so called enzyme based logic) which code for two-inputs logic gates and mimic the stochastic AND (and NAND) as well as the stochastic OR (and NOR). This accomplishment, together with the already-known single-input gates (performing as YES and NOT), provides a logic base and paves the way to the development of powerful biotechnological devices. However, as biochemical systems are always affected by the presence of noise (e.g. thermal), standard logic is not the correct theoretical reference framework, rather we show that statistical mechanics can work for this scope: here we formulate a complete statistical mechanical description of the Monod-Wyman-Changeaux allosteric model for both single and double ligand systems, with the purpose of exploring their practical capabilities to express noisy logical operators and/or perform stochastic logical operations. Mixing statistical mechanics with logics, and testing quantitatively the resulting findings on the available biochemical data, we successfully revise the concept of cooperativity (and anti-cooperativity) for allosteric systems, with particular emphasis on its computational capabilities, the related ranges and scaling of the involved parameters and its differences with classical cooperativity (and anti-cooperativity).

  9. Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics.

    Science.gov (United States)

    Arcisio-Miranda, Manoel; Muroi, Yukiko; Chowdhury, Sandipan; Chanda, Baron

    2010-11-01

    The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.

  10. Enzyme activity and allosteric characteristics of gamma-irradiated solid aspartate transcarbamylase

    International Nuclear Information System (INIS)

    Bigler, W.N.; Tolbert, B.M.

    1977-01-01

    Aspartate transcarbamylase purified from E. coli was lyophilized, irradiated in vacuo with γ radiation from a cesium-137 source, redissolved in buffer under a nitrogen atmosphere, and assayed for enzyme activity. Lyophilized and redissolved enzyme had normal catalytic and allosteric kinetic characteristics. The average D 37 observed with saturating substrate, 25 mM aspartate, was 4.1 Mrad. With less than saturating substrate, 5 mM aspartate, the activity increases from zero to 1.6 Mrad and then decreases with a D 37 of 7.2 Mrad. Inclusion of 1 mM CTP, an allosteric inhibitor, in the 5 mM aspartate assays results in a more pronounced maximum in the activity curve occurring at slightly higher dose, 2.2 Mrad. Inhibitability by CTP has a D 37 of 2.3 Mrad with doses below the activity maximum. Enzyme lyophilized in the presence of 1 mM CTP has a D 37 of 2.9 Mrad. ATCase activity changes caused by irradiation of lyophylized bacteria were qualitatively like the changes observed in the detailed studies with the purified enzyme. Apparent radiation sensitivities of ATCase in lyophilized bacteria were observed to vary with the technique used to disrupt the resuspended bacteria

  11. Allosteric substrate inhibition of Arabidopsis NAD-dependent malic enzyme 1 is released by fumarate.

    Science.gov (United States)

    Tronconi, Marcos Ariel; Wheeler, Mariel Claudia Gerrard; Martinatto, Andrea; Zubimendi, Juan Pablo; Andreo, Carlos Santiago; Drincovich, María Fabiana

    2015-03-01

    Plant mitochondria can use L-malate and fumarate, which accumulate in large levels, as respiratory substrates. In part, this property is due to the presence of NAD-dependent malic enzymes (NAD-ME) with particular biochemical characteristics. Arabidopsis NAD-ME1 exhibits a non-hyperbolic behavior for the substrate L-malate, and its activity is strongly stimulated by fumarate. Here, the possible structural connection between these properties was explored through mutagenesis, kinetics, and fluorescence studies. The results indicated that NAD-ME1 has a regulatory site for L-malate that can also bind fumarate. L-Malate binding to this site elicits a sigmoidal and low substrate-affinity response, whereas fumarate binding turns NAD-ME1 into a hyperbolic and high substrate affinity enzyme. This effect was also observed when the allosteric site was either removed or altered. Hence, fumarate is not really an activator, but suppresses the inhibitory effect of l-malate. In addition, residues Arg50, Arg80 and Arg84 showed different roles in organic acid binding. These residues form a triad, which is the basis of the homo and heterotrophic effects that characterize NAD-ME1. The binding of L-malate and fumarate at the same allosteric site is herein reported for a malic enzyme and clearly indicates an important role of NAD-ME1 in processes that control flow of C4 organic acids in Arabidopsis mitochondrial metabolism. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Colored Contact Lens Dangers

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  13. Colored Contact Lens Dangers

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  14. Colored Contact Lens Dangers

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  15. Colored Contact Lens Dangers

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  16. Colored Contact Lens Dangers

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    Full Text Available ... people with high myopia? Mar 29, 2017 New Technology Helps the Legally Blind Be More Independent Oct ... Multimedia Public & Patients: Contact Us About the Academy Jobs at the Academy Financial Relationships with Industry Medical ...

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  18. Colored Contact Lens Dangers

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  19. Ergonomics SA: Contact

    African Journals Online (AJOL)

    Principal Contact. Mrs June McDougall. Rhodes University. Department of Human Kinetics and Ergonomics. P.O. Box 94. Rhodes University. Grahamstown. 6140. Phone: +27 46 6038471. Email: j.mcdougall@ru.ac.za ...

  20. Colored Contact Lens Dangers

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  1. Tomato contact dermatitis

    DEFF Research Database (Denmark)

    Paulsen, Evy; Christensen, Lars P; Andersen, Klaus Ejner

    2012-01-01

    The tomato plant (Solanum lycopersicum) is an important crop worldwide. Whereas immediate-type reactions to tomato fruits are well known, contact dermatitis caused by tomatoes or tomato plants is rarely reported. The aims of this study were to present new data on contact sensitization to tomato...... plants and review the literature on contact dermatitis caused by both plants and fruits. An ether extract of tomato plants made as the original oleoresin plant extracts, was used in aimed patch testing, and between 2005 and 2011. 8 of 93 patients (9%) tested positive to the oleoresin extracts....... This prevalence is in accordance with the older literature that reports tomato plants as occasional sensitizers. The same applies to tomato fruits, which, in addition, may cause protein contact dermatitis. The allergens of the plant are unknown, but both heat-stable and heat-labile constituents seem...

  2. Fragrance allergic contact dermatitis.

    Science.gov (United States)

    Cheng, Judy; Zug, Kathryn A

    2014-01-01

    Fragrances are a common cause of allergic contact dermatitis in Europe and in North America. They can affect individuals at any age and elicit a spectrum of reactions from contact urticaria to systemic contact dermatitis. Growing recognition of the widespread use of fragrances in modern society has fueled attempts to prevent sensitization through improved allergen identification, labeling, and consumer education. This review provides an overview and update on fragrance allergy. Part 1 discusses the epidemiology and evaluation of suspected fragrance allergy. Part 2 reviews screening methods, emerging fragrance allergens, and management of patients with fragrance contact allergy. This review concludes by examining recent legislation on fragrances and suggesting potential additions to screening series to help prevent and detect fragrance allergy.

  3. Colored Contact Lens Dangers

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    Full Text Available ... Cleveland. "This is far from the truth." Real People, Real Problems with Colored Contact Lenses Julian: Teenager ... About the Academy Jobs at the Academy Financial Relationships with Industry Medical Disclaimer Privacy Policy Terms of ...

  4. Colored Contact Lens Dangers

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    Full Text Available ... Follow The Academy Professionals: Education Guidelines News Multimedia Public & Patients: Contact Us About the Academy Jobs at the Academy Financial Relationships with Industry Medical Disclaimer Privacy Policy Terms of Service For Advertisers For Media Ophthalmology ...

  5. Colored Contact Lens Dangers

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  6. Colored Contact Lens Dangers

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    Full Text Available ... an impulsive buy from a souvenir shop, but 10 hours after she first put in a pair ... Prescription Contact Lens Laura: Vision Loss After Just 10 Hours Robyn: Blurry Vision and Daily Eye Drops ...

  7. Colored Contact Lens Dangers

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    Full Text Available ... in beauty salons, novelty shops or in pop-up Halloween stores are not FDA-approved and are ... share contact lenses with another person. Get follow up exams with your eye care provider. If you ...

  8. Colored Contact Lens Dangers

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    Full Text Available ... videos on your website Promotional materials for eye health observances EyeSmart resources are also available in Spanish . Follow The Academy Professionals: Education Guidelines News Multimedia Public & Patients: Contact Us About the Academy Jobs at ...

  9. GAS-FOVEAL CONTACT

    DEFF Research Database (Denmark)

    Alberti, Mark; la Cour, Morten

    2018-01-01

    PURPOSE: To compare gas-foveal contact in face-down positioning (FDP) and nonsupine positioning (NSP), to analyze causes of gas-foveal separation and to determine how gas-foveal contact affects clinical outcome after idiopathic macular hole repair. METHODS: Single center, randomized controlled...... study. Participants with an idiopathic macular hole were allocated to either FDP or NSP. Primary outcome was gas-foveal contact, calculated by analyzing positioning in relation to intraocular gas fill. Positioning was measured with an electronic device recording positioning for 72 hours postoperatively....... RESULTS: Positioning data were available for 33/35 in the FDP group and 35/37 in the NSP group, thus results are based on 68 analyzed participants. Median gas-foveal contact was 99.82% (range 73.6-100.0) in the FDP group and 99.57% (range 85.3-100.0) in the NSP group (P = 0.22). In a statistical model...

  10. Colored Contact Lens Dangers

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  11. Colored Contact Lens Dangers

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  12. Colored Contact Lens Dangers

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  13. Colored Contact Lens Dangers

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  14. Colored Contact Lens Dangers

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  15. Colored Contact Lens Dangers

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  16. Colored Contact Lens Dangers

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  17. Colored Contact Lens Dangers

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    Full Text Available ... also available in Spanish . Follow The Academy Professionals: Education Guidelines News Multimedia Public & Patients: Contact Us About the Academy Jobs at the Academy Financial Relationships with Industry Medical Disclaimer Privacy Policy Terms ...

  18. Colored Contact Lens Dangers

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  19. Colored Contact Lens Dangers

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  20. Contact Lens Risks

    Science.gov (United States)

    ... Tap and distilled water have been associated with Acanthamoeba keratitis, a corneal infection that is resistant to ... to: Advice for Patients With Soft Contact Lenses: Acanthamoeba Keratitis Infections Related to Complete® MoisturePlus Multi Purpose ...