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Sample records for reveals f-actin dynamics

  1. A dynamic formin-dependent deep F-actin network in axons

    PubMed Central

    Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe

    2015-01-01

    Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902

  2. Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering*

    PubMed Central

    Jayasundar, Jayant James; Ju, Jeong Ho; He, Lilin; Liu, Dazhi; Meilleur, Flora; Zhao, Jinkui; Callaway, David J. E.; Bu, Zimei

    2012-01-01

    Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP2. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP2 binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP2 binding. The study provides the first structural view of the activated ezrin bound to PIP2 and to F-actin. PMID:22927432

  3. Dynamic and elastic properties of F-actin: a normal-modes analysis.

    PubMed Central

    ben-Avraham, D; Tirion, M M

    1995-01-01

    We examine the dynamic, elastic, and mechanical consequences of the proposed atomic models of F-actin, using a normal mode analysis. This initial analysis is done in vacuo and assumes that all monomers are rigid and equivalent. Our computation proceeds from the atomic level and, relying on a single fitting parameter, reproduces various experimental results, including persistence lengths, elastic moduli, and contact energies. The computations reveal modes of motion characteristic to all polymers, such as longitudinal pressure waves, torsional waves, and bending, as well as motions unique to F-actin. Motions typical to actin include a "groove-swinging" motion of the two long-pitch helices, as well as an axial slipping motion of the two strands. We prepare snapshots of thermally activated filaments and quantify the accumulation of azimuthal angular "disorder," variations in cross-over lengths, and various other fluctuations. We find that the orientation of a small number of select residues has a surprisingly large effect on the filament flexibility and elasticity characteristics. PMID:7787015

  4. Cofilin 1-Mediated Biphasic F-Actin Dynamics of Neuronal Cells Affect Herpes Simplex Virus 1 Infection and Replication

    PubMed Central

    Xiang, Yangfei; Zheng, Kai; Ju, Huaiqiang; Wang, Shaoxiang; Pei, Ying; Ding, Weichao; Chen, Zhenping; Wang, Qiaoli; Qiu, Xianxiu; Zhong, Meigong; Zeng, Fanli; Ren, Zhe; Qian, Chuiwen; Liu, Ge

    2012-01-01

    Herpes simplex virus 1 (HSV-1) invades the nervous system and causes pathological changes. In this study, we defined the remodeling of F-actin and its possible mechanisms during HSV-1 infection of neuronal cells. HSV-1 infection enhanced the formation of F-actin-based structures in the early stage of infection, which was followed by a continuous decrease in F-actin during the later stages of infection. The disruption of F-actin dynamics by chemical inhibitors significantly reduced the efficiency of viral infection and intracellular HSV-1 replication. The active form of the actin-depolymerizing factor cofilin 1 was found to increase at an early stage of infection and then to continuously decrease in a manner that corresponded to the remodeling pattern of F-actin, suggesting that cofilin 1 may be involved in the biphasic F-actin dynamics induced by HSV-1 infection. Knockdown of cofilin 1 impaired HSV-1-induced F-actin assembly during early infection and inhibited viral entry; however, overexpression of cofilin 1 did not affect F-actin assembly or viral entry during early infection but decreased intracellular viral reproduction efficiently. Our results, for the first time, demonstrated the biphasic F-actin dynamics in HSV-1 neuronal infection and confirmed the association of F-actin with the changes in the expression and activity of cofilin 1. These results may provide insight into the mechanism by which HSV-1 productively infects neuronal cells and causes pathogenesis. PMID:22623803

  5. In vivo imaging of cell behaviors and F-actin reveals LIM-HD transcription factor regulation of peripheral versus central sensory axon development

    PubMed Central

    2011-01-01

    Background Development of specific neuronal morphology requires precise control over cell motility processes, including axon formation, outgrowth and branching. Dynamic remodeling of the filamentous actin (F-actin) cytoskeleton is critical for these processes; however, little is known about the mechanisms controlling motile axon behaviors and F-actin dynamics in vivo. Neuronal structure is specified in part by intrinsic transcription factor activity, yet the molecular and cellular steps between transcription and axon behavior are not well understood. Zebrafish Rohon-Beard (RB) sensory neurons have a unique morphology, with central axons that extend in the spinal cord and a peripheral axon that innervates the skin. LIM homeodomain (LIM-HD) transcription factor activity is required for formation of peripheral RB axons. To understand how neuronal morphogenesis is controlled in vivo and how LIM-HD transcription factor activity differentially regulates peripheral versus central axons, we used live imaging of axon behavior and F-actin distribution in vivo. Results We used an F-actin biosensor containing the actin-binding domain of utrophin to characterize actin rearrangements during specific developmental processes in vivo, including axon initiation, consolidation and branching. We found that peripheral axons initiate from a specific cellular compartment and that F-actin accumulation and protrusive activity precede peripheral axon initiation. Moreover, disruption of LIM-HD transcriptional activity has different effects on the motility of peripheral versus central axons; it inhibits peripheral axon initiation, growth and branching, while increasing the growth rate of central axons. Our imaging revealed that LIM-HD transcription factor activity is not required for F-actin based protrusive activity or F-actin accumulation during peripheral axon initiation, but can affect positioning of F-actin accumulation and axon formation. Conclusion Our ability to image the dynamics of

  6. Entangled F-actin displays a unique crossover to microscale nonlinearity dominated by entanglement segment dynamics.

    PubMed

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

    2015-06-14

    We drive optically trapped microspheres through entangled F-actin at constant speeds and distances well beyond the linear regime, and measure the microscale force response of the entangled filaments during and following strain. Our results reveal a unique crossover to appreciable nonlinearity at a strain rate of [small gamma, Greek, dot above]c ≈ 3 s(-1) which corresponds remarkably well with the theoretical rate of relaxation of entanglement length deformations 1/τent. Above [small gamma, Greek, dot above]c, we observe stress stiffening which occurs over very short time scales comparable to the predicted timescale over which mesh size deformations relax. Stress softening then takes over, yielding to an effectively viscous regime over a timescale comparable to the entanglement length relaxation time, τent. The viscous regime displays shear thinning but with a less pronounced viscosity scaling with strain rate compared to flexible polymers. The relaxation of induced force on filaments following strain shows that the relative relaxation proceeds more quickly for increasing strain rates; and for rates greater than [small gamma, Greek, dot above]c, the relaxation displays a complex power-law dependence on time. Our collective results reveal that molecular-level nonlinear viscoelasticity is driven by non-classical dynamics of individual entanglement segments that are unique to semiflexible polymers. PMID:25920523

  7. In vivo dynamics of the F-actin-binding protein neurabin-II.

    PubMed Central

    Stephens, D J; Banting, G

    2000-01-01

    Neurabin-II (spinophilin) is a ubiquitously expressed F-actin-binding protein containing an N-terminal actin-binding domain, a PDZ (PSD95/discs large/ZO-1) domain and a C-terminal domain predicted to form a coiled-coil structure. We have stably expressed a green fluorescent protein (GFP)-tagged version of neurabin-II in PC12 cells, and characterized the in vivo dynamics of this actin-binding protein using confocal fluorescence microscopy. We show that GFP-neurabin-II localizes to actin filaments, especially at cortical sites and areas underlying sites of active membrane remodelling. GFP-neurabin-II labels only a subset of F-actin within these cells, as indicated by rhodamine-phalloidin staining. Both actin filaments and small, highly motile structures within the cell body are seen. Photobleaching experiments show that GFP-neurabin-II also exhibits highly dynamic behaviour when bound to actin filaments. Latrunculin B treatment results in rapid relocalization of GFP-neurabin-II to the cytosol, whereas cytochalasin D treatment causes the collapse of GFP-neurabin-II fluorescence to intensely fluorescent foci of F-actin within the cell body. This collapse is reversed on cytochalasin D removal, recovery from which is greatly accelerated by stimulation of cells with epidermal growth factor (EGF). Furthermore, we show that this EGF-induced relocalization of GFP-neurabin-II is dependent on the activity of the small GTPase Rac1 but not the activity of ADP-ribosylation factor 6. PMID:10620493

  8. Live imaging provides new insights on dynamic F-actin filopodia and differential endocytosis during myoblast fusion in Drosophila.

    PubMed

    Haralalka, Shruti; Shelton, Claude; Cartwright, Heather N; Guo, Fengli; Trimble, Rhonda; Kumar, Ram P; Abmayr, Susan M

    2014-01-01

    The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the

  9. Docking, molecular dynamics and QM/MM studies to delineate the mode of binding of CucurbitacinE to F-actin.

    PubMed

    Kumar, R Pravin; Roopa, L; Nongthomba, Upendra; Sudheer Mohammed, M M; Kulkarni, Naveen

    2016-01-01

    CucurbitacinE (CurE) has been known to bind covalently to F-actin and inhibit depolymerization. However, the mode of binding of CurE to F-actin and the consequent changes in the F-actin dynamics have not been studied. Through quantum mechanical/molecular mechanical (QM/MM) and density function theory (DFT) simulations after the molecular dynamics (MD) simulations of the docked complex of F-actin and CurE, a detailed transition state (TS) model for the Michael reaction is proposed. The TS model shows nucleophilic attack of the sulphur of Cys257 at the β-carbon of Michael Acceptor of CurE producing an enol intermediate that forms a covalent bond with CurE. The MD results show a clear difference between the structure of the F-actin in free form and F-actin complexed with CurE. CurE affects the conformation of the nucleotide binding pocket increasing the binding affinity between F-actin and ADP, which in turn could affect the nucleotide exchange. CurE binding also limits the correlated displacement of the relatively flexible domain 1 of F-actin causing the protein to retain a flat structure and to transform into a stable "tense" state. This structural transition could inhibit depolymerization of F-actin. In conclusion, CurE allosterically modulates ADP and stabilizes F-actin structure, thereby affecting nucleotide exchange and depolymerization of F-actin. PMID:26615469

  10. F-actin remodeling defects as revealed in primary immunodeficiency disorders.

    PubMed

    Janssen, W J M; Geluk, H C A; Boes, M

    2016-03-01

    Primary immunodeficiencies (PIDs) are a heterogeneous group of immune-related diseases. PIDs develop due to defects in gene-products that have consequences to immune cell function. A number of PID-proteins is involved in the remodeling of filamentous actin (f-actin) to support the generation of a contact zone between the antigen-specific T cell and antigen presenting cell (APC): the immunological synapse (IS). IS formation is the first step towards T-cell activation and essential for clonal expansion and acquisition of effector function. We here evaluated PIDs in which aberrant f-actin-driven IS formation may contribute to the PID disease phenotypes as seen in patients. We review examples of such contributions to PID phenotypes from literature, and highlight cases in which PID-proteins were evaluated for a role in f-actin polymerization and IS formation. We conclude with the proposition that patient groups might benefit from stratifying them in distinct functional groups in regard to their f-actin remodeling phenotypes in lymphocytes. PMID:26802313

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

    PubMed

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

    2015-12-01

    Azimuthal movement of tropomyosin around the F-actin thin filament is responsible for muscle activation and relaxation. Recently a model of αα-tropomyosin, derived from molecular-mechanics and electron microscopy of different contractile states, showed that tropomyosin is rather stiff and pre-bent to present one specific face to F-actin during azimuthal transitions. However, a new model based on cryo-EM of troponin- and myosin-free filaments proposes that the interacting-face of tropomyosin can differ significantly from that in the original model. Because resolution was insufficient to assign tropomyosin side-chains, the interacting-face could not be unambiguously determined. Here, we use structural analysis and energy landscapes to further examine the proposed models. The observed bend in seven crystal structures of tropomyosin is much closer in direction and extent to the original model than to the new model. Additionally, we computed the interaction map for repositioning tropomyosin over the F-actin surface, but now extended over a much larger surface than previously (using the original interacting-face). This map shows two energy minima-one corresponding to the "blocked-state" as in the original model, and the other related by a simple 24 Å translation of tropomyosin parallel to the F-actin axis. The tropomyosin-actin complex defined by the second minimum fits perfectly into the recent cryo-EM density, without requiring any change in the interacting-face. Together, these data suggest that movement of tropomyosin between regulatory states does not require interacting-face rotation. Further, they imply that thin filament assembly may involve an interplay between initially seeded tropomyosin molecules growing from distinct binding-site regions on actin. PMID:26286845

  12. Disentangling Membrane Dynamics and Cell Migration; Differential Influences of F-actin and Cell-Matrix Adhesions

    PubMed Central

    Kowalewski, Jacob M.; Shafqat-Abbasi, Hamdah; Jafari-Mamaghani, Mehrdad; Endrias Ganebo, Bereket; Gong, Xiaowei

    2015-01-01

    Cell migration is heavily interconnected with plasma membrane protrusion and retraction (collectively termed “membrane dynamics”). This makes it difficult to distinguish regulatory mechanisms that differentially influence migration and membrane dynamics. Yet such distinctions may be valuable given evidence that cancer cell invasion in 3D may be better predicted by 2D membrane dynamics than by 2D cell migration, implying a degree of functional independence between these processes. Here, we applied multi-scale single cell imaging and a systematic statistical approach to disentangle regulatory associations underlying either migration or membrane dynamics. This revealed preferential correlations between membrane dynamics and F-actin features, contrasting with an enrichment of links between cell migration and adhesion complex properties. These correlative linkages were often non-linear and therefore context-dependent, strengthening or weakening with spontaneous heterogeneity in cell behavior. More broadly, we observed that slow moving cells tend to increase in area, while fast moving cells tend to shrink, and that the size of dynamic membrane domains is independent of cell area. Overall, we define macromolecular features preferentially associated with either cell migration or membrane dynamics, enabling more specific interrogation and targeting of these processes in future. PMID:26248038

  13. Characterization of ring-like F-actin structure as a mechanical partner for spindle positioning in mitosis.

    PubMed

    Lu, Huan; Zhao, Qun; Jiang, Hao; Zhu, Tongge; Xia, Peng; Seffens, William; Aikhionbare, Felix; Wang, Dongmei; Dou, Zhen; Yao, Xuebiao

    2014-01-01

    Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin). Although mounting evidence demonstrates the role of F-actin in cortical cytoskeleton dynamics, it remains elusive as to the structure and function of F-actin-based networks in spindle geometry. Here we showed a ring-like F-actin structure surrounding the mitotic spindle which forms since metaphase and maintains in MG132-arrested metaphase HeLa cells. This cytoplasmic F-actin structure is relatively isotropic and less dynamic. Our computational modeling of spindle position process suggests a possible mechanism by which the ring-like F-actin structure can regulate astral microtubule dynamics and thus mitotic spindle orientation. We further demonstrated that inhibiting Plk1, Mps1 or Myosin, and disruption of microtubules or F-actin polymerization perturbs the formation of the ring-like F-actin structure and alters spindle position and symmetric division. These findings reveal a previously unrecognized but important link between mitotic spindle and ring-like F-actin network in accurate mitosis and enables the development of a method to theoretically illustrate the relationship between mitotic spindle and cytoplasmic F-actin. PMID:25299690

  14. ROP Gtpase–Dependent Dynamics of Tip-Localized F-Actin Controls Tip Growth in Pollen Tubes

    PubMed Central

    Fu, Ying; Wu, Guang; Yang, Zhenbiao

    2001-01-01

    Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein–tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase–activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants. PMID:11238457

  15. Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

    SciTech Connect

    Solecki, Dr. David; Trivedi, Dr. Niraj; Govek, Eve-Ellen; Kerekes, Ryan A; Gleason, Shaun Scott; Hatten, Mary E

    2009-01-01

    Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6{alpha} localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6{alpha} inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to 'pull' the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6{alpha}.

  16. The Actin-binding Domain of Cortactin is Dynamic and Unstructured and Affects Lateral and Longitudinal Contacts in F-actin

    PubMed Central

    Shvetsov, Alexander; Berkane, Emir; Chereau, David; Dominguez, Roberto; Reisler, Emil

    2011-01-01

    Cortactin is an F-actin- and Arp2/3 complex-binding protein, implicated in the regulation of cytoskeleton dynamics and cortical actin-assembly. The actin-binding domain of cortactin consists of a 6.5 tandem repeat of a 37-amino acid sequence known as the cortactin repeat (residues 80-325). Using a combination of structure prediction, circular dichroism and cysteine crosslinking, we tested a recently published three-dimensional model of the cortactin molecule in which the cortactin repeat is folded as a globular helical domain (Zhang et al., 2007). We show that the cortactin repeat is unstructured in solution. Thus, wild type and mutant constructs of the cortactin repeat, containing pairs of cysteines at positions 112 and 246, 83 and 112, 83 and 246, and 83 and 306, could be readily crosslinked with reagents of varying lengths (0–9.6 Å). Using yeast actin cysteine mutants, we also show that cortactin inhibits disulfide and dibromobimane crosslinking across the lateral and longitudinal interfaces of actin subunits in the filament, suggesting a weakening of inter-subunits contacts. Our results are in disagreement with the proposed model of the cortactin molecule and have important implications for our understanding of cortactin regulation of cytoskeleton dynamics. PMID:19089942

  17. The actin-binding domain of cortactin is dynamic and unstructured and affects lateral and longitudinal contacts in F-actin.

    PubMed

    Shvetsov, Alexander; Berkane, Emir; Chereau, David; Dominguez, Roberto; Reisler, Emil

    2009-02-01

    Cortactin is an F-actin- and Arp2/3 complex-binding protein, implicated in the regulation of cytoskeleton dynamics and cortical actin-assembly. The actin-binding domain of cortactin consists of a 6.5 tandem repeat of a 37-amino acid sequence known as the cortactin repeat (residues 80-325). Using a combination of structure prediction, circular dichroism, and cysteine crosslinking, we tested a recently published three-dimensional model of the cortactin molecule in which the cortactin repeat is folded as a globular helical domain [Zhang et al., 2007, Mol Cell 27:197-213]. We show that the cortactin repeat is unstructured in solution. Thus, wild type and mutant constructs of the cortactin repeat, containing pairs of cysteines at positions 112 and 246, 83 and 112, 83 and 246, and 83 and 306, could be readily crosslinked with reagents of varying lengths (0-9.6 A). Using yeast actin cysteine mutants, we also show that cortactin inhibits disulfide and dibromobimane crosslinking across the lateral and longitudinal interfaces of actin subunits in the filament, suggesting a weakening of intersubunits contacts. Our results are in disagreement with the proposed model of the cortactin molecule and have important implications for our understanding of cortactin regulation of cytoskeleton dynamics. PMID:19089942

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

    PubMed

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

    2015-03-01

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

  19. Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

    PubMed Central

    Gao, Ying; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES. PMID:27358069

  20. Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells.

    PubMed

    Gao, Ying; Lui, Wing-Yee; Lee, Will M; Cheng, C Yan

    2016-01-01

    Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES. PMID:27358069

  1. F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling

    PubMed Central

    Johnson, Heath E.; King, Samantha J.; Asokan, Sreeja B.; Rotty, Jeremy D.; Bear, James E.

    2015-01-01

    Mesenchymal cells such as fibroblasts are weakly polarized and reorient directionality by a lamellipodial branching mechanism that is stabilized by phosphoinositide 3-kinase (PI3K) signaling. However, the mechanisms by which new lamellipodia are initiated and directed are unknown. Using total internal reflection fluorescence microscopy to monitor cytoskeletal and signaling dynamics in migrating cells, we show that peripheral F-actin bundles/filopodia containing fascin-1 serve as templates for formation and orientation of lamellipodia. Accordingly, modulation of fascin-1 expression tunes cell shape, quantified as the number of morphological extensions. Ratiometric imaging reveals that F-actin bundles/filopodia play both structural and signaling roles, as they prime the activation of PI3K signaling mediated by integrins and focal adhesion kinase. Depletion of fascin-1 ablated fibroblast haptotaxis on fibronectin but not platelet-derived growth factor chemotaxis. Based on these findings, we conceptualize haptotactic sensing as an exploration, with F-actin bundles directing and lamellipodia propagating the process and with signaling mediated by adhesions playing the role of integrator. PMID:25666809

  2. Piccolo Directs Activity Dependent F-Actin Assembly from Presynaptic Active Zones via Daam1

    PubMed Central

    Wagh, Dhananjay; Terry-Lorenzo, Ryan; Waites, Clarissa L.; Leal-Ortiz, Sergio A.; Maas, Christoph; Reimer, Richard J.; Garner, Craig C.

    2015-01-01

    The dynamic assembly of filamentous (F) actin plays essential roles in the assembly of presynaptic boutons, the fusion, mobilization and recycling of synaptic vesicles (SVs), and presynaptic forms of plasticity. However, the molecular mechanisms that regulate the temporal and spatial assembly of presynaptic F-actin remain largely unknown. Similar to other F-actin rich membrane specializations, presynaptic boutons contain a set of molecules that respond to cellular cues and trans-synaptic signals to facilitate activity-dependent assembly of F-actin. The presynaptic active zone (AZ) protein Piccolo has recently been identified as a key regulator of neurotransmitter release during SV cycling. It does so by coordinating the activity-dependent assembly of F-Actin and the dynamics of key plasticity molecules including Synapsin1, Profilin and CaMKII. The multidomain structure of Piccolo, its exquisite association with the AZ, and its ability to interact with a number of actin-associated proteins suggest that Piccolo may function as a platform to coordinate the spatial assembly of F-actin. Here we have identified Daam1, a Formin that functions with Profilin to drive F-actin assembly, as a novel Piccolo binding partner. We also found that within cells Daam1 activation promotes Piccolo binding, an interaction that can spatially direct the polymerization of F-Actin. Moreover, similar to Piccolo and Profilin, Daam1 loss of function impairs presynaptic-F-actin assembly in neurons. These data suggest a model in which Piccolo directs the assembly of presynaptic F-Actin from the AZ by scaffolding key actin regulatory proteins including Daam1. PMID:25897839

  3. Membrane Supply and Demand Regulates F-Actin in a Cell Surface Reservoir.

    PubMed

    Figard, Lauren; Wang, Mengyu; Zheng, Liuliu; Golding, Ido; Sokac, Anna Marie

    2016-05-01

    Cells store membrane in surface reservoirs of pits and protrusions. These membrane reservoirs facilitate cell shape change and buffer mechanical stress, but we do not know how reservoir dynamics are regulated. During cellularization, the first cytokinesis in Drosophila embryos, a reservoir of microvilli unfolds to fuel cleavage furrow ingression. We find that regulated exocytosis adds membrane to the reservoir before and during unfolding. Dynamic F-actin deforms exocytosed membrane into microvilli. Single microvilli extend and retract in ∼20 s, while the overall reservoir is depleted in sync with furrow ingression over 60-70 min. Using pharmacological and genetic perturbations, we show that exocytosis promotes microvillar F-actin assembly, while furrow ingression controls microvillar F-actin disassembly. Thus, reservoir F-actin and, consequently, reservoir dynamics are regulated by membrane supply from exocytosis and membrane demand from furrow ingression. PMID:27165556

  4. F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex

    PubMed Central

    Murrell, Michael P.; Gardel, Margaret L.

    2012-01-01

    Here we develop a minimal model of the cell actomyosin cortex by forming a quasi-2D cross-linked filamentous actin (F-actin) network adhered to a model cell membrane and contracted by myosin thick filaments. Myosin motors generate both compressive and tensile stresses on F-actin and consequently induce large bending fluctuations, which reduces their effective persistence length to <1 μm. Over a large range of conditions, we show the extent of network contraction corresponds exactly to the extent of individual F-actin shortening via buckling. This demonstrates an essential role of buckling in breaking the symmetry between tensile and compressive stresses to facilitate mesoscale network contraction of up to 80% strain. Portions of buckled F-actin with a radius of curvature ∼300 nm are prone to severing and thus compressive stresses mechanically coordinate contractility with F-actin severing, the initial step of F-actin turnover. Finally, the F-actin curvature acquired by myosin-induced stresses can be further constrained by adhesion of the network to a membrane, accelerating filament severing but inhibiting the long-range transmission of the stresses necessary for network contractility. Thus, the extent of membrane adhesion can regulate the coupling between network contraction and F-actin severing. These data demonstrate the essential role of the nonlinear response of F-actin to compressive stresses in potentiating both myosin-mediated contractility and filament severing. This may serve as a general mechanism to mechanically coordinate contractility and cortical dynamics across diverse actomyosin assemblies in smooth muscle and nonmuscle cells. PMID:23213249

  5. Phospholipase Cη2 Activation Redirects Vesicle Trafficking by Regulating F-actin*

    PubMed Central

    Yamaga, Masaki; Kielar-Grevstad, D. Michelle; Martin, Thomas F. J.

    2015-01-01

    PI(4,5)P2 localizes to sites of dense core vesicle exocytosis in neuroendocrine cells and is required for Ca2+-triggered vesicle exocytosis, but the impact of local PI(4,5)P2 hydrolysis on exocytosis is poorly understood. Previously, we reported that Ca2+-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P2 hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (also known as CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca2+-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 or 95 mm KCl buffers increased peak Ca2+ levels to ∼400 or ∼800 nm, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mm K+ preferentially elicited the exocytosis of plasma membrane-resident vesicles, whereas 95 mm K+ preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mm K+ but not with 56 mm K+ activated PLCη2 to catalyze PI(4,5)P2 hydrolysis. The decrease in PI(4,5)P2 promoted F-actin disassembly, which increased exocytosis of newly arriving vesicles. Consistent with its role as a Ca2+-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P2 for coordinating cytoskeletal dynamics with vesicle exocytosis and reveal a new role for PLCη2 as a Ca2+-dependent regulator of F-actin dynamics and vesicle trafficking. PMID:26432644

  6. Double localization of F-actin in chemoattractant-stimulated polymorphonuclear leucocytes.

    PubMed

    Lepidi, H; Benoliel, A M; Mege, J L; Bongrand, P; Capo, C

    1992-09-01

    Uniform concentrations of chemoattractants such as formylpeptides induced a morphological polarization of human polymorphonuclear leucocytes (PMNs) and a concentration of F-actin at the cell front. They also induced a transient increase in filamentous actin (F-actin) which preceded the cell shape change. We combined fluorescence microscopy and image analysis to study the localization of F-actin, as revealed by a specific probe (bodipyTM phallacidin) in suspended PMNs stimulated by chemoattractants. F-actin exhibited remarkable concentration in focal points after a 30 s exposure to 10(-8) M formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), although no shape change of PMNs was detectable. A 10-min incubation with formylpeptide (10(-6) to 10(-9) M) induced the morphological polarization of PMNs and the appearance of a principal focus of F-actin in the cell head region and a secondary focus in the cell posterior end. The distribution of F-actin-associated fluorescence in 2D images of polarized PMNs might be due to an actual concentration of F-actin in privileged areas, to a local concentration of plasma membrane drawing filamentous actin or to variations in the cell volume. Then, we studied the distribution of a cytoplasmic marker, fluorescein diacetate and a membrane probe, TMA-DPH, in unstimulated rounded PMNs and in spherical and morphologically polarized PMNs stimulated by formylpeptide. The distribution of neither of these probes was correlated with F-actin distribution, especially in rounded PMNs stimulated 30 s with 10(-8) M fMet-Leu-Phe, suggesting that F-actin was concentrated in two foci located in the cell head region and in the cell posterior end. In addition, zymosan-activated serum induced the morphological polarization of PMNs and the appearance of two foci of filamentous actin, demonstrating that binding of formylpeptide to its specific receptor was not required for F-actin reorganization. We conclude that the accumulation of F-actin probably

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

    PubMed

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

    2014-08-01

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

  8. Short Stop provides an essential link between F-actin and microtubules during axon extension.

    PubMed

    Lee, Seungbok; Kolodziej, Peter A

    2002-03-01

    Coordination of F-actin and microtubule dynamics is important for cellular motility and morphogenesis, but little is known about underlying mechanisms. short stop (shot) encodes an evolutionarily conserved, neuronally expressed family of rod-like proteins required for sensory and motor axon extension in Drosophila melanogaster. We identify Shot isoforms that contain N-terminal F-actin and C-terminal microtubule-binding domains, and that crosslink F-actin and microtubules in cultured cells. The F-actin- and microtubule-binding domains of Shot are required in the same molecule for axon extension, though the length of the connecting rod domain can be dramatically reduced without affecting activity. Shot therefore functions as a cytoskeletal crosslinker in axon extension, rather than mediating independent interactions with F-actin and microtubules. A Ca(2+)-binding motif located adjacent to the microtubule-binding domain is also required for axon extension, suggesting that intracellular Ca(2+) release may regulate Shot activity. These results suggest that Shot coordinates regulated interactions between F-actin and microtubules that are crucial for neuronal morphogenesis. PMID:11874915

  9. Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

    PubMed Central

    Huang, Junqi; Huang, Yinyi; Yu, Haochen; Subramanian, Dhivya; Padmanabhan, Anup; Thadani, Rahul; Tao, Yaqiong; Tang, Xie; Wedlich-Soldner, Roland

    2012-01-01

    In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly. PMID:23185032

  10. Supercoiling of f-actin filaments.

    PubMed

    Lednev, V V; Popp, D

    1990-05-01

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

  11. The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility.

    PubMed

    Faix, J; Clougherty, C; Konzok, A; Mintert, U; Murphy, J; Albrecht, R; Mühlbauer, B; Kuhlmann, J

    1998-10-01

    DGAP1 of Dictyostelium discoideum is a cell cortex associated 95 kDa protein that shows homology to both RasGTPase-activating proteins (RasGAPs) and RasGAP-related proteins. When tested for RasGAP activity, recombinant DGAP1 protein did not promote the GTPase activity of human H-Ras or of Dictyostelium RasG in vitro. Instead, DGAP1 bound to Dictyostelium Rac1A and human Rac1, but not to human Cdc42. DGAP1 preferentially interacted with the activated GTP-bound forms of Rac1 and Rac1A, but did not affect the GTPase activities. Since Rho-type GTPases are implicated in the formation of specific F-actin structures and in the control of cell morphology, the microfilament system of mutants that either lack or overexpress DGAP1 has been analysed. DGAP1-null mutants showed elevated levels of F-actin that was organised in large leading edges, membrane ruffles or numerous large filopods. Expression of actin fused to green fluorescent protein (GFP) was used to monitor the actin dynamics in these cells, and revealed that the F-actin cytoskeleton of DGAP1-null cells was rapidly re-arranged to form ruffles and filopods. Conversely, in DGAP1-overexpressing cells, the formation of cellular projections containing F-actin was largely suppressed. Measurement of cell migration demonstrated that DGAP1 expression is inversely correlated with the speed of cell motility. PMID:9739079

  12. Drebrin inhibits cofilin-induced severing of F-actin.

    PubMed

    Grintsevich, Elena E; Reisler, Emil

    2014-08-01

    Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer's disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor's binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments. PMID:25047716

  13. Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

    SciTech Connect

    Aoyama, Kazumasa; Yuki, Ryuzaburo; Horiike, Yasuyoshi; Kubota, Sho; Yamaguchi, Noritaka; Morii, Mariko; Ishibashi, Kenichi; Nakayama, Yuji; Kuga, Takahisa; Hashimoto, Yuuki; Tomonaga, Takeshi; Yamaguchi, Naoto

    2013-12-10

    The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus. - Highlights: • We show the involvement of c-Abl tyrosine kinase in nuclear actin dynamics. • Nuclear F-actin is formed by nuclear-localized c-Abl and its kinase-dead version. • The c-Abl actin-binding domain is prerequisite for nuclear F-actin formation. • Formation of long nuclear F-actin bundles requires nuclear c-Abl kinase activity. • We discuss a role for nuclear F-actin bundle formation in chromatin regulation.

  14. Hierarchical Cross-linked F-actin Networks: Understanding Structure and Assembly

    NASA Astrophysics Data System (ADS)

    Hirst, Linda; Nguyen, Lam

    2009-11-01

    The protein, F-actin provides us with an interesting system in which to investigate the assembly properties of semi-flexible filaments in the presence of cross-linkers. Recently it was observed that F-actin, in the presence of the cross-linker alpha-actinin at high molar ratios will generate a novel hierarchical network of filament bundles. We investigate this system using coarse-grained molecular dynamics (MD) simulation, confocal microscopy and x-ray scattering. We have studied the F-actin/alpha-actinin system in detail with different actin conc. (C) and alpha-actinin/actin molar ratios (gamma). Confocal microscopy and analysis shows that the assembled systems fall into one of 3 phases depending on C and gamma: (1) loosely connected network of F-actin and bundles, (2) loosely connected network of dense domains and (3) uniform network of bundles. This can be explained and replicated using MD simulation. We have also examined different types of cross-linkers to represent the proteins, fascin and filamin. Results show that phase formation is related to the flexibility in binding between F-actin and cross-linkers. This degree of freedom, possible with longer cross-linkers allows the formation of branch points and thus bundle networks.

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

    PubMed

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

    2001-09-01

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

  16. Three cotton genes preferentially expressed in flower tissues encode actin-depolymerizing factors which are involved in F-actin dynamics in cells

    PubMed Central

    Li, Xue-Bao; Xu, Dan; Wang, Xiu-Lan; Huang, Geng-Qing; Luo, Juan; Li, Deng-Di; Zhang, Ze-Ting; Xu, Wen-Liang

    2010-01-01

    To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5′-flanking region, including the promoter and 5′-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination. PMID:19861654

  17. Self-organized Gels in DNA/F-Actin mixtures without Crosslinkers

    NASA Astrophysics Data System (ADS)

    Butler, John; Hwee Lai, Ghee; Zribi, Olena; Smalyukh, Ivan; Angelini, Thomas; Purdy, Kirstin; Golestanian, Ramin; Wong, Gerard C. L.

    2009-03-01

    Interactions between flexible chains and rigid rods govern a broad range of soft matter systems. As a model system of like-charged rigid rods and flexible chains, we examine mixtures of DNA and filamentous actin (F-actin). Confocal microscopy reveals the formation of elongated nematic F-actin domains reticulated via defect-free vertices into a network embedded in a mesh of random DNA. Synchrotron small-angle x-ray scattering (SAXS) indicates that the DNA mesh squeezes the F-actin domains into a nematic state with an inter-actin spacing that decreases with increasing DNA concentration. Salt strongly influences the domain sizes and transitions the system from a counterion-controlled regime to a depletion-controlled regime, both mechanisms of which are entropic in origin.

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  19. Structural Transitions of F-Actin:Espin Bundles

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin; Bartles, James; Wong, Gerard

    2006-03-01

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

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

    PubMed Central

    Borejdo, J; Ortega, H

    1989-01-01

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

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

    PubMed

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

    1992-11-01

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

  2. Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo

    PubMed Central

    Tran, Duy T.; Masedunskas, Andrius; Weigert, Roberto; Ten Hagen, Kelly G.

    2015-01-01

    The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. However, the mechanisms controlling F-actin dynamics in this process are largely unknown. Through 3D time-lapse imaging in a secreting organ, we show that F-actin is actively disassembled along the apical plasma membrane at the site of secretory vesicle fusion and re-assembled directionally on vesicle membranes. Moreover, we show that fusion pore formation and PIP2 redistribution precedes actin and myosin recruitment to secretory vesicle membranes. Finally, we show essential roles for the branched actin nucleators Arp2/3- and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Our results highlight previously unknown roles for branched actin in exocytosis and provide a genetically tractable system to image the temporal and spatial dynamics of polarized secretion in vivo. PMID:26639106

  3. Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo.

    PubMed

    Tran, Duy T; Masedunskas, Andrius; Weigert, Roberto; Ten Hagen, Kelly G

    2015-01-01

    The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. However, the mechanisms controlling F-actin dynamics in this process are largely unknown. Through 3D time-lapse imaging in a secreting organ, we show that F-actin is actively disassembled along the apical plasma membrane at the site of secretory vesicle fusion and re-assembled directionally on vesicle membranes. Moreover, we show that fusion pore formation and PIP2 redistribution precedes actin and myosin recruitment to secretory vesicle membranes. Finally, we show essential roles for the branched actin nucleators Arp2/3- and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Our results highlight previously unknown roles for branched actin in exocytosis and provide a genetically tractable system to image the temporal and spatial dynamics of polarized secretion in vivo. PMID:26639106

  4. Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells

    PubMed Central

    Ueda, Haruko; Yokota, Etsuo; Kutsuna, Natsumaro; Shimada, Tomoo; Tamura, Kentaro; Shimmen, Teruo; Hasezawa, Seiichiro; Dolja, Valerian V.; Hara-Nishimura, Ikuko

    2010-01-01

    Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to ∼3.5 μm/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming. PMID:20351265

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

    PubMed Central

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

    2012-01-01

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

  6. Strong Dependence of Hydration State of F-Actin on the Bound Mg(2+)/Ca(2+) Ions.

    PubMed

    Suzuki, Makoto; Imao, Asato; Mogami, George; Chishima, Ryotaro; Watanabe, Takahiro; Yamaguchi, Takaya; Morimoto, Nobuyuki; Wazawa, Tetsuichi

    2016-07-21

    Understanding of the hydration state is an important issue in the chemomechanical energetics of versatile biological functions of polymerized actin (F-actin). In this study, hydration-state differences of F-actin by the bound divalent cations are revealed through precision microwave dielectric relaxation (DR) spectroscopy. G- and F-actin in Ca- and Mg-containing buffer solutions exhibit dual hydration components comprising restrained water with DR frequency f2 (fw). The hydration state of F-actin is strongly dependent on the ionic composition. In every buffer tested, the HMW signal Dhyme (≡ (f1 - fw)δ1/(fwδw)) of F-actin is stronger than that of G-actin, where δw is DR-amplitude of bulk solvent and δ1 is that of HMW in a fixed-volume ellipsoid containing an F-actin and surrounding water in solution. Dhyme value of F-actin in Ca2.0-buffer (containing 2 mM Ca(2+)) is markedly higher than in Mg2.0-buffer (containing 2 mM Mg(2+)). Moreover, in the presence of 2 mM Mg(2+), the hydration state of F-actin is changed by adding a small fraction of Ca(2+) (∼0.1 mM) and becomes closer to that of the Ca-bound form in Ca2.0-buffer. This is consistent with the results of the partial specific volume and the Cotton effect around 290 nm in the CD spectra, indicating a change in the tertiary structure and less apparent change in the secondary structure of actin. The number of restrained water molecules per actin (N2) is estimated to be 1600-2100 for Ca2.0- and F-buffer and ∼2500 for Mg2.0-buffer at 10-15 °C. These numbers are comparable to those estimated from the available F-actin atomic structures as in the first water layer. The number of HMW molecules is roughly explained by the volume between the equipotential surface of -kT/2e and the first water layer of the actin surface by solving the Poisson-Boltzmann equation using UCSF Chimera. PMID:27332748

  7. F-actin mechanics control spindle centring in the mouse zygote

    NASA Astrophysics Data System (ADS)

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S.; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition.

  8. F-actin mechanics control spindle centring in the mouse zygote.

    PubMed

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition. PMID:26727405

  9. F-actin mechanics control spindle centring in the mouse zygote

    PubMed Central

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S.; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition. PMID:26727405

  10. Self-Organized Gels in DNA/F-Actin Mixtures without Crosslinkers: Networks of Induced Nematic Domains with Tunable Density

    NASA Astrophysics Data System (ADS)

    Lai, Ghee Hwee; Butler, John C.; Zribi, Olena V.; Smalyukh, Ivan I.; Angelini, Thomas E.; Purdy, Kirstin R.; Golestanian, Ramin; Wong, Gerard C. L.

    2008-11-01

    We examine mixtures of DNA and filamentous actin (F-actin) as a model system of like-charged rigid rods and flexible chains. Confocal microscopy reveals the formation of elongated nematic F-actin domains reticulated via defect-free vertices into a network embedded in a mesh of random DNA. Synchrotron x-ray scattering results indicate that the DNA mesh squeezes the F-actin domains into a nematic state with an interactin spacing that decreases with increasing DNA concentration as dactin∝ρDNA-1/2. Interestingly, the system changes from a counterion-controlled regime to a depletion-controlled regime with added salt, with drastic consequences for the osmotic pressure induced phase behavior.

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

    NASA Astrophysics Data System (ADS)

    Kim, Taeyoon; Jung, Wonyeong; Murrell, Michael

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

  12. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  13. Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis.

    PubMed

    Dambournet, Daphné; Machicoane, Mickael; Chesneau, Laurent; Sachse, Martin; Rocancourt, Murielle; El Marjou, Ahmed; Formstecher, Etienne; Salomon, Rémi; Goud, Bruno; Echard, Arnaud

    2011-08-01

    Abscission is the least understood step of cytokinesis. It consists of the final cut of the intercellular bridge connecting the sister cells at the end of mitosis, and is thought to involve membrane trafficking as well as lipid and cytoskeleton remodelling. We previously identified the Rab35 GTPase as a regulator of a fast recycling endocytic pathway that is essential for post-furrowing cytokinesis stages. Here, we report that the phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) 5-phosphatase OCRL, which is mutated in Lowe syndrome patients, is an effector of the Rab35 GTPase in cytokinesis abscission. GTP-bound (active) Rab35 directly interacts with OCRL and controls its localization at the intercellular bridge. Depletion of Rab35 or OCRL inhibits cytokinesis abscission and is associated with local abnormal PtdIns(4,5)P2 and F-actin accumulation in the intercellular bridge. These division defects are also found in cell lines derived from Lowe patients and can be corrected by the addition of low doses of F-actin depolymerization drugs. Our data demonstrate that PtdIns(4,5)P2 hydrolysis is important for normal cytokinesis abscission to locally remodel the F-actin cytoskeleton in the intercellular bridge. They also reveal an unexpected role for the phosphatase OCRL in cell division and shed new light on the pleiotropic phenotypes associated with Lowe disease. PMID:21706022

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

    PubMed Central

    Walter, Nadine; Holweg, Carola L

    2008-01-01

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

  15. The catalytic domain of inositol-1,4,5-trisphosphate 3-kinase-a contributes to ITPKA-induced modulation of F-actin.

    PubMed

    Ashour, Dina Julia; Pelka, Benjamin; Jaaks, Patricia; Wundenberg, Torsten; Blechner, Christine; Zobiak, Bernd; Failla, Antonio Virgilio; Windhorst, Sabine

    2015-02-01

    Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3 Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3 Kinase activity is doubled and the product of InsP3 Kinase activity, Ins(1,3,4,5)P4 , inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3 Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3 Kinase domain substantially contributes to this process. PMID:25620569

  16. Stable high capacity, F-actin affinity column

    SciTech Connect

    Luna, E.J.; Wang, Y.L.; Voss, E.W. Jr.; Branton, D.; Taylor, D.L.

    1982-11-10

    A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds /sup 125/I-heavy meromyosin and /sup 125/I-tropomyosin. The associations between the F-actin-binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

  17. A Theoretical Model for F-actin Remodeling in Vascular Smooth Muscle Cells Subjected to Cyclic Stretch

    PubMed Central

    Na, S.; Meininger, G.A.; Humphrey, J.D.

    2007-01-01

    A constrained mixture theory model was developed and used to estimate remodeling of F-actin in vascular smooth muscle cells that were subjected to 10% equibiaxial stretching for up to 30 minutes. The model was based on a synthesis of data on time-dependent changes in atomic force microscopy measured cell stiffness and immunofluorescence measured focal adhesion associated vinculin as well as data on stress fiber stiffness and pre-stretch. Results suggest that an observed acute (after 2 minutes of stretching) increase in cell stiffness is consistent with an increased stretch of the originally present F-actin plus an assembly of new F-actin having nearly homeostatic values of stretch. Moreover, the subsequent (after 30 minutes of stretching) decrease in cell stiffness back towards the baseline value is consistent with a replacement of the overstretched original filaments with the new (reassembled), less stretched filaments. That is, overall cell response is consistent with a recently proposed concept of “tensional homeostasis” whereby cells seek to maintain constant certain mechanical factors via a remodeling of intracellular and transmembrane proteins. Although there is a need to refine the model based on more comprehensive data sets, using multiple experimental approaches, the present results suggest that a constrained mixture theory can capture salient features of the dynamics of F-actin remodeling and that it offers some advantages over many past methods of modeling, particularly those based on classical linearized viscoelasticity. PMID:17240401

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    1987-10-01

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

  20. Global architecture of F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction

    PubMed Central

    Shao, Yue; Mann, Jennifer M.; Chen, Weiqiang; Fu, Jianping

    2014-01-01

    Uniaxial stretch is an important biophysical regulator of cell morphology (or shape) and functions of vascular endothelial cells (ECs). However, it is unclear whether and how cell shape can independently regulate EC mechanotransductive properties under uniaxial stretch. Herein, utilizing a novel uniaxial cell-stretching device integrated with micropost force sensors, we reported the first experimental evidence showing cell shape-dependent EC mechanotransduction via cytoskeleton (CSK) contractile forces in response to uniaxial stretch. Combining experiments and theoretical modeling from first principles, we showed that it was the global architecture of the F-actin CSK that instructed the cell shape-dependent EC mechanotransductive process. Furthermore, a cell shape-dependent nature was relayed in EC mechanotransduction via dynamic focal adhesion (FA) assembly. Our results suggested a novel mechanotransductive process in ECs wherein the global architecture of the F-actin CSK, governed by cell shape, controls mechanotransduction via CSK contractile forces and force-dependent FA assembly under uniaxial stretch. PMID:24435061

  1. Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation.

    PubMed

    Mathur, Jaideep; Mathur, Neeta; Kirik, Victor; Kernebeck, Birgit; Srinivas, Bhylahalli Purushottam; Hülskamp, Martin

    2003-07-01

    The generation of a specific cell shape requires differential growth, whereby specific regions of the cell expand more relative to others. The Arabidopsis crooked mutant exhibits aberrant cell shapes that develop because of mis-directed expansion, especially during a rapid growth phase. GFP-aided visualization of the F-actin cytoskeleton and the behavior of subcellular organelles in different cell-types in crooked and wild-type Arabidopsis revealed that localized expansion is promoted in cellular regions with fine F-actin arrays but is restricted in areas that maintain dense F-actin. This suggested that a spatiotemporal distinction between fine versus dense F-actin in a growing cell could determine the final shape of the cell. CROOKED was molecularly identified as the plant homolog of ARPC5, the smallest sub-unit of the ARP2/3 complex that in other organisms is renowned for its role in creating dendritic arrays of fine F-actin. Rescue of crooked phenotype by the human ortholog provides the first molecular evidence for the presence and functional conservation of the complex in higher plants. Our cell-biological and molecular characterization of CROOKED suggests a general actin-based mechanism for regulating differential growth and generating cell shape diversity. PMID:12783786

  2. Direct interaction of beta-dystroglycan with F-actin.

    PubMed Central

    Chen, Yun-Ju; Spence, Heather J; Cameron, Jacqueline M; Jess, Thomas; Ilsley, Jane L; Winder, Steven J

    2003-01-01

    Dystroglycans are essential transmembrane adhesion receptors for laminin. Alpha-dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of beta-dystroglycan. Beta-dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin-glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of beta-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells. PMID:12892561

  3. Disrupted dynamics of F-actin and insulin granule fusion in INS-1 832/13 beta-cells exposed to glucotoxicity: partial restoration by glucagon-like peptide 1.

    PubMed

    Quinault, Aurore; Gausseres, Blandine; Bailbe, Danielle; Chebbah, Nella; Portha, Bernard; Movassat, Jamileh; Tourrel-Cuzin, Cecile

    2016-08-01

    Actin dynamics in pancreatic β-cells is involved in insulin exocytosis but the molecular mechanisms of this dynamics and its role in biphasic insulin secretion in pancreatic β-cells is largely unknown. Moreover, the impact of a glucotoxic environment on the sub-cortical actin network dynamics is poorly studied. In this study, we investigate the behavior of insulin granules and the subcortical actin network dynamics in INS-1 832/13 β-cells submitted to a normal or glucotoxic environment. Our results show that glucose stimulation leads to a reorganization of the subcortical actin network with a rupture of its interactions with t-SNARE proteins (Syntaxin 1A and SNAP-25), promoting insulin secretion in INS-1 832/13 β-cells. Prolonged exposure of INS-1 832/13 β-cells to high-glucose levels (glucotoxicity) leads to the densification of the cortical actin network, which prevents its reorganization under acute glucose, and diminishes the glucose-stimulated insulin secretion, as shown by the decreased number of fusion events. The most interesting in our results is the partial restoration by GLP-1 of the insulin secretion ability from high-glucose treated INS-1 832/13 cells. This improved insulin exocytosis is associated with partial restored actin dynamics and fusion events during the two phases of the secretion, with a preferential involvement of Epac2 signaling in the first phase and a rather involvement of PKA signaling in the second phase of insulin exocytosis. All these data provide some new insights into the mechanism by which current therapeutics may be improving insulin secretion. PMID:27101990

  4. Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear.

    PubMed

    Drummond, Meghan C; Barzik, Melanie; Bird, Jonathan E; Zhang, Duan-Sun; Lechene, Claude P; Corey, David P; Cunningham, Lisa L; Friedman, Thomas B

    2015-01-01

    The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24-48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of (15)N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips. PMID:25898120

  5. Claudin 28b and F-actin are involved in rainbow trout gill pavement cell tight junction remodeling under osmotic stress.

    PubMed

    Sandbichler, Adolf Michael; Egg, Margit; Schwerte, Thorsten; Pelster, Bernd

    2011-05-01

    Permeability of rainbow trout gill pavement cells cultured on permeable supports (single seeded inserts) changes upon exposure to freshwater or treatment with cortisol. The molecular components of this change are largely unknown, but tight junctions that regulate the paracellular pathway are prime candidates in this adaptational process. Using differential display polymerase chain reaction we found a set of 17 differentially regulated genes in trout pavement cells that had been exposed to freshwater apically for 24 h. Five genes were related to the cell-cell contact. One of these genes was isolated and identified as encoding claudin 28b, an integral component of the tight junction. Immunohistochemical reactivity to claudin 28b protein was concentrated in a circumferential ring colocalized to the cortical F-actin ring. To study the contribution of this isoform to changes in transepithelial resistance and Phenol Red diffusion under apical hypo-or hyperosmotic exposure we quantified the fluorescence signal of this claudin isoform in immunohistochemical stainings together with the fluorescence of phalloidin-probed F-actin. Upon hypo-osmotic stress claudin 28b fluorescence and epithelial tightness remained stable. Under hyperosmotic stress, the presence of claudin 28b at the junction significantly decreased, and epithelial tightness was severely reduced. Cortical F-actin fluorescence increased upon hypo-osmotic stress, whereas hyperosmotic stress led to a separation of cortical F-actin rings and the number of apical crypt-like pores increased. Addition of cortisol to the basolateral medium attenuated cortical F-actin separation and pore formation during hyperosmotic stress and reduced claudin 28b in junctions except after recovery of cells from exposure to freshwater. Our results showed that short-term salinity stress response in cultured trout gill cells was dependent on a dynamic remodeling of tight junctions, which involves claudin 28b and the supporting F-actin ring

  6. F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

    PubMed Central

    Gu, Yanping; Wang, Congying; Li, GuangWen

    2016-01-01

    Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund’s adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund’s adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors. PMID:27385722

  7. F-actin dismantling through a redox-driven synergy between Mical and cofilin.

    PubMed

    Grintsevich, Elena E; Yesilyurt, Hunkar Gizem; Rich, Shannon K; Hung, Ruei-Jiun; Terman, Jonathan R; Reisler, Emil

    2016-08-01

    Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF (actin-depolymerizing factor)/cofilins (encoded by the twinstar gene in Drosophila), sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. Here we uncover a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. We find that Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared with either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodelling, axon guidance and Semaphorin-Plexin repulsion. Mical and cofilin, therefore, form a redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties. PMID:27454820

  8. Dolastatin 11 connects two long-pitch strands in F-actin to stabilize microfilaments.

    PubMed

    Oda, Toshiro; Crane, Zackary D; Dicus, Christopher W; Sufi, Bilal A; Bates, Robert B

    2003-04-25

    Dolastatin 11, a drug isolated from the Indian Ocean sea hare Dolabella auricularia, arrests cytokinesis in vivo and increases the amount of F-actin to stabilize F-actin in vitro, like phalloidin and jasplakinolide. However, according to the previous biochemical study, the binding of dolastatin 11 to F-actin does not compete with that of phalloidin, suggesting that the binding sites are different. To understand the mechanism of F-actin stabilization by dolastatin 11, we determined the position of bound dolastatin 11 in F-actin using the X-ray fiber diffraction from oriented filament sols. Our analysis shows that the position of dolastatin 11 is clearly different from that of phalloidin. However, these bound drugs are present in the gap between the two long-pitch F-actin strands in a similar way. The result suggests that the connection between the two long-pitch F-actin strands might be a key for the control of F-actin stabilization. PMID:12691743

  9. Prestressed F-actin networks cross-linked by hinged filamins replicate mechanical properties of cells

    NASA Astrophysics Data System (ADS)

    Gardel, M. L.; Nakamura, F.; Hartwig, J. H.; Crocker, J. C.; Stossel, T. P.; Weitz, D. A.

    2006-02-01

    We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditionsis a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces. cytoskeleton | cell mechanics | nonlinear rheology

  10. Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

    PubMed Central

    Sanders, Lori K.; Xian, Wujing; Guáqueta, Camilo; Strohman, Michael J.; Vrasich, Chuck R.; Luijten, Erik; Wong, Gerard C. L.

    2007-01-01

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin–lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin–lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin–lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity. PMID:17911256

  11. Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

    SciTech Connect

    Sanders, Lori K.; Xian, Wujing; Guaqueta, Camilo; Strohman, Michael J.; Vrasich, Chuck R.; Luijten, Erik; Wong, Gerard C.L.

    2008-07-11

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  12. Control of Electrostatic Interactions Between F-Actin And Genetically Modified Lysozyme in Aqueous Media

    SciTech Connect

    Sanders, L.K.; Xian, W.; Guaqueta, C.; Strohman, M.; Vrasich, C.R.; Luijten, E.; Wong, G.C.L.

    2009-06-04

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  13. Activation of F-Actin Binding Capacity of Ezrin: Synergism of PIP2 Interaction and Phosphorylation

    PubMed Central

    Bosk, Sabine; Braunger, Julia A.; Gerke, Volker; Steinem, Claudia

    2011-01-01

    Ezrin is a membrane-cytoskeleton linker protein that can bind F-actin in its active conformation. Several means of regulation of ezrin's activity have been described including phosphorylation of Thr-567 and binding of L-α-phosphatidylinositol-4,5-bisphosphate (PIP2). However, the relative contributions of these events toward activation of the protein and their potential interdependence are not known. We developed an assay based on solid-supported membranes, to which different ezrin mutants (ezrin T567A (inactive mutant), wild-type, and T567D (active pseudophosphorylated mutant)) were bound, that enabled us to analyze the influence of phosphorylation and PIP2 binding on ezrin's activation state in vitro. The lipid bilayers employed contained either DOGS-NTA-Ni to bind the proteins via an N-terminal His-tag, or PIP2, to which ezrin binds via specific binding sites located in the N-terminal region of the protein. Quantitative analysis of the binding behavior of all three proteins to the two different receptor lipids revealed that all three bind with high affinity and specificity to the two receptor lipids. Fluorescence microscopy on ezrin-decorated solid-supported membranes showed that, dependent on the mode of binding and the phosphorylation state, ezrin is capable of binding actin filaments. A clear synergism between phosphorylation and the receptor lipid PIP2 was observed, suggesting a conformational switch from the dormant to the active, F-actin binding state by recognition of PIP2, which is enhanced by the phosphorylation. PMID:21463584

  14. Mechanosensitive systems at the cadherin-F-actin interface.

    PubMed

    Huveneers, Stephan; de Rooij, Johan

    2013-01-15

    Cells integrate biochemical and mechanical information to function within multicellular tissue. Within developing and remodeling tissues, mechanical forces contain instructive information that governs important cellular processes that include stem cell maintenance, differentiation and growth. Although the principles of signal transduction (protein phosphorylation, allosteric regulation of enzymatic activity and binding sites) are the same for biochemical and mechanical-induced signaling, the first step of mechanosensing, in which protein complexes under tension transduce changes in physical force into cellular signaling, is very different, and the molecular mechanisms are only beginning to be elucidated. In this Commentary, we focus on mechanotransduction at cell-cell junctions, aiming to comprehend the molecular mechanisms involved. We describe how different junction structures are associated with the actomyosin cytoskeleton and how this relates to the magnitude and direction of forces at cell-cell junctions. We discuss which cell-cell adhesion receptors have been shown to take part in mechanotransduction. Then we outline the force-induced molecular events that might occur within a key mechanosensitive system at cell-cell junctions; the cadherin-F-actin interface, at which α-catenin and vinculin form a central module. Mechanotransduction at cell-cell junctions emerges as an important signaling mechanism, and we present examples of its potential relevance for tissue development and disease. PMID:23524998

  15. Cooperative and non-cooperative conformational changes of F-actin induced by cofilin

    SciTech Connect

    Aihara, Tomoki; Oda, Toshiro

    2013-05-31

    Highlights: •Mobility of MTSL attached to C374 in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to C374 with cofilin-binding was cooperative. •Mobility of MTSL attached to V43C in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to V43C with cofilin-binding was linear. -- Abstract: Cofilin is an actin-binding protein that promotes F-actin depolymerization. It is well-known that cofilin-coated F-actin is more twisted than naked F-actin, and that the protomer is more tilted. However, the means by which the local changes induced by the binding of individual cofilin proteins proceed to the global conformational changes of the whole F-actin molecule remain unknown. Here we investigated the cofilin-induced changes in several parts of F-actin, through site-directed spin-label electron paramagnetic resonance spectroscopy analyses of recombinant actins containing single reactive cysteines. We found that the global, cooperative conformational changes induced by cofilin-binding, which were detected by the spin-label attached to the Cys374 residue, occurred without the detachment of the D-loop in subdomain 2 from the neighboring protomer. The two processes of local and global changes do not necessarily proceed in sequence.

  16. A synaptic F-actin network controls otoferlin-dependent exocytosis in auditory inner hair cells

    PubMed Central

    Vincent, Philippe FY; Bouleau, Yohan; Petit, Christine; Dulon, Didier

    2015-01-01

    We show that a cage-shaped F-actin network is essential for maintaining a tight spatial organization of Cav1.3 Ca2+ channels at the synaptic ribbons of auditory inner hair cells. This F-actin network is also found to provide mechanosensitivity to the Cav1.3 channels when varying intracellular hydrostatic pressure. Furthermore, this F-actin mesh network attached to the synaptic ribbons directly influences the efficiency of otoferlin-dependent exocytosis and its sensitivity to intracellular hydrostatic pressure, independently of its action on the Cav1.3 channels. We propose a new mechanistic model for vesicle exocytosis in auditory hair cells where the rate of vesicle recruitment to the ribbons is directly controlled by a synaptic F-actin network and changes in intracellular hydrostatic pressure. DOI: http://dx.doi.org/10.7554/eLife.10988.001 PMID:26568308

  17. A synaptic F-actin network controls otoferlin-dependent exocytosis in auditory inner hair cells.

    PubMed

    Vincent, Philippe Fy; Bouleau, Yohan; Petit, Christine; Dulon, Didier

    2015-01-01

    We show that a cage-shaped F-actin network is essential for maintaining a tight spatial organization of Cav1.3 Ca(2+) channels at the synaptic ribbons of auditory inner hair cells. This F-actin network is also found to provide mechanosensitivity to the Cav1.3 channels when varying intracellular hydrostatic pressure. Furthermore, this F-actin mesh network attached to the synaptic ribbons directly influences the efficiency of otoferlin-dependent exocytosis and its sensitivity to intracellular hydrostatic pressure, independently of its action on the Cav1.3 channels. We propose a new mechanistic model for vesicle exocytosis in auditory hair cells where the rate of vesicle recruitment to the ribbons is directly controlled by a synaptic F-actin network and changes in intracellular hydrostatic pressure. PMID:26568308

  18. Electrostatic self-assembly between biological polymers & macroions: Interactions of F-actin & DNA with lysozyme

    NASA Astrophysics Data System (ADS)

    Sanders, Lori K.; Matthews, Brian W.; Wong, Gerard C. L.

    2005-03-01

    The pathological self-assembly of polyelectrolytes such as DNA and F-actin with cationic antimicrobial proteins such as lysozyme may have significant clinical consequences in Cystic Fibrosis (CF) lung infections. Wild-type lysozyme is a compact, cationic, globular protein which carries a net charge of +9e at neutral pH. Our Small Angle X-ray Scattering (SAXS) experiments on F-actin-lysozyme complexes indicate that the wild-type lysozyme close packs into 1-D columns between hexagonally organized F-actin filaments. We will present SAXS results of the interactions of F-actin and DNA with genetically engineered lysozyme mutants that carry a reduced charge of +5e. We have also used fluorescence microscopy to investigate the morphologies and sizes of such bundles induced with divalent cations, wild-type lysozyme, and mutant lysozymes.

  19. Calcium storage and release properties of F-actin: evidence for the involvement of F-actin in cellular calcium signaling.

    PubMed

    Lange, K; Brandt, U

    1996-10-21

    Preceding studies have shown that the bulk of the ATP-dependent, inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store of hamster insulinoma (HIT) cells is located in microvilli on the cell surface. Similar results were obtained with isolated rat hepatocytes. Moreover, in vesicles of microvillar origin, passive fluxes of Ca2+, ATP, and IP3 occur through cation and anion channels, respectively, suggesting that Ca2+ storage is due to ATP-dependent Ca2+ binding to an intravesicular component. Here we demonstrate that F-actin may be a possible candidate for this function. ATP-actin monomers bind Ca2+ with high affinity (Kd = 2-8 nM) to their divalent cation binding sites. Polymerization of actin monomers decreases the rate constant for divalent cation exchange at this binding site by more than 3 orders of magnitude rendering bound cations nearly unavailable. F-actin-bound Ca2+ can be released by depolymerization and dissociation from Ca(2+)-ADP-actin monomers (Kd = 375 nM). We now provide additional evidence for the possible involvement of actin in Ca2+ storage. (1) Preincubation of surface-derived Ca(2+)-storing vesicles from HIT cells with the F-actin stabilizer, phalloidin, strongly inhibited ATP-dependent Ca2+ uptake, reducing the IP3-sensitive Ca2+ pool by 70%. Phalloidin, when added after the loading process, affected neither the amount of stored Ca2+ nor IP3 action on the store. (2) F-actin polymerized in the presence of Mg2+ in nominally Ca(2+)-free buffer still contained about half of the high affinity sites occupied with Ca2+ (Mg/Ca-F-actin). (3) Using the fura-2 technique, we found that in the presence of ATP, Mg/Ca-F-actin incorporated free Ca2+ at a relatively low rate. Short pulses of ultrasound (3-10 s) strongly accelerated Ca2+ uptake, decreasing free Ca2+ from 500 nM to below 100 nM. (4) In the presence of physiological levels of Mg2+ (0.5 mM), sonication liberated large amounts of Ca2+ from Mg/Ca-F-actin. (5) Ca-F-actin released bound Ca2+ at a very

  20. Mechanical force-induced polymerization and depolymerization of F-actin at water/solid interfaces

    NASA Astrophysics Data System (ADS)

    Zhang, Xueqiang; Hu, Xiuyuan; Lei, Haozhi; Hu, Jun; Zhang, Yi

    2016-03-01

    polymerization and depolymerization behaviors at water/solid interfaces using an atomic force microscope (AFM) operated in liquid. By raster scanning an AFM probe on a substrate surface with a certain load, it was found that actin monomers could polymerize into filaments without the help of actin related proteins (ARPs). Further study indicated that actin monomers were inclined to form filaments only under a small scanning load. The polymerized actin filaments would be depolymerized when the mechanical force was stronger. A possible mechanism has been suggested to explain the mechanical force induced actin polymerization. Electronic supplementary information (ESI) available: The height histograms of Fig. 1b-1d, the effect of G-actin concentration on the mechanical-force-induced F-actin formation, and the effect of different mechanical forces on the depolymerization of F-actin. See DOI: 10.1039/c5nr08713a

  1. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

    2014-01-01

    STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by

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

    PubMed Central

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

    2005-01-01

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

  3. Self-organization of chemoattractant waves in Dictyostelium depends on F-actin and cell-substrate adhesion.

    PubMed

    Fukujin, Fumihito; Nakajima, Akihiko; Shimada, Nao; Sawai, Satoshi

    2016-06-01

    In the social amoeba Dictyostelium discoideum, travelling waves of extracellular cyclic adenosine monophosphate (cAMP) self-organize in cell populations and direct aggregation of individual cells to form multicellular fruiting bodies. In contrast to the large body of studies that addressed how movement of cells is determined by spatial and temporal cues encoded in the dynamic cAMP gradients, how cell mechanics affect the formation of a self-generated chemoattractant field has received less attention. Here, we show, by live cell imaging analysis, that the periodicity of the synchronized cAMP waves increases in cells treated with the actin inhibitor latrunculin. Detail analysis of the extracellular cAMP-induced transients of cytosolic cAMP (cAMP relay response) in well-isolated cells demonstrated that their amplitude and duration were markedly reduced in latrunculin-treated cells. Similarly, in cells strongly adhered to a poly-l-lysine-coated surface, the response was suppressed, and the periodicity of the population-level oscillations was markedly lengthened. Our results suggest that cortical F-actin is dispensable for the basic low amplitude relay response but essential for its full amplification and that this enhanced response is necessary to establish high-frequency signalling centres. The observed F-actin dependence may prevent aggregation centres from establishing in microenvironments that are incompatible with cell migration. PMID:27358278

  4. F-actin forms mobile and unwinding ring-shaped structures in germinating Arabidopsis pollen expressing Lifeact

    PubMed Central

    Vogler, Frank; Sprunck, Stefanie

    2015-01-01

    The flowering plant pollen tube is the fastest elongating plant cell and transports the sperm cells for double fertilization. The highly dynamic formation and reorganization of the actin cytoskeleton is essential for pollen germination and pollen tube growth. To drive pollen-specific expression of fluorescent marker proteins, commonly the strong Lat52 promoter is used. Here we show by quantitative fluorescent analysis that the gametophyte-specific ARO1 promoter from Arabidopsis drives an about 3.5 times weaker transgene expression than the Lat52 promoter. In one third of the pollen of F-actin-labeled ARO1p:tagRFP-T-Lifeact transgenic lines we observed mobile ring-shaped actin structures in pollen grains and pollen tubes. Pollen tube growth, transgene transmission and seed production were not affected by tagRFP-T-Lifeact expression. F-actin rings were able to integrate into emerging actin filaments and they may reflect a particular physiological state of the pollen or a readily available storage form provided for rapid actin network remodeling. PMID:26337326

  5. Self-organization of chemoattractant waves in Dictyostelium depends on F-actin and cell–substrate adhesion

    PubMed Central

    Fukujin, Fumihito; Nakajima, Akihiko; Shimada, Nao; Sawai, Satoshi

    2016-01-01

    In the social amoeba Dictyostelium discoideum, travelling waves of extracellular cyclic adenosine monophosphate (cAMP) self-organize in cell populations and direct aggregation of individual cells to form multicellular fruiting bodies. In contrast to the large body of studies that addressed how movement of cells is determined by spatial and temporal cues encoded in the dynamic cAMP gradients, how cell mechanics affect the formation of a self-generated chemoattractant field has received less attention. Here, we show, by live cell imaging analysis, that the periodicity of the synchronized cAMP waves increases in cells treated with the actin inhibitor latrunculin. Detail analysis of the extracellular cAMP-induced transients of cytosolic cAMP (cAMP relay response) in well-isolated cells demonstrated that their amplitude and duration were markedly reduced in latrunculin-treated cells. Similarly, in cells strongly adhered to a poly-l-lysine-coated surface, the response was suppressed, and the periodicity of the population-level oscillations was markedly lengthened. Our results suggest that cortical F-actin is dispensable for the basic low amplitude relay response but essential for its full amplification and that this enhanced response is necessary to establish high-frequency signalling centres. The observed F-actin dependence may prevent aggregation centres from establishing in microenvironments that are incompatible with cell migration. PMID:27358278

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-05-01

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

  8. Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas.

    PubMed

    Onishi, Masayuki; Pringle, John R; Cross, Frederick R

    2016-03-01

    Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses. PMID:26715672

  9. Passive and active microrheology for cross-linked F-actin networks in vitro.

    PubMed

    Lee, Hyungsuk; Ferrer, Jorge M; Nakamura, Fumihiko; Lang, Matthew J; Kamm, Roger D

    2010-04-01

    Actin filament (F-actin) is one of the dominant structural constituents in the cytoskeleton. Orchestrated by various actin-binding proteins (ABPs), F-actin is assembled into higher-order structures such as bundles and networks that provide mechanical support for the cell and play important roles in numerous cellular processes. Although mechanical properties of F-actin networks have been extensively studied, the underlying mechanisms for network elasticity are not fully understood, in part because different measurements probe different length and force scales. Here, we developed both passive and active microrheology techniques using optical tweezers to estimate the mechanical properties of F-actin networks at a length scale comparable to cells. For the passive approach we tracked the motion of a thermally fluctuating colloidal sphere to estimate the frequency-dependent complex shear modulus of the network. In the active approach, we used an optical trap to oscillate an embedded microsphere and monitored the response in order to obtain network viscoelasticity over a physiologically relevant force range. While both active and passive measurements exhibit similar results at low strain, the F-actin network subject to high strain exhibits non-linear behavior which is analogous to the strain-hardening observed in macroscale measurements. Using confocal and total internal reflection fluorescent microscopy, we also characterize the microstructure of reconstituted F-actin networks in terms of filament length, mesh size and degree of bundling. Finally, we propose a model of network connectivity by investigating the effect of filament length on the mechanical properties and structure. PMID:19883801

  10. Myosin IIB and F-actin control apical vacuolar morphology and histamine-induced trafficking of H-K-ATPase-containing tubulovesicles in gastric parietal cells

    PubMed Central

    Crothers, James M.; Rosen, Jared E.; Nakada, Stephanie L.; Rakholia, Milap; Okamoto, Curtis T.; Forte, John G.; Machen, Terry E.

    2014-01-01

    Selective inhibitors of myosin or actin function and confocal microscopy were used to test the role of an actomyosin complex in controlling morphology, trafficking, and fusion of tubulovesicles (TV) containing H-K-ATPase with the apical secretory canaliculus (ASC) of primary-cultured rabbit gastric parietal cells. In resting cells, myosin IIB and IIC, ezrin, and F-actin were associated with ASC, whereas H-K-ATPase localized to intracellular TV. Histamine caused fusion of TV with ASC and subsequent expansion resulting from HCl and water secretion; F-actin and ezrin remained associated with ASC whereas myosin IIB and IIC appeared to dissociate from ASC and relocalize to the cytoplasm. ML-7 (inhibits myosin light chain kinase) caused ASC of resting cells to collapse and most myosin IIB, F-actin, and ezrin to dissociate from ASC. TV were unaffected by ML-7. Jasplakinolide (stabilizes F-actin) caused ASC to develop large blebs to which actin, myosin II, and ezrin, as well as tubulin, were prominently localized. When added prior to stimulation, ML-7 and jasplakinolide prevented normal histamine-stimulated transformations of ASC/TV and the cytoskeleton, but they did not affect cells that had been previously stimulated with histamine. These results indicate that dynamic pools of actomyosin are required for maintenance of ASC structure in resting cells and for trafficking of TV to ASC during histamine stimulation. However, the dynamic pools of actomyosin are not required once the histamine-stimulated transformation of TV/ASC and cytoskeleton has occurred. These results also show that vesicle trafficking in parietal cells shares mechanisms with similar processes in renal collecting duct cells, neuronal synapses, and skeletal muscle. PMID:24578340

  11. Tracer diffusion in F-actin and Ficoll mixtures. Toward a model for cytoplasm.

    PubMed Central

    Hou, L; Lanni, F; Luby-Phelps, K

    1990-01-01

    We have previously reported that self-diffusion of inert tracer particles in the cytoplasm of living Swiss 3T3 cells is hindered in a size-dependent manner (Luby-Phelps, K., D.L. Taylor, and F. Lanni. 1986. J. Cell Biol. 102:2015-2022; Luby-Phelps, K., P.E. Castle, D.L. Taylor, and F. Lanni. 1987. Proc Natl. Acad. Sci. USA. 84:4910-4913). Lacking a theory that completely explains our data, we are attempting to understand the molecular architecture responsible for this phenomenon by studying tracer diffusion in simple, reconstituted model systems. This report contains our findings on tracer diffusion in concentrated solutions of Ficoll 70 or Ficoll 400, in solutions of entangled F-actin filaments, and in solutions of entangled F-actin containing a background of concentrated Ficoll particles or concentrated bovine serum albumin (BSA). A series of size-fractionated fluorescein-Ficolls were used as tracer particles. By fluorescence recovery after photobleaching (FRAP), we obtained the mean diffusion coefficients in a dilute, aqueous reference phase (Do), the mean diffusion coefficients in the model matrices (D), and the mean hydrodynamic radii (RH) for selected tracer fractions. For each model matrix, the results were compared with similar data obtained from living cells. As in concentrated solutions of globular proteins (Luby-Phelps et al., 1987), D/Do was not significantly size-dependent in concentrated solutions of Ficoll 400 or Ficoll 70. In contrast, D/Do decreased monotonically with increasing RH in solutions of F-actin ranging in concentration from 1 to 12 mg/ml. This size dependence was most pronounced at higher F-actin concentrations. However, the shape of the curve and the extrapolated value of D/Do in the limit, RH----O did not closely resemble the cellular data for tracers in the same size range (3 less than RH less than 30 nm). In mixtures of F-actin and Ficoll or F-actin and BSA, D/Do was well approximated by D/Do for the same concentration of F-actin

  12. F-Actin Organization and Pollen Tube Tip Growth in Arabidopsis Are Dependent on the Gametophyte-Specific Armadillo Repeat Protein ARO1[W

    PubMed Central

    Gebert, Marina; Dresselhaus, Thomas; Sprunck, Stefanie

    2008-01-01

    The signal-mediated and spatially controlled assembly and dynamics of actin are crucial for maintaining shape, motility, and tip growth of eukaryotic cells. We report that a novel Armadillo repeat protein in Arabidopsis thaliana, ARMADILLO REPEAT ONLY1 (ARO1), is of fundamental importance for polar growth and F-actin organization in tip-growing pollen tubes. ARO1 is specifically expressed in the vegetative cell of pollen as well as in the egg cell. ARO1-GFP (for green fluorescent protein) fusion proteins accumulate most notably in pollen tube tips and partially colocalize with F-actin in the shank of pollen tubes. ARO1 knockout results in a highly disorganized actin cytoskeleton, growth depolarization, and ultimately tube growth arrest. Tip-localized ARO1-GFP is spatially shifted toward the future site of tip growth, indicating a role of ARO1 in the signaling network controlling tip growth and regulating actin organization. After the pollen tube discharges its contents into the receptive synergid, ARO1-GFP colocalizes with emerging F-actin structures near the site of sperm cell fusion, suggesting additional participation in the mechanism of sperm cell tracking toward the female gametes. The variable localization of ARO1 in the cytoplasm, the nucleus, and at the plasma membrane, however, indicates a multifunctional role like that of β-catenin/Armadillo and the p120 catenins. PMID:18931021

  13. F-actin distribution and function during sexual development in Eimeria maxima.

    PubMed

    Frölich, Sonja; Wallach, Michael

    2015-06-01

    To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima. PMID:25800683

  14. Rapid non-equilibrium turnover fluidizes entangled F-actin solutions

    NASA Astrophysics Data System (ADS)

    McCall, Patrick M.; Kovar, David R.; Gardel, Margaret L.

    The actin cytoskeleton of living cells is a semiflexible polymer network which regulates cell division, motility, and morphogenesis by controlling cell shape. These complex shape-changing processes require both mechanical deformation and remodeling of the actin cytoskeleton. Molecular motors generate internal forces to drive deformation, while cytoskeletal remodeling is regulated by non-equilibrium polymer turnover. Although the mechanical properties of equilibrium actin filament (F-actin) networks are well-described by theories of semiflexible polymers, these theories do not incorporate the effects of non-equilibrium turnover. To address this experimentally, we developed a model system in which both the turnover rate and the length distribution of purified F-actin can be tuned independently at steady-state through the combined action of actin regulatory proteins. Specifically we tune the concentrations of cofilin, profilin, and formin to regulate F-actin severing, recycling, and nucleation, respectively. We find that the actin turnover rate can be tuned by cofilin up to 25-fold (31 +/- 2 subunits/sec/filament). Surprisingly, changes in turnover rate have no effect on the steady-state F-actin length distribution, which is instead set by formin concentration. Passive microrheology measurements show that increased turnover leads to striking fluidization in both entangled and crosslinked networks. Non-equilibrium turnover thus enables modulation of network mechanics, which impacts force transmission and material deformation.

  15. A Role for Nuclear F-Actin Induction in Human Cytomegalovirus Nuclear Egress

    PubMed Central

    Wilkie, Adrian R.; Lawler, Jessica L.

    2016-01-01

    ABSTRACT Herpesviruses, which include important pathogens, remodel the host cell nucleus to facilitate infection. This remodeling includes the formation of structures called replication compartments (RCs) in which herpesviruses replicate their DNA. During infection with the betaherpesvirus, human cytomegalovirus (HCMV), viral DNA synthesis occurs at the periphery of RCs within the nuclear interior, after which assembled capsids must reach the inner nuclear membrane (INM) for translocation to the cytoplasm (nuclear egress). The processes that facilitate movement of HCMV capsids to the INM during nuclear egress are unknown. Although an actin-based mechanism of alphaherpesvirus capsid trafficking to the INM has been proposed, it is controversial. Here, using a fluorescently-tagged, nucleus-localized actin-binding peptide, we show that HCMV, but not herpes simplex virus 1, strongly induced nuclear actin filaments (F-actin) in human fibroblasts. Based on studies using UV inactivation and inhibitors, this induction depended on viral gene expression. Interestingly, by 24 h postinfection, nuclear F-actin formed thicker structures that appeared by super-resolution microscopy to be bundles of filaments. Later in infection, nuclear F-actin primarily localized along the RC periphery and between the RC periphery and the nuclear rim. Importantly, a drug that depolymerized nuclear F-actin caused defects in production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization near the nuclear rim, without decreasing capsid accumulation in the nucleus. Thus, our results suggest that for at least one herpesvirus, nuclear F-actin promotes capsid movement to the nuclear periphery and nuclear egress. We discuss our results in terms of competing models for these processes. PMID:27555312

  16. Quantitation of liquid-crystalline ordering in F-actin solutions.

    PubMed

    Coppin, C M; Leavis, P C

    1992-09-01

    Actin filaments (F-actin) are important determinants of cellular shape and motility. These functions depend on the collective organization of numerous filaments with respect to both position and orientation in the cytoplasm. Much of the orientational organization arises spontaneously through liquid crystal formation in concentrated F-actin solutions. In studying this phenomenon, we found that solutions of purified F-actin undergo a continuous phase transition, from the isotropic state to a liquid crystalline state, when either the mean filament length or the actin concentration is increased above its respective threshold value. The phase diagram representing the threshold filament lengths and concentrations at which the phase transition occurs is consistent with that predicted by Flory's theory on solutions of noninteracting, rigid cylinders (Flory, 1956b). However, in contrast to other predictions based on this model, we found no evidence for the coexistence of isotropic and anisotropic phases. Furthermore, the phase transition proved to be temperature dependent, which suggests the existence of orientation-dependent interfilament interactions or of a temperature-dependent filament flexibility. We developed a simple method for growing undistorted fluorescent acrylodan-labeled F-actin liquid crystals; and we derived a simple theoretical treatment by which polarization-of-fluorescence measurements could be used to quantitate, for the first time, the degree of spontaneous filament ordering (nematic order parameter) in these F-actin liquid crystals. This order parameter was found to increase monotonically with both filament length and concentration. Actin liquid crystals can readily become distorted by a process known as "texturing." Zigzaging and helicoidal liquid crystalline textures which persisted in the absence of ATP were observed through the polarizing microscope. Possible texturing mechanisms are discussed. PMID:1330036

  17. F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    PubMed Central

    Burger, Danielle; Fickentscher, Céline; de Moerloose, Philippe; Brandt, Karim J.

    2016-01-01

    NLRP3 and ASC are able to form a large multimeric complex called inflammasome in response to a number danger signals. The NLRP3 inflammasome is required for the activation of caspase-1 and subsequent maturation of pro-IL-1β into active IL-1β. Although the mechanisms regulating the formation and activity of NLRP3 inflammasome are yet not fully elucidated, data suggest that the assembly of NLRP3 inflammasome requires microtubules to induce the proximity of ASC and NLRP3. In this study we show that microfilaments (F-actin) inhibit NLRP3 inflammasome activity and interact with NLRP3 and ASC. We demonstrate that the inhibition depends on the actin polymerization state but not on the active polymerization process. In ATP- or nigericin-activated macrophages, our data further indicate that Flightless-I (FliI) and leucine-rich repeat FliI-interaction protein 2 (LRRFIP2) are required for the co-localization of NLRP3, ASC and F-actin. We also established that the ability of Ca2+ to accentuate the activity of NLRP3 inflammasome is abrogated in FliI- and LRRFIP2-knockdown macrophages, suggesting that Ca2+ signaling requires the presence of FliI and LRRFIP2. Accordingly, we observed that Ca2+/FliI-dependent severing of F-actin suppresses F-actin/FliI/LRRFIP2-dependent NLRP3 inflammasome inhibition leading to increase IL-1β production. Altogether, our results unveil a new function of F-actin in the regulation of NLRP3 inflammasome activity strengthening the importance of cytoskeleton in the regulation of inflammation. PMID:27431477

  18. Morphogenetic role of F-actin meshwork in chamber formation: immunolabeling results from symbiont bearing benthic foraminifera

    NASA Astrophysics Data System (ADS)

    Tyszka, Jaroslaw; Raitzsch, Markus; Bijma, Jelle; Höher, Nicole; Bickmeyer, Ulf; Rivera-Ingraham, Georginia; Topa, Paweł; Kaczmarek, Karina; Mewes, Antje; Bowser, Samuel; Travis, Jeffrey

    2015-04-01

    Foraminifera are excellent tracers of palaeoceanographic conditions recorded in their shell (test) morphology and chemical composition. Understanding foraminiferal morphology controlled by chamberwise growth can be reduced to processes of chamber formation. However, little is known about how foraminifera control the shape of the chamber wall to be biosynthesized and precipitated. Searching for fundamental morphogenetic features involved in biomineralization, we focused on foraminifers, which belong to the class Globothalamea. The most critical condition to run experiments was to have convenient access to early stages of chamber formation in any species of cultured benthic foraminifers. We have tested small foraminifers collected from the tidal flats of the North Sea. All species, including Ammonia, Haynesina, and Elphidium, turned out to be unsuitable due to their reproduction seasonality and/or unpredictability. The problem was solved by using symbiont bearing Amphistegina lessonii cultured in small aquaria. In well treated cultures, such foraminifera often reproduce on a glass wall surface, serving as a continuous source of juveniles. They tend to regularly construct chambers. Another important point is that symbiont bearing foraminifers usually do not construct opaque protective cysts from detritus that disturb observations. All these features facilitate immunolabeling experiments observed under confocal microscopy. Therefore, for the first time, we managed to label cytoskeleton proteins during the chamber formation in Foraminifera. The results show that the shape of chamber is predefined by a meshwork of F-actin, which acts as a dynamic organic scaffold most likely responsible for distribution and docking of biomineralizing molecules (glycoproteins). The F-actin meshwork interacts with microtubules and all associated proteins, which are involved in the morphogenesis of biomineralized structures. Foraminifera, like other eukaryotic cells, can form active

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

    PubMed

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

    2016-04-01

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

  20. Mitochondrial Dysfunction, Disruption of F-Actin Polymerization, and Transcriptomic Alterations in Zebrafish Larvae Exposed to Trichloroethylene.

    PubMed

    Wirbisky, Sara E; Damayanti, Nur P; Mahapatra, Cecon T; Sepúlveda, Maria S; Irudayaraj, Joseph; Freeman, Jennifer L

    2016-02-15

    Trichloroethylene (TCE) is primarily used as an industrial degreasing agent and has been in use since the 1940s. TCE is released into the soil, surface, and groundwater. From an environmental and regulatory standpoint, more than half of Superfund hazardous waste sites on the National Priority List are contaminated with TCE. Occupational exposure to TCE occurs primarily via inhalation, while environmental TCE exposure also occurs through ingestion of contaminated drinking water. Current literature links TCE exposure to various adverse health effects including cardiovascular toxicity. Current studies aiming to address developmental cardiovascular toxicity utilized rodent and avian models, with the majority of studies using relatively higher parts per million (mg/L) doses. In this study, to further investigate developmental cardiotoxicity of TCE, zebrafish embryos were treated with 0, 10, 100, or 500 parts per billion (ppb; μg/L) TCE during embryogenesis and/or through early larval stages. After the appropriate exposure period, angiogenesis, F-actin, and mitochondrial function were assessed. A significant dose-response decrease in angiogenesis, F-actin, and mitochondrial function was observed. To further complement this data, a transcriptomic profile of zebrafish larvae was completed to identify gene alterations associated with the 10 ppb TCE exposure. Results from the transcriptomic data revealed that embryonic TCE exposure caused significant changes in genes associated with cardiovascular disease, cancer, and organismal injury and abnormalities with a number of targets in the FAK signaling pathway. Overall, results from our study support TCE as a developmental cardiovascular toxicant, provide molecular targets and pathways for investigation in future studies, and indicate a need for continued priority for environmental regulation. PMID:26745549

  1. Measuring F-actin properties in dendritic spines

    PubMed Central

    Koskinen, Mikko; Hotulainen, Pirta

    2014-01-01

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

  2. Coronin 3 involvement in F-actin-dependent processes at the cell cortex

    SciTech Connect

    Rosentreter, Andre; Hofmann, Andreas; Xavier, Charles-Peter; Stumpf, Maria; Noegel, Angelika A.; Clemen, Christoph S. . E-mail: christoph.clemen@uni-koeln.de

    2007-03-10

    The actin interaction of coronin 3 has been mainly documented by in vitro experiments. Here, we discuss coronin 3 properties in the light of new structural information and focus on assays that reflect in vivo roles of coronin 3 and its impact on F-actin-associated functions. Using GFP-tagged coronin 3 fusion proteins and RNAi silencing we show that coronin 3 has roles in wound healing, protrusion formation, cell proliferation, cytokinesis, endocytosis, axonal growth, and secretion. During formation of cell protrusions actin accumulation precedes the focal enrichment of coronin 3 suggesting a role for coronin 3 in events that follow the initial F-actin assembly. Moreover, we show that coronin 3 similar to other coronins interacts with the Arp2/3-complex and cofilin indicating that this family in general is involved in regulating Arp2/3-mediated events.

  3. Ordered stacking of F-actin layers and mixed lipid bilayers: a columnar liquid crystal.

    PubMed

    Caillé, A; Artzner, F; Amblard, F

    2013-01-25

    In this Letter, we show how the grooved helical structure of actin microfilaments (F-actin) interacting with mixed fluid lipid bilayers leads to handedness-independent 1D lipid bilayer undulations coupled to longitudinal in-plane ordering of the microfilaments. This longitudinal ordering is forced by the emerging in-plane compression and curvature energy terms of the straight 1D bilayer undulation wave fronts. Thereby, adjacent helices are set into registry along their long axis in their monolayer and π shifted between adjacent monolayers. An ordered composite multilamellar structure emerges by alternate stacking of these lipid bilayers and monolayers of F-actin. This two-dimensionally ordered system has the symmetries of a centered rectangular columnar liquid crystal, the straight 1D wave fronts playing the role of the classical molecular columns. PMID:25166203

  4. Concomitant binding of Afadin to LGN and F-actin directs planar spindle orientation.

    PubMed

    Carminati, Manuel; Gallini, Sara; Pirovano, Laura; Alfieri, Andrea; Bisi, Sara; Mapelli, Marina

    2016-02-01

    Polarized epithelia form by oriented cell divisions in which the mitotic spindle aligns parallel to the epithelial plane. To orient the mitotic spindle, cortical cues trigger the recruitment of NuMA-dynein-based motors, which pull on astral microtubules via the protein LGN. We demonstrate that the junctional protein Afadin is required for spindle orientation and correct epithelial morphogenesis of Caco-2 cysts. Molecularly, Afadin binds directly and concomitantly to F-actin and to LGN. We determined the crystallographic structure of human Afadin in complex with LGN and show that it resembles the LGN-NuMA complex. In mitosis, Afadin is necessary for cortical accumulation of LGN and NuMA above the spindle poles, in an F-actin-dependent manner. Collectively, our results depict Afadin as a molecular hub governing the enrichment of LGN and NuMA at the cortex. To our knowledge, Afadin is the first-described mechanical anchor between dynein and cortical F-actin. PMID:26751642

  5. Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis.

    PubMed

    Wollert, Torsten; Weiss, Dieter G; Gerdes, Hans-Hermann; Kuznetsov, Sergei A

    2002-11-25

    It is widely believed that microtubule- and F-actin-based transport of cytoplasmic organelles and membrane fusion is down-regulated during mitosis. Here we show that during the transition of Xenopus egg extracts from interphase to metaphase myosin V-driven movement of small globular vesicles along F-actin is strongly inhibited. In contrast, the movement of ER and ER network formation on F-actin is up-regulated in metaphase extracts. Our data demonstrate that myosin V-driven motility of distinct organelles is differently controlled during the cell cycle and suggest an active role of F-actin in partitioning, positioning, and membrane fusion of the ER during cell division. PMID:12438410

  6. F-actin is intermolecularly crosslinked by N,N'-p-phenylenedimaleimide through lysine-191 and cysteine-374.

    PubMed

    Elzinga, M; Phelan, J J

    1984-11-01

    The bifunctional reagent N,N'-p-phenylenedimaleimide (PDM) is being used in an attempt to measure distances between specific side chains in adjacent monomers within F-actin. [14C]PDM was synthesized and was used to crosslink F-actin. Uncrosslinked actin was removed by gel filtration, and, from an arginine-specific tryptic digest of the covalently crosslinked dimers and higher oligomers, one radioactive crosslinked peptide was obtained in high yield. Amino acid composition and sequence analysis indicated that it comprises residues 184-196 of one monomer and 373-375 of an adjacent actin molecule, bridged by PDM through Cys-374 and Lys-191. Thus, these groups are shown to be 1.2-1.4 nm apart in adjacent actin monomers in F-actin. This information may be crucial in establishing the orientation of actin monomers within F-actin. PMID:6436818

  7. Chromaffin granule membrane-F-actin interactions and spectrin-like protein of subcellular organelles: a possible relationship.

    PubMed

    Aunis, D; Perrin, D

    1984-06-01

    The membrane of chromaffin granule, the secretory vesicle of adrenal medullary cells storing catecholamines, enkephalins, and many other components, interacts with F-actin. Using low shear falling ball viscometry to estimate actin binding to membranes, we demonstrated that mitochondrial and plasma membranes from chromaffin cells also provoked large increases in viscosity of F-actin solutions. Mitochondrial membranes also had the capacity to cause complete gelation of F-actin. In addition, vasopressin-containing granules from neurohypophysial tissue were shown to bind F-actin and to increase the viscosity of F-actin solutions. Using an antibody directed against human erythrocyte spectrin, it was found that a spectrin-like protein was associated with secretory granule membrane, mitochondrial membrane, and plasma membrane. The chromaffin granule membrane-associated spectrin-like protein faces the cytoplasmic side, is composed of two subunits (240 kD and 235kD ), the alpha-subunit (240 kD, pHi5 .5) being recognized by the antibody. Nonionic detergents such as Triton X-100 or Nonidet P40 failed to release fully active spectrin-like protein. In contrast, Kyro EOB , a different nonionic detergent, was found to release spectrin-like protein while keeping intact F-actin binding capacity, at least below 0.5% Kyro EOB concentration. Chromaffin cells in culture were stained with antispectrin antibody, showing the presence of spectrin-like protein in the cell periphery close to the cell membrane but also in the cytoplasm. We conclude that in living cells the interaction of F-actin with chromaffin granule membrane spectrin observed in vitro is important in controlling the potential function of secretory vesicles. PMID:6374036

  8. Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis.

    PubMed Central

    Szymanski, D B; Marks, M D; Wick, S M

    1999-01-01

    Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin-disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern. PMID:10590162

  9. Motion in partially and fully cross-linked F-actin networks

    NASA Astrophysics Data System (ADS)

    Morris, Eliza; Ehrlicher, Allen; Weitz, David

    2012-02-01

    Single molecule experiments have measured stall forces and procession rates of molecular motors on isolated cytoskeletal fibers in Newtonian fluids. But in the cell, these motors are transporting cargo through a highly complex cytoskeletal network. To compare these single molecule results to the forces exerted by motors within the cell, an evaluation of the response of the cytoskeletal network is needed. Using magnetic tweezers and fluorescence confocal microscopy we observe and quantify the relationship between bead motion and filament response in F-actin networks both partially and fully cross-linked with filamin We find that when the transition from full to partial cross-linking is brought about by a decrease in cross-linker concentration there is a simultaneous decline in the elasticity of the network, but the response of the bead remains qualitatively similar. However, when the cross-linking is reduced through a shortening of the F-actin filaments the bead response is completely altered. The characteristics of the altered bead response will be discussed here.

  10. Cadherin 6 promotes neural crest cell detachment via F-actin regulation and influences active Rho distribution during epithelial-to-mesenchymal transition

    PubMed Central

    Clay, Matthew R.; Halloran, Mary C.

    2014-01-01

    The epithelial-to-mesenchymal transition (EMT) is a complex change in cell phenotype that is important for cell migration, morphogenesis and carcinoma metastasis. Loss of epithelial cell adhesion and tight regulation of cadherin adhesion proteins are crucial for EMT. Cells undergoing EMT often display cadherin switching, where they downregulate one cadherin and induce expression of another. However, the functions of the upregulated cadherins and their effects on cell motility are poorly understood. Neural crest cells (NCCs), which undergo EMT during development, lose N-cadherin and upregulate Cadherin 6 (Cdh6) prior to EMT. Cdh6 has been suggested to suppress EMT via cell adhesion, but also to promote EMT by mediating pro-EMT signals. Here, we determine novel roles for Cdh6 in generating cell motility during EMT. We use live imaging of NCC behavior in vivo to show that Cdh6 promotes detachment of apical NCC tails, an important early step of EMT. Furthermore, we show that Cdh6 affects spatiotemporal dynamics of F-actin and active Rho GTPase, and that Cdh6 is required for accumulation of F-actin in apical NCC tails during detachment. Moreover, Cdh6 knockdown alters the subcellular distribution of active Rho, which is known to promote localized actomyosin contraction that is crucial for apical NCC detachment. Together, these data suggest that Cdh6 is an important determinant of where subcellular actomyosin forces are generated during EMT. Our results also identify mechanisms by which an upregulated cadherin can generate cell motility during EMT. PMID:24917505

  11. Vacuole formation in mast cells responding to osmotic stress and to F-actin disassembly.

    PubMed

    Koffer, Anna; Williams, Mark; Johansen, Torben

    2002-01-01

    Fluorescent probes were used to visualize the morphology of membranes and of F-actin in rat peritoneal mast cells, exposed to hyperosmotic medium and consequently reversed to isotonicity. Hypertonicity induced cell shrinkage followed by a regulatory volume increase, and cell alkalinization that was sensitive to amiloride, an inhibitor of the Na(+)/H(+) exchanger (NHE), but not to Latrunculin B, an inhibitor of actin polymerization. Using Bodipy-Sphingomyelin, we have observed formation of vacuole-like dilations (VLDs), primarily at or close to the adhesion plane, following the reversal from hyper- to isotonic medium. VLD formation was not inhibited by Latrunculin B or by amiloride. Phalloidin staining has shown that actin filaments do not surround the vacuoles and latrunculin-induced depolymerization of actin has actually promoted vacuole formation, even in isotonic conditions. The results support the idea that a decrease in membrane tension promotes the internalization of the plasma membrane. PMID:12421579

  12. High-Resolution Crystal Structures of Villin Headpiece nad Mutants with Reduced F-Actin Binding Activity

    SciTech Connect

    Meng,J.; Vardar, D.; Wang, Y.; Guo, H.; Head, J.; McKnight, C.

    2005-01-01

    Villin-type headpiece domains are approximately 70 amino acid modular motifs found at the C terminus of a variety of actin cytoskeleton-associated proteins. The headpiece domain of villin, a protein found in the actin bundles of the brush border epithelium, is of interest both as a compact F-actin binding domain and as a model folded protein. We have determined the high-resolution crystal structures of chicken villin headpiece (HP67) at 1.4 Angstrom resolution as well as two mutants, R37A and W64Y, at 1.45 and 1.5 Angstrom resolution, respectively. Replacement of R37 causes a 5-fold reduction in F-actin binding affinity in sedimentation assays. Replacement of W64 results in a much more drastic reduction in F-actin binding affinity without significant changes in headpiece structure or stability. The detailed comparison of these crystal structures with each other and to our previously determined NMR structures of HP67 and the 35-residue autonomously folding subdomain in villin headpiece, HP35, provides the details of the headpiece fold and further defines the F-actin binding site of villin-type headpiece domains.

  13. Plasma membrane-associated SCAR complex subunits promote cortical F-actin accumulation and normal growth characteristics in Arabidopsis roots

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ARP2/3 complex, a highly conserved nucleator of F-actin polymerization, and its activator, the SCAR complex, have been shown to play important roles in leaf epidermal cell morphogenesis in Arabidopsis. However, the intracellular site(s) and function(s) of SCAR complex and ARP2/3 complex-depende...

  14. Structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum.

    PubMed

    Kim, Min-Kyu; Kim, Ji-Hye; Kim, Ji-Sun; Kang, Sa-Ouk

    2015-09-01

    The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions. PMID:26327373

  15. Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol).

    PubMed

    Walsh, J L; Knull, H R

    1988-01-01

    Interactions of glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.3), enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP:Pyruvate O2-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase [S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were observed. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice. PMID:3334856

  16. Spatial Association of Signaling Proteins and F-Actin Effects on Cluster Assembly Analyzed via Photoactivation Localization Microscopy in T Cells

    PubMed Central

    Hsu, Chih-Jung; Baumgart, Tobias

    2011-01-01

    Recognition of antigens by T cell receptors (TCRs) triggers cellular signaling cascades initiated by recruitment to the plasma membrane of numerous effector molecules to form signaling microclusters (MCs). Here we show that the method of high-resolution photoactivation localization microscopy (PALM) imaging can be used to analyze the spatial correlation between kinase ZAP70 and adaptor SLP76 MCs at the cell periphery and the effects of F-actin on MC assembly. We first determined the photophysical rate constants of Dronpa and tdEos fluorescence probes, which allowed us to optimize our dual-color PALM imaging method. We next analyzed the degrees of spatial association through determination of Mander's colocalization coefficients from PALM images, which revealed increasing spatial segregation of ZAP70 and SLP76 MCs at the cell periphery after initiation of signaling. We showed that this spatial segregation at the cell periphery occurred in parallel with the reduction of MC phosphorylation levels. Furthermore, we used Ripley's K function to analyze spatial randomness, and determined average radii of clusters as a function of activation time. The average radii of SLP76 and LAT MCs were found to decrease, whereas ZAP70 MC radii remained relatively constant. Finally, effects of F-actin depolymerization on MC morphology were studied by determining radial distributions of cluster circularity. Our data suggest that MC morphology is affected by actin polymerization. The quantitative analysis of sub-diffraction PALM images may provide a starting point for a molecular interpretation of cluster-cluster interactions and of the regulation of T cell signaling MCs by the cytoskeleton. PMID:21887278

  17. Effect of ADP on binding of skeletal S1 to F-actin.

    PubMed

    Andreev, O A; Ushakov, D S; Borejdo, J

    1998-12-22

    The proximity of skeletal myosin subfragment-1 (S1) to actin, and its orientation with respect to thin filaments of single muscle fibers, were compared in the presence and in the absence of ADP. The proximity was assessed by the efficiency of carbodiimide-induced cross-linking and the orientation by polarization of fluorescence of probes attached to the essential light chains. ADP made no difference in proximity or orientation when the molar ratio of S1 to actin was low or high. However, at the intermediate ratios, ADP made a significant difference. Strong dissociating agents, AMP-PNP and PPi, made significant differences at all ratios. To explain this behavior, it is unnecessary to invoke the ADP-induced "swinging" of the tail of S1. Rather, it is simply explained by the "two-state" model which we proposed earlier, in which S1 binds to one or to two actin protomers, depending on the saturation of the filaments with S1s. The dissociation induced by the ADP shifts the equilibrium between the two bound states. At high and low degrees of saturation, ADP is unable to significantly decrease the amount of S1 bound to F-actin. However, at intermediate saturation levels, ADP causes significantly more S1s to bind to two actins. These results suggest that the ADP-induced changes seen at the intermediate molar ratios are due to the dissociation-induced reorientation of S1. PMID:9922150

  18. F-actin cross-linking enhances the stability of force generation in disordered actomyosin networks

    NASA Astrophysics Data System (ADS)

    Jung, Wonyeong; Murrell, Michael P.; Kim, Taeyoon

    2015-12-01

    Myosin molecular motors and actin cross-linking proteins (ACPs) are known to mediate the generation and transmission of mechanical forces within the cortical F-actin cytoskeleton that drive major cellular processes such as cell division and migration. However, how motors and ACPs interact collectively over diverse timescales to modulate the time-dependent mechanical properties of the cytoskeleton remains unclear. In this study, we present a three-dimensional agent-based computational model of the cortical actomyosin network to quantitatively determine the effects of motor activity and the density and kinetics of ACPs on the accumulation and maintenance of mechanical tension within a disordered actomyosin network. We found that motors accumulate large stress quickly by behaving as temporary cross-linkers although this stress is relaxed over time unless there are sufficient passive ACPs to stabilize the network. Stabilization by ACPs helps motors to generate forces up to their maximum potential, leading to significant enhancement of the efficiency and stability of stress generation. Thus, we demonstrated that the force-dependent kinetics of ACP dissociation plays a critical role for the accumulation and sustainment of stress and the structural remodeling of networks.

  19. Street rabies virus causes dendritic injury and F-actin depolymerization in the hippocampus

    PubMed Central

    Song, Yan; Hou, Jinli; Qiao, Bin; Li, Yanchao; Xu, Ye; Duan, Ming; Guan, Zhenhong; Sun, Liankun

    2013-01-01

    Rabies is an acute viral infection of the central nervous system and is typically fatal in humans and animals; however, its pathogenesis remains poorly understood. In this study, the morphological changes of dendrites and dendritic spines in the CA1 region of the hippocampus were investigated in mice that were infected intracerebrally with an MRV strain of the street rabies virus. Haematoxylin and eosin and fluorescence staining analysis of brain sections from the infected mice showed very few morphological changes in the neuronal bodies and neuronal processes. However, we found a significant decrease in the number of dendritic spines. Primary neuronal cultures derived from the hippocampus of mice (embryonic day 16.5) that were infected with the virus also showed an obvious decrease in the number of dendritic spines. Furthermore, the decrease in the number of dendritic spines was related to the depolymerization of actin filaments (F-actin). We propose that the observed structural changes can partially explain the severe clinical disease that was found in experimental models of street rabies virus infections. PMID:23114630

  20. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  1. Mutations in the Gene That Encodes the F-Actin Binding Protein Anillin Cause FSGS

    PubMed Central

    Hall, Gentzon; Adeyemo, Adebowale; Hanke, Nils; Tossidou, Irini; Burchette, James; Wu, Guanghong; Homstad, Alison; Sparks, Matthew A.; Gomez, Jose; Jiang, Ruiji; Alonso, Andrea; Lavin, Peter; Conlon, Peter; Korstanje, Ron; Stander, M. Christine; Shamsan, Ghaidan; Barua, Moumita; Spurney, Robert; Singhal, Pravin C.; Kopp, Jeffrey B.; Haller, Hermann; Howell, David; Pollak, Martin R.; Shaw, Andrey S.; Schiffer, Mario; Winn, Michelle P.

    2014-01-01

    FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1–associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm–associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton. PMID:24676636

  2. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding

    PubMed Central

    Avery, Adam W.; Crain, Jonathan; Thomas, David D.; Hays, Thomas S.

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  3. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

    PubMed

    Avery, Adam W; Crain, Jonathan; Thomas, David D; Hays, Thomas S

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  4. Stimulation of regulatory volume decrease (RVD) by isolated bovine articular chondrocytes following F-actin disruption using latrunculin B.

    PubMed

    Kerrigan, Mark J P; Hall, Andrew C

    2005-01-01

    Articular chondrocytes are exposed to significant changes in extracellular osmolarity during normal joint activity, which can lead to changes in cell volume and metabolism of the extracellular matrix (ECM). Chondrocytes can respond to cell swelling/shrinking by volume regulatory pathways, but the signalling pathways are poorly understood although a role for the cytoskeleton is frequently implicated. Here, we have investigated the effects of disruption of the chondrocyte F-actin cytoskeleton on the recovery of cell volume by RVD. The cytoskeleton was perturbed using the relatively specific agent latrunculin B (5 microM; 30 min) and loss of F-actin integrity quantified using fluorescent phalloidin-labelling and confocal laser scanning microscopy (CLSM). Imaging of isolated chondrocytes labelled with Fura-2 to measure the fluorescence associated with cell volume changes, showed that the extent of hypo-osmotic swelling was unaffected by latrunculin B treatment. Two categories of the chondrocyte RVD response were observed: 'fast' RVD where at 3 min post-osmotic challenge there was a recovery in cell fluorescence of >or=80%, whereas other cells exhibited 'slow' RVD. Latrunculin B increased the proportion of chondrocytes demonstrating 'fast' RVD by approximately 10 fold and reduced those cells showing 'slow' RVD. An inhibitor of chondrocyte RVD (REV 5901) had no significant effect on the integrity of the cytoskeleton showing that the RVD response could be inhibited independent of the state of the F-actin cytoskeleton. These results suggest that the intact cortical F-actin cytoskeleton has a restraining effect on the RVD response of isolated bovine articular chondrocytes. PMID:16227656

  5. An Arp2/3 nucleated F-actin shell fragments nuclear membranes at nuclear envelope breakdown in starfish oocytes.

    PubMed

    Mori, Masashi; Somogyi, Kálmán; Kondo, Hiroshi; Monnier, Nilah; Falk, Henning J; Machado, Pedro; Bathe, Mark; Nédélec, François; Lénárt, Péter

    2014-06-16

    Animal cells disassemble and reassemble their nuclear envelopes (NEs) upon each division. Nuclear envelope breakdown (NEBD) serves as a major regulatory mechanism by which mixing of cytoplasmic and nuclear compartments drives the complete reorganization of cellular architecture, committing the cell for division. Breakdown is initiated by phosphorylation-driven partial disassembly of the nuclear pore complexes (NPCs), increasing their permeability but leaving the overall NE structure intact. Subsequently, the NE is rapidly broken into membrane fragments, defining the transition from prophase to prometaphase and resulting in complete mixing of cyto- and nucleoplasm. However, the mechanism underlying this rapid NE fragmentation remains largely unknown. Here, we show that NE fragmentation during NEBD in starfish oocytes is driven by an Arp2/3 complex-nucleated F-actin "shell" that transiently polymerizes on the inner surface of the NE. Blocking the formation of this F-actin shell prevents membrane fragmentation and delays entry of large cytoplasmic molecules into the nucleus. We observe spike-like protrusions extending from the F-actin shell that appear to "pierce" the NE during the fragmentation process. Finally, we show that NE fragmentation is essential for successful reproduction, because blocking this process in meiosis leads to formation of aneuploid eggs. PMID:24909322

  6. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation

    PubMed Central

    Chen, Zhe; Luo, Qing; Lin, Chuanchuan; Kuang, Dongdong; Song, Guanbin

    2016-01-01

    Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity. PMID:27444891

  7. Nuclear DNA helicase II (RNA helicase A) binds to an F-actin containing shell that surrounds the nucleolus.

    PubMed

    Zhang, Suisheng; Köhler, Carsten; Hemmerich, Peter; Grosse, Frank

    2004-02-15

    Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), is an F-actin binding protein that is particularly enriched in the nucleolus of mouse cells. Here, we show that the nucleolar localization of NDH II of murine 3T3 cells depended on an ongoing rRNA synthesis. NDH II migrated out of the nucleolus after administration of 0.05 microg/ml actinomycin D, while nucleolin and the upstream binding factor (UBF) remained there. In S phase-arrested mouse cells, NDH II was frequently found at the nucleolar periphery, where it was accompanied by newly synthesized nucleolar RNA. Human NDH II was mainly distributed through the whole nucleoplasm and not enriched in the nucleoli. However, in the human breast carcinoma cell line MCF-7, NDH II was also found at the nucleolar periphery, together with the tumor suppressor protein p53. Both NDH II and p53 were apparently attached to the F-actin-based filamentous network that surrounded the nucleoli. Accordingly, this subnuclear structure was sensitive to F-actin depolymerizing agents. Depolymerization with gelsolin led to a striking accumulation of NDH II in the nucleoli of MCF-7 cells. This effect was abolished by RNase, which extensively released nucleolus-bound NDH II when added together with gelsolin. Taken together, these results support the idea that an actin-based filamentous network may anchor NDH II at the nucleolar periphery for pre-ribosomal RNA processing, ribosome assembly, and/or transport. PMID:14729462

  8. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation.

    PubMed

    Chen, Zhe; Luo, Qing; Lin, Chuanchuan; Kuang, Dongdong; Song, Guanbin

    2016-01-01

    Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity. PMID:27444891

  9. Hypotonicity and cell volume regulation in shark rectal gland: role of organic osmolytes and F-actin.

    PubMed

    Ziyadeh, F N; Mills, J W; Kleinzeller, A

    1992-03-01

    Hypotonic stress (reduction of external tonicity from approximately 900 mosM and 295 mM NaCl to approximately 600 mosM and 135 mM NaCl) produced a relatively slow regulatory volume decrease (RVD) in dogfish shark (Squalus acanthias) rectal gland cells. During the 5-h experiment, cell K+ content remained unchanged; cell content of Na+ and Cl- dropped in the initial swelling phase by some 50% (reflecting the corresponding reduction in medium NaCl), and then remained unchanged during volume recovery phase. Also, cellular fluxes of 86Rb+ and urea were not affected by hypotonic stress. However, hypotonicity enhanced 10- to 20-fold the efflux of organic cell osmolytes taurine, betaine, and trimethyloxamine, and this accounted for the loss of osmotically obliged water during RVD. Enhancement of osmolyte efflux by hypotonic stress was abolished by readjusting the low-Na+ saline to isotonicity (approximately 900 mosM) with innocuous cations (choline+, Li+, or N-methylglucamine+). The results suggest that reduction of medium tonicity may be the determinant for the RVD response to hypotonic stress. The above properties of the observed RVD were also displayed when studying changes on cell F-actin at the basolateral cell face; hypotonic stress (medium with 135 mM NaCl) produced a rapid disappearance of fluorescence related to this cytoskeletal component, whereas no such changes were seen in low-Na+ salines made isotonic with choline or N-methylglucamine chloride nor in a saline made hyposmolar by omitting urea. Hence, hypotonicity is required to affect F-actin organization (depolymerization?). These changes of F-actin fluorescence are transient; they were completed within 5-10 min of hypotonic stress, and afterwards a gradual reconstitution of cell F-actin organization was seen. The above observations are consistent with the assumption that, in shark rectal gland cells, transient loss of cytoskeleton (F-actin) organization at the basolateral cell face, induced by hypotonicity

  10. Effect of Flumorph on F-Actin Dynamics in the Potato Late Blight Pathogen Phytophthora infestans.

    PubMed

    Hua, Chenlei; Kots, Kiki; Ketelaar, Tijs; Govers, Francine; Meijer, Harold J G

    2015-04-01

    Oomycetes are fungal-like pathogens that cause notorious diseases. Protecting crops against oomycetes requires regular spraying with chemicals, many with an unknown mode of action. In the 1990s, flumorph was identified as a novel crop protection agent. It was shown to inhibit the growth of oomycete pathogens including Phytophthora spp., presumably by targeting actin. We recently generated transgenic Phytophthora infestans strains that express Lifeact-enhanced green fluorescent protein (eGFP), which enabled us to monitor the actin cytoskeleton during hyphal growth. For analyzing effects of oomicides on the actin cytoskeleton in vivo, the P. infestans Lifeact-eGFP strain is an excellent tool. Here, we confirm that flumorph is an oomicide with growth inhibitory activity. Microscopic analyses showed that low flumorph concentrations provoked hyphal tip swellings accompanied by accumulation of actin plaques in the apex, a feature reminiscent of tips of nongrowing hyphae. At higher concentrations, swelling was more pronounced and accompanied by an increase in hyphal bursting events. However, in hyphae that remained intact, actin filaments were indistinguishable from those in nontreated, nongrowing hyphae. In contrast, in hyphae treated with the actin depolymerizing drug latrunculin B, no hyphal bursting was observed but the actin filaments were completely disrupted. This difference demonstrates that actin is not the primary target of flumorph. PMID:25496300

  11. Ultrastructural localization of F-actin using phalloidin and quantum dots in HL-60 promyelocytic leukemia cell line after cell death induction by arsenic trioxide.

    PubMed

    Izdebska, Magdalena; Gagat, Maciej; Grzanka, Dariusz; Grzanka, Alina

    2013-06-01

    Quantum dots (QDs) are fluorescent nanocrystals whose unique properties are fundamentally different from organic fluorophores. Moreover, their cores display sufficient electron density to be visible under transmission electron microscopy (TEM). Here, we report a technique for phalloidin-based TEM detection of F-actin. The ultrastructural reorganization of F-actin after arsenic trioxide (ATO) treatment was estimated using a combination of pre- and post-embedding techniques with biotinylated phalloidin and QD-streptavidin conjugates or colloidal gold (AU) conjugated to streptavidin. Ultrastructural studies showed ATO-induced apoptosis of HL-60 cells. Moreover, different patterns of QD-labeled F-actin after ATO treatment were seen. In the case of AU labeling, only a few gold particles were seen and it was impossible to see any difference in F-actin distribution. TEM imaging experiments using QDs and colloidal gold (AU) showed that the strategy of bioconjugation of nanoprobes is the most important factor in biotinylated phalloidin detection of F-actin using streptavidin-coated nanoparticles, especially at the ultrastructural level. Additionally, the results presented in present study confirm the essential role of F-actin in chromatin reorganization during cell death processes. PMID:23312591

  12. Nuclear F-actin enhances the transcriptional activity of β-catenin by increasing its nuclear localization and binding to chromatin.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; de Lanerolle, Primal; Harata, Masahiko

    2016-04-01

    Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor β-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of β-catenin. Our results also show that the nuclear F-actin colocalizes with β-catenin and enhances the binding of β-catenin to the downstream target genes of the Wnt/β-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/β-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/β-catenin signaling. PMID:26900020

  13. A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis

    PubMed Central

    2013-01-01

    Introduction Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit. Methods We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading. Results With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS). Conclusions CapG inhibition strongly reduces breast cancer

  14. The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.

    PubMed Central

    Root, D. D.; Reisler, E.

    1992-01-01

    Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1304380

  15. Experimental and computational assessment of F-actin influence in regulating cellular stiffness and relaxation behaviour of fibroblasts.

    PubMed

    Fallqvist, Björn; Fielden, Matthew L; Pettersson, Torbjörn; Nordgren, Niklas; Kroon, Martin; Gad, Annica K B

    2016-06-01

    In biomechanics, a complete understanding of the structures and mechanisms that regulate cellular stiffness at a molecular level remain elusive. In this paper, we have elucidated the role of filamentous actin (F-actin) in regulating elastic and viscous properties of the cytoplasm and the nucleus. Specifically, we performed colloidal-probe atomic force microscopy (AFM) on BjhTERT fibroblast cells incubated with Latrunculin B (LatB), which results in depolymerisation of F-actin, or DMSO control. We found that the treatment with LatB not only reduced cellular stiffness, but also greatly increased the relaxation rate for the cytoplasm in the peripheral region and in the vicinity of the nucleus. We thus conclude that F-actin is a major determinant in not only providing elastic stiffness to the cell, but also in regulating its viscous behaviour. To further investigate the interdependence of different cytoskeletal networks and cell shape, we provided a computational model in a finite element framework. The computational model is based on a split strain energy function of separate cellular constituents, here assumed to be cytoskeletal components, for which a composite strain energy function was defined. We found a significant influence of cell geometry on the predicted mechanical response. Importantly, the relaxation behaviour of the cell can be characterised by a material model with two time constants that have previously been found to predict mechanical behaviour of actin and intermediate filament networks. By merely tuning two effective stiffness parameters, the model predicts experimental results in cells with a partly depolymerised actin cytoskeleton as well as in untreated control. This indicates that actin and intermediate filament networks are instrumental in providing elastic stiffness in response to applied forces, as well as governing the relaxation behaviour over shorter and longer time-scales, respectively. PMID:26766328

  16. Eye Movements Reveal Dynamics of Task Control

    ERIC Educational Resources Information Center

    Mayr, Ulrich; Kuhns, David; Rieter, Miranda

    2013-01-01

    With the goal to determine the cognitive architecture that underlies flexible changes of control settings, we assessed within-trial and across-trial dynamics of attentional selection by tracking of eye movements in the context of a cued task-switching paradigm. Within-trial dynamics revealed a switch-induced, discrete delay in onset of…

  17. Fluid Shear Stress Upregulates E-Tmod41 via miR-23b-3p and Contributes to F-Actin Cytoskeleton Remodeling during Erythropoiesis

    PubMed Central

    Mu, Weiyun; Wang, Xifu; Zhang, Xiaolan; Zhu, Sida; Sun, Dagong; Ka, Weibo; Sung, Lanping Amy; Yao, Weijuan

    2015-01-01

    The membrane skeleton of mature erythrocyte is formed during erythroid differentiation. Fluid shear stress is one of the main factors that promote embryonic hematopoiesis, however, its effects on erythroid differentiation and cytoskeleton remodeling are unclear. Erythrocyte tropomodulin of 41 kDa (E-Tmod41) caps the pointed end of actin filament (F-actin) and is critical for the formation of hexagonal topology of erythrocyte membrane skeleton. Our study focused on the regulation of E-Tmod41 and its role in F-actin cytoskeleton remodeling during erythroid differentiation induced by fluid shear stress. Mouse erythroleukemia (MEL) cells and embryonic erythroblasts were subjected to fluid shear stress (5 dyn/cm2) and erythroid differentiation was induced in both cells. F-actin content and E-Tmod41 expression were significantly increased in MEL cells after shearing. E-Tmod41 overexpression resulted in a significant increase in F-actin content, while the knockdown of E-Tmod41 generated the opposite result. An E-Tmod 3’UTR targeting miRNA, miR-23b-3p, was found suppressed by shear stress. When miR-23b-3p level was overexpressed / inhibited, both E-Tmod41 protein level and F-actin content were reduced / augmented. Furthermore, among the two alternative promoters of E-Tmod, PE0 (upstream of exon 0), which mainly drives the expression of E-Tmod41, was found activated by shear stress. In conclusion, our results suggest that fluid shear stress could induce erythroid differentiation and F-actin cytoskeleton remodeling. It upregulates E-Tmod41 expression through miR-23b-3p suppression and PE0 promoter activation, which, in turn, contributes to F-actin cytoskeleton remodeling. PMID:26308647

  18. Fluid Shear Stress Upregulates E-Tmod41 via miR-23b-3p and Contributes to F-Actin Cytoskeleton Remodeling during Erythropoiesis.

    PubMed

    Mu, Weiyun; Wang, Xifu; Zhang, Xiaolan; Zhu, Sida; Sun, Dagong; Ka, Weibo; Sung, Lanping Amy; Yao, Weijuan

    2015-01-01

    The membrane skeleton of mature erythrocyte is formed during erythroid differentiation. Fluid shear stress is one of the main factors that promote embryonic hematopoiesis, however, its effects on erythroid differentiation and cytoskeleton remodeling are unclear. Erythrocyte tropomodulin of 41 kDa (E-Tmod41) caps the pointed end of actin filament (F-actin) and is critical for the formation of hexagonal topology of erythrocyte membrane skeleton. Our study focused on the regulation of E-Tmod41 and its role in F-actin cytoskeleton remodeling during erythroid differentiation induced by fluid shear stress. Mouse erythroleukemia (MEL) cells and embryonic erythroblasts were subjected to fluid shear stress (5 dyn/cm2) and erythroid differentiation was induced in both cells. F-actin content and E-Tmod41 expression were significantly increased in MEL cells after shearing. E-Tmod41 overexpression resulted in a significant increase in F-actin content, while the knockdown of E-Tmod41 generated the opposite result. An E-Tmod 3'UTR targeting miRNA, miR-23b-3p, was found suppressed by shear stress. When miR-23b-3p level was overexpressed / inhibited, both E-Tmod41 protein level and F-actin content were reduced / augmented. Furthermore, among the two alternative promoters of E-Tmod, PE0 (upstream of exon 0), which mainly drives the expression of E-Tmod41, was found activated by shear stress. In conclusion, our results suggest that fluid shear stress could induce erythroid differentiation and F-actin cytoskeleton remodeling. It upregulates E-Tmod41 expression through miR-23b-3p suppression and PE0 promoter activation, which, in turn, contributes to F-actin cytoskeleton remodeling. PMID:26308647

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

    PubMed

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

    1993-10-01

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

  20. Difference in hydration structures between F-actin and myosin subfragment-1 detected by small-angle X-ray and neutron scattering

    PubMed Central

    Matsuo, Tatsuhito; Arata, Toshiaki; Oda, Toshiro; Fujiwara, Satoru

    2013-01-01

    Hydration structures around F-actin and myosin subfragment-1 (S1), which play central roles as counterparts in muscle contraction, were investigated by small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). The radius of gyration of chymotryptic S1 was evaluated to be 41.3±1.1 Å for SAXS, 40.1±3.0 Å for SANS in H2O, and 37.8±0.8 Å for SANS in D2O, respectively. The values of the cross-sectional radius of gyration of F-actin were 25.4±0.03 Å for SAXS, 23.4±2.4 Å for SANS in H2O, and 22.6 ± 0.6 Å for SANS in D2O, respectively. These differences arise from different contributions of the hydration shell to the scattering curves. Analysis by model calculations showed that the hydration shell of S1 has the average density 10–15% higher than bulk water, being the typical hydration shell. On the other hand, the hydration shell of F-actin has the average density more than 19% higher than bulk water, indicating that F-actin has a denser, unusual hydration structure. The results indicate a difference in the hydration structures around F-actin and S1. The unusual hydration structure around F-actin may have the structural property of so-called “hyper-mobile water” around F-actin. PMID:27493547

  1. Changes in F-actin organization induced by hard metal particle exposure in rat pulmonary epithelial cells using laser scanning confocal microscopy.

    PubMed

    Antonini, J M; Starks, K; Roberts, J R; Millecchia, L; Yang, H M; Rao, K M

    2000-01-01

    Chronic inhalation of hard metal (WC-Co) particles causes alveolitis and the eventual development of pulmonary fibrosis. The initial inflammatory response includes a change in the alveolar epithelial cell-capillary barrier, which has been shown to be regulated by the state of assembly and organization of the actin cytoskeletal network. The objective of this study was to evaluate the effect WC-Co particles have on F-actin organization of lung epithelial cells in an in vitro culture system. Rat lung epithelial (L2) cells were exposed to 5, 25, and 100 microg/mL of WC-Co particles, as well as the individual components (Co and WC) of the hard metal mixture particles for 24 h. The effect on F-actin organization was visualized by laser scanning confocal microscopy (LSCM) following Bodipy-Phallacidin staining. Minimal changes in the F-actin microfilaments of L2 cells were observed by LSCM after exposure to WC and WC-Co at 5 and 25 microg/mL, while at 100 microg/mL, there was a noticeable disruption in the uniform distribution of L2 cell F-actin microfilaments. After exposure to Co, a dose-dependent change in the F-actin organization of the L2 cells was observed. Little change in F-actin assembly was observed after treatment with 5 microg/mL of Co (the concentration equivalent to the 5% amount of Co commonly present in 100 microg/mL of the WC-Co sample mixture). However, at 100 microg/mL of Co, the microfilaments aggregated into homogeneous masses within the cells, and a significant loss in the organization of L2 F-actin was observed. These dramatic alterations in F-actin organization seen after exposure to the higher doses of Co were attributed to an increase in L2 cell injury as measured by lactate dehydrogenase and trypan blue exclusion. We conclude the pulmonary response evoked in the lung by inhalation of high levels of WC-Co particles is unlikely due to alterations in the F-actin microfilaments of lung-epithelial cells. PMID:10900403

  2. Plasma Gelsolin Levels Decrease in Diabetic State and Increase upon Treatment with F-Actin Depolymerizing Versions of Gelsolin

    PubMed Central

    Khatri, Neeraj; Sagar, Amin; Peddada, Nagesh; Choudhary, Vikas; Chopra, Bhupinder Singh; Garg, Veena; Ashish

    2014-01-01

    The study aims to map plasma gelsolin (pGSN) levels in diabetic humans and mice models of type II diabetes and to evaluate the efficacy of gelsolin therapy in improvement of diabetes in mice. We report that pGSN values decrease by a factor of 0.45 to 0.5 in the blood of type II diabetic humans and mice models. Oral glucose tolerance test in mice models showed that subcutaneous administration of recombinant pGSN and its F-actin depolymerizing competent versions brought down blood sugar levels comparable to Sitagliptin, a drug used to manage hyperglycemic condition. Further, daily dose of pGSN or its truncated versions to diabetic mice for a week kept sugar levels close to normal values. Also, diabetic mice treated with Sitagliptin for 7 days, showed increase in their pGSN values with the decrease in blood glucose as compared to their levels at the start of treatment. Gelsolin helped in improving glycemic control in diabetic mice. We propose that gelsolin level monitoring and replacement of F-actin severing capable gelsolin(s) should be considered in diabetic care. PMID:25478578

  3. F-actin-rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local RhoA signalling

    PubMed Central

    Heemskerk, Niels; Schimmel, Lilian; Oort, Chantal; van Rijssel, Jos; Yin, Taofei; Ma, Bin; van Unen, Jakobus; Pitter, Bettina; Huveneers, Stephan; Goedhart, Joachim; Wu, Yi; Montanez, Eloi; Woodfin, Abigail; van Buul, Jaap D.

    2016-01-01

    During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation. PMID:26814335

  4. Prevalence of IgG autoantibody against F-actin in patients suspected of having autoimmune or acute viral hepatitis.

    PubMed

    Jaskowski, Troy D; Konnick, Eric Q; Ashwood, Edward R; Litwin, Christine M; Hill, Harry R

    2007-01-01

    Our objectives in this study were to compare results obtained by an enzyme immunoassay (EIA) for F-actin antibody (FAA) immunoglobulin G (IgG) to those determined by an indirect fluorescent antibody (IFA) assay for smooth muscle antibody (SMA) IgG, and to determine the prevalence of FAA in patient sera having serologic evidence of acute viral hepatitis. Sera from 415 patients suspected of having autoimmune hepatitis (AIH), 208 patients suspected of having acute viral hepatitis A, B, or C, and 100 healthy blood donors (HBD) were included in the study. Only one of 100 HBD showed low levels (20-30 Units) of F-actin IgG. In patients suspected of having AIH, the prevalence of FAA increased as SMA titers increased and all sera with SMA titers of >or=1:160 were FAA-positive. In contrast, there were many sera with negative (<1:20) or low (1:20-1:40) SMA titers that contained moderate to high levels (>30 Units) of FAA; many exceeding 80 Units. Moreover, 51.4% of these sera were also positive for anti-nuclear antibody (ANA), which is also utilized in diagnosing type 1 AIH. FAA was detected in 25% of viral hepatitis antibody-positive sera, with the majority (59.3%) containing low levels, and all were ANA-negative. PMID:17621360

  5. F-actin-rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local RhoA signalling.

    PubMed

    Heemskerk, Niels; Schimmel, Lilian; Oort, Chantal; van Rijssel, Jos; Yin, Taofei; Ma, Bin; van Unen, Jakobus; Pitter, Bettina; Huveneers, Stephan; Goedhart, Joachim; Wu, Yi; Montanez, Eloi; Woodfin, Abigail; van Buul, Jaap D

    2016-01-01

    During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation. PMID:26814335

  6. Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells.

    PubMed

    Rudolf, Rüdiger; Kögel, Tanja; Kuznetsov, Sergei A; Salm, Thorsten; Schlicker, Oliver; Hellwig, Andrea; Hammer, John A; Gerdes, Hans-Hermann

    2003-04-01

    Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the trans-Golgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells. PMID:12615975

  7. Formin3 is required for assembly of the F-actin structure that mediates tracheal fusion in Drosophila.

    PubMed

    Tanaka, Hiromasa; Takasu, Etsuko; Aigaki, Toshiro; Kato, Kagayaki; Hayashi, Shigeo; Nose, Akinao

    2004-10-15

    During tracheal development in Drosophila, some branches join to form a continuous luminal network. Specialized cells at the branch tip, called fusion cells, extend filopodia to make contact and become doughnut shaped to allow passage of the lumen. These morphogenetic processes accompany the highly regulated cytoskeletal reorganization of fusion cells. We identified the Drosophila formin3 (form3) gene that encodes a novel formin and plays a role in tracheal fusion. Formins are a family of proteins characterized by highly conserved formin homology (FH) domains. The formin family functions in various actin-based processes, including cytokinesis and cell polarity. During embryogenesis, form3 mRNA is expressed mainly in the tracheal system. In form3 mutant embryos, the tracheal fusion does not occur at some points. This phenotype is rescued by the forced expression of form3 in the trachea. We used live imaging of GFP-moesin during tracheal fusion to show that an F-actin structure that spans the adjoining fusion cells and mediates the luminal connection does not form at abnormal anastomosis sites in form3 mutants. These results suggested that Form3 plays a role in the F-actin assembly, which is essential for cellular rearrangement during tracheal fusion. PMID:15385168

  8. ARF6 promotes the formation of Rac1 and WAVE-dependent ventral F-actin rosettes in breast cancer cells in response to epidermal growth factor.

    PubMed

    Marchesin, Valentina; Montagnac, Guillaume; Chavrier, Philippe

    2015-01-01

    Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF) stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration. PMID:25799492

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

    PubMed Central

    Anderson, Tom W.; Vaughan, Andrew N.

    2008-01-01

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

  10. Nuclear F-actin Formation and Reorganization upon Cell Spreading*♦

    PubMed Central

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

    2015-01-01

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

  11. Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

    PubMed Central

    Zheng, Wenjun; Barua, Bipasha; Hitchcock-DeGregori, Sarah E.

    2013-01-01

    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. PMID:24138864

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

    PubMed

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

    2009-11-13

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

  13. The PCH family member MAYP/PSTPIP2 directly regulates F-actin bundling and enhances filopodia formation and motility in macrophages.

    PubMed

    Chitu, Violeta; Pixley, Fiona J; Macaluso, Frank; Larson, Daniel R; Condeelis, John; Yeung, Yee-Guide; Stanley, E Richard

    2005-06-01

    Macrophage actin-associated tyrosine phosphorylated protein (MAYP) belongs to the Pombe Cdc15 homology (PCH) family of proteins involved in the regulation of actin-based functions including cell adhesion and motility. In mouse macrophages, MAYP is tyrosine phosphorylated after activation of the colony-stimulating factor-1 receptor (CSF-1R), which also induces actin reorganization, membrane ruffling, cell spreading, polarization, and migration. Because MAYP associates with F-actin, we investigated the function of MAYP in regulating actin organization in macrophages. Overexpression of MAYP decreased CSF-1-induced membrane ruffling and increased filopodia formation, motility and CSF-1-mediated chemotaxis. The opposite phenotype was observed with reduced expression of MAYP, indicating that MAYP is a negative regulator of CSF-1-induced membrane ruffling and positively regulates formation of filopodia and directional migration. Overexpression of MAYP led to a reduction in total macrophage F-actin content but was associated with increased actin bundling. Consistent with this, purified MAYP bundled F-actin and regulated its turnover in vitro. In addition, MAYP colocalized with cortical and filopodial F-actin in vivo. Because filopodia are postulated to increase directional motility by acting as environmental sensors, the MAYP-stimulated increase in directional movement may be at least partly explained by enhancement of filopodia formation. PMID:15788569

  14. Tropomyosin-1 protects transformed alveolar epithelial cells against cigaret smoke extract through the stabilization of F-actin-dependent cell-cell junctions.

    PubMed

    Gagat, Maciej; Grzanka, Dariusz; Izdebska, Magdalena; Sroka, Wiktor Dariusz; Hałas-Wiśniewska, Marta; Grzanka, Alina

    2016-04-01

    The aim of the study was to estimate the effect of tropomyosin-1-based structural stabilization of F-actin in transformed human alveolar epithelial line H1299 cells subjected to high oxidative stress induced by cigaret smoke extract. We demonstrated here that cigaret smoke extract induces cell shrinking and detachment as a consequence of F-actin cytoskeleton degradation in H1299 cells not overexpressing tropomyosin-1. Furthermore, the treatment of these cells with cigaret smoke extract resulted in the loss of peripheral localization of ZO-1 and initiated apoptosis. In contrast, structural stabilization of F-actin, by overexpression of tropomyosin-1, preserved cell to cell interactions through the attenuation of cortical actin organization into thin fibers and thus protected these cells against oxidative stress-induced degradation of actin cytoskeleton and cell death. In conclusion, we suggest that structural stabilization of thin cortical F-actin fibers increases link between tight junctions proteins and actin cytoskeleton and thus protects H1299 cells against cigaret smoke extract. PMID:26805581

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

    SciTech Connect

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki

    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

  16. Reconstitution of Actin-based Motility by Vasodilator-stimulated Phosphoprotein (VASP) Depends on the Recruitment of F-actin Seeds from the Solution Produced by Cofilin*

    PubMed Central

    Siton, Orit; Bernheim-Groswasser, Anne

    2014-01-01

    Vasodilator-stimulated phosphoprotein (VASP) is active in many filopodium-based and cytoskeleton reorganization processes. It is not fully understood how VASP directly functions in actin-based motility and how regulatory proteins affect its function. Here, we combine bead motility assay and single filament experiments. In the presence of a bundling component, actin bundles that grow from the surface of WT-VASP-coated beads induced movement of the beads. VASP promotes actin-based movement alone, in the absence of other actin nucleators. We propose that at physiological salt conditions VASP nucleation activity is too weak to promote motility and bundle formation. Rather, VASP recruits F-actin seeds from the solution and promotes their elongation. Cofilin has a crucial role in the nucleation of these F-actin seeds, notably under conditions of unfavorable spontaneous actin nucleation. We explored the role of multiple VASP variants. We found that the VASP-F-actin binding domain is required for the recruitment of F-actin seeds from the solution. We also found that the interaction of profilin-actin complexes with the VASP-proline-rich domain and the binding of the VASP-F-actin binding domain to the side of growing filaments is critical for transforming actin polymerization into motion. At the single filament level, profilin mediates both filament elongation rate and VASP anti-capping activity. Binding of profilin-actin complexes increases the polymerization efficiency by VASP but decreases its efficiency as an anti-capper; binding of free profilin creates the opposite effect. Finally, we found that an additional component such as methylcellulose or fascin is required for actin bundle formation and motility mediated by VASP. PMID:25246528

  17. Subcellular localisation of the p40phox component of NADPH oxidase involves direct interactions between the Phox homology domain and F-actin

    PubMed Central

    Shao, Dongmin; Segal, Anthony W.; Dekker, Lodewijk V.

    2010-01-01

    Cytosolic components of the NADPH oxidase interact with the actin cytoskeleton. These interactions are thought to be important for the activation of this enzyme system but they are poorly characterised at the molecular level. Here we have explored the interaction between the actin cytoskeleton and p40phox, one of the cytosolic components of NADPH oxidase. Full length p40phox expressed in COS cells co-localised with F-actin in a peripheral lamellar compartment. The co-localisation was lost after deletion of the Phox homology (PX) domain and the PX domain in isolation (p40PX) showed the same F-actin co-localisation as the full length protein. PX domains are known lipid-binding modules however, a mutant p40PX which did not bind lipids still co-localised with F-actin suggesting that lipid-independent interactions underlie the localisation. Affinity chromatography identified actin as a binding partner for p40PX in neutrophil extracts. Pure actin interacted with both p40phox and with p40PX suggesting it is a direct interaction. Disruption of the actin cytoskeleton with cytochalasin D resulted in actin rearrangement and concomitantly the localisation of full length p40phox proteins and that of p40PX changed. Thus p40PX is a dual F-actin/lipid-binding module and F-actin interactions with the PX domain dictate at least in part the intracellular localisation of the cytosolic p40phox subunit of the NADPH oxidase. PMID:20637895

  18. Growth-arrest-specific 7C protein inhibits tumor metastasis via the N-WASP/FAK/F-actin and hnRNP U/β-TrCP/β-catenin pathways in lung cancer

    PubMed Central

    Chang, Jer-Wei; Mao, Jiou-Shan; Tsai, Charng-Dar; Wu, Pei-Chen; Lin, Cuei-Jyuan; Lu, Yi-Lin; Liao, Sheng-You; Cheng, Hung-Chi; Hsu, Han-Shui

    2015-01-01

    Growth-arrest-specific 7 (GAS7) belongs to a group of adaptor proteins that coordinate the actin cytoskeleton. Among human GAS7 isoforms, only GAS7C possesses a Src homology 3 domain. We report here that GAS7C acts as a migration suppressor and can serve as a prognostic biomarker in lung cancer. GAS7C overexpression reduces lung cancer migration, whereas GAS7C knockdown enhances cancer cell migration. Importantly, ectopically overexpressed GAS7C binds tightly with N-WASP thus inactivates the fibronectin/integrin/FAK pathway, which in turn leads to the suppression of F-actin dynamics. In addition, overexpression of GAS7C sequesters hnRNP U and thus decreases the level of β-catenin protein via the β-TrCP ubiquitin-degradation pathway. The anti-metastatic effect of GAS7C overexpression was also confirmed using lung cancer xenografts. Our clinical data indicated that 23.6% (25/106) of lung cancer patients showed low expression of GAS7C mRNA which correlated with a poorer overall survival. In addition, low GAS7C mRNA expression was detected in 60.0% of metastatic lung cancer patients, indicating an association between low GAS7C expression and cancer progression. A significant inverse correlation between mRNA expression and promoter hypermethylation was also found, which suggests that the low level of GAS7C expression was partly due to promoter hypermethylation. Our results provide novel evidence that low GAS7C correlates with poor prognosis and promotes metastasis in lung cancer. Low GAS7C increases cancer cell motility by promoting N-WASP/FAK/F-actin cytoskeleton dynamics. It also enhances β-catenin stability via hnRNP U/β-TrCP complex formation. Therefore, GAS7C acts as a metastasis suppressor in lung cancer. PMID:26506240

  19. Dynamic Environmental Photosynthetic Imaging Reveals Emergent Phenotypes.

    PubMed

    Cruz, Jeffrey A; Savage, Linda J; Zegarac, Robert; Hall, Christopher C; Satoh-Cruz, Mio; Davis, Geoffry A; Kovac, William Kent; Chen, Jin; Kramer, David M

    2016-06-22

    Understanding and improving the productivity and robustness of plant photosynthesis requires high-throughput phenotyping under environmental conditions that are relevant to the field. Here we demonstrate the dynamic environmental photosynthesis imager (DEPI), an experimental platform for integrated, continuous, and high-throughput measurements of photosynthetic parameters during plant growth under reproducible yet dynamic environmental conditions. Using parallel imagers obviates the need to move plants or sensors, reducing artifacts and allowing simultaneous measurement on large numbers of plants. As a result, DEPI can reveal phenotypes that are not evident under standard laboratory conditions but emerge under progressively more dynamic illumination. We show examples in mutants of Arabidopsis of such "emergent phenotypes" that are highly transient and heterogeneous, appearing in different leaves under different conditions and depending in complex ways on both environmental conditions and plant developmental age. These emergent phenotypes appear to be caused by a range of phenomena, suggesting that such previously unseen processes are critical for plant responses to dynamic environments. PMID:27336966

  20. Intrastrand cross-linked actin between Gln-41 and Cys-374. I. Mapping of sites cross-linked in F-actin by N-(4-azido-2-nitrophenyl) putrescine.

    PubMed

    Hegyi, G; Mák, M; Kim, E; Elzinga, M; Muhlrad, A; Reisler, E

    1998-12-22

    A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40-50 (or 40-62 in the arginine limited digest) and residues 373-375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 A, constrains to that range the distance between the gamma-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin [Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44-49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836]. The effect of cross-linking on the function of actin is described in the companion papers. PMID:9922144

  1. An affine continuum mechanical model for cross-linked F-actin networks with compliant linker proteins.

    PubMed

    Holzapfel, Gerhard A; Unterberger, Michael J; Ogden, Ray W

    2014-10-01

    Cross-linked actin networks are important building blocks of the cytoskeleton. In order to gain deeper insight into the interpretation of experimental data on actin networks, adequate models are required. In this paper we introduce an affine constitutive network model for cross-linked F-actin networks based on nonlinear continuum mechanics, and specialize it in order to reproduce the experimental behavior of in vitro reconstituted model networks. The model is based on the elastic properties of single filaments embedded in an isotropic matrix such that the overall properties of the composite are described by a free-energy function. In particular, we are able to obtain the experimentally determined shear and normal stress responses of cross-linked actin networks typically observed in rheometer tests. In the present study an extensive analysis is performed by applying the proposed model network to a simple shear deformation. The single filament model is then extended by incorporating the compliance of cross-linker proteins and further extended by including viscoelasticity. All that is needed for the finite element implementation is the constitutive model for the filaments, the linkers and the matrix, and the associated elasticity tensor in either the Lagrangian or Eulerian formulation. The model facilitates parameter studies of experimental setups such as micropipette aspiration experiments and we present such studies to illustrate the efficacy of this modeling approach. PMID:25043658

  2. Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

    NASA Technical Reports Server (NTRS)

    Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

    1995-01-01

    The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

  3. Disassembly of F-Actin Cytoskeleton after Interaction of Bacillus cereus with Fully Differentiated Human Intestinal Caco-2 Cells

    PubMed Central

    Minnaard, Jessica; Lievin-Le Moal, Vanessa; Coconnier, Marie-Helene; Servin, Alain L.; Pérez, Pablo F.

    2004-01-01

    In the present study, the role of direct procaryote-eucaryote interactions in the virulence of Bacillus cereus was investigated. As a model of human enterocytes, differentiated Caco-2 cells were used. Infection of fully differentiated Caco-2 cells with B. cereus in the exponential phase of growth, in order to minimize the concentration of spores or sporulating microorganisms, shows that a strain-dependent cytopathic effect develops. Interestingly, addition of 3-h-old cultures of some strains resulted in complete detachment of the cultured cells after a 3-h infection whereas no such effect was found after a 3-h infection with 16-h-old cultures. Infection of enterocyte-like cells with B. cereus leads to disruption of the F-actin network and necrosis. Even though the effect of secreted factors cannot be ruled out, direct eucaryote-procaryote interaction seems to be necessary. In addition, we observed that some B. cereus strains were able to be internalized in Caco-2 cells. Our findings add a new insight into the mechanisms of virulence of B. cereus in the context of intestinal infection. PMID:15155611

  4. Neutron Imaging Reveals Internal Plant Hydraulic Dynamics

    SciTech Connect

    Warren, Jeffrey; Bilheux, Hassina Z; Kang, Misun; Voisin, Sophie; Cheng, Chu-Lin; Horita, Jusuke; Perfect, Edmund

    2013-01-01

    Many terrestrial ecosystem processes are constrained by water availability and transport within the soil. Knowledge of plant water fluxes is thus critical for assessing mechanistic processes linked to biogeochemical cycles, yet resolution of root structure and xylem water transport dynamics has been a particularly daunting task for the ecologist. Through neutron imaging, we demonstrate the ability to non-invasively monitor individual root functionality and water fluxes within Zea mays L. (maize) and Panicum virgatum L. (switchgrass) seedlings growing in a sandy medium. Root structure and growth were readily imaged by neutron radiography and neutron computed tomography. Seedlings were irrigated with water or deuterium oxide and imaged through time as a growth lamp was cycled on to alter leaf demand for water. Sub-millimeter scale resolution reveals timing and magnitudes of root water uptake, redistribution within the roots, and root-shoot hydraulic linkages, relationships not well characterized by other techniques.

  5. Mechanical force-induced polymerization and depolymerization of F-actin at water/solid interfaces.

    PubMed

    Zhang, Xueqiang; Hu, Xiuyuan; Lei, Haozhi; Hu, Jun; Zhang, Yi

    2016-03-21

    Actin molecules are among the three main cytoskeleton proteins of cells and undergo rapid cycling to regulate critical processes such as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. Although extensive studies have been carried out on the dynamics as well as biological functions of actin polymerization and depolymerization both in vivo and in vitro, the molecular mechanisms by which cells sense and respond to mechanical signals are not fully understood. In particular, little attention has been paid to the effect of a physical force that is exerted directly on the actin cytoskeleton. In this paper, we have explored how the mechanical force affects the actin polymerization and depolymerization behaviors at water/solid interfaces using an atomic force microscope (AFM) operated in liquid. By raster scanning an AFM probe on a substrate surface with a certain load, it was found that actin monomers could polymerize into filaments without the help of actin related proteins (ARPs). Further study indicated that actin monomers were inclined to form filaments only under a small scanning load. The polymerized actin filaments would be depolymerized when the mechanical force was stronger. A possible mechanism has been suggested to explain the mechanical force induced actin polymerization. PMID:26928199

  6. Enterohaemorrhagic E. coli (EHEC) exploits a tryptophan switch to hijack host F-actin assembly

    PubMed Central

    Aitio, Olli; Hellman, Maarit; Skehan, Brian; Kesti, Tapio; Leong, John M.; Saksela, Kalle; Permi, Perttu

    2012-01-01

    SUMMARY Intrinsically disordered protein (IDP)-mediated interactions are often characterized by low affinity but high specificity. These traits are essential in signaling and regulation that require reversibility. Enterohaemorrhagic Escherichia coli (EHEC) exploit this situation by commandeering host cytoskeletal signaling to stimulate actin assembly beneath bound bacteria, generating ‘pedestals’ that promote intestinal colonization. EHEC translocates into the host cell two proteins, EspFU and Tir, which form a complex with the host protein IRTKS. The interaction of this complex with N-WASP triggers localized actin polymerization. We show that EspFU is an IDP that contains a transiently α-helical N-terminus and dynamic C-terminus. Our structure shows that single EspFU repeat is capable of forming a high-affinity trimolecular complex with N-WASP and IRTKS. We demonstrate that bacterial and cellular ligands interact with IRTKS SH3 in a similar fashion but the bacterial protein has evolved to outcompete cellular targets by utilizing a tryptophan switch that offers superior binding affinity enabling EHEC-induced pedestal formation. PMID:22921828

  7. Two-headed binding of a processive myosin to F-actin.

    PubMed

    Walker, M L; Burgess, S A; Sellers, J R; Wang, F; Hammer, J A; Trinick, J; Knight, P J

    2000-06-15

    Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation. PMID:10866203

  8. Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration

    PubMed Central

    Xavier, Charles-Peter; Rastetter, Raphael H.; Blömacher, Margit; Stumpf, Maria; Himmel, Mirko; Morgan, Reginald O.; Fernandez, Maria-Pilar; Wang, Conan; Osman, Asiah; Miyata, Yoshihiko; Gjerset, Ruth A.; Eichinger, Ludwig; Hofmann, Andreas; Linder, Stefan; Noegel, Angelika A.; Clemen, Christoph S.

    2012-01-01

    CRN2 (synonyms: coronin 1C, coronin 3) functions in the re-organization of the actin network and is implicated in cellular processes like protrusion formation, secretion, migration and invasion. We demonstrate that CRN2 is a binding partner and substrate of protein kinase CK2, which phosphorylates CRN2 at S463 in its C-terminal coiled coil domain. Phosphomimetic S463D CRN2 loses the wild-type CRN2 ability to inhibit actin polymerization, to bundle F-actin, and to bind to the Arp2/3 complex. As a consequence, S463D mutant CRN2 changes the morphology of the F-actin network in the front of lamellipodia. Our data imply that CK2-dependent phosphorylation of CRN2 is involved in the modulation of the local morphology of complex actin structures and thereby inhibits cell migration. PMID:22355754

  9. Three-dimensional reconstruction of a co-complex of F-actin with antibody Fab fragments to actin's NH2 terminus.

    PubMed Central

    Orlova, A; Yu, X; Egelman, E H

    1994-01-01

    We have decorated F-actin with Fab fragments of antibodies to actin residues 1-7. These antibody fragments do not strongly affect the rigor binding of myosin S-1 to actin, but do affect the binding of S-1 to actin in the presence of nucleotide (DasGupta, G., and E. Reisler, 1989. J. Mol. Biol. 207:833-836; 1991. Biochemistry. 30:9961-9966; 1992. Biochemistry. 31:1836-1841). Although the binding constant is rather low, we estimate that we have achieved about 85% occupancy of the actin sites. Three-dimensional reconstructions from electron micrographs of both negatively stained and frozen-hydrated filaments show that the Fab fragment is bound at the location of the NH2 terminus in the model of Holmes et al. (Holmes, K.C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature. 347:37-44) for F-actin, excluding very different orientations of the actin subunit in the filament. Most of the mass of the antibody is not visualized, which is due to the large mobility of the NH2 terminus in F-actin, differences in binding angle within the polyclonal antibody population, or a combination of both of these possibilities. Images FIGURE 1 FIGURE 5 FIGURE 7 FIGURE 10 PMID:8161679

  10. Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1997-01-01

    A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

  11. Analysis of rhodamine and fluorescein-labeled F-actin diffusion in vitro by fluorescence photobleaching recovery.

    PubMed Central

    Simon, J R; Gough, A; Urbanik, E; Wang, F; Lanni, F; Ware, B R; Taylor, D L

    1988-01-01

    Properties of filamentous acetamidofluorescein-labeled actin and acetamidotetramethylrhodamine-labeled actin (AF and ATR-actin, respectively) were examined to resolve discrepancies in the reported translational diffusion coefficients of F-actin measured in vitro by FPR and other techniques. Using falling-ball viscometry and two independent versions of fluorescence photobleaching recovery (FPR), the present data indicate that several factors are responsible for these discrepancies. Gel filtration chromatography profoundly affects the viscosity of actin solutions and filament diffusion coefficients. ATR-actin and, to a lesser degree, AF-actin show a reduction in viscosity in proportion to the fraction labeled, presumably due to filament shortening. Actin filaments containing AF-actin or ATR-actin are susceptible to photoinduced damage, including a covalent cross-linking of actin protomers within filaments and an apparent cleavage of filaments detected by a decrease of the measured viscosity and an increase in the measured filament diffusion coefficients. Quantum yields of the two photoinduced effects are quite different. Multiple cross-links are produced relative to each photobleaching event, whereas less than 1% filament cleavage occurs. Substantial differences in the filament diffusion coefficients measured by FPR are also the result of differences in illumination geometry and sampling time. However, under controlled conditions, FPR can be used as a quantitative tool for measuring the hydrodynamic properties of actin filaments. Incremented filament shortening caused by photoinduced cleavage or incremental addition of filament capping proteins produces a continuous and approximately linear increase of filament diffusion coefficients, indicating that filaments are not associated in solution. Our results indicate that actin filaments exhibit low mobilities and it is inferred that actin filaments formed in vitro by column-purified actin, under standard conditions, are

  12. A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic β cells

    PubMed Central

    Kalwat, Michael A.; Yoder, Stephanie M.; Wang, Zhanxiang; Thurmond, Debbie C.

    2012-01-01

    Human islet studies implicate an important signaling role for the Cdc42 effector protein p21-activated kinase (PAK1) in the sustained/second-phase of insulin secretion. Because human islets from type 2 diabetic donors lack ~80% of normal PAK1 protein levels, the mechanistic requirement for PAK1 signaling in islet function was interrogated. Similar to MIN6 β cells, human islets elicited glucose-stimulated PAK1 activation that was sensitive to the PAK1 inhibitor, IPA3. Given that sustained insulin secretion has been correlated with glucose-induced filamentous actin (F-actin) remodeling, we tested the hypothesis that a Cdc42-activated PAK1 signaling cascade is required to elicit F-actin remodeling to mobilize granules to the cell surface. Live-cell imaging captured the glucose-induced cortical F-actin remodeling in MIN6 β cells; IPA3-mediated inhibition of PAK1 abolished this remodeling. IPA3 also ablated glucose-stimulated insulin granule accumulation at the plasma membrane, consistent with its role in sustained/second-phase insulin release. Both IPA3 and a selective inhibitor of the Cdc42 GTPase, ML-141, blunted the glucose-stimulated activation of Raf-1, suggesting Raf-1 to be downstream of Cdc42→PAK1. IPA3 also inhibited MEK1/2 activation, implicating the MEK1/2→ERK1/2 cascade to occur downstream of PAK1. Importantly, PD0325901, a new selective inhibitor of MEK1/2→ERK1/2 activation, impaired F-actin remodeling and the sustained/amplification pathway of insulin release. Taken together, these data suggest that glucose-mediated activation of Cdc42 leads to activation of PAK1 and prompts activation of its downstream targets Raf-1, MEK1/2 and ERK1/2 to elicit F-actin remodeling and recruitment of insulin granules to the plasma membrane to support the sustained phase of insulin release. PMID:23246867

  13. Neutron Imaging Reveals Internal Plant Hydraulic Dynamics

    NASA Astrophysics Data System (ADS)

    Warren, J.; Bilheux, H.; Kang, M.; Voisin, S.; Cheng, C.; Horita, J.; Perfect, E.

    2011-12-01

    In situ quantification of soil-plant water fluxes have not been fully successful due to a lack of non-destructive techniques capable of revealing roots or water fluxes at relevant spatial scales. Neutron imaging is a unique non-invasive tool that can assess sub-millimeter scale material properties and transport in situ, and which has been successfully applied to characterize soil and plant water status. Here, we have applied neutron radiography and tomography to quantify water transport through individual maize roots in response to internal plant demand. Zea mays seedlings were grown for 10 days in Flint silica sand within 2.6 cm diameter Al chambers. Using a reactor-based neutron source at Oak Ridge National Laboratory (HFIR), water fluxes were tracked through the maize soil-root systems by collecting consecutive neutron radiographs over a 12 h period following irrigation with D2O. D has a much lower neutron attenuation than H, thus D2O displacement of existing H2O within the plant vascular system, or influx of D2O into previously dry tissue or soil is readily tracked by changes in image intensity through time. Plant water release and uptake was regulated by periodically cycling on a high-intensity grow light. From each maize replicate, selected regions of interest (ROI) were delineated around individual roots, root free soil, stem and leaf segments. Changes in ROI were tracked through time to reveal patterns of water flux. The hydration of root and stem tissue cycled in response to illumination; root water content often increased during darkness, then decreased with illumination as water was transported from the root into the stem. Relative root-shoot hydration through time illustrates the balance between demand, storage capacity and uptake, which varies depending on root characteristics and its localized soil environment. The dynamic transport of water between soil, individual roots, stems and leaves was readily visualized and quantified illustrating the value

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

    PubMed

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

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

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

    PubMed Central

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

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

  16. Dispersion of Response Times Reveals Cognitive Dynamics

    PubMed Central

    Holden, John G.; Van Orden, Guy C.; Turvey, Michael T.

    2013-01-01

    Trial to trial variation in word pronunciation times exhibits 1/f scaling. One explanation is that human performances are consequent on multiplicative interactions among interdependent processes – interaction dominant dynamics. This article describes simulated distributions of pronunciation times in a further test for multiplicative interactions and interdependence. Individual participant distributions of ≈1100 word pronunciation times are successfully mimicked for each participant in combinations of lognormal and power law behavior. Successful hazard function simulations generalize these results to establish interaction dominant dynamics, in contrast with component dominant dynamics, as a likely mechanism for cognitive activity. PMID:19348544

  17. [A study of quantitative dynamics of F-actin during oocyte maturation in the starfish Asterias amurensis].

    PubMed

    Lamash, N E; Eliseĭkina, M G

    2006-01-01

    We studied the actin cytoskeleton state in Asterias amurensis oocytes within 30 min after the 1-methyladenine-induced maturation until the germinal vesicle breakdown. The total amount of actin remained unchanged during oocyte maturation. In immature oocytes, the major part of actin is not a part of filaments, but in the presence of 1-methyladenine massive actin polymerization began already within 20 min. Electron immunocytochemistry methods demonstrated joint localization of actin and alpha-protein in the cytoplasm. They were redistributed from the cortex to the cytoplasm in the presence of 1-methyladenine. A possible involvement of actin cytoskeleton in transmembrane transduction of the hormonal signal at the postreceptor stages is discussed. PMID:17022441

  18. Dispersion of Response Times Reveals Cognitive Dynamics

    ERIC Educational Resources Information Center

    Holden, John G.; Van Orden, Guy C.; Turvey, Michael T.

    2009-01-01

    Trial-to-trial variation in word-pronunciation times exhibits 1/f scaling. One explanation is that human performances are consequent on multiplicative interactions among interdependent processes-interaction dominant dynamics. This article describes simulated distributions of pronunciation times in a further test for multiplicative interactions and…

  19. Peptide crystal simulations reveal hidden dynamics

    PubMed Central

    Janowski, Pawel A.; Cerutti, David S.; Holton, James; Case, David A.

    2013-01-01

    Molecular dynamics simulations of biomolecular crystals at atomic resolution have the potential to recover information on dynamics and heterogeneity hidden in the X-ray diffraction data. We present here 9.6 microseconds of dynamics in a small helical peptide crystal with 36 independent copies of the unit cell. The average simulation structure agrees with experiment to within 0.28 Å backbone and 0.42 Å all-atom rmsd; a model refined against the average simulation density agrees with the experimental structure to within 0.20 Å backbone and 0.33 Å all-atom rmsd. The R-factor between the experimental structure factors and those derived from this unrestrained simulation is 23% to 1.0 Å resolution. The B-factors for most heavy atoms agree well with experiment (Pearson correlation of 0.90), but B-factors obtained by refinement against the average simulation density underestimate the coordinate fluctuations in the underlying simulation where the simulation samples alternate conformations. A dynamic flow of water molecules through channels within the crystal lattice is observed, yet the average water density is in remarkable agreement with experiment. A minor population of unit cells is characterized by reduced water content, 310 helical propensity and a gauche(−) side-chain rotamer for one of the valine residues. Careful examination of the experimental data suggests that transitions of the helices are a simulation artifact, although there is indeed evidence for alternate valine conformers and variable water content. This study highlights the potential for crystal simulations to detect dynamics and heterogeneity in experimental diffraction data, as well as to validate computational chemistry methods. PMID:23631449

  20. Properties and catalytic activities of MICAL1, the flavoenzyme involved in cytoskeleton dynamics, and modulation by its CH, LIM and C-terminal domains.

    PubMed

    Vitali, Teresa; Maffioli, Elisa; Tedeschi, Gabriella; Vanoni, Maria A

    2016-03-01

    MICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a ∼ 10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10-50 μM) and increases kcat (10-25 s(-1)) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is ∼ 10-fold higher than that of the other forms (3-5 μM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues. PMID:26845023

  1. 2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

    PubMed

    Conejeros, I; Velásquez, Z D; Carretta, M D; Alarcón, P; Hidalgo, M A; Burgos, R A

    2012-01-15

    2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 μM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 μM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle. PMID:22226550

  2. N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements

    PubMed Central

    Nandadasa, Sumeda; Tao, Qinghua; Menon, Nikhil R.; Heasman, Janet; Wylie, Christopher

    2009-01-01

    Summary Transmembrane cadherins are calcium-dependent intercellular adhesion molecules. Recently, they have also been shown to be sites of actin assembly during adhesive contact formation. However, the roles of actin assembly on transmembrane cadherins during development are not fully understood. We show here, using the developing ectoderm of the Xenopus embryo as a model, that F-actin assembly is a primary function of both N-cadherin in the neural ectoderm and E-cadherin in the non-neural (epidermal) ectoderm, and that each cadherin is essential for the characteristic morphogenetic movements of these two tissues. However, depletion of N-cadherin and E-cadherin did not cause dissociation in these tissues at the neurula stage, probably owing to the expression of C-cadherin in each tissue. Depletion of each of these cadherins is not rescued by the other, nor by the expression of C-cadherin, which is expressed in both tissues. One possible reason for this is that each cadherin is expressed in a different domain of the cell membrane. These data indicate the combinatorial nature of cadherin function, the fact that N- and E-cadherin play primary roles in F-actin assembly in addition to roles in cell adhesion, and that this function is specific to individual cadherins. They also show how cell adhesion and motility can be combined in morphogenetic tissue movements that generate the form and shape of the embryonic organs. PMID:19279134

  3. Mutations in the Drosophila orthologs of the F-actin capping protein alpha- and beta-subunits cause actin accumulation and subsequent retinal degeneration.

    PubMed

    Delalle, Ivana; Pfleger, Cathie M; Buff, Eugene; Lueras, Paula; Hariharan, Iswar K

    2005-12-01

    The progression of several human neurodegenerative diseases is characterized by the appearance of intracellular inclusions or cytoskeletal abnormalities. An important question is whether these abnormalities actually contribute to the degenerative process or whether they are merely manifestations of cells that are already destined for degeneration. We have conducted a large screen in Drosophila for mutations that alter the growth or differentiation of cells during eye development. We have used mitotic recombination to generate patches of homozygous mutant cells. In our entire screen, mutations in only two different loci, burned (bnd) and scorched (scrd), resulted in eyes in which the mutant patches appeared black and the mutant tissue appeared to have undergone degeneration. In larval imaginal discs, growth and cell fate specification occur normally in mutant cells, but there is an accumulation of F-actin. Mutant cells degenerate much later during the pupal phase of development. burned mutations are allelic to mutations in the previously described cpb locus that encodes the beta-subunit of the F-actin capping protein, while scorched mutations disrupt the gene encoding its alpha-subunit (cpa). The alpha/beta-heterodimer caps the barbed ends of an actin filament and restricts its growth. In its absence, cells progressively accumulate actin filaments and eventually die. A possible role for their human orthologs in neurodegenerative disease merits further investigation. PMID:16143599

  4. F-Actin-Dependent Endocytosis of Cell Wall Pectins in Meristematic Root Cells. Insights from Brefeldin A-Induced Compartments1

    PubMed Central

    Baluška, František; Hlavacka, Andrej; Šamaj, Jozef; Palme, Klaus; Robinson, David G.; Matoh, Toru; McCurdy, David W.; Menzel, Diedrik; Volkmann, Dieter

    2002-01-01

    Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1→4)-β-d-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner. PMID:12226521

  5. Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

    PubMed Central

    Volceanov, Larisa; Herbst, Katharina; Biniossek, Martin; Schilling, Oliver; Haller, Dirk; Nölke, Thilo; Subbarayal, Prema; Rudel, Thomas; Zieger, Barbara

    2014-01-01

    ABSTRACT Chlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens. PMID:25293760

  6. AVHRR imagery reveals Antarctic ice dynamics

    SciTech Connect

    Bindschadler, R.A.; Vornberger, P.L. STX Corp., Lanham, MD )

    1990-06-01

    A portion of AVHRR data taken on December 5, 1987 at 06:15 GMT over a part of Antarctica is used here to show that many of the most significant dynamic features of ice sheets can be identified by a careful examination of AVHRR imagery. The relatively low resolution of this instrument makes it ideal for obtaining a broad view of the ice sheets, while its wide swath allows coverage of areas beyond the reach of high-resolution imagers either currently in orbit or planned. An interpretation is given of the present data, which cover the area of ice streams that drain the interior of the West Antarctic ice sheet into the Ross Ice Shelf. 21 refs.

  7. Chain networking revealed by molecular dynamics simulation

    NASA Astrophysics Data System (ADS)

    Zheng, Yexin; Tsige, Mesfin; Wang, Shi-Qing

    Based on Kremer-Grest model for entangled polymer melts, we demonstrate how the response of a polymer glass depends critically on the chain length. After quenching two melts of very different chain lengths (350 beads per chain and 30 beads per chain) into deeply glassy states, we subject them to uniaxial extension. Our MD simulations show that the glass of long chains undergoes stable necking after yielding whereas the system of short chains is unable to neck and breaks up after strain localization. During ductile extension of the polymer glass made of long chain significant chain tension builds up in the load-bearing strands (LBSs). Further analysis is expected to reveal evidence of activation of the primary structure during post-yield extension. These results lend support to the recent molecular model 1 and are the simulations to demonstrate the role of chain networking. This work is supported, in part, by a NSF Grant (DMR-EAGER-1444859)

  8. Revealing protein dynamics by photobleaching techniques.

    PubMed

    van Drogen, Frank; Peter, Matthias

    2004-01-01

    Green fluorescent proteins (GFPs) are widely used tools to visualize proteins and study their intracellular distribution. One feature of working with GFP variants, photobleaching, has recently been combined with an older technique known as fluorescence recovery after photobleaching (FRAP) to study protein kinetics in vivo. During photobleaching, fluorochromes get destroyed irreversibly by repeated excitation with an intensive light source. When the photobleaching is applied to a restricted area or structure, recovery of fluorescence will be the result of active or passive diffusion from fluorescent molecules from unbleached surrounding areas. Fluorescence loss in photobleaching (FLIP) is a variant of FRAP where an area is bleached, and loss of fluorescence in surrounding areas is observed. FLIP can be used to study the dynamics of different pools of a protein or can show how a protein diffuses, or is transported through a cell or cellular structure. Here, we discuss these photobleaching fluorescent imaging techniques, illustrated with examples of these techniques applied to proteins of the Saccharomyces cerevisiae pheromone response MAPK pathway. PMID:15173624

  9. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes

    PubMed Central

    1987-01-01

    F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N- ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin- membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge. PMID:3312238

  10. Specific Conserved C-terminal Amino Acids of Caenorhabditis elegans HMP-1/α-Catenin Modulate F-actin Binding Independently of Vinculin*

    PubMed Central

    Maiden, Stephanie L.; Harrison, Neale; Keegan, Jack; Cain, Brian; Lynch, Allison M.; Pettitt, Jonathan; Hardin, Jeff

    2013-01-01

    Stable intercellular adhesions formed through the cadherin-catenin complex are important determinants of proper tissue architecture and help maintain tissue integrity during morphogenetic movements in developing embryos. A key regulator of this stability is α-catenin, which connects the cadherin-catenin complex to the actin cytoskeleton. Although the C-terminal F-actin-binding domain of α-catenin has been shown to be crucial for its function, a more detailed in vivo analysis of discrete regions and residues required for actin binding has not been performed. Using Caenorhabditis elegans as a model system, we have characterized mutations in hmp-1/α-catenin that identify HMP-1 residues 687–742 and 826–927, as well as amino acid 802, as critical to the localization of junctional proximal actin during epidermal morphogenesis. We also find that the S823F transition in a hypomorphic allele, hmp-1(fe4), decreases actin binding in vitro. Using hmp-1(fe4) animals in a mutagenesis screen, we were then able to identify 11 intragenic suppressors of hmp-1(fe4) that revert actin binding to wild-type levels. Using homology modeling, we show that these amino acids are positioned at key conserved sites within predicted α-helices in the C terminus. Through the use of transgenic animals, we also demonstrate that HMP-1 residues 315–494, which correspond to a putative mechanotransduction domain that binds vinculin in vertebrate αE-catenin, are not required during epidermal morphogenesis but may aid efficient recruitment of HMP-1 to the junction. Our studies are the first to identify key conserved amino acids in the C terminus of α-catenin that modulate F-actin binding in living embryos of a simple metazoan. PMID:23271732

  11. Selenium nanoparticles induced membrane bio-mechanical property changes in MCF-7 cells by disturbing membrane molecules and F-actin.

    PubMed

    Pi, Jiang; Yang, Fen; Jin, Hua; Huang, Xun; Liu, Ruiying; Yang, Peihui; Cai, Jiye

    2013-12-01

    Selenium nanoparticles (Se NPs) have been served as promising materials for biomedical applications, especially for cancer treatment. The anti-cancer effects of Se NPs against cancer cells have been widely studied in recent years, but whether Se NPs can induce the changes of cell membrane bio-mechanical properties in cancer cells still remain unexplored. In this Letter, we prepared Se NPs for investigating the intracellular localization of Se NPs in MCF-7 cells and determined the effects of Se NPs on apoptosis and necrosis in MCF-7 cells. Especially, we reported for the first time about the effects of Se NPs on the bio-mechanical properties of cancer cells and found that Se NPs could remarkably decrease the adhesion force and Young's modulus of MCF-7 cells. To further understand the potential mechanisms about how Se NPs affect the bio-mechanical properties of MCF-7 cells, we also investigated the expression of CD44 molecules, the structure and the amounts of F-actin. The results indicated that the decreased adhesion force between AFM tip and cell membrane was partially due to the changes of membrane molecules induced by Se NPs, such as the down-regulation of trans-membrane CD44 molecules. Additionally, the decrease of Young's modulus of MCF-7 cells was due to the dis-organization and down-regulation of F-actin induced by Se NPs. These results collectively suggested that cell membrane was of vital importance in Se NPs induced toxicity in cancer cells, which could be served as a potential target for cancer treatment by Se NPs. PMID:24140445

  12. Coupling of the hydration water dynamics and the internal dynamics of actin detected by quasielastic neutron scattering

    SciTech Connect

    Fujiwara, Satoru; Plazanet, Marie; Oda, Toshiro

    2013-02-15

    Highlights: ► Quasielastic neutron scattering spectra of F-actin and G-actin were measured. ► Analysis of the samples in D{sub 2}O and H{sub 2}O provided the spectra of hydration water. ► The first layer hydration water around F-actin is less mobile than around G-actin. ► This difference in hydration water is in concert with the internal dynamics of actin. ► Water outside the first layer behaves bulk-like but influenced by the first layer. -- Abstract: In order to characterize dynamics of water molecules around F-actin and G-actin, quasielastic neutron scattering experiments were performed on powder samples of F-actin and G-actin, hydrated either with D{sub 2}O or H{sub 2}O, at hydration ratios of 0.4 and 1.0. By combined analysis of the quasielastic neutron scattering spectra, the parameter values characterizing the dynamics of the water molecules in the first hydration layer and those of the water molecules outside of the first layer were obtained. The translational diffusion coefficients (D{sub T}) of the hydration water in the first layer were found to be 1.2 × 10{sup −5} cm{sup 2}/s and 1.7 × 10{sup −5} cm{sup 2}/s for F-actin and G-actin, respectively, while that for bulk water was 2.8 × 10{sup −5} cm{sup 2}/s. The residence times were 6.6 ps and 5.0 ps for F-actin and G-actin, respectively, while that for bulk water was 0.62 ps. These differences between F-actin and G-actin, indicating that the hydration water around G-actin is more mobile than that around F-actin, are in concert with the results of the internal dynamics of F-actin and G-actin, showing that G-actin fluctuates more rapidly than F-actin. This implies that the dynamics of the hydration water is coupled to the internal dynamics of the actin molecules. The D{sub T} values of the water molecules outside of the first hydration layer were found to be similar to that of bulk water though the residence times are strongly affected by the first hydration layer. This supports the

  13. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes.

    PubMed

    Wuestehube, L J; Luna, E J

    1987-10-01

    F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge. PMID:3312238

  14. Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin.

    PubMed Central

    Bernardini, M L; Mounier, J; d'Hauteville, H; Coquis-Rondon, M; Sansonetti, P J

    1989-01-01

    The capacity of Shigella to spread within the cytosol of infected epithelial cells and to infect adjacent cells is critical for the development of infection foci, which lead to mucosal abscesses. Shigella is a nonmotile microorganism that appears to utilize host cell microfilaments to generate intra- as well as intercellular movements, since this movement was inhibited by cytochalasin D and involvement of F-actin was demonstrated by direct labeling of infected cells with the specific dye N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin. Such movements led to the formation of extracellular protrusions, which may explain cell to cell spread. icsA, a locus necessary for intra- and intercellular spread, was identified on the Shigella flexneri virulence plasmid pWR100. This locus was cloned and shown to express a 120-kDa outer membrane protein, which plays an important role in the interactions established between host cell microfilaments and the bacterial surface, thus leading to intracellular movement. Images PMID:2542950

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

    PubMed Central

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

    2008-01-01

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

  16. Topological structure dynamics revealing collective evolution in active nematics

    PubMed Central

    Shi, Xia-qing; Ma, Yu-qiang

    2013-01-01

    Topological defects frequently emerge in active matter like bacterial colonies, cytoskeleton extracts on substrates, self-propelled granular or colloidal layers and so on, but their dynamical properties and the relations to large-scale organization and fluctuations in these active systems are seldom touched. Here we reveal, through a simple model for active nematics using self-driven hard elliptic rods, that the excitation, annihilation and transportation of topological defects differ markedly from those in non-active media. These dynamical processes exhibit strong irreversibility in active nematics in the absence of detailed balance. Moreover, topological defects are the key factors in organizing large-scale dynamic structures and collective flows, resulting in multi-spatial temporal effects. These findings allow us to control the self-organization of active matter through topological structures. PMID:24346733

  17. Electro-optical imaging of F-actin and endoplasmic reticulum in living and fixed plant cells.

    PubMed

    Allen, N S; Bennett, M N

    1996-01-01

    Confocal and video micrographs of living and fixed alfalfa roots, onion epithelial and pear pollen cells illustrate the architecture of the cytoskeleton and endoplasmic reticulum in plant cells. Fixation of plant tissues to preserve cytoplasmic structure poses special problems. When possible, emphasis should be placed on the imaging of structures in stained living cells over time. The early events that occur when Nod factors or bacteria elicit nodule formation in alfalfa roots will illustrate several approaches to plant cell fixation, staining and imaging. The first observable events after Nod factor stimulation occur in root hairs and are changes in rates of cytoplasmic streaming, nuclear movements, and changes in the shape of the vacuole. Within ten minutes, the endoplasmic reticulum shifts position towards the tip of the root hair. For comparison, the endoplasmic reticulum localization in pollen tubes and onion epithelial cells will be illustrated. The actin cytoskeleton undergoes a series of changes over a twelve hour period. These changes in the cytoskeleton are spatially and temporally correlated with the observed growth changes of the root hairs. This dynamic change of the actin filament and endoplasmic reticulum and associated secretory vesicles in these root hairs suggests a mechanism for the observed root hair growth changes. PMID:9601538

  18. The F-actin capping proteins of Physarum polycephalum: cap42(a) is very similar, if not identical, to fragmin and is structurally and functionally very homologous to gelsolin; cap42(b) is Physarum actin.

    PubMed Central

    Ampe, C; Vandekerckhove, J

    1987-01-01

    We have carried out a primary structure analysis of the F-actin capping proteins of Physarum polycephalum. Cap42(b) was completely sequenced and was found to be identical with Physarum actin. Approximately 88% of the sequence of cap42(a) was determined. Cap42(a) and fragmin were found to be identical by amino acid composition, isoelectric point, mol. wt, elution time on reversed-phase chromatography and amino acid sequence of their tryptic peptides. The available sequence of cap42(a) is greater than 36% homologous with the NH2-terminal 42-kd domain of human gelsolin. A highly homologous region of 16 amino acids is also shared between cap42(a), gelsolin and the Acanthamoeba profilins. Cap42(a) binds two actin molecules in a similar way to gelsolin suggesting a mechanism of F-actin modulation that has been conserved during evolution. Images Fig. 1. Fig. 3. Fig. 4. PMID:2832154

  19. Characterization of F-Actin Tryptophan Phosphorescence in the Presence and Absence of Tryptophan-Free Myosin Motor Domain

    PubMed Central

    Bódis, Emöke; Strambini, Giovanni B.; Gonnelli, Margherita; Málnási-Csizmadia, András; Somogyi, Béla

    2004-01-01

    The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140–293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (τ1 and τ2) and increasing by ∼20% the longest component (τ3). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (τ4, twice as long as τ3) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique. PMID:15298917

  20. Personal Omics Profiling Reveals Dynamic Molecular and Medical Phenotypes

    PubMed Central

    Chen, Rui; Mias, George I.; Li-Pook-Than, Jennifer; Jiang, Lihua; Lam, Hugo Y. K.; Chen, Rong; Miriami, Elana; Karczewski, Konrad J.; Hariharan, Manoj; Dewey, Frederick E.; Cheng, Yong; Clark, Michael J.; Im, Hogune; Habegger, Lukas; Balasubramanian, Suganthi; O'Huallachain, Maeve; Dudley, Joel T.; Hillenmeyer, Sara; Haraksingh, Rajini; Sharon, Donald; Euskirchen, Ghia; Lacroute, Phil; Bettinger, Keith; Boyle, Alan P.; Kasowski, Maya; Grubert, Fabian; Seki, Scott; Garcia, Marco; Whirl-Carrillo, Michelle; Gallardo, Mercedes; Blasco, Maria A.; Greenberg, Peter L.; Snyder, Phyllis; Klein, Teri E.; Altman, Russ B.; Butte, Atul; Ashley, Euan A.; Nadeau, Kari C.; Gerstein, Mark; Tang, Hua; Snyder, Michael

    2012-01-01

    SUMMARY Personalized medicine is expected to benefit from combining genomic information with regular monitoring of physiological states by multiple high-throughput methods. Here we present an integrative Personal Omics Profile (iPOP), an analysis that combines genomic, transcriptomic, proteomic, metabolomic, and autoantibody profiles from a single individual over a 14-month period. Our iPOP analysis revealed various medical risks, including Type II diabetes. It also uncovered extensive, dynamic changes in diverse molecular components and biological pathways across healthy and diseased conditions. Extremely high coverage genomic and transcriptomic data, which provide the basis of our iPOP, discovered extensive heteroallelic changes during healthy and diseased states and an unexpected RNA editing mechanism. This study demonstrates that longitudinal iPOP can be used to interpret healthy and disease states by connecting genomic information with additional dynamic omics activity. PMID:22424236

  1. Ultrafast cooling reveals microsecond-scale biomolecular dynamics.

    PubMed

    Polinkovsky, Mark E; Gambin, Yann; Banerjee, Priya R; Erickstad, Michael J; Groisman, Alex; Deniz, Ashok A

    2014-01-01

    The temperature-jump technique, in which the sample is rapidly heated by a powerful laser pulse, has been widely used to probe the fast dynamics of folding of proteins and nucleic acids. However, the existing temperature-jump setups tend to involve sophisticated and expensive instrumentation, while providing only modest temperature changes of ~10-15 °C, and the temperature changes are only rapid for heating, but not cooling. Here we present a setup comprising a thermally conductive sapphire substrate with light-absorptive nano-coating, a microfluidic device and a rapidly switched moderate-power infrared laser with the laser beam focused on the nano-coating, enabling heating and cooling of aqueous solutions by ~50 °C on a 1-μs time scale. The setup is used to probe folding and unfolding dynamics of DNA hairpins after direct and inverse temperature jumps, revealing low-pass filter behaviour during periodic temperature variations. PMID:25517430

  2. Counterions between charged polymers exhibit liquid-like organization and dynamics

    PubMed Central

    Angelini, Thomas E.; Golestanian, Ramin; Coridan, Robert H.; Butler, John C.; Beraud, Alexandre; Krisch, Michael; Sinn, Harald; Schweizer, Kenneth S.; Wong, Gerard C. L.

    2006-01-01

    Current understanding of electrostatics in water is based on mean-field theories like the Poisson–Boltzmann formalism and its approximations, which are routinely used in colloid science and computational biology. This approach, however, breaks down for highly charged systems, which exhibit counterintuitive phenomena such as overcharging and like-charge attraction. Models of counterion correlations have been proposed as possible explanations, but no experimental comparisons are available. Here, collective dynamics of counterions that mediate like-charge attraction between F-actin filaments have been directly observed in aqueous solution using high-resolution inelastic x-ray scattering down to molecular length-scales. We find a previously undescribed acoustic-like phonon mode associated with correlated counterions. The excitation spectra at high wave-vector Q reveal unexpected dynamics due to ions interacting with their “cages” of nearest neighbors. We examine this behavior in the context of intrinsic charge density variations on F-actin. The measured speed of sound and collective relaxation rates in this liquid agree surprisingly well with simple model calculations. PMID:16690742

  3. Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

    PubMed Central

    Gary, R; Bretscher, A

    1995-01-01

    Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking. Images PMID:7579708

  4. The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span

    PubMed Central

    Lejskova, Renata; Malcova, Ivana

    2015-01-01

    Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth. PMID:26351139

  5. The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast.

    PubMed

    Li, Yujie; Christensen, Jenna R; Homa, Kaitlin E; Hocky, Glen M; Fok, Alice; Sees, Jennifer A; Voth, Gregory A; Kovar, David R

    2016-06-01

    The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction. PMID:27075176

  6. The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

    PubMed Central

    Li, Yujie; Christensen, Jenna R.; Homa, Kaitlin E.; Hocky, Glen M.; Fok, Alice; Sees, Jennifer A.; Voth, Gregory A.; Kovar, David R.

    2016-01-01

    The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction. PMID:27075176

  7. Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy.

    PubMed

    Pagano, Philip; Guo, Qi; Kohen, Amnon; Cheatum, Christopher M

    2016-07-01

    Enzymes move on a variety of length and time scales. While much is known about large structural fluctuations that impact binding of the substrates and release of products, little is known about faster motions of enzymes and how these motions may influence enzyme-catalyzed reactions. This Letter reports frequency fluctuations of the azide anion bound to the active site of formate dehydrogenase measured via 2D IR spectroscopy. These measurements reveal an underdamped oscillatory component to the frequency-frequency correlation function when the azide is bound to the NAD(+) ternary complex. This oscillation disappears when the reduced cofactor is added, indicating that the oscillating contributions most likely come from the charged nicotinamide ring. These oscillatory motions may be relevant to donor-acceptor distance sampling of the catalyzed hydride transfer and therefore may give future insights into the dynamic behavior involved in enzyme catalysis. PMID:27305279

  8. Stochastic heart-rate model can reveal pathologic cardiac dynamics

    NASA Astrophysics Data System (ADS)

    Kuusela, Tom

    2004-03-01

    A simple one-dimensional Langevin-type stochastic difference equation can simulate the heart-rate fluctuations in a time scale from minutes to hours. The model consists of a deterministic nonlinear part and a stochastic part typical of Gaussian noise, and both parts can be directly determined from measured heart-rate data. Data from healthy subjects typically exhibit the deterministic part with two or more stable fixed points. Studies of 15 congestive heart-failure subjects reveal that the deterministic part of pathologic heart dynamics has no clear stable fixed points. Direct simulations of the stochastic model for normal and pathologic cases can produce statistical parameters similar to those of real subjects. Results directly indicate that pathologic situations simplify the heart-rate control system.

  9. Identification and characterization of espin, an actin-binding protein localized to the F-actin-rich junctional plaques of Sertoli cell ectoplasmic specializations.

    PubMed

    Bartles, J R; Wierda, A; Zheng, L

    1996-06-01

    Ectoplasmic specializations are membrane-cytoskeletal assemblages found in Sertoli cells at sites of attachment to elongate spermatids or neighboring Sertoli cells. They are characterized in part by the presence of a unique junctional plaque which contains a narrow layer of parallel actin bundles sandwiched between the Sertoli cell plasma membrane and an affiliated cistern of endoplasmic reticulum. Using a monoclonal antibody, we have identified 'espin,' a novel actin-binding protein localized to ectoplasmic specializations. By immunogold electron microscopy, espin was localized to the parallel actin bundles of ectoplasmic specializations at sites where Sertoli cells contacted the heads of elongate spermatids. The protein was also detected at the sites of ectoplasmic specializations between neighboring Sertoli cells. Espin exhibits an apparent molecular mass of approximately 110 kDa in SDS gels. It is encoded by an approximately 2.9 kb mRNA, which was found to be specific to testis among the 11 rat organs and tissues examined. On the basis of cDNA sequence, espin is predicted to be an 836 amino acid protein which contains 8 ankyrin-like repeats in its N-terminal third, a potential P-loop, two proline-rich peptides and two peptides which contain clusters of multiple glutamates bracketed by arginines, lysines and glutamines in a pattern reminiscent of the repetitive motif found in the protein trichohyalin. The ankyrin-like repeats and a 66 amino acid peptide in the C terminus show significant sequence similarity to proteins encoded by the forked gene of Drosophila. A fusion protein containing the C-terminal 378 amino acids of espin was found to bind with high affinity (Kd = approximately 10 nM) to F-actin in vitro with a stoichiometry of approximately 1 espin per 6 actin monomers. When expressed by transfected NRK fibroblasts, the same C-terminal fragment of espin was observed to decorate actin fibers or cables. On the basis of its structure, localization and

  10. Spectrins in axonal cytoskeletons: Dynamics revealed by extensions and fluctuations

    NASA Astrophysics Data System (ADS)

    Lai, Lipeng; Cao, Jianshu

    2014-07-01

    The macroscopic properties, the properties of individual components, and how those components interact with each other are three important aspects of a composited structure. An understanding of the interplay between them is essential in the study of complex systems. Using axonal cytoskeleton as an example system, here we perform a theoretical study of slender structures that can be coarse-grained as a simple smooth three-dimensional curve. We first present a generic model for such systems based on the fundamental theorem of curves. We use this generic model to demonstrate the applicability of the well-known worm-like chain (WLC) model to the network level and investigate the situation when the system is stretched by strong forces (weakly bending limit). We specifically studied recent experimental observations that revealed the hitherto unknown periodic cytoskeleton structure of axons and measured the longitudinal fluctuations. Instead of focusing on single molecules, we apply analytical results from the WLC model to both single molecule and network levels and focus on the relations between extensions and fluctuations. We show how this approach introduces constraints to possible local dynamics of the spectrin tetramers in the axonal cytoskeleton and finally suggests simple but self-consistent dynamics of spectrins in which the spectrins in one spatial period of axons fluctuate in-sync.

  11. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

    PubMed

    Shankaran, Mahalakshmi; King, Chelsea L; Angel, Thomas E; Holmes, William E; Li, Kelvin W; Colangelo, Marc; Price, John C; Turner, Scott M; Bell, Christopher; Hamilton, Karyn L; Miller, Benjamin F; Hellerstein, Marc K

    2016-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  12. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

    PubMed Central

    Shankaran, Mahalakshmi; King, Chelsea L.; Angel, Thomas E.; Holmes, William E.; Li, Kelvin W.; Colangelo, Marc; Price, John C.; Turner, Scott M.; Bell, Christopher; Hamilton, Karyn L.; Miller, Benjamin F.; Hellerstein, Marc K.

    2015-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  13. Unsupervised Deconvolution of Dynamic Imaging Reveals Intratumor Vascular Heterogeneity and Repopulation Dynamics

    PubMed Central

    Chen, Li; Choyke, Peter L.; Wang, Niya; Clarke, Robert; Bhujwalla, Zaver M.; Hillman, Elizabeth M. C.; Wang, Ge; Wang, Yue

    2014-01-01

    With the existence of biologically distinctive malignant cells originated within the same tumor, intratumor functional heterogeneity is present in many cancers and is often manifested by the intermingled vascular compartments with distinct pharmacokinetics. However, intratumor vascular heterogeneity cannot be resolved directly by most in vivo dynamic imaging. We developed multi-tissue compartment modeling (MTCM), a completely unsupervised method of deconvoluting dynamic imaging series from heterogeneous tumors that can improve vascular characterization in many biological contexts. Applying MTCM to dynamic contrast-enhanced magnetic resonance imaging of breast cancers revealed characteristic intratumor vascular heterogeneity and therapeutic responses that were otherwise undetectable. MTCM is readily applicable to other dynamic imaging modalities for studying intratumor functional and phenotypic heterogeneity, together with a variety of foreseeable applications in the clinic. PMID:25379705

  14. Cytoplasmic Dynamics Reveals Two Modes of Nucleoid-Dependent Mobility

    PubMed Central

    Stylianidou, Stella; Kuwada, Nathan J.; Wiggins, Paul A.

    2014-01-01

    It has been proposed that forces resulting from the physical exclusion of macromolecules from the bacterial nucleoid play a central role in organizing the bacterial cell, yet this proposal has not been quantitatively tested. To investigate this hypothesis, we mapped the generic motion of large protein complexes in the bacterial cytoplasm through quantitative analysis of thousands of complete cell-cycle trajectories of fluorescently tagged ectopic MS2-mRNA complexes. We find the motion of these complexes in the cytoplasm is strongly dependent on their spatial position along the long axis of the cell, and that their dynamics are consistent with a quantitative model that requires only nucleoid exclusion and membrane confinement. This analysis also reveals that the nucleoid increases the mobility of MS2-mRNA complexes, resulting in a fourfold increase in diffusion coefficients between regions of the lowest and highest nucleoid density. These data provide strong quantitative support for two modes of nucleoid action: the widely accepted mechanism of nucleoid exclusion in organizing the cell and a newly proposed mode, in which the nucleoid facilitates rapid motion throughout the cytoplasm. PMID:25468347

  15. Substrate Channel in Nitrogenase Revealed by a Molecular Dynamics Approach

    SciTech Connect

    Smith, Dayle; Danyal, Karamatullah; Raugei, Simone; Seefeldt, Lance C.

    2014-03-22

    Mo-dependent nitrogenase catalyzes the biological reduction of N2 to 2NH3 at the FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized sub-microsecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel not previously reported. The viability of the proposed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, with discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment that approaches a face of FeMo-cofactor earlier implicated in substrate binding.

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

    PubMed Central

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

    2015-01-01

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

  17. Accumulation of glucosylceramide in the absence of the beta-glucosidase GBA2 alters cytoskeletal dynamics.

    PubMed

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

    2015-03-01

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

  18. Revealing the dynamic heterogeneity of PMMA/PVDF blends: from microscopic dynamics to macroscopic properties.

    PubMed

    Lu, Bo; Lamnawar, Khalid; Maazouz, Abderrahim; Zhang, Huagui

    2016-04-01

    An effort was made to demonstrate the dynamic heterogeneity of poly(methyl methacrylate) (PMMA)/poly(vinylidene fluoride) (PVDF) blends, where its composition dependence and the role of interphase were probed. Firstly, the composition dependence of thermorheological complexity of PMMA/PVDF blends in the melt was revealed. The molecular entanglement state involving intra- and interchain entanglements was found to govern the scenario of thermorheological complexity. Intriguingly, local heterogeneity was further demonstrated to exist in the melt-state blends with intermediate compositions, and its origin was depicted to be the interphase. The interphase, coupled with unfavourable interchain entanglements in those blends, could explain the reduced viscosity and speed-up relaxations, contributing to the overall thermorheological complexity. Besides, two experimental glass transition temperatures of blends were resolved in view of segment motions in the miscible phase and the crystal-amorphous interphase, and further assessed via the "self-concentration" concept. The presence of a crystal-amorphous interphase, likely leading to three distinct dynamics of segments in blends, was supposed to contribute to the dynamic heterogeneity in segment relaxations for PMMA/PVDF blends in the solid state. Lastly, effects of dynamic heterogeneity on dynamic mechanical properties were also evaluated. PMID:26932245

  19. Organization and dynamics of the actin cytoskeleton during dendritic spine morphological remodeling.

    PubMed

    Chazeau, Anaël; Giannone, Grégory

    2016-08-01

    In the central nervous system, most excitatory post-synapses are small subcellular structures called dendritic spines. Their structure and morphological remodeling are tightly coupled to changes in synaptic transmission. The F-actin cytoskeleton is the main driving force of dendritic spine remodeling and sustains synaptic plasticity. It is therefore essential to understand how changes in synaptic transmission can regulate the organization and dynamics of actin binding proteins (ABPs). In this review, we will provide a detailed description of the organization and dynamics of F-actin and ABPs in dendritic spines and will discuss the current models explaining how the actin cytoskeleton sustains both structural and functional synaptic plasticity. PMID:27105623

  20. Laser altimetry reveals complex pattern of Greenland Ice Sheet dynamics.

    PubMed

    Csatho, Beata M; Schenk, Anton F; van der Veen, Cornelis J; Babonis, Gregory; Duncan, Kyle; Rezvanbehbahani, Soroush; van den Broeke, Michiel R; Simonsen, Sebastian B; Nagarajan, Sudhagar; van Angelen, Jan H

    2014-12-30

    We present a new record of ice thickness change, reconstructed at nearly 100,000 sites on the Greenland Ice Sheet (GrIS) from laser altimetry measurements spanning the period 1993-2012, partitioned into changes due to surface mass balance (SMB) and ice dynamics. We estimate a mean annual GrIS mass loss of 243 ± 18 Gt ⋅ y(-1), equivalent to 0.68 mm ⋅ y(-1) sea level rise (SLR) for 2003-2009. Dynamic thinning contributed 48%, with the largest rates occurring in 2004-2006, followed by a gradual decrease balanced by accelerating SMB loss. The spatial pattern of dynamic mass loss changed over this time as dynamic thinning rapidly decreased in southeast Greenland but slowly increased in the southwest, north, and northeast regions. Most outlet glaciers have been thinning during the last two decades, interrupted by episodes of decreasing thinning or even thickening. Dynamics of the major outlet glaciers dominated the mass loss from larger drainage basins, and simultaneous changes over distances up to 500 km are detected, indicating climate control. However, the intricate spatiotemporal pattern of dynamic thickness change suggests that, regardless of the forcing responsible for initial glacier acceleration and thinning, the response of individual glaciers is modulated by local conditions. Recent projections of dynamic contributions from the entire GrIS to SLR have been based on the extrapolation of four major outlet glaciers. Considering the observed complexity, we question how well these four glaciers represent all of Greenland's outlet glaciers. PMID:25512537

  1. Dynamic Coupling among Protein Binding, Sliding, and DNA Bending Revealed by Molecular Dynamics.

    PubMed

    Tan, Cheng; Terakawa, Tsuyoshi; Takada, Shoji

    2016-07-13

    Protein binding to DNA changes the DNA's structure, and altered DNA structure can, in turn, modulate the dynamics of protein binding. This mutual dependency is poorly understood. Here we investigated dynamic couplings among protein binding to DNA, protein sliding on DNA, and DNA bending by applying a coarse-grained simulation method to the bacterial architectural protein HU and 14 other DNA-binding proteins. First, we verified our method by showing that the simulated HU exhibits a weak preference for A/T-rich regions of DNA and a much higher affinity for gapped and nicked DNA, consistent with biochemical experiments. The high affinity was attributed to a local DNA bend, but not the specific chemical moiety of the gap/nick. The long-time dynamic analysis revealed that HU sliding is associated with the movement of the local DNA bending site. Deciphering single sliding steps, we found the coupling between HU sliding and DNA bending is akin to neither induced-fit nor population-shift; instead they moved concomitantly. This is reminiscent of a cation transfer on DNA and can be viewed as a protein version of polaron-like sliding. Interestingly, on shorter time scales, HU paused when the DNA was highly bent at the bound position and escaped from pauses once the DNA spontaneously returned to a less bent structure. The HU sliding is largely regulated by DNA bending dynamics. With 14 other proteins, we explored the generality and versatility of the dynamic coupling and found that 6 of the 15 assayed proteins exhibit the polaron-like sliding. PMID:27309278

  2. Cytoskeletal F-actin polymerization from cytosolic G-actin occurs in the phagocytosing immunocytes of arthropods (Limulus polyphemus and Gromphadorhina portentosa): does [cAMP]i play any role?

    PubMed

    Gupta, A P; Campenot, E S

    1996-09-01

    Phagocytosis is a major defense reaction in arthropods and is accomplished by two blood cells (hemocytes), the granulocyte (GRs) and plasmatocytes (PLs), collectively called immunocytes. Immunocytes (principally the GRs) from two arthropods, Limulus polyphemus (horseshoe crab) and Gromphadorhina portentosa (Madagascar hissing cockroach) effectively phagocytose fluorescein isothiocyanate (FITC)-conjugated fluoresbrite microspheres (FITC-FM) and chicken (Gallus domesticus) erythrocytes within 1 hr of incubation. Although actin polymerization and changes in intracellular cAMP ([cAMP]i) levels occur during the early stages of phagocytosis in vertebrates, these two phenomena have not been studied in arthropod immunocytes. Using the DNase I inhibition assay, we found a decrease in cytosolic G-actin and an increase in the cytoskeletal F-actin in the phagocytosing immunocytes; the total actin in both resting and phagocytosing immunocytes remained constant. These results showed an 86% increase in F-actin in G. portentosa immunocytes and a 29% increase in those of L. polyphemus after 1 hr of initial incubation with FITC-FM. As in some vertebrates, the role of [cAMP]i in the early stages of phagocytosis in these two animals- and perhaps in arthropods in general-is variable; although we detected some negligible amounts of [cAMP]i (0.10-0.80 pmol/cell at different time intervals) in L. polyphemus immunocytes, it was inconclusive whether those in G. portentosa also contained [cAMP]i. Even in L. polyphemus, the difference in the amounts of [cAMP]i in resting and phagocytosing cells was insignificant (P > 0.05). It was also inconclusive whether [Ca2+]i and/or [Mg2+]i play any roles in the early stages of phagocytosis in the two arthropods in this study. These results suggest that the two phenomena (F-actin polymerization and levels of [cAMP]i in arthropods) are basically similar to those in vertebrate neutrophils and macrophages, which suggests that certain immunological

  3. SGR9, a RING type E3 ligase, modulates amyloplast dynamics important for gravity sensing.

    NASA Astrophysics Data System (ADS)

    Morita, Miyo T.; Nakamura, Moritaka; Tasaka, Masao

    Gravitropism is triggered when the directional change of gravity is sensed in the specific cells, called statocytes. In higher plants, statocytes contain sinking heavier amyloplasts which are particular plastids accumulating starch granules. The displacement of amyloplasts within the statocytes is thought to be the initial event of gravity perception. We have demonstrated that endodermal cells are most likely to be the statocytes in Arabidop-sis shoots. Live cell imaging of the endodermal cell of stem has shown that most amyloplasts are sediment to the direction of gravity but they are not static. Several amyloplasts move dynamically in an actin filament (F-actin) dependent manner. In the presence of actin poly-merization inhibitor, all amyloplasts become static and sediment to the direction of gravity. In addition, stems treated with the inhibitor can exhibit gravitropism. These results suggest that F-actin-dependent dynamic movement of amyloplasts is not essential for gravity sensing. sgr (shoot gravitropism) 9 mutant exhibits greatly reduced shoot gravitropism. In endodermal cells of sgr9, dynamic amyloplast movement was predominantly observed and amyloplasts did not sediment to the direction of gravity. Interestingly, inhibition of actin polymerization re-stored both gravitropism and amyloplast sedimentation in sgr9. The SGR9 encodes a novel RING finger protein, which is localized to amyloplasts in endodermal cells. SGR9 showed ubiq-uitin E3 ligase activity in vitro. Together with live cell imaging of amyloplasts and F-actin, our data suggest that SGR9 modulate interaction between amyloplasts and F-actin on amylo-plasts. SGR9 positively act on amyloplasts sedimentation, probably by releasing amyloplasts from F-actin. SGR9 that is localized to amyloplast, possibly degrades unknown substrates by its E3 ligase activity, and this might promote release of amyloplasts from F-actin.

  4. Rhodopsin Photoactivation Dynamics Revealed by Quasi-Elastic Neutron Scattering

    NASA Astrophysics Data System (ADS)

    Bhowmik, Debsindhu; Shrestha, Utsab; Perera, Suchhithranga M. C. D.; Chawla, Udeep; Mamontov, Eugene; Brown, Michael; Chu, Xiang-Qiang

    2015-03-01

    Rhodopsin is a G-protein-coupled receptor (GPCR) responsible for vision. During photoactivation, the chromophore retinal dissociates from protein yielding the opsin apoprotein. What are the changes in protein dynamics that occur during the photoactivation process? Here, we studied the microscopic dynamics of dark-state rhodopsin and the ligand-free opsin using quasielastic neutron scattering (QENS). The QENS technique tracks individual hydrogen atom motion because of the much higher neutron scattering cross-section of hydrogen than other atoms. We used protein with CHAPS detergent hydrated with heavy water. The activation of proteins is confirmed at low temperatures up to 300 K by mean-square displacement (MSD) analysis. The QENS experiments at temperatures ranging from 220 K to 300 K clearly indicate an increase in protein dynamic behavior with temperature. The relaxation time for the ligand-bound protein rhodopsin is faster compared to opsin, which can be correlated with the photoactivation. Moreover, the protein dynamics are orders of magnitude slower than the accompanying CHAPS detergent, which unlike protein, manifests localized motions.

  5. OLIGOMERIZATION STATE OF RUBISCO ACTIVASE REVEALED BY DYNAMIC LIGHT SCATTERING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The self-association of Rubisco activase has been suggested to be required for Rubisco activation via ATP hydrolysis. To study the oligmerization patterns in detail, we initially measured the size of each isoform (42 KDa and 45 KDa) of recombinant spinach activase using dynamic light scattering spec...

  6. Structure and Dynamics of Four-way DNA Junctions Dynamics Revealed by Single-Molecule AFM

    NASA Astrophysics Data System (ADS)

    Lyubchenko, Yuri

    2004-03-01

    For-way DNA junctions (Holliday junctions) are critical intermediates for homologous, site-specific recombination, DNA repair and replication. A wealth of structural information is available for immobile four-way junctions. However, these data cannot give the answer on the mechanism of branch migration, the major property of the Holliday junction. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal, exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration. Note that it was the first time when the dynamics of the four-way DNA junction capable of branch migration had been analyzed. We applied time-lapse atomic force microscopy (single molecule dynamics AFM) to image directly loosely bound DNA at liquid-surface interface. These experiments show that mobile Holliday junctions adopt an unfolded conformation during branch migration. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction. The analysis of the Holliday junctions dynamics at conditions limiting branch migration revealed a broad movement of the arms suggesting that the range of mobility of these junctions is much wider than detected before. Further applications of the time-lapse AFM approach in attempt to resolve the subpopulations of the junctions conformers and the prospects for analyses of dynamics of complex biological systems will be discussed.

  7. Dynamics of Membrane Tethers Reveal Novel Aspects of Cytoskeleton-Membrane Interactions in Axons

    PubMed Central

    Datar, Anagha; Bornschlögl, Thomas; Bassereau, Patricia; Prost, Jacques; Pullarkat, Pramod A.

    2015-01-01

    Mechanical properties of cell membranes are known to be significantly influenced by the underlying cortical cytoskeleton. The technique of pulling membrane tethers from cells is one of the most effective ways of studying the membrane mechanics and the membrane-cortex interaction. In this article, we show that axon membranes make an interesting system to explore as they exhibit both free membrane-like behavior where the tether-membrane junction is movable on the surface of the axons (unlike many other cell membranes) as well as cell-like behavior where there are transient and spontaneous eruptions in the tether force that vanish when F-actin is depolymerized. We analyze the passive and spontaneous responses of axonal membrane tethers and propose theoretical models to explain the observed behavior. PMID:25650917

  8. Charge-dependent conformations and dynamics of pamam dendrimers revealed by neutron scattering and molecular dynamics

    NASA Astrophysics Data System (ADS)

    Wu, Bin

    spatial instrumental scales, understanding experimental results involves extensive and difficult data analysis based on liquid theory and condensed matter physics. Therefore, a model that successfully describes the inter- and intra-dendrimer correlations is crucial in obtaining and delivering reliable information. On the other hand, making meaningful comparisons between molecular dynamics and neutron scattering is a fundamental challenge to link simulations and experiments at the nano-scale. This challenge stems from our approach to utilize MD simulation to explain the underlying mechanism of experimental observation. The SANS measurements were conducted on a series of SANS spectrometers including the Extended Q-Range Small-Angle Neutron Scattering Diffractometer (EQ-SANS) and the General-Purpose Small-Angle Neutron Scattering Diffractometer (GP-SANS) at the Oak Ridge National Laboratory (ORNL), and NG7 Small Angle Neutron Scattering Spectrometer at National Institute of Standards (NIST) and Technology in U.S.A., large dynamic range small-angle diffractometer D22 at Institut Laue-Langevin (ILL) in France, and 40m-SANS Spectrometer at Korea Atomic Energy Research Institute (KAERI) in Korea. On the other hand, the Amber molecular dynamics simulation package is utilized to carry out the computational study. In this dissertation, the following observations have been revealed. The previously developed theoretical model for polyelectrolyte dendrimers are adopted to analyze SANS measurements and superb model fitting quality is found. Coupling with advanced contrast variation small angle neutron scattering (CVSANS) data analysis scheme reported recently, the intra-dendrimer hydration and hydrocarbon components distributions are revealed experimentally. The results indeed indicate that the maximum density is located in the molecular center rather than periphery, which is consistent to previous SANS studies and the back-folding picture of PAMAM dendrimers. According to this picture

  9. Dynamical transition of myoglobin revealed by inelastic neutron scattering

    NASA Astrophysics Data System (ADS)

    Doster, Wolfgang; Cusack, Stephen; Petry, Winfried

    1989-02-01

    Structural fluctuations in proteins on the picosecond timescale have been studied in considerable detail by theoretical methods such as molecular dynamics simulation1,2, but there exist very few experimental data with which to test the conclusions. We have used the technique of inelastic neutron scattering to investigate atomic motion in hydrated myoglobin over the temperature range 4 350 K and on the molecular dynamics timescale 0.1 100 ps. At temperatures below 180 K myglobin behaves as a harmonic solid, with essentially only vibrational motion. Above 180 K there is a striking dynamic transition arising from the excitation of non-vibrational motion, which we interpret as corresponding to tor-sional jumps between states of different energy, with a mean energy asymmetry of KJ mol -1. This extra mobility is reflected in a strong temperature dependence of the mean-square atomic displacements, a phenomenon previously observed specifically for the heme iron by Mossbauer spectroscopy3 5, but on a much slower timescale (10-7 s). It also correlates with a glass-like transition in the hydration shell of myoglobin6 and with the temperature-dependence of ligand-binding rates at the heme iron, as monitored by flash photolysis7. In contrast, the crystal structure of myoglobin determined down to 80 K shows no significant structural transition8 10. The dynamical behaviour we find for myoglobin (and other globular proteins) suggests a coupling of fast local motions to slower collective motions, which is a characteristic feature of other dense glass-forming systems.

  10. Fluctuation power spectra reveal dynamical heterogeneity of peptides

    NASA Astrophysics Data System (ADS)

    Khatri, Bhavin; Yew, Zu Thur; Krivov, Sergei; McLeish, Tom; Paci, Emanuele

    2010-07-01

    Characterizing the conformational properties and dynamics of biopolymers and their relation to biological activity and function is an ongoing challenge. Single molecule techniques have provided a rich experimental window on these properties, yet they have often relied on simple one-dimensional projections of a multidimensional free energy landscape for a practical interpretation of the results. Here, we study three short peptides with different structural propensity (α helical, β hairpin, and random coil) in the presence (or absence) of a force applied to their ends using Langevin dynamics simulation and an all-atom model with implicit solvation. Each peptide produces fluctuation power spectra with a characteristic dynamic fingerprint consistent with persistent structural motifs of helices, hairpins, and random coils. The spectra for helix formation shows two well-defined relaxation modes, corresponding to local relaxation and cooperative coil to uncoil interconversion. In contrast, both the hairpin and random coil are polymerlike, showing a broad and continuous range of relaxation modes giving characteristic power laws of ω-5/4 and ω-3/2, respectively; the -5/4 power law for hairpins is robust and has not been previously observed. Langevin dynamics simulations of diffusers on a potential of mean force derived from the atomistic simulations fail to reproduce the fingerprints of each peptide motif in the power spectral density, demonstrating explicitly that such information is lacking in such one-dimensional projections. Our results demonstrate the yet unexploited potential of single molecule fluctuation spectroscopy to probe more fine scaled properties of proteins and biological macromolecules and how low dimensional projections may cause the loss of relevant information.

  11. Optogenetic perturbations reveal the dynamics of an oculomotor integrator

    PubMed Central

    Gonçalves, Pedro J.; Arrenberg, Aristides B.; Hablitzel, Bastian; Baier, Herwig; Machens, Christian K.

    2014-01-01

    Many neural systems can store short-term information in persistently firing neurons. Such persistent activity is believed to be maintained by recurrent feedback among neurons. This hypothesis has been fleshed out in detail for the oculomotor integrator (OI) for which the so-called “line attractor” network model can explain a large set of observations. Here we show that there is a plethora of such models, distinguished by the relative strength of recurrent excitation and inhibition. In each model, the firing rates of the neurons relax toward the persistent activity states. The dynamics of relaxation can be quite different, however, and depend on the levels of recurrent excitation and inhibition. To identify the correct model, we directly measure these relaxation dynamics by performing optogenetic perturbations in the OI of zebrafish expressing halorhodopsin or channelrhodopsin. We show that instantaneous, inhibitory stimulations of the OI lead to persistent, centripetal eye position changes ipsilateral to the stimulation. Excitatory stimulations similarly cause centripetal eye position changes, yet only contralateral to the stimulation. These results show that the dynamics of the OI are organized around a central attractor state—the null position of the eyes—which stabilizes the system against random perturbations. Our results pose new constraints on the circuit connectivity of the system and provide new insights into the mechanisms underlying persistent activity. PMID:24616666

  12. Dynamic Monitoring Reveals Motor Task Characteristics in Prehistoric Technical Gestures

    PubMed Central

    Pfleging, Johannes; Stücheli, Marius; Iovita, Radu; Buchli, Jonas

    2015-01-01

    Reconstructing ancient technical gestures associated with simple tool actions is crucial for understanding the co-evolution of the human forelimb and its associated control-related cognitive functions on the one hand, and of the human technological arsenal on the other hand. Although the topic of gesture is an old one in Paleolithic archaeology and in anthropology in general, very few studies have taken advantage of the new technologies from the science of kinematics in order to improve replicative experimental protocols. Recent work in paleoanthropology has shown the potential of monitored replicative experiments to reconstruct tool-use-related motions through the study of fossil bones, but so far comparatively little has been done to examine the dynamics of the tool itself. In this paper, we demonstrate that we can statistically differentiate gestures used in a simple scraping task through dynamic monitoring. Dynamics combines kinematics (position, orientation, and speed) with contact mechanical parameters (force and torque). Taken together, these parameters are important because they play a role in the formation of a visible archaeological signature, use-wear. We present our new affordable, yet precise methodology for measuring the dynamics of a simple hide-scraping task, carried out using a pull-to (PT) and a push-away (PA) gesture. A strain gage force sensor combined with a visual tag tracking system records force, torque, as well as position and orientation of hafted flint stone tools. The set-up allows switching between two tool configurations, one with distal and the other one with perpendicular hafting of the scrapers, to allow for ethnographically plausible reconstructions. The data show statistically significant differences between the two gestures: scraping away from the body (PA) generates higher shearing forces, but requires greater hand torque. Moreover, most benchmarks associated with the PA gesture are more highly variable than in the PT gesture

  13. Dynamic Monitoring Reveals Motor Task Characteristics in Prehistoric Technical Gestures.

    PubMed

    Pfleging, Johannes; Stücheli, Marius; Iovita, Radu; Buchli, Jonas

    2015-01-01

    Reconstructing ancient technical gestures associated with simple tool actions is crucial for understanding the co-evolution of the human forelimb and its associated control-related cognitive functions on the one hand, and of the human technological arsenal on the other hand. Although the topic of gesture is an old one in Paleolithic archaeology and in anthropology in general, very few studies have taken advantage of the new technologies from the science of kinematics in order to improve replicative experimental protocols. Recent work in paleoanthropology has shown the potential of monitored replicative experiments to reconstruct tool-use-related motions through the study of fossil bones, but so far comparatively little has been done to examine the dynamics of the tool itself. In this paper, we demonstrate that we can statistically differentiate gestures used in a simple scraping task through dynamic monitoring. Dynamics combines kinematics (position, orientation, and speed) with contact mechanical parameters (force and torque). Taken together, these parameters are important because they play a role in the formation of a visible archaeological signature, use-wear. We present our new affordable, yet precise methodology for measuring the dynamics of a simple hide-scraping task, carried out using a pull-to (PT) and a push-away (PA) gesture. A strain gage force sensor combined with a visual tag tracking system records force, torque, as well as position and orientation of hafted flint stone tools. The set-up allows switching between two tool configurations, one with distal and the other one with perpendicular hafting of the scrapers, to allow for ethnographically plausible reconstructions. The data show statistically significant differences between the two gestures: scraping away from the body (PA) generates higher shearing forces, but requires greater hand torque. Moreover, most benchmarks associated with the PA gesture are more highly variable than in the PT gesture

  14. Shapiro like steps reveals molecular nanomagnets' spin dynamics

    NASA Astrophysics Data System (ADS)

    Abdollahipour, Babak; Abouie, Jahanfar; Ebrahimi, Navid

    2015-09-01

    We present an accurate way to detect spin dynamics of a nutating molecular nanomagnet by inserting it in a tunnel Josephson junction and studying the current voltage (I-V) characteristic. The spin nutation of the molecular nanomagnet is generated by applying two circularly polarized magnetic fields. We demonstrate that modulation of the Josephson current by the nutation of the molecular nanomagnet's spin appears as a stepwise structure like Shapiro steps in the I-V characteristic of the junction. Width and heights of these Shapiro-like steps are determined by two parameters of the spin nutation, frequency and amplitude of the nutation, which are simply tuned by the applied magnetic fields.

  15. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    NASA Astrophysics Data System (ADS)

    Stingaciu, Laura-Roxana; O'Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  16. Photon echo spectroscopy reveals structure-dynamics relationships in carotenoids

    NASA Astrophysics Data System (ADS)

    Christensson, N.; Polivka, T.; Yartsev, A.; Pullerits, T.

    2009-06-01

    Based on simultaneous analysis of the frequency-resolved transient grating, peak shift, and echo width signals, we present a model for the third-order optical response of carotenoids including population dynamics and system-bath interactions. Our frequency-resolved photon echo experiments show that the model needs to incorporate the excited-state absorption from both the S2 and the S1 states. We apply our model to analyze the experimental results on astaxanthin and lycopene, aiming to elucidate the relation between structure and system-bath interactions. Our analysis allows us to relate structural motifs to changes in the energy-gap correlation functions. We find that the terminal rings of astaxanthin lead to increased coupling between slow molecular motions and the electronic transition. We also find evidence for stronger coupling to higher frequency overdamped modes in astaxanthin, pointing to the importance of the functional groups in providing coupling to fluctuations influencing the dynamics in the passage through the conical intersection governing the S2-S1 relaxation.

  17. Genomic analysis of regulatory network dynamics reveals large topological changes

    NASA Astrophysics Data System (ADS)

    Luscombe, Nicholas M.; Madan Babu, M.; Yu, Haiyuan; Snyder, Michael; Teichmann, Sarah A.; Gerstein, Mark

    2004-09-01

    Network analysis has been applied widely, providing a unifying language to describe disparate systems ranging from social interactions to power grids. It has recently been used in molecular biology, but so far the resulting networks have only been analysed statically. Here we present the dynamics of a biological network on a genomic scale, by integrating transcriptional regulatory information and gene-expression data for multiple conditions in Saccharomyces cerevisiae. We develop an approach for the statistical analysis of network dynamics, called SANDY, combining well-known global topological measures, local motifs and newly derived statistics. We uncover large changes in underlying network architecture that are unexpected given current viewpoints and random simulations. In response to diverse stimuli, transcription factors alter their interactions to varying degrees, thereby rewiring the network. A few transcription factors serve as permanent hubs, but most act transiently only during certain conditions. By studying sub-network structures, we show that environmental responses facilitate fast signal propagation (for example, with short regulatory cascades), whereas the cell cycle and sporulation direct temporal progression through multiple stages (for example, with highly inter-connected transcription factors). Indeed, to drive the latter processes forward, phase-specific transcription factors inter-regulate serially, and ubiquitously active transcription factors layer above them in a two-tiered hierarchy. We anticipate that many of the concepts presented here-particularly the large-scale topological changes and hub transience-will apply to other biological networks, including complex sub-systems in higher eukaryotes.

  18. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    DOE PAGESBeta

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-21

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolutionmore » inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. We find our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. Lastly, these observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.« less

  19. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    PubMed Central

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture. PMID:26790980

  20. Invisible Electronic States and Their Dynamics Revealed by Perturbations

    NASA Astrophysics Data System (ADS)

    Merer, Anthony J.

    2011-06-01

    Sooner or later everyone working in the field of spectroscopy encounters perturbations. These can range in size from a small shift of a single rotational level to total destruction of the vibrational and rotational patterns of an electronic state. To some workers perturbations are a source of terror, but to others they are the most fascinating features of molecular spectra, because they give information about molecular dynamics, and about states that would otherwise be invisible as a result of unfavorable selection rules. An example of the latter is the essentially complete characterization of the tilde{b}^3A_2 state of SO_2 from the vibronic perturbations it causes in the tilde{a}^3B_1 state. The S_1-trans state of acetylene is a beautiful example of dynamics in action. The level patterns of the three bending vibrations change dramatically with increasing vibrational excitation as a result of the vibrational angular momentum and the approach to the isomerization barrier. Several vibrational levels of the S_1-cis isomer, previously thought to be unobservable, can now be assigned. They obtain their intensity through interactions with nearby levels of the trans isomer.

  1. Population dynamics of flaviviruses revealed by molecular phylogenies.

    PubMed Central

    Zanotto, P M; Gould, E A; Gao, G F; Harvey, P H; Holmes, E C

    1996-01-01

    The phylogeny of 123 complete envelope gene sequences was reconstructed in order to understand the evolution of tick- and mosquito-borne flaviviruses. An analysis of phylogenetic tree structure reveals a continual and asymmetric branching process in the tick-borne flaviviruses, compared with an explosive radiation in the last 200 years in viruses transmitted by mosquitoes. The distinction between these two viral groups probably reflects differences in modes of dispersal, propagation, and changes in the size of host populations. The most serious implication of this work is that growing human populations are being exposed to an expanding range of increasingly diverse viral strains. PMID:8570593

  2. Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics.

    PubMed

    Jang, Joon Hee; Huang, Yu; Zheng, Peilin; Jo, Myeong Chan; Bertolet, Grant; Zhu, Michael Xi; Qin, Lidong; Liu, Dongfang

    2015-08-01

    The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell-cell conjugates. In this study, we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single-cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically "stacked" cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1, and the cytotoxic synapse at the single-cell level. In addition to the technique innovation, we have demonstrated novel biological findings by this VCP device, including novel distribution of F-actin and cytolytic granules at the IS, programed death protein-1 microclusters at the NK IS, and kinetics of cytotoxicity. We propose that this high-throughput, cost-effective, easy-to-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines. PMID:26123352

  3. Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics

    PubMed Central

    Jang, Joon Hee; Huang, Yu; Zheng, Peilin; Jo, Myeong Chan; Bertolet, Grant; Qin, Lidong; Liu, Dongfang

    2015-01-01

    The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell-cell conjugates. Here we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically “stacked” cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1 (PD-1) and the cytotoxic synapse at the single cell level. In addition to the technique innovation, we demonstrated novel biological findings by this VCP device, including novel distribution of F-actin and cytolytic granules at the IS, PD-1 microclusters in the NK IS, and kinetics of cytotoxicity. We propose that this high-throughput, cost-effective, easy-to-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines. PMID:26123352

  4. Excitable actin dynamics in lamellipodial protrusion and retraction.

    PubMed

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

    2012-04-01

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

  5. Shapiro like steps reveals molecular nanomagnets’ spin dynamics

    SciTech Connect

    Abdollahipour, Babak; Abouie, Jahanfar Ebrahimi, Navid

    2015-09-15

    We present an accurate way to detect spin dynamics of a nutating molecular nanomagnet by inserting it in a tunnel Josephson junction and studying the current voltage (I-V) characteristic. The spin nutation of the molecular nanomagnet is generated by applying two circularly polarized magnetic fields. We demonstrate that modulation of the Josephson current by the nutation of the molecular nanomagnet’s spin appears as a stepwise structure like Shapiro steps in the I-V characteristic of the junction. Width and heights of these Shapiro-like steps are determined by two parameters of the spin nutation, frequency and amplitude of the nutation, which are simply tuned by the applied magnetic fields.

  6. Blood Sugar Measurement in Zebrafish Reveals Dynamics of Glucose Homeostasis

    PubMed Central

    Eames, Stefani C.; Philipson, Louis H.; Prince, Victoria E.

    2010-01-01

    Abstract The adult zebrafish has the potential to become an important model for diabetes-related research. To realize this potential, small-scale methods for analyzing pancreas function are required. The measurement of blood glucose level is a commonly used method for assessing β-cell function, but the small size of the zebrafish presents challenges both for collecting blood samples and for measuring glucose. We have developed methods for collecting microsamples of whole blood and plasma for the measurement of hematocrit and blood glucose. We demonstrate that two hand-held glucose meters designed for use by human diabetics return valid results with zebrafish blood. Additionally, we present methods for fasting and for performing postprandial glucose and intraperitoneal glucose tolerance tests. We find that the dynamics of zebrafish blood glucose homeostasis are consistent with patterns reported for other omnivorous teleost fish. PMID:20515318

  7. Bacterial associations reveal spatial population dynamics in Anopheles gambiae mosquitoes.

    PubMed

    Buck, Moritz; Nilsson, Louise K J; Brunius, Carl; Dabiré, Roch K; Hopkins, Richard; Terenius, Olle

    2016-01-01

    The intolerable burden of malaria has for too long plagued humanity and the prospect of eradicating malaria is an optimistic, but reachable, target in the 21(st) century. However, extensive knowledge is needed about the spatial structure of mosquito populations in order to develop effective interventions against malaria transmission. We hypothesized that the microbiota associated with a mosquito reflects acquisition of bacteria in different environments. By analyzing the whole-body bacterial flora of An. gambiae mosquitoes from Burkina Faso by 16 S amplicon sequencing, we found that the different environments gave each mosquito a specific bacterial profile. In addition, the bacterial profiles provided precise and predicting information on the spatial dynamics of the mosquito population as a whole and showed that the mosquitoes formed clear local populations within a meta-population network. We believe that using microbiotas as proxies for population structures will greatly aid improving the performance of vector interventions around the world. PMID:26960555

  8. Bacterial associations reveal spatial population dynamics in Anopheles gambiae mosquitoes

    PubMed Central

    Buck, Moritz; Nilsson, Louise K. J.; Brunius, Carl; Dabiré, Roch K.; Hopkins, Richard; Terenius, Olle

    2016-01-01

    The intolerable burden of malaria has for too long plagued humanity and the prospect of eradicating malaria is an optimistic, but reachable, target in the 21st century. However, extensive knowledge is needed about the spatial structure of mosquito populations in order to develop effective interventions against malaria transmission. We hypothesized that the microbiota associated with a mosquito reflects acquisition of bacteria in different environments. By analyzing the whole-body bacterial flora of An. gambiae mosquitoes from Burkina Faso by 16 S amplicon sequencing, we found that the different environments gave each mosquito a specific bacterial profile. In addition, the bacterial profiles provided precise and predicting information on the spatial dynamics of the mosquito population as a whole and showed that the mosquitoes formed clear local populations within a meta-population network. We believe that using microbiotas as proxies for population structures will greatly aid improving the performance of vector interventions around the world. PMID:26960555

  9. COMBINED DELAY AND GRAPH EMBEDDING OF EPILEPTIC DISCHARGES IN EEG REVEALS COMPLEX AND RECURRENT NONLINEAR DYNAMICS

    PubMed Central

    Erem, B.; Hyde, D.E.; Peters, J.M.; Duffy, F.H.; Brooks, D.H.; Warfield, S.K.

    2015-01-01

    The dynamical structure of the brain’s electrical signals contains valuable information about its physiology. Here we combine techniques for nonlinear dynamical analysis and manifold identification to reveal complex and recurrent dynamics in interictal epileptiform discharges (IEDs). Our results suggest that recurrent IEDs exhibit some consistent dynamics, which may only last briefly, and so individual IED dynamics may need to be considered in order to understand their genesis. This could potentially serve to constrain the dynamics of the inverse source localization problem. PMID:26366250

  10. Single-cell dynamics reveals sustained growth during diauxic shifts.

    PubMed

    Boulineau, Sarah; Tostevin, Filipe; Kiviet, Daniel J; ten Wolde, Pieter Rein; Nghe, Philippe; Tans, Sander J

    2013-01-01

    Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of E. coli cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring lac expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic lac expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between lac expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, lac expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival. PMID:23637881

  11. Phase Resetting Reveals Network Dynamics Underlying a Bacterial Cell Cycle

    PubMed Central

    Lin, Yihan; Li, Ying; Crosson, Sean; Dinner, Aaron R.; Scherer, Norbert F.

    2012-01-01

    Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS). PMID:23209388

  12. Dynamic Zebrafish Interactome Reveals Transcriptional Mechanisms of Dioxin Toxicity

    PubMed Central

    Alexeyenko, Andrey; Wassenberg, Deena M.; Lobenhofer, Edward K.; Yen, Jerry; Linney, Elwood; Sonnhammer, Erik L. L.; Meyer, Joel N.

    2010-01-01

    Background In order to generate hypotheses regarding the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) causes toxicity, we analyzed global gene expression changes in developing zebrafish embryos exposed to this potent toxicant in the context of a dynamic gene network. For this purpose, we also computationally inferred a zebrafish (Danio rerio) interactome based on orthologs and interaction data from other eukaryotes. Methodology/Principal Findings Using novel computational tools to analyze this interactome, we distinguished between dioxin-dependent and dioxin-independent interactions between proteins, and tracked the temporal propagation of dioxin-dependent transcriptional changes from a few genes that were altered initially, to large groups of biologically coherent genes at later times. The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes). Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a) as well as other genes that were dioxin-regulated one day after exposure. This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research. Conclusions/Significance Thus, a powerful microarray experiment coupled with a flexible interactome and multi-pronged interactome tools (which are now made publicly available for microarray analysis and related work) suggest the hypothesis that dioxin, best known in fish as a potent cardioteratogen, has many other targets. Many of these types of toxicity have been observed in mammalian species and are potentially caused by alterations to cyp1a. PMID:20463971

  13. Revealing the dynamics of Class 0 protostellar discs with ALMA

    NASA Astrophysics Data System (ADS)

    Seifried, D.; Sánchez-Monge, Á.; Walch, S.; Banerjee, R.

    2016-06-01

    We present synthetic ALMA observations of Keplerian, protostellar discs in the Class 0 stage studying the emission of molecular tracers like 13CO, C18O, HCO+, H13CO+, N2H+, and H2CO. We model the emission of discs around low- and intermediate-mass protostars. We show that under optimal observing conditions ALMA is able to detect the discs already in the earliest stage of protostellar evolution, although the emission is often concentrated to the innermost 50 au. Therefore, a resolution of a few 0.1 arcsec might be too low to detect Keplerian discs around Class 0 objects. We also demonstrate that under optimal conditions for edge-on discs Keplerian rotation signatures are recognisable, from which protostellar masses can be inferred. For this we here introduce a new approach, which allows us to determine protostellar masses with higher fidelity than before. Furthermore, we show that it is possible to reveal Keplerian rotation even for strongly inclined discs and that ALMA should be able to detect possible signs of fragmentation in face-on discs. In order to give some guidance for future ALMA observations, we investigate the influence of varying observing conditions and source distances. We show that it is possible to probe Keplerian rotation in inclined discs with an observing time of 2 h and a resolution of 0.1 arcsec, even in the case of moderate weather conditions. Furthermore, we demonstrate that under optimal conditions, Keplerian discs around intermediate-mass protostars should be detectable up to kpc distances.

  14. Oman metamorphic sole formation reveals early subduction dynamics

    NASA Astrophysics Data System (ADS)

    Soret, Mathieu; Agard, Philippe; Dubacq, Benoît; Plunder, Alexis; Ildefonse, Benoît; Yamato, Philippe; Prigent, Cécile

    2016-04-01

    Metamorphic soles correspond to m to ~500m thick tectonic slices welded beneath most of the large-scale ophiolites. They typically show a steep inverted metamorphic structure where the pressure and temperature conditions of crystallization increase upward (from 500±100°C at 0.5±0.2 GPa to 800±100°C at 1.0±0.2 GPa), with isograds subparallel to the contact with the overlying ophiolitic peridotite. The proportion of mafic rocks in metamorphic soles also increases from the bottom (meta-sediments rich) to the top (approaching the ophiolite peridotites). These soles are interpreted as the result of heat transfer from the incipient mantle wedge toward the nascent slab (associated with large-scale fluid transfer and possible shear heating) during the first My of intra-oceanic subduction (as indicated by radiometric ages). Metamorphic soles provide therefore major constraints on early subduction dynamics (i.e., thermal structure, fluid migration and rheology along the nascent slab interface). We present a detailed structural and petrological study of the metamorphic sole from 4 major cross-sections along the Oman ophiolite. We show precise pressure-temperature estimates obtained by pseudosection modelling and EBSD measurements performed on both the garnet-bearing and garnet-free high-grade sole. Results allow quantification of the micro-scale deformation and highlight differences in pressure-temperature-deformation conditions between the 4 different locations, showing that the inverted metamorphic gradient through the sole is not continuous in all locations. Based on these new constraints, we suggest a new tectonic-petrological model for the formation of metamorphic soles below ophiolites. This model involves the stacking of several homogeneous slivers of oceanic crust leading to the present-day structure of the sole. In this view, these thrusts are the result of rheological contrasts between the sole and the peridotite as the plate interface progressively cools down

  15. HUBBLE IMAGES REVEAL A YOUNG STAR'S DYNAMIC DISK AND JETS

    NASA Technical Reports Server (NTRS)

    2002-01-01

    These images of HH 30 show changes over only a five-year period in the disk and jets of this newborn star, which is about half a million years old. The pictures were taken between 1995 and 2000 with the Wide Field and Planetary Camera 2 aboard NASA's Hubble Space Telescope. Astronomers are interested in the disk because it is probably similar to the one from which the Sun and the planets in our solar system formed. Hubble reveals an edge-on disk (located at the bottom of the images), which appears as a flattened cloud of dust split into two halves by a dark lane. The disk blocks light from the central star. All that is visible is the reflection of the star's light by dust above and below the plane of the disk. The disk's diameter is 450 astronomical units (one astronomical unit equals the Earth-Sun distance). Shadows billions of miles in size can be seen moving across the disk. In 1995 and 2000, the left and right sides of the disk were about the same brightness, but in 1998 the right side was brighter. These patterns may be caused by bright spots on the star or variations in the disk near the star. The dust cloud near the top of these frames is illuminated by the star and reflects changes in its brightness. The star's magnetic field plays a major role in forming the jets (located above and below the disk), which look like streams of water from a fire hose. The powerful magnetic field creates the jets by channeling gas from the disk along the magnetic poles above and below the star. The gaps between the compact knots of gas seen in the jet above the disk indicate that this is a sporadic process. By tracking the motion of these knots over time, astronomers have measured the jet's speed at between 200,000 to 600,000 miles per hour (160,000 and 960,000 kilometers per hour). Oddly, the jet below the disk is moving twice as fast as the one above it. Credits: NASA, Alan Watson (Universidad Nacional Autonoma de Mexico), Karl Stapelfeldt (Jet Propulsion Laboratory), John

  16. Self-similar multiscale structure of lignin revealed by neutron scattering and molecular dynamics simulation

    SciTech Connect

    Petridis, Loukas; Pingali, Sai Venkatesh; Urban, Volker; Heller, William T; O'Neill, Hugh Michael; Foston, Marcus B; Ragauskas, Arthur J; Smith, Jeremy C

    2011-01-01

    Lignin, a major polymeric component of plant cell walls, forms aggregates in vivo and poses a barrier to cellulosic ethanol production. Here, neutron scattering experiments and molecular dynamics simulations reveal that lignin aggregates are characterized by a surface fractal dimension that is invariant under change of scale from 1 1000 A. The simulations also reveal extensive water penetration of the aggregates and heterogeneous chain dynamics corresponding to a rigid core with a fluid surface.

  17. A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

    SciTech Connect

    Zhang, Bingyu; Luo, Qing; Mao, Xinjian; Xu, Baiyao; Yang, Li; Ju, Yang; Song, Guanbin

    2014-03-10

    Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway. - Highlights: • Mechano-growth factor E peptide (MGF-C25E) promotes migration of rat tenocytes. • MGF-C25E activates the FAK-ERK1/2 pathway in rat tenocytes. • MGF-C25E induces the actin remodeling and the formation of pseudopodia, and decreases the stiffness in rat tenocytes. • MGF-C25E promotes tenocyte migration via altering stiffness and forming pseudopodia by the activation of the FAK-ERK1

  18. Slow dynamics of nanocomposite polymer aerogels as revealed by X-ray photocorrelation spectroscopy (XPCS)

    SciTech Connect

    Hernández, Rebeca E-mail: aurora.nogales@csic.es; Mijangos, Carmen; Nogales, Aurora E-mail: aurora.nogales@csic.es; Ezquerra, Tiberio A.; Sprung, Michael

    2014-01-14

    We report on a novel slow dynamics of polymer xerogels, aerogels, and nanocomposite aerogels with iron oxide nanoparticles, as revealed by X-ray photon correlation spectroscopy. The polymer aerogel and its nanocomposite aerogels, which are porous in nature, exhibit hyper-diffusive dynamics at room temperature. In contrast, non-porous polymer xerogels exhibit an absence of this peculiar dynamics. This slow dynamical process has been assigned to a relaxation of the characteristic porous structure of these materials and not to the presence of nanoparticles.

  19. Mammalian target of rapamycin complex (mTOR) pathway modulates blood-testis barrier (BTB) function through F-actin organization and gap junction.

    PubMed

    Li, Nan; Cheng, C Yan

    2016-09-01

    mTOR (mammalian target of rapamycin) is one of the most important signaling molecules in mammalian cells which regulates an array of cellular events, ranging from cell metabolism to cell proliferation. Based on the association of mTOR with the core component proteins, such as Raptor or Rictor, mTOR can become the mTORC1 (mammalian target of rapamycin complex 1) or mTORC2, respectively. Studies have shown that during the epithelial cycle of spermatogenesis, mTORC1 promotes remodeling and restructuring of the blood-testis barrier (BTB) in vitro and in vivo, making the Sertoli cell tight junction (TJ)-permeability barrier "leaky"; whereas mTORC2 promotes BTB integrity, making the Sertoli cell TJ-barrier "tighter". These contrasting effects, coupled with the spatiotemporal expression of the core signaling proteins at the BTB that confer the respective functions of mTORC1 vs. mTORC2 thus provide a unique mechanism to modulate BTB dynamics, allowing or disallowing the transport of biomolecules and also preleptotene spermatocytes across the immunological barrier. More importantly, studies have shown that these changes to BTB dynamics conferred by mTORC1 and mTORC2 are mediated by changes in the organization of the actin microfilament networks at the BTB, and involve gap junction (GJ) intercellular communication. Since GJ has recently been shown to be crucial to reboot spermatogenesis and meiosis following toxicant-induced aspermatogenesis, these findings thus provide new insightful information regarding the integration of mTOR and GJ to regulate spermatogenesis. PMID:26957088

  20. In vivo imaging of growth cone and filopodial dynamics: evidence for contact-mediated retraction of filopodia leading to the tiling of sibling processes.

    PubMed

    Baker, Michael W; Macagno, Eduardo R

    2007-02-10

    In the leech embryo, the peripheral comb cell (CC) sends out many nonoverlapping, growth cone-tipped processes that grow in parallel and serve as a scaffold for the migrating myocytes of the later-developing oblique muscle layer. To explore how the parallel arrangement is generated we first examined the arrangement of CC cytoskeletal components by expressing a tubulin-binding protein and actin, both tagged with fluorescent reporters. This revealed that the growth cones were compartmentalized into F-actin-rich filopodia and a microtubule-rich central region. Time-lapse analysis with a 2-photon laser scanning microscope revealed that the growth cones of the CC are highly dynamic, undergoing rapid filopodial extension and retraction. Measurements of filopodial lifespan and length revealed that most filopodia at the leading edge of the growth cone achieved significantly longer lifespans and length than lateral filopodia. Furthermore, for the short-lived lateral filopodia, apparent interaction with a neighboring process was found to be a significant predictor of their nearly immediate (within 2-4 minutes) retraction. When contact was experimentally prevented by ablating individual CCs, the filopodia from the growth cones of adjacent segmental neighbors were found to be significantly lengthened in the direction of the removed homolog. Treatment with low doses of cytochalasin D to disrupt F-actin assembly led to filopodial retraction and growth cone collapse and resulted in the bifurcation of many CC processes, numerous crossover errors, and the loss of parallelism. These findings indicate the existence of a contact-mediated repulsive interaction between processes of the CC. PMID:17177256

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

    PubMed Central

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

    2011-01-01

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

  2. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    SciTech Connect

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; Howell, Danielle; Kerekes, Ryan A.; Solecki, David J.

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia are motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.

  3. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    DOE PAGESBeta

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; Howell, Danielle; Kerekes, Ryan A.; Solecki, David J.

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

  4. RNA-dependent association with myosin IIA promotes F-actin-guided trafficking of the ELAV-like protein HuR to polysomes

    PubMed Central

    Doller, Anke; Schulz, Sebastian; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2013-01-01

    The role of the mRNA-binding protein human antigen R (HuR) in stabilization and translation of AU-rich elements (ARE) containing mRNAs is well established. However, the trafficking of HuR and bound mRNA cargo, which comprises a fundamental requirement for the aforementioned HuR functions is only poorly understood. By administering different cytoskeletal inhibitors, we found that the protein kinase Cδ (PKCδ)-triggered accumulation of cytoplasmic HuR by Angiotensin II (AngII) is an actin-myosin driven process functionally relevant for stabilization of ARE-bearing mRNAs. Furthermore, we show that the AngII-induced recruitment of HuR and its bound mRNA from ribonucleoprotein particles to free and cytoskeleton bound polysomes strongly depended on an intact actomyosin cytoskeleton. In addition, HuR allocation to free and cytoskeletal bound polysomes is highly sensitive toward RNase and PPtase and structurally depends on serine 318 (S318) located within the C-terminal RNA recognition motif (RRM3). Conversely, the trafficking of the phosphomimetic HuRS318D, mimicking HuR phosphorylation at S318 by the PKCδ remained PPtase resistant. Co-immunoprecipitation experiments with truncated HuR proteins revealed that the stimulus-induced association of HuR with myosin IIA is strictly RNA dependent and mediated via the RRM3. Our data implicate a microfilament dependent transport of HuR, which is relevant for stimulus-induced targeting of ARE-bearing mRNAs from translational inactive ribonucleoprotein particles to polysomes. PMID:23921630

  5. Molecular Dynamics Simulation Reveals Correlated Inter-Lobe Motion in Protein Lysine Methyltransferase SMYD2

    PubMed Central

    Spellmon, Nicholas; Sun, Xiaonan; Sirinupong, Nualpun; Edwards, Brian; Li, Chunying; Yang, Zhe

    2015-01-01

    SMYD proteins are an exciting field of study as they are linked to many types of cancer-related pathways. Cardiac and skeletal muscle development and function also depend on SMYD proteins opening a possible avenue for cardiac-related treatment. Previous crystal structure studies have revealed that this special class of protein lysine methyltransferases have a bilobal structure, and an open–closed motion may regulate substrate specificity. Here we use the molecular dynamics simulation to investigate the still-poorly-understood SMYD2 dynamics. Cross-correlation analysis reveals that SMYD2 exhibits a negative correlated inter-lobe motion. Principle component analysis suggests that this correlated dynamic is contributed to by a twisting motion of the C-lobe with respect to the N-lobe and a clamshell-like motion between the lobes. Dynamical network analysis defines possible allosteric paths for the correlated dynamics. There are nine communities in the dynamical network with six in the N-lobe and three in the C-lobe, and the communication between the lobes is mediated by a lobe-bridging β hairpin. This study provides insight into the dynamical nature of SMYD2 and could facilitate better understanding of SMYD2 substrate specificity. PMID:26717235

  6. A novel dynamics combination model reveals the hidden information of community structure

    NASA Astrophysics Data System (ADS)

    Li, Hui-Jia; Li, Huiying; Jia, Chuanliang

    2015-09-01

    The analysis of the dynamic details of community structure is an important question for scientists from many fields. In this paper, we propose a novel Markov-Potts framework to uncover the optimal community structures and their stabilities across multiple timescales. Specifically, we model the Potts dynamics to detect community structure by a Markov process, which has a clear mathematical explanation. Then the local uniform behavior of spin values revealed by our model is shown that can naturally reveal the stability of hierarchical community structure across multiple timescales. To prove the validity, phase transition of stochastic dynamic system is used to indicate that the stability of community structure we proposed is able to describe the significance of community structure based on eigengap theory. Finally, we test our framework on some example networks and find it does not have resolute limitation problem at all. Results have shown the model we proposed is able to uncover hierarchical structure in different scales effectively and efficiently.

  7. Structural dynamics of potassium channel gating revealed by single molecule FRET

    PubMed Central

    Borschel, William F.; Ha, Taekjip; Nichols, Colin G.

    2016-01-01

    Crystallography has provided invaluable insights to ion channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites, and examined structural dynamics within lipid membranes by single molecule FRET. We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and for cation channels in general. PMID:26641713

  8. Global dynamic topography observations reveal limited influence of large-scale mantle flow

    NASA Astrophysics Data System (ADS)

    Hoggard, M. J.; White, N.; Al-Attar, D.

    2016-06-01

    Convective circulation of the Earth's mantle maintains some fraction of surface topography that varies with space and time. Most predictive models show that this dynamic topography has peak amplitudes of about +/-2 km, dominated by wavelengths of 104 km. Here, we test these models against our comprehensive observational database of 2,120 spot measurements of dynamic topography that were determined by analysing oceanic seismic surveys. These accurate measurements have typical peak amplitudes of +/-1 km and wavelengths of approximately 103 km, and are combined with limited continental constraints to generate a global spherical harmonic model, the robustness of which has been carefully tested and benchmarked. Our power spectral analysis reveals significant discrepancies between observed and predicted dynamic topography. At longer wavelengths (such as 104 km), observed dynamic topography has peak amplitudes of about +/-500 m. At shorter wavelengths (such as 103 km), significant dynamic topography is still observed. We show that these discrepancies can be explained if short-wavelength dynamic topography is generated by temperature-driven density anomalies within a sub-plate asthenospheric channel. Stratigraphic observations from adjacent continental margins show that these dynamic topographic signals evolve quickly with time. More rapid temporal and spatial changes in vertical displacement of the Earth's surface have direct consequences for fields as diverse as mantle flow, oceanic circulation and long-term climate change.

  9. Dynamic changes in network synchrony reveal resting-state functional networks

    NASA Astrophysics Data System (ADS)

    Vuksanović, Vesna; Hövel, Philipp

    2015-02-01

    Experimental functional magnetic resonance imaging studies have shown that spontaneous brain activity, i.e., in the absence of any external input, exhibit complex spatial and temporal patterns of co-activity between segregated brain regions. These so-called large-scale resting-state functional connectivity networks represent dynamically organized neural assemblies interacting with each other in a complex way. It has been suggested that looking at the dynamical properties of complex patterns of brain functional co-activity may reveal neural mechanisms underlying the dynamic changes in functional interactions. Here, we examine how global network dynamics is shaped by different network configurations, derived from realistic brain functional interactions. We focus on two main dynamics measures: synchrony and variations in synchrony. Neural activity and the inferred hemodynamic response of the network nodes are simulated using a system of 90 FitzHugh-Nagumo neural models subject to system noise and time-delayed interactions. These models are embedded into the topology of the complex brain functional interactions, whose architecture is additionally reduced to its main structural pathways. In the simulated functional networks, patterns of correlated regional activity clearly arise from dynamical properties that maximize synchrony and variations in synchrony. Our results on the fast changes of the level of the network synchrony also show how flexible changes in the large-scale network dynamics could be.

  10. Revealing glacier flow and surge dynamics from animated satellite image sequences: examples from the Karakoram

    NASA Astrophysics Data System (ADS)

    Paul, F.

    2015-04-01

    Although animated images are very popular on the Internet, they have so far found only limited use for glaciological applications. With long time-series of satellite images becoming increasingly available and glaciers being well recognized for their rapid changes and variable flow dynamics, animated sequences of multiple satellite images reveal glacier dynamics in a time-lapse mode, making the otherwise slow changes of glacier movement visible and understandable for a wide public. For this study animated image sequences were created from freely available image quick-looks of orthorectified Landsat scenes for four regions in the central Karakoram mountain range. The animations play automatically in a web-browser and might help to demonstrate glacier flow dynamics for educational purposes. The animations revealed highly complex patterns of glacier flow and surge dynamics over a 15-year time period (1998-2013). In contrast to other regions, surging glaciers in the Karakoram are often small (around 10 km2), steep, debris free, and advance for several years at comparably low annual rates (a few hundred m a-1). The advance periods of individual glaciers are generally out of phase, indicating a limited climatic control on their dynamics. On the other hand, nearly all other glaciers in the region are either stable or slightly advancing, indicating balanced or even positive mass budgets over the past few years to decades.

  11. Dynamics of methane ebullition from a peat monolith revealed from a dynamic flux chamber system

    NASA Astrophysics Data System (ADS)

    Yu, Zhongjie; Slater, Lee D.; Schäfer, Karina V. R.; Reeve, Andrew S.; Varner, Ruth K.

    2014-09-01

    Methane (CH4) ebullition in northern peatlands is poorly quantified in part due to its high spatiotemporal variability. In this study, a dynamic flux chamber (DFC) system was used to continuously measure CH4 fluxes from a monolith of near-surface Sphagnum peat at the laboratory scale to understand the complex behavior of CH4 ebullition. Coincident transmission ground penetrating radar measurements of gas content were also acquired at three depths within the monolith. A graphical method was developed to separate diffusion, steady ebullition, and episodic ebullition fluxes from the total CH4 flux recorded and to identify the timing and CH4 content of individual ebullition events. The results show that the application of the DFC had minimal disturbance on air-peat CH4 exchange and estimated ebullition fluxes were not sensitive to the uncertainties associated with the graphical model. Steady and episodic ebullition fluxes were estimated to be averagely 36 ± 24% and 38 ± 24% of the total fluxes over the study period, respectively. The coupling between episodic CH4 ebullition and gas content within the three layers supports the existence of a threshold gas content regulating CH4 ebullition. However, the threshold at which active ebullition commenced varied between peat layers with a larger threshold (0.14 m3 m-3) observed in the deeper layers, suggesting that the peat physical structure controls gas bubble dynamics in peat. Temperature variation (23°C to 27°C) was likely only responsible for small episodic ebullition events from the upper peat layer, while large ebullition events from the deeper layers were most likely triggered by drops in atmospheric pressure.

  12. An analysis toolbox to explore mesenchymal migration heterogeneity reveals adaptive switching between distinct modes

    PubMed Central

    Shafqat-Abbasi, Hamdah; Kowalewski, Jacob M; Kiss, Alexa; Gong, Xiaowei; Hernandez-Varas, Pablo; Berge, Ulrich; Jafari-Mamaghani, Mehrdad; Lock, John G; Strömblad, Staffan

    2016-01-01

    Mesenchymal (lamellipodial) migration is heterogeneous, although whether this reflects progressive variability or discrete, 'switchable' migration modalities, remains unclear. We present an analytical toolbox, based on quantitative single-cell imaging data, to interrogate this heterogeneity. Integrating supervised behavioral classification with multivariate analyses of cell motion, membrane dynamics, cell-matrix adhesion status and F-actin organization, this toolbox here enables the detection and characterization of two quantitatively distinct mesenchymal migration modes, termed 'Continuous' and 'Discontinuous'. Quantitative mode comparisons reveal differences in cell motion, spatiotemporal coordination of membrane protrusion/retraction, and how cells within each mode reorganize with changed cell speed. These modes thus represent distinctive migratory strategies. Additional analyses illuminate the macromolecular- and cellular-scale effects of molecular targeting (fibronectin, talin, ROCK), including 'adaptive switching' between Continuous (favored at high adhesion/full contraction) and Discontinuous (low adhesion/inhibited contraction) modes. Overall, this analytical toolbox now facilitates the exploration of both spontaneous and adaptive heterogeneity in mesenchymal migration. DOI: http://dx.doi.org/10.7554/eLife.11384.001 PMID:26821527

  13. Combined TMS and FMRI reveal dissociable cortical pathways for dynamic and static face perception.

    PubMed

    Pitcher, David; Duchaine, Bradley; Walsh, Vincent

    2014-09-01

    Faces contain structural information, for identifying individuals, as well as changeable information, which can convey emotion and direct attention. Neuroimaging studies reveal brain regions that exhibit preferential responses to invariant [1, 2] or changeable [3-5] facial aspects but the functional connections between these regions are unknown. We addressed this issue by causally disrupting two face-selective regions with thetaburst transcranial magnetic stimulation (TBS) and measuring the effects of this disruption in local and remote face-selective regions with functional magnetic resonance imaging (fMRI). Participants were scanned, over two sessions, while viewing dynamic or static faces and objects. During these sessions, TBS was delivered over the right occipital face area (rOFA) or right posterior superior temporal sulcus (rpSTS). Disruption of the rOFA reduced the neural response to both static and dynamic faces in the downstream face-selective region in the fusiform gyrus. In contrast, the response to dynamic and static faces was doubly dissociated in the rpSTS. Namely, disruption of the rOFA reduced the response to static but not dynamic faces, while disruption of the rpSTS itself reduced the response to dynamic but not static faces. These results suggest that dynamic and static facial aspects are processed via dissociable cortical pathways that begin in early visual cortex, a conclusion inconsistent with current models of face perception [6-9]. PMID:25131678

  14. Light-induced nuclear export reveals rapid dynamics of epigenetic modifications.

    PubMed

    Yumerefendi, Hayretin; Lerner, Andrew Michael; Zimmerman, Seth Parker; Hahn, Klaus; Bear, James E; Strahl, Brian D; Kuhlman, Brian

    2016-06-01

    We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation. PMID:27089030

  15. Ideal probe single-molecule experiments reveal the intrinsic dynamic heterogeneity of a supercooled liquid

    PubMed Central

    Paeng, Keewook; Park, Heungman; Hoang, Dat Tien; Kaufman, Laura J.

    2015-01-01

    The concept of dynamic heterogeneity and the picture of the supercooled liquid as a mosaic of environments with distinct dynamics that interchange in time have been invoked to explain the nonexponential relaxations measured in these systems. The spatial extent and temporal persistence of these regions of distinct dynamics have remained challenging to identify. Here, single-molecule fluorescence measurements using a probe similar in size and mobility to the host o-terphenyl unambiguously reveal exponential relaxations distributed in time and space and directly demonstrate ergodicity of the system down to the glass transition temperature. In the temperature range probed, at least 200 times the structural relaxation time of the host is required to recover ensemble-averaged relaxation at every spatial region in the system. PMID:25825739

  16. Multiple Differential Networks Strategy Reveals Carboplatin and Melphalan-Induced Dynamic Module Changes in Retinoblastoma.

    PubMed

    Chen, Cui; Ma, Feng-Wei; Du, Cui-Yun; Wang, Ping

    2016-01-01

    BACKGROUND Retinoblastoma (RB) is the most common malignant tumor of the eye in childhood. The objective of this paper was to investigate carboplatin (CAR)- and melphalan (MEL)-induced dynamic module changes in RB based on multiple (M) differential networks, and to generate systems-level insights into RB progression. MATERIAL AND METHODS To achieve this goal, we constructed M-differential co-expression networks (DCNs), assigned a weight to each edge, and identified seed genes in M DCNs by ranking genes based on their topological features. Starting with seed genes, a module search was performed to explore candidate modules in CAR and MEL condition. M-DMs were detected according to significance evaluations of M-modules, which originated from refinement of candidate modules. Further, we revealed dynamic changes in M-DM activity and connectivity on the basis of significance of Module Connectivity Dynamic Score (MCDS). RESULTS In the present study, M=2, a total of 21 seed genes were obtained. By assessing module search, refinement, and evaluation, we gained 18 2-DMs. Moreover, 3 significant 2-DMs (Module 1, Module 2, and Module 3) with dynamic changes across CAR and MEL condition were determined, and we denoted them as dynamic modules. Module 1 had 27 nodes of which 6 were seed genes and 56 edges. Module 2 was composed of 28 nodes and 54 edges. A total of 28 nodes interacted with 45 edges presented in Module 3. CONCLUSIONS We have identified 3 dynamic modules with changes induced by CAR and MEL in RB, which might give insights in revealing molecular mechanism for RB therapy. PMID:27144687

  17. Dynamic functional network connectivity reveals unique and overlapping profiles of insula subdivisions.

    PubMed

    Nomi, Jason S; Farrant, Kristafor; Damaraju, Eswar; Rachakonda, Srinivas; Calhoun, Vince D; Uddin, Lucina Q

    2016-05-01

    The human insular cortex consists of functionally diverse subdivisions that engage during tasks ranging from interoception to cognitive control. The multiplicity of functions subserved by insular subdivisions calls for a nuanced investigation of their functional connectivity profiles. Four insula subdivisions (dorsal anterior, dAI; ventral, VI; posterior, PI; middle, MI) derived using a data-driven approach were subjected to static- and dynamic functional network connectivity (s-FNC and d-FNC) analyses. Static-FNC analyses replicated previous work demonstrating a cognition-emotion-interoception division of the insula, where the dAI is functionally connected to frontal areas, the VI to limbic areas, and the PI and MI to sensorimotor areas. Dynamic-FNC analyses consisted of k-means clustering of sliding windows to identify variable insula connectivity states. The d-FNC analysis revealed that the most frequently occurring dynamic state mirrored the cognition-emotion-interoception division observed from the s-FNC analysis, with less frequently occurring states showing overlapping and unique subdivision connectivity profiles. In two of the states, all subdivisions exhibited largely overlapping profiles, consisting of subcortical, sensory, motor, and frontal connections. Two other states showed the dAI exhibited a unique connectivity profile compared with other insula subdivisions. Additionally, the dAI exhibited the most variable functional connections across the s-FNC and d-FNC analyses, and was the only subdivision to exhibit dynamic functional connections with regions of the default mode network. These results highlight how a d-FNC approach can capture functional dynamics masked by s-FNC approaches, and reveal dynamic functional connections enabling the functional flexibility of the insula across time. Hum Brain Mapp 37:1770-1787, 2016. © 2016 Wiley Periodicals, Inc. PMID:26880689

  18. Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

    PubMed Central

    Zhou, Zhenzhen; Shi, Haifan; Chen, Binqing; Zhang, Ruihui; Huang, Shanjin; Fu, Ying

    2015-01-01

    Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca2+ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth. PMID:25804540

  19. Effects of pressure on the dynamics of a hyperthermophilic protein revealed by quasielastic neutron scattering

    NASA Astrophysics Data System (ADS)

    Shrestha, U. R.; Bhowmik, D.; Copley, J. R. D.; Tyagi, M.; Leao, J. B.; Chu, X.-Q.

    Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is nearly 100 MPa. Here we study the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range using quasielastic neutron scattering (QENS) technique. Two spectrometers were used to investigate the β-relaxation dynamics of proteins in time ranges from 2 to 25 ps, and from 100 ps to 2 ns. Our results reveal that, under the pressure of 100 MPa, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), opposite to what we observed previously under the ambient pressure. These contradictory observations imply that high pressure affects the dynamical properties of proteins by distorting their energy landscapes. Accordingly, we derived a general schematic denaturation phase diagram that can be used as a general picture to understand the effects of pressure on protein dynamics and activities Wayne State Univ Startup Fund.

  20. Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles.

    PubMed

    Fornili, Arianna; Pandini, Alessandro; Lu, Hui-Chun; Fraternali, Franca

    2013-11-12

    The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein

  1. Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

    PubMed Central

    2013-01-01

    The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein

  2. Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling.

    PubMed

    Fusco, Ludovico; Lefort, Riwal; Smith, Kevin; Benmansour, Fethallah; Gonzalez, German; Barillari, Caterina; Rinn, Bernd; Fleuret, Francois; Fua, Pascal; Pertz, Olivier

    2016-01-01

    Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth. PMID:26728857

  3. Dynamics of water-in-oil nanoemulsions revealed by fluorescence lifetime correlation spectroscopy.

    PubMed

    Orte, Angel; Ruedas-Rama, Maria J; Paredes, Jose M; Crovetto, Luis; Alvarez-Pez, Jose M

    2011-11-01

    The size, diffusional properties, and dynamics of reverse water-in-oil nanoemulsions, or reverse micelles (RMs), have been widely investigated because of interest in this system as a model for biological compartmentalization. Here, we have employed fluorescence lifetime correlation spectroscopy (FLCS) to reveal the dynamics and sizes of aerosol-OT (AOT)/isooctane RMs using a fluorescent xanthene derivative called Tokyo Green II (TG-II). The dye undergoes a partition and a shift in its tautomeric equilibrium such that the TG-II anion remains in the inner micellar aqueous core, and the neutral quinoid form lies in the interfacial region. By applying FLCS, we specifically obtained the lifetime filtered autocorrelation curves of the anionic TG-II, which shows a characteristic lifetime of approximately 4 ns. Analysis of the FLCS curves provides the diffusion coefficient and hydrodynamic radius of the RMs as well as micelle dynamics in the same experiment. The FLCS curves show dynamics in the microsecond time range, which represents an interconversion rate that changes the distribution of the TG-II neutral and anionic forms in the hydrophobic interface and the water core. PMID:21913723

  4. Capturing Arabidopsis root architecture dynamics with ROOT-FIT reveals diversity in responses to salinity.

    PubMed

    Julkowska, Magdalena M; Hoefsloot, Huub C J; Mol, Selena; Feron, Richard; de Boer, Gert-Jan; Haring, Michel A; Testerink, Christa

    2014-11-01

    The plant root is the first organ to encounter salinity stress, but the effect of salinity on root system architecture (RSA) remains elusive. Both the reduction in main root (MR) elongation and the redistribution of the root mass between MRs and lateral roots (LRs) are likely to play crucial roles in water extraction efficiency and ion exclusion. To establish which RSA parameters are responsive to salt stress, we performed a detailed time course experiment in which Arabidopsis (Arabidopsis thaliana) seedlings were grown on agar plates under different salt stress conditions. We captured RSA dynamics with quadratic growth functions (root-fit) and summarized the salt-induced differences in RSA dynamics in three growth parameters: MR elongation, average LR elongation, and increase in number of LRs. In the ecotype Columbia-0 accession of Arabidopsis, salt stress affected MR elongation more severely than LR elongation and an increase in LRs, leading to a significantly altered RSA. By quantifying RSA dynamics of 31 different Arabidopsis accessions in control and mild salt stress conditions, different strategies for regulation of MR and LR meristems and root branching were revealed. Different RSA strategies partially correlated with natural variation in abscisic acid sensitivity and different Na(+)/K(+) ratios in shoots of seedlings grown under mild salt stress. Applying root-fit to describe the dynamics of RSA allowed us to uncover the natural diversity in root morphology and cluster it into four response types that otherwise would have been overlooked. PMID:25271266

  5. Automatic generation of predictive dynamic models reveals nuclear phosphorylation as the key Msn2 control mechanism.

    PubMed

    Sunnåker, Mikael; Zamora-Sillero, Elias; Dechant, Reinhard; Ludwig, Christina; Busetto, Alberto Giovanni; Wagner, Andreas; Stelling, Joerg

    2013-05-28

    Predictive dynamical models are critical for the analysis of complex biological systems. However, methods to systematically develop and discriminate among systems biology models are still lacking. We describe a computational method that incorporates all hypothetical mechanisms about the architecture of a biological system into a single model and automatically generates a set of simpler models compatible with observational data. As a proof of principle, we analyzed the dynamic control of the transcription factor Msn2 in Saccharomyces cerevisiae, specifically the short-term mechanisms mediating the cells' recovery after release from starvation stress. Our method determined that 12 of 192 possible models were compatible with available Msn2 localization data. Iterations between model predictions and rationally designed phosphoproteomics and imaging experiments identified a single-circuit topology with a relative probability of 99% among the 192 models. Model analysis revealed that the coupling of dynamic phenomena in Msn2 phosphorylation and transport could lead to efficient stress response signaling by establishing a rate-of-change sensor. Similar principles could apply to mammalian stress response pathways. Systematic construction of dynamic models may yield detailed insight into nonobvious molecular mechanisms. PMID:23716718

  6. Dynamics and Organization of Cortical Microtubules as Revealed by Superresolution Structured Illumination Microscopy1[W

    PubMed Central

    Komis, George; Mistrik, Martin; Šamajová, Olga; Doskočilová, Anna; Ovečka, Miroslav; Illés, Peter; Bartek, Jiri; Šamaj, Jozef

    2014-01-01

    Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for the optimal documentation of Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells, dynamic cortical microtubules labeled with green fluorescent protein fused to the microtubule-binding domain of the mammalian microtubule-associated protein MAP4 and with green fluorescent protein-fused to the alpha tubulin6 were comparatively recorded in wild-type Arabidopsis plants and in the mitogen-activated protein kinase mutant mpk4 possessing the former microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of the microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at the subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles. Time-lapse SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid, and nondynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. In conclusion, this study demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization. PMID:24686112

  7. Fractal dynamics in self-evaluation reveal self-concept clarity.

    PubMed

    Wong, Alexander E; Vallacher, Robin R; Nowak, Andrzej

    2014-10-01

    The structural account of self-esteem and self-evaluation maintains that they are distinct constructs. Trait self-esteem is stable and is expressed over macro timescales, whereas state self-evaluation is unstable and experienced on micro timescales. We compared predictions based on the structural account with those derived from a dynamical systems perspective on the self, which maintains that self-esteem and self-evaluation are hierarchically related and share basic dynamic properties. Participants recorded a 3-minute narrative about themselves, then used the mouse paradigm (Vallacher, Nowak, Froehlich, & Rockloff, 2002) to track the momentary self-evaluation in their narrative. Multiple methods converged to reveal fractal patterns in the resultant temporal patterns, indicative of nested timescales that link micro and macro selfevaluation and thus supportive of the dynamical account. The fractal dynamics were associated with participants' self-concept clarity, suggesting that the hierarchical relation between macro self-evaluation (self-esteem) and momentary self-evaluation is predicted by the coherence of self-concept organization. PMID:25196705

  8. Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

    PubMed Central

    Fusco, Ludovico; Lefort, Riwal; Smith, Kevin; Benmansour, Fethallah; Gonzalez, German; Barillari, Caterina; Rinn, Bernd; Fleuret, Francois; Fua, Pascal

    2016-01-01

    Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth. PMID:26728857

  9. Dynamic localization of electronic excitation in photosynthetic complexes revealed with chiral two-dimensional spectroscopy

    NASA Astrophysics Data System (ADS)

    Fidler, Andrew F.; Singh, Ved P.; Long, Phillip D.; Dahlberg, Peter D.; Engel, Gregory S.

    2014-02-01

    Time-resolved ultrafast optical probes of chiral dynamics provide a new window allowing us to explore how interactions with such structured environments drive electronic dynamics. Incorporating optical activity into time-resolved spectroscopies has proven challenging because of the small signal and large achiral background. Here we demonstrate that two-dimensional electronic spectroscopy can be adapted to detect chiral signals and that these signals reveal how excitations delocalize and contract following excitation. We dynamically probe the evolution of chiral electronic structure in the light-harvesting complex 2 of purple bacteria following photoexcitation by creating a chiral two-dimensional mapping. The dynamics of the chiral two-dimensional signal directly reports on changes in the degree of delocalization of the excitonic states following photoexcitation. The mechanism of energy transfer in this system may enhance transfer probability because of the coherent coupling among chromophores while suppressing fluorescence that arises from populating delocalized states. This generally applicable spectroscopy will provide an incisive tool to probe ultrafast transient molecular fluctuations that are obscured in non-chiral experiments.

  10. Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

    PubMed Central

    He, Yufan; Haque, Mohammad Mahfuzul; Stuehr, Dennis J.; Lu, H. Peter

    2015-01-01

    Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS. PMID:26311846

  11. Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics.

    PubMed

    He, Yufan; Haque, Mohammad Mahfuzul; Stuehr, Dennis J; Lu, H Peter

    2015-09-22

    Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS. PMID:26311846

  12. Dynamic changes in brewing yeast cells in culture revealed by statistical analyses of yeast morphological data.

    PubMed

    Ohnuki, Shinsuke; Enomoto, Kenichi; Yoshimoto, Hiroyuki; Ohya, Yoshikazu

    2014-03-01

    The vitality of brewing yeasts has been used to monitor their physiological state during fermentation. To investigate the fermentation process, we used the image processing software, CalMorph, which generates morphological data on yeast mother cells and bud shape, nuclear shape and location, and actin distribution. We found that 248 parameters changed significantly during fermentation. Successive use of principal component analysis (PCA) revealed several important features of yeast, providing insight into the dynamic changes in the yeast population. First, PCA indicated that much of the observed variability in the experiment was summarized in just two components: a change with a peak and a change over time. Second, PCA indicated the independent and important morphological features responsible for dynamic changes: budding ratio, nucleus position, neck position, and actin organization. Thus, the large amount of data provided by imaging analysis can be used to monitor the fermentation processes involved in beer and bioethanol production. PMID:24012106

  13. Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes.

    PubMed

    Dean, Samuel; Moreira-Leite, Flavia; Varga, Vladimir; Gull, Keith

    2016-08-30

    The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics. PMID:27519801

  14. Something in the way she moves--movement trajectories reveal dynamics of self-control.

    PubMed

    Dignath, David; Pfister, Roland; Eder, Andreas B; Kiesel, Andrea; Kunde, Wilfried

    2014-06-01

    This study examined the dynamic impact of self-control conflict on action execution. We reasoned that the tug-of-war between antagonistic action tendencies is not ultimately solved before movement initiation but leaks into action execution. To this end, we measured mouse trajectories to quantify the dynamic competition between initial temptations and the struggle to overcome them. Participants moved the mouse cursor from a start location to one of two targets. Each target represented a gain or a loss of points. Although participants earned points on the majority of the trials, they also had to make movements to the loss target on some trials to prevent an even higher loss. Two experiments found that movement trajectories on these loss trials deviate toward the tempting stimulus: The way we move reveals self-control conflicts that have not been resolved prior to action execution. PMID:24254808

  15. Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

    PubMed Central

    Streets, Aaron M.; Sourigues, Yannick; Kopito, Ron R.; Melki, Ronald; Quake, Stephen R.

    2013-01-01

    An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation. PMID:23349924

  16. Endothelial cells undergo morphological, biomechanical, and dynamic changes in response to tumor necrosis factor-α.

    PubMed

    Stroka, Kimberly M; Vaitkus, Janina A; Aranda-Espinoza, Helim

    2012-11-01

    The immune response triggers a complicated sequence of events, one of which is release of the cytokine tumor necrosis factor-α (TNF-α) from stromal cells, for example monocytes and macrophages. In this work we investigated the biophysical effects of TNF-α on endothelial cells (ECs), including changes in cell morphology, biomechanics, migration, and cytoskeletal dynamics. We found that TNF-α induces a wide distribution of cell area and aspect ratio, with these properties increasing on average during treatment. Interestingly, aspect ratio peaks after approximately 10 h of exposure to TNF-α, corresponding also to a peak in exerted traction forces. Meanwhile, ECs treated with TNF-α soften, and we associate this with significant increases in estimated cellular volume. In addition, our evaluation of migratory dynamics revealed an inverse correlation between cell aspect ratio and migration speed after TNF-α treatment, suggesting that cell shape may be an important functional regulator of EC migration during an inflammatory response. Finally, we addressed the basic mechanics of how the reorganization of F-actin filaments occurs during TNF-α treatment, and observed a dynamic shift of existing actin filaments. Together, our results suggest a functional link between EC morphology, biomechanics, migration, and cytoskeletal dynamics during an inflammatory response. PMID:22940754

  17. Revealing glacier flow and surge dynamics from animated satellite image sequences: examples from the Karakoram

    NASA Astrophysics Data System (ADS)

    Paul, F.

    2015-11-01

    Although animated images are very popular on the internet, they have so far found only limited use for glaciological applications. With long time series of satellite images becoming increasingly available and glaciers being well recognized for their rapid changes and variable flow dynamics, animated sequences of multiple satellite images reveal glacier dynamics in a time-lapse mode, making the otherwise slow changes of glacier movement visible and understandable to the wider public. For this study, animated image sequences were created for four regions in the central Karakoram mountain range over a 25-year time period (1990-2015) from freely available image quick-looks of orthorectified Landsat scenes. The animations play automatically in a web browser and reveal highly complex patterns of glacier flow and surge dynamics that are difficult to obtain by other methods. In contrast to other regions, surging glaciers in the Karakoram are often small (10 km2 or less), steep, debris-free, and advance for several years to decades at relatively low annual rates (about 100 m a-1). These characteristics overlap with those of non-surge-type glaciers, making a clear identification difficult. However, as in other regions, the surging glaciers in the central Karakoram also show sudden increases of flow velocity and mass waves travelling down glacier. The surges of individual glaciers are generally out of phase, indicating a limited climatic control on their dynamics. On the other hand, nearly all other glaciers in the region are either stable or slightly advancing, indicating balanced or even positive mass budgets over the past few decades.

  18. Early embryonic development in the Djungarian hamster (Phodopus sungorus) is accompanied by alterations in the distribution and intensity of an estrogen (E2)-dependent oviduct glycoprotein in the blastomere membrane and zona pellucida and in its association with F-actin.

    PubMed

    Murray, M; Messinger, S M

    1994-12-01

    The luminal environment of the estrogen (E2)-dominated mammalian oviduct generates and sustains the environment in which the first embryonic cleavages take place. The objective of this study was to determine, by use of an antiserum against an E2-dependent sheep oviduct secretory glycoprotein (M(r) 90,000-92,000), whether the E2-dominated and pregnant oviduct of the Djungarian hamster (Phodopus sungorus) releases an antigenically related protein. If the protein was present, a secondary objective was to define its fate and association with filamentous-actin (f-actin) and chromatin patterns in early cleavage-stage embryos. Oviduct flushings containing embryos (1-cell fertilized, 2-, 4-, and 8-cells), and uterine flushings (> 16 cell embryos) were obtained from pregnant hamsters. Embryos were removed from flushings, and oviduct secretions were analyzed by Western blotting. The zona pellucida was removed with acid Tyrode's solution from approximately half of the 2-, 4-, and 8-cell embryos. Zona-intact and zona-free embryos were then fixed and subjected to triple immunofluorescence staining with an antiserum to the sheep oviduct protein, rhodamine phalloidin, and Hoechst 33258. An antigenically related protein M(r) 200,000) was detected in oviduct secretions of E2-treated, ovariectomized, and pregnant hamsters, and not in secretions from ovariectomized controls. In the zona pellucida of 1- and 2-cell embryos, the oviduct protein displayed an intertwining, reticular organization that was replaced by a diffuse and more intense accumulation in 4-, 8-, and > 16-cell embryos. In 2-cell embryos, punctate foci of the oviduct protein were distributed unevenly over the apical blastomere plasma membrane, forming patches in regions of f-actin exclusion, which were absent at later development stages. At the 4- and 8-cell stage of development, as blastomeres lost their spherical form by minimizing intercellular spaces, the oviduct protein took on a polarized arrangement and was

  19. Intricate phase diagram of a prevalent visual circuit reveals universal dynamics, phase transitions, and resonances.

    PubMed

    Caudill, Matthew S; Brandt, Sebastian F; Nussinov, Zohar; Wessel, Ralf

    2009-11-01

    Neural feedback-triads consisting of two feedback loops with a nonreciprocal lateral connection from one loop to the other are ubiquitous in the brain. We show analytically that the dynamics of this network topology are determined by algebraic combinations of its five synaptic weights. Exploration of network activity over the parameter space demonstrates the importance of the nonreciprocal lateral connection and reveals intricate behavior involving continuous transitions between qualitatively different activity states. In addition, we show that the response to periodic inputs is narrowly tuned around a center frequency determined by the effective synaptic parameters. PMID:20365022

  20. Intravital imaging technology reveals immune system dynamics in vivo.

    PubMed

    Ishii, Masaru

    2016-07-01

    Fluorescent 'intravital' imaging is a new research technique by which the interior of living tissues and organs (in living bodies, if possible) can be observed, revealing the kinetics of cell and molecular processes in real time. Recent technological innovations in optical equipment and fluorescence imaging techniques have enabled a variety of cellular phenomena in different tissues and organs to be characterized under completely native conditions. This shift from static to dynamic biology constitutes the beginning of a new era in biomedical sciences. PMID:27238377

  1. Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation.

    PubMed

    Maier, Ezekiel J; Haynes, Brian C; Gish, Stacey R; Wang, Zhuo A; Skowyra, Michael L; Marulli, Alyssa L; Doering, Tamara L; Brent, Michael R

    2015-05-01

    Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus-its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and "foreman" TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes. PMID:25644834

  2. Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation

    PubMed Central

    Maier, Ezekiel J.; Haynes, Brian C.; Gish, Stacey R.; Wang, Zhuo A.; Skowyra, Michael L.; Marulli, Alyssa L.; Doering, Tamara L.; Brent, Michael R.

    2015-01-01

    Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus—its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and “foreman” TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes. PMID:25644834

  3. EEG microstate sequences in healthy humans at rest reveal scale-free dynamics.

    PubMed

    Van de Ville, Dimitri; Britz, Juliane; Michel, Christoph M

    2010-10-19

    Recent findings identified electroencephalography (EEG) microstates as the electrophysiological correlates of fMRI resting-state networks. Microstates are defined as short periods (100 ms) during which the EEG scalp topography remains quasi-stable; that is, the global topography is fixed but strength might vary and polarity invert. Microstates represent the subsecond coherent activation within global functional brain networks. Surprisingly, these rapidly changing EEG microstates correlate significantly with activity in fMRI resting-state networks after convolution with the hemodynamic response function that constitutes a strong temporal smoothing filter. We postulate here that microstate sequences should reveal scale-free, self-similar dynamics to explain this remarkable effect and thus that microstate time series show dependencies over long time ranges. To that aim, we deploy wavelet-based fractal analysis that allows determining scale-free behavior. We find strong statistical evidence that microstate sequences are scale free over six dyadic scales covering the 256-ms to 16-s range. The degree of long-range dependency is maintained when shuffling the local microstate labels but becomes indistinguishable from white noise when equalizing microstate durations, which indicates that temporal dynamics are their key characteristic. These results advance the understanding of temporal dynamics of brain-scale neuronal network models such as the global workspace model. Whereas microstates can be considered the "atoms of thoughts," the shortest constituting elements of cognition, they carry a dynamic signature that is reminiscent at characteristic timescales up to multiple seconds. The scale-free dynamics of the microstates might be the basis for the rapid reorganization and adaptation of the functional networks of the brain. PMID:20921381

  4. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin.

    PubMed

    Janssen, Aniek; Breuer, Gregory A; Brinkman, Eva K; van der Meulen, Annelot I; Borden, Sean V; van Steensel, Bas; Bindra, Ranjit S; LaRocque, Jeannine R; Karpen, Gary H

    2016-07-15

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context.Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains. PMID:27474442

  5. Proteome Dynamics Reveals Pro-Inflammatory Remodeling of Plasma Proteome in a Mouse Model of NAFLD.

    PubMed

    Li, Ling; Bebek, Gurkan; Previs, Stephen F; Smith, Jonathan D; Sadygov, Rovshan G; McCullough, Arthur J; Willard, Belinda; Kasumov, Takhar

    2016-09-01

    Nonalcoholic fatty liver disease (NAFLD) is associated with an increased risk of cardiovascular disease. Because the liver is the major source of circulatory proteins, it is not surprising that hepatic disease could lead to alterations in the plasma proteome, which are therein implicated in atherosclerosis. The current study used low-density lipoprotein receptor-deficient (LDLR(-/-)) mice to examine the impact of Western diet (WD)-induced NAFLD on plasma proteome homeostasis. Using a (2)H2O-metabolic labeling method, we found that a WD led to a proinflammatory distribution of circulatory proteins analyzed in apoB-depleted plasma, which was attributed to an increased production. The fractional turnover rates of short-lived proteins that are implicated in stress-response, lipid metabolism, and transport functions were significantly increased with WD (P < 0.05). Pathway analyses revealed that alterations in plasma proteome dynamics were related to the suppression of hepatic PPARα, which was confirmed based on reduced gene and protein expression of PPARα in mice fed a WD. These changes were associated with ∼4-fold increase (P < 0.0001) in the proinflammatory property of apoB-depleted plasma. In conclusion, the proteome dynamics method reveals proinflammatory remodeling of the plasma proteome relevant to liver disease. The approach used herein may provide a useful metric of in vivo liver function and better enable studies of novel therapies surrounding NAFLD and other diseases. PMID:27439437

  6. Glassy dynamics of polymers confined to nanoporous glasses revealed by relaxational and scattering experiments.

    PubMed

    Schönhals, A; Goering, H; Schick, Ch; Frick, B; Zorn, R

    2003-09-01

    The glassy dynamics of poly(propylene glycol) (PPG) and poly(dimethyl siloxane) (PDMS) confined to a nanoporous host system revealed by dielectric spectroscopy, temperature-modulated DSC and neutron scattering is compared. For both systems the relaxation rates estimated from dielectric spectroscopy and temperature-modulated DSC agree quantitatively indicating that both experiments sense the glass transition. For PPG the segmental dynamics is determined by a counterbalance of adsorption and confinement effect. The former results form an interaction of the confined macromolecules with the internal surfaces. A confinement effect originates from an inherent length scale on which the underlying molecular motions take place. The increment of the specific-heat capacity [Formula: see text] at the glass transition vanishes at a finite length scale of 1.8 nm. Both results support the conception that a characteristic length scale is relevant for glassy dynamics. For PDMS only a confinement effect is observed which is much stronger than that for PPG. Down to a pore size of 7.5 nm, the temperature dependence of the relaxation times follows the Vogel-Fulcher-Tammann dependence. At a pore size of 5 nm this changes to an Arrhenius-like behaviour with a low activation energy. At the same pore size [Formula: see text] vanishes for PDMS. Quasielastic neutron scattering experiments reveal that also the diffusive character of the relevant molecular motions --found to be characteristic above the glass transition-- seems to disappear at this length scale. These results gives further strong support that the glass transition has to be characterised by an inherent length scale of the relevant molecular motions. PMID:15007697

  7. Dynamics of natural killer cell receptor revealed by quantitative analysis of photoswitchable protein.

    PubMed

    Pageon, Sophie V; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G; Davis, Daniel M

    2013-11-01

    Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies that rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report an apparently novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, after a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 ± 0.06 μm(2) s(-1)) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse, and remains surprisingly stable there. Unexpectedly, however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected intersynaptic exchange of protein. PMID:24209843

  8. Complex Contact-Based Dynamics of Microsphere Monolayers Revealed by Resonant Attenuation of Surface Acoustic Waves.

    PubMed

    Hiraiwa, M; Abi Ghanem, M; Wallen, S P; Khanolkar, A; Maznev, A A; Boechler, N

    2016-05-13

    Contact-based vibrations play an essential role in the dynamics of granular materials. Significant insights into vibrational granular dynamics have previously been obtained with reduced-dimensional systems containing macroscale particles. We study contact-based vibrations of a two-dimensional monolayer of micron-sized spheres on a solid substrate that forms a microscale granular crystal. Measurements of the resonant attenuation of laser-generated surface acoustic waves reveal three collective vibrational modes that involve displacements and rotations of the microspheres, as well as interparticle and particle-substrate interactions. To identify the modes, we tune the interparticle stiffness, which shifts the frequency of the horizontal-rotational resonances while leaving the vertical resonance unaffected. From the measured contact resonance frequencies we determine both particle-substrate and interparticle contact stiffnesses and find that the former is an order of magnitude larger than the latter. This study paves the way for investigating complex contact-based dynamics of microscale granular crystals and yields a new approach to studying micro- to nanoscale contact mechanics in multiparticle networks. PMID:27232047

  9. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.

    PubMed

    Hanna, David A; Harvey, Raven M; Martinez-Guzman, Osiris; Yuan, Xiaojing; Chandrasekharan, Bindu; Raju, Gheevarghese; Outten, F Wayne; Hamza, Iqbal; Reddi, Amit R

    2016-07-01

    Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20-40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines. PMID:27247412

  10. Ligand induced conformational changes of the human serotonin transporter revealed by molecular dynamics simulations.

    PubMed

    Koldsø, Heidi; Autzen, Henriette Elisabeth; Grouleff, Julie; Schiøtt, Birgit

    2013-01-01

    The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design. PMID:23776432

  11. Transcriptome-wide mapping reveals reversible and dynamic N(1)-methyladenosine methylome.

    PubMed

    Li, Xiaoyu; Xiong, Xushen; Wang, Kun; Wang, Lixia; Shu, Xiaoting; Ma, Shiqing; Yi, Chengqi

    2016-05-01

    N(1)-Methyladenosine (m(1)A) is a prevalent post-transcriptional RNA modification, yet little is known about its abundance, topology and dynamics in mRNA. Here, we show that m(1)A is prevalent in Homo sapiens mRNA, which shows an m(1)A/A ratio of ∼0.02%. We develop the m(1)A-ID-seq technique, based on m(1)A immunoprecipitation and the inherent ability of m(1)A to stall reverse transcription, as a means for transcriptome-wide m(1)A profiling. m(1)A-ID-seq identifies 901 m(1)A peaks (from 600 genes) in mRNA and noncoding RNA and reveals a prominent feature, enrichment in the 5' untranslated region of mRNA transcripts, that is distinct from the pattern for N(6)-methyladenosine, the most abundant internal mammalian mRNA modification. Moreover, m(1)A in mRNA is reversible by ALKBH3, a known DNA/RNA demethylase. Lastly, we show that m(1)A methylation responds dynamically to stimuli, and we identify hundreds of stress-induced m(1)A sites. Collectively, our approaches allow comprehensive analysis of m(1)A modification and provide tools for functional studies of potential epigenetic regulation via the reversible and dynamic m(1)A methylation. PMID:26863410

  12. Function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

    SciTech Connect

    Nutt, David; Smith, Jeremy C

    2008-10-01

    Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.

  13. Complex Contact-Based Dynamics of Microsphere Monolayers Revealed by Resonant Attenuation of Surface Acoustic Waves

    NASA Astrophysics Data System (ADS)

    Hiraiwa, M.; Abi Ghanem, M.; Wallen, S. P.; Khanolkar, A.; Maznev, A. A.; Boechler, N.

    2016-05-01

    Contact-based vibrations play an essential role in the dynamics of granular materials. Significant insights into vibrational granular dynamics have previously been obtained with reduced-dimensional systems containing macroscale particles. We study contact-based vibrations of a two-dimensional monolayer of micron-sized spheres on a solid substrate that forms a microscale granular crystal. Measurements of the resonant attenuation of laser-generated surface acoustic waves reveal three collective vibrational modes that involve displacements and rotations of the microspheres, as well as interparticle and particle-substrate interactions. To identify the modes, we tune the interparticle stiffness, which shifts the frequency of the horizontal-rotational resonances while leaving the vertical resonance unaffected. From the measured contact resonance frequencies we determine both particle-substrate and interparticle contact stiffnesses and find that the former is an order of magnitude larger than the latter. This study paves the way for investigating complex contact-based dynamics of microscale granular crystals and yields a new approach to studying micro- to nanoscale contact mechanics in multiparticle networks.

  14. Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

    PubMed Central

    Grouleff, Julie; Schiøtt, Birgit

    2013-01-01

    The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design. PMID:23776432

  15. Using State Space Methods to Reveal Dynamical Associations Between Cortisol and Depression.

    PubMed

    Toonen, Roelof B; Wardenaar, Klaas J; van Ockenburg, Sonja L; Bos, Elisabeth H; de Jonge, Peter

    2016-01-01

    Despite extensive research, the link between etiological factors and depression remains poorly understood. This may in part be due to a focus on strictly linear definitions of causality, derived at the group level. However, etiological relations in depression are likely to be dynamical, nonlinear and potentially unquantifiable with traditional statistics. Therefore the aim of this study was to evaluate the use of the convergent cross-mapping (CCM) method in investigating possible nonlinear relationships between supposed etiological factors and depressive symptomatology. Time series data from six healthy individuals were used to model the relationship between 24-h urinary free cortisol and negative affect using CCM and dewdrop embeddings. CCM is a nonlinear measure of causality, based on state space reconstruction with lagged coordinate embeddings. The results showed that nonlinear dynamical relationships between cortisol and negative affect may be present within participants, as demonstrated by a positive cross-map convergence from negative affect to cortisol. However, analyses also showed that noise and influential points had considerable impact on the results. Convergent crossmapping can be used to reveal possible nonlinear dynamical relationships between etiological factors and psychopathology that may remain undetected with traditional linear causality measures. PMID:26639919

  16. In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

    PubMed Central

    Bajanca, Fernanda; Gonzalez-Perez, Vinicio; Gillespie, Sean J; Beley, Cyriaque; Garcia, Luis; Theveneau, Eric; Sear, Richard P; Hughes, Simon M

    2015-01-01

    Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmdta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics. DOI: http://dx.doi.org/10.7554/eLife.06541.001 PMID:26459831

  17. Dynamic Transcription Factor Activity Profiles Reveal Key Regulatory Interactions During Megakaryocytic and Erythroid Differentiation

    PubMed Central

    Duncan, Mark T.; Shin, Seungjin; Wu, Jia J.; Mays, Zachary; Weng, Stanley; Bagheri, Neda; Miller, William M.; Shea, Lonnie D.

    2014-01-01

    The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation. PMID:24853077

  18. Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation.

    PubMed

    Duncan, Mark T; Shin, Seungjin; Wu, Jia J; Mays, Zachary; Weng, Stanley; Bagheri, Neda; Miller, William M; Shea, Lonnie D

    2014-10-01

    The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation. PMID:24853077

  19. Dynamic Localization of G-actin During Membrane Protrusion in Neuronal Motility

    PubMed Central

    Lee, Chi Wai; Vitriol, Eric A.; Shim, Sangwoo; Wise, Ariel L.; Velayutham, Radhi P.; Zheng, James Q.

    2013-01-01

    Summary Background Actin-based cell motility is fundamental for the development, function, and malignant events of eukaryotic organisms. During neural development, axonal growth cones depend on rapid assembly and disassembly of actin filaments (F-actin) for their guided extension to specific targets for wiring. Monomeric globular actin (G-actin) is the building block for F-actin but is not considered to play a direct role in spatiotemporal control of actin dynamics in cell motility. Results Here we report that a pool of G-actin dynamically localizes to the leading edge of growth cones and neuroblastoma cells to spatially elevate the G-/F-actin ratio that drives membrane protrusion and cell movement. Loss of G-actin localization leads to the cessation and retraction of membrane protrusions. Moreover, G-actin localization occurs asymmetrically in growth cones during attractive turning. Finally, we identify the actin monomer binding proteins profilin and thymosin β4 as key molecules that localize actin monomers to the leading edge of lamellipodia for their motility. Conclusions Our results suggest that dynamic localization of G-actin provides a novel mechanism to regulate the spatiotemporal actin dynamics underlying membrane protrusion in cell locomotion and growth cone chemotaxis. PMID:23746641

  20. Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis

    PubMed Central

    2010-01-01

    Background Surface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE), a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F) or produced with an old-young smearing process (M). Results TTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei), the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans) that developed early in ripening (day 14 to 20), shortly after the growth of staphylococci (day 7). A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 × 102 CFU cm-2, when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (105 CFU cm-2) on cheeses smeared with a defined surface culture. Conclusions This work reports

  1. Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse

    PubMed Central

    Zampieri, Alexandre; Champagne, Julien; Auzemery, Baptiste; Fuentes, Ivanna; Maurel, Benjamin; Bienvenu, Frédéric

    2015-01-01

    We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed “Tandem-HTRF”, can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material. PMID:26503526

  2. Cortical mechanisms of smooth eye movements revealed by dynamic covariations of neural and behavioral responses.

    PubMed

    Schoppik, David; Nagel, Katherine I; Lisberger, Stephen G

    2008-04-24

    Neural activity in the frontal eye fields controls smooth pursuit eye movements, but the relationship between single neuron responses, cortical population responses, and eye movements is not well understood. We describe an approach to dynamically link trial-to-trial fluctuations in neural responses to parallel variations in pursuit and demonstrate that individual neurons predict eye velocity fluctuations at particular moments during the course of behavior, while the population of neurons collectively tiles the entire duration of the movement. The analysis also reveals the strength of correlations in the eye movement predictions derived from pairs of simultaneously recorded neurons and suggests a simple model of cortical processing. These findings constrain the primate cortical code for movement, suggesting that either a few neurons are sufficient to drive pursuit at any given time or that many neurons operate collectively at each moment with remarkably little variation added to motor command signals downstream from the cortex. PMID:18439409

  3. Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.

    PubMed

    Schmidt, Jens C; Zaug, Arthur J; Cech, Thomas R

    2016-08-25

    Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end. PMID:27523609

  4. Nanomechanical Behavior of Single Crystalline SiC Nanotubes Revealed by Molecular Dynamics Simulations

    SciTech Connect

    Wang, Zhiguo; Zu, Xiaotao T.; Gao, Fei; Weber, William J.

    2008-11-01

    Molecular dynamics simulations with Tersoff potentials were used to study the response of single crystalline SiC nanotubes under tensile, compressive, torsional, combined tension-torsional and combined compression-torsional strains. The simulation results reveal that the nanotubes deform through bond-stretching and breaking and exhibit brittle properties under uniaxial tensile strain, except for the thinnest nanotube at high temperatures, which fails in a ductile manner. Under uniaxial compressive strain, the SiC nanotubes buckle with two modes, i.e. shell buckling and column buckling, depending on the length of the nanotubes. Under torsional strain, the nanotubes buckle either collapse in the middle region into a dumbbell-like structure for thinner wall thicknesses or fail by bond breakage for the largest wall thickness. Both the tensile failure stress and buckling stress decrease under combined tension-torsional and combined compression-torsional strain, and they decrease with increasing torsional rate under combined loading.

  5. Lagrangian Descriptors: A Method for Revealing Phase Space Structures of General Time Dependent Dynamical Systems

    NASA Astrophysics Data System (ADS)

    Mancho, Ana M.; Wiggins, Stephen; Curbelo, Jezabel; Mendoza, Carolina

    2013-11-01

    Lagrangian descriptors are a recent technique which reveals geometrical structures in phase space and which are valid for aperiodically time dependent dynamical systems. We discuss a general methodology for constructing them and we discuss a ``heuristic argument'' that explains why this method is successful. We support this argument by explicit calculations on a benchmark problem. Several other benchmark examples are considered that allow us to assess the performance of Lagrangian descriptors with both finite time Lyapunov exponents (FTLEs) and finite time averages of certain components of the vector field (``time averages''). In all cases Lagrangian descriptors are shown to be both more accurate and computationally efficient than these methods. We thank CESGA for computing facilities. This research was supported by MINECO grants: MTM2011-26696, I-Math C3-0104, ICMAT Severo Ochoa project SEV-2011-0087, and CSIC grant OCEANTECH. SW acknowledges the support of the ONR (Grant No. N00014-01-1-0769).

  6. Revealing the flux: Using processed Husimi maps to visualize dynamics of bound systems and mesoscopic transport

    NASA Astrophysics Data System (ADS)

    Mason, Douglas J.; Borunda, Mario F.; Heller, Eric J.

    2015-04-01

    We elaborate upon the "processed Husimi map" representation for visualizing quantum wave functions using coherent states as a measurement of the local phase space to produce a vector field related to the probability flux. Adapted from the Husimi projection, the processed Husimi map is mathematically related to the flux operator under certain limits but offers a robust and flexible alternative since it can operate away from these limits and in systems that exhibit zero flux. The processed Husimi map is further capable of revealing the full classical dynamics underlying a quantum wave function since it reverse engineers the wave function to yield the underlying classical ray structure. We demonstrate the capabilities of processed Husimi maps on bound systems with and without electromagnetic fields, as well as on open systems on and off resonance, to examine the relationship between closed system eigenstates and mesoscopic transport.

  7. Histone H3 Dynamics Reveal Domains with Distinct Proliferation Potential in the Arabidopsis Root.

    PubMed

    Otero, Sofía; Desvoyes, Bénédicte; Peiró, Ramón; Gutierrez, Crisanto

    2016-06-01

    A coordinated transition from cell proliferation to differentiation is crucial for organogenesis. We found that extensive chromatin reorganization, shown here for histone H3 proteins, characterizes cell population dynamics in the root developmental compartments. The canonical H3.1 protein, incorporated during S-phase, is maintained at high levels in cells dividing at a high rate but is massively evicted in cells undergoing their last cell cycle before exit to differentiation. A similar pattern was observed in the quadruple mutant for the H3.1-encoding genes HTR1, HTR2, HTR3, and HTR9 (htr1,2,3,9), in which H3.1 is expressed only from the HTR13 gene. H3 eviction is a fast process occurring within the G2 phase of the last cell cycle, which is longer than G2 in earlier cell cycles. This longer G2 likely contributes to lower the H3.1/H3.3 ratio in cells leaving the root meristem. The high H3.1/H3.3 ratio and H3.1 eviction process also occurs in endocycling cells before differentiation, revealing a common principle of H3 eviction in the proliferating and endocycling domains of the root apex. Mutants in the H3.1 chaperone CAF-1 (fas1-4) maintain a pattern similar to that of wild-type roots. Our studies reveal that H3 incorporation and eviction dynamics identify cells with different cell division potential during organ patterning. PMID:27207857

  8. Degradation dynamics of microRNAs revealed by a novel pulse-chase approach.

    PubMed

    Marzi, Matteo J; Ghini, Francesco; Cerruti, Benedetta; de Pretis, Stefano; Bonetti, Paola; Giacomelli, Chiara; Gorski, Marcin M; Kress, Theresia; Pelizzola, Mattia; Muller, Heiko; Amati, Bruno; Nicassio, Francesco

    2016-04-01

    The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2> 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2= 4-14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions. PMID:26821571

  9. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    PubMed Central

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-01-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy. PMID:26893143

  10. Multifractal analysis of Barkhausen noise reveals the dynamic nature of criticality at hysteresis loop

    NASA Astrophysics Data System (ADS)

    Tadić, Bosiljka

    2016-06-01

    The field-driven magnetisation reversal processes in disordered systems exhibit a collective behaviour that is manifested in the scale-invariance of avalanches, closely related to underlying dynamical mechanisms. Using the multifractal time series analysis, we study the structure of fluctuations at different scales in the accompanying Barkhausen noise. The stochastic signal represents the magnetisation discontinuities along the hysteresis loop of a three-dimensional random field Ising model simulated for varied disorder strength and driving rates. The analysis of the spectrum of the generalised Hurst exponents reveals that the dominant segments of the signal with large fluctuations represent two distinct classes of stochastic processes in weak and strong pinning regimes. Furthermore, in the weak pinning regime, the part of the signal originating from the beginning of the hysteresis loop has a different multifractal spectrum than the signal near the coercive field. The enhanced fluctuations (primarily in the central part of the hysteresis loop) for increased driving rate and larger system size, lead to a further broadening of the spectrum. The analysed Barkhausen signals are also shown to exhibit temporal correlations and power-law distributions of the magnetisation discontinuity and avalanche sizes, in agreement with previous studies. The multifractal properties of Barkhausen noise describe the dynamical state of domains and precisely discriminate the weak pinning, permitting the motion of individual walls, from the mechanisms occurring in strongly disordered systems.

  11. Molecular dynamics simulations reveal specific interactions of post-translational palmitoyl modifications with rhodopsin in membranes

    PubMed Central

    Olausson, Bjoern E.S.; Grossfield, Alan; Pitman, Michael C.; Brown, Michael F.; Feller, Scott E.; Vogel, Alexander

    2012-01-01

    We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations—as well as their extensive fluctuations—suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin. PMID:22280374

  12. Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

    PubMed Central

    Niu, Kai-Yang; Liao, Hong-Gang; Zheng, Haimei

    2012-01-01

    The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment. PMID:23287885

  13. Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics

    PubMed Central

    Helassa, Nordine; Zhang, Xiao-hua; Conte, Ianina; Scaringi, John; Esposito, Elric; Bradley, Jonathan; Carter, Thomas; Ogden, David; Morad, Martin; Török, Katalin

    2015-01-01

    Faithful reporting of temporal patterns of intracellular Ca2+ dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca2+ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca2+ affinities and accelerated kinetics by weakening the Ca2+-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca2+ decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca2+ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca2+ at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca2+ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes. PMID:26527405

  14. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells.

    PubMed

    Trapnell, Cole; Cacchiarelli, Davide; Grimsby, Jonna; Pokharel, Prapti; Li, Shuqiang; Morse, Michael; Lennon, Niall J; Livak, Kenneth J; Mikkelsen, Tarjei S; Rinn, John L

    2014-04-01

    Defining the transcriptional dynamics of a temporal process such as cell differentiation is challenging owing to the high variability in gene expression between individual cells. Time-series gene expression analyses of bulk cells have difficulty distinguishing early and late phases of a transcriptional cascade or identifying rare subpopulations of cells, and single-cell proteomic methods rely on a priori knowledge of key distinguishing markers. Here we describe Monocle, an unsupervised algorithm that increases the temporal resolution of transcriptome dynamics using single-cell RNA-Seq data collected at multiple time points. Applied to the differentiation of primary human myoblasts, Monocle revealed switch-like changes in expression of key regulatory factors, sequential waves of gene regulation, and expression of regulators that were not known to act in differentiation. We validated some of these predicted regulators in a loss-of function screen. Monocle can in principle be used to recover single-cell gene expression kinetics from a wide array of cellular processes, including differentiation, proliferation and oncogenic transformation. PMID:24658644

  15. Postnatal changes in the growth dynamics of the human face revealed from bone modelling patterns

    PubMed Central

    Martinez-Maza, Cayetana; Rosas, Antonio; Nieto-Díaz, Manuel

    2013-01-01

    Human skull morphology results from complex processes that involve the coordinated growth and interaction of its skeletal components to keep a functional and structural balance. Previous histological works have studied the growth of different craniofacial regions and their relationship to functional spaces in humans up to 14 years old. Nevertheless, how the growth dynamics of the facial skeleton and the mandible are related and how this relationship changes through the late ontogeny remain poorly understood. To approach these two questions, we have compared the bone modelling activities of the craniofacial skeleton from a sample of subadult and adult humans. In this study, we have established for the first time the bone modelling pattern of the face and the mandible from adult humans. Our analyses reveal a patchy distribution of the bone modelling fields (overemphasized by the presence of surface islands with no histological information) reflecting the complex growth dynamics associated to the individual morphology. Subadult and adult specimens show important differences in the bone modelling patterns of the anterior region of the facial skeleton and the posterior region of the mandible. These differences indicate developmental changes in the growth directions of the whole craniofacial complex, from a predominantly downward growth in subadults that turns to a forward growth observed in the adult craniofacial skeleton. We hypothesize that these ontogenetic changes would respond to the physiological and physical requirements to enlarge the oral and nasal cavities once maturation of the brain and the closure of the cranial sutures have taken place during craniofacial development. PMID:23819603

  16. Visualization of the Serratia Type VI Secretion System Reveals Unprovoked Attacks and Dynamic Assembly

    PubMed Central

    Gerc, Amy J.; Diepold, Andreas; Trunk, Katharina; Porter, Michael; Rickman, Colin; Armitage, Judith P.; Stanley-Wall, Nicola R.; Coulthurst, Sarah J.

    2015-01-01

    Summary The Type VI secretion system (T6SS) is a bacterial nanomachine that fires toxic proteins into target cells. Deployment of the T6SS represents an efficient and widespread means by which bacteria attack competitors or interact with host organisms and may be triggered by contact from an attacking neighbor cell as a defensive strategy. Here, we use the opportunist pathogen Serratia marcescens and functional fluorescent fusions of key components of the T6SS to observe different subassemblies of the machinery simultaneously and on multiple timescales in vivo. We report that the localization and dynamic behavior of each of the components examined is distinct, revealing a multi-stage and dynamic assembly process for the T6SS machinery. We also show that the T6SS can assemble and fire without needing a cell contact trigger, defining an aggressive strategy that broadens target range and suggesting that activation of the T6SS is tailored to survival in specific niches. PMID:26387948

  17. Visualization of the Serratia Type VI Secretion System Reveals Unprovoked Attacks and Dynamic Assembly.

    PubMed

    Gerc, Amy J; Diepold, Andreas; Trunk, Katharina; Porter, Michael; Rickman, Colin; Armitage, Judith P; Stanley-Wall, Nicola R; Coulthurst, Sarah J

    2015-09-29

    The Type VI secretion system (T6SS) is a bacterial nanomachine that fires toxic proteins into target cells. Deployment of the T6SS represents an efficient and widespread means by which bacteria attack competitors or interact with host organisms and may be triggered by contact from an attacking neighbor cell as a defensive strategy. Here, we use the opportunist pathogen Serratia marcescens and functional fluorescent fusions of key components of the T6SS to observe different subassemblies of the machinery simultaneously and on multiple timescales in vivo. We report that the localization and dynamic behavior of each of the components examined is distinct, revealing a multi-stage and dynamic assembly process for the T6SS machinery. We also show that the T6SS can assemble and fire without needing a cell contact trigger, defining an aggressive strategy that broadens target range and suggesting that activation of the T6SS is tailored to survival in specific niches. PMID:26387948

  18. Health trajectories reveal the dynamic contributions of host genetic resistance and tolerance to infection outcome

    PubMed Central

    Lough, Graham; Kyriazakis, Ilias; Bergmann, Silke; Lengeling, Andreas; Doeschl-Wilson, Andrea B.

    2015-01-01

    Resistance and tolerance are two alternative strategies hosts can adopt to survive infections. Both strategies may be genetically controlled. To date, the relative contribution of resistance and tolerance to infection outcome is poorly understood. Here, we use a bioluminescent Listeria monocytogenes (Lm) infection challenge model to study the genetic determination and dynamic contributions of host resistance and tolerance to listeriosis in four genetically diverse mouse strains. Using conventional statistical analyses, we detect significant genetic variation in both resistance and tolerance, but cannot capture the time-dependent relative importance of either host strategy. We overcome these limitations through the development of novel statistical tools to analyse individual infection trajectories portraying simultaneous changes in infection severity and health. Based on these tools, early expression of resistance followed by expression of tolerance emerge as important hallmarks for surviving Lm infections. Our trajectory analysis further reveals that survivors and non-survivors follow distinct infection paths (which are also genetically determined) and provides new survival thresholds as objective endpoints in infection experiments. Future studies may use trajectories as novel traits for mapping and identifying genes that control infection dynamics and outcome. A Matlab script for user-friendly trajectory analysis is provided. PMID:26582028

  19. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    NASA Astrophysics Data System (ADS)

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-02-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

  20. Degradation dynamics of microRNAs revealed by a novel pulse-chase approach

    PubMed Central

    Marzi, Matteo J.; Ghini, Francesco; Cerruti, Benedetta; de Pretis, Stefano; Bonetti, Paola; Giacomelli, Chiara; Gorski, Marcin M.; Kress, Theresia; Pelizzola, Mattia; Muller, Heiko; Amati, Bruno; Nicassio, Francesco

    2016-01-01

    The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2 > 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2 = 4–14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions. PMID:26821571

  1. Insight into the dynamics of lanthanide-DTPA complexes as revealed by oxygen-17 NMR.

    PubMed

    Fusaro, Luca; Mocci, Francesca; Muller, Robert N; Luhmer, Michel

    2012-08-01

    DTPA chelates of various diamagnetic and paramagnetic lanthanide(III) metal ions, as well as the chemically similar DTPA chelate of Y(3+), were studied in aqueous solution by variable temperature (17)O NMR with the aim of characterizing their internal dynamics. As a consequence of poor chemical shift dispersion and fast quadrupole relaxation, no dynamic exchange process could be detected for the diamagnetic complexes nor for the Sm-DTPA complex. In contrast, the spectra recorded for the Eu-DTPA complex show chemical exchange due to the well-known racemization process and, at high temperature, feature signal broadening that reveals a fluxional process involving the interchange of the coordinated and noncoordinated oxygen atoms of the carboxylate groups. The spectra recorded for the Pr-DTPA complex feature coalescence events due to such a fluxional process, which is ascribable to the rotation of the carboxylate groups. The activation free energy barriers determined experimentally are remarkably lower than the calculated activation barriers recently reported for the rotation of the carboxylate groups of various Ln-DOTA complexes. Furthermore, the smallest activation free energy measured for the Pr-DTPA complex, about 45 kJ mol(-1), is significantly lower than the activation free energy characterizing the racemization process. The fluxional behavior of the carboxylate groups is, however, not expected to significantly affect the residence time of the water molecule coordinated to the metal ion. PMID:22817329

  2. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations

    PubMed Central

    Hertig, Samuel

    2016-01-01

    Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest. PMID:27285999

  3. Ocean time-series reveals recurring seasonal patterns of virioplankton dynamics in the northwestern Sargasso Sea.

    PubMed

    Parsons, Rachel J; Breitbart, Mya; Lomas, Michael W; Carlson, Craig A

    2012-02-01

    There are an estimated 10(30) virioplankton in the world oceans, the majority of which are phages (viruses that infect bacteria). Marine phages encompass enormous genetic diversity, affect biogeochemical cycling of elements, and partially control aspects of prokaryotic production and diversity. Despite their importance, there is a paucity of data describing virioplankton distributions over time and depth in oceanic systems. A decade of high-resolution time-series data collected from the upper 300 m in the northwestern Sargasso Sea revealed recurring temporal and vertical patterns of virioplankton abundance in unprecedented detail. An annual virioplankton maximum developed between 60 and 100 m during periods of summer stratification and eroded during winter convective mixing. The timing and vertical positioning of this seasonal pattern was related to variability in water column stability and the dynamics of specific picophytoplankton and heterotrophic bacterioplankton lineages. Between 60 and 100 m, virioplankton abundance was negatively correlated to the dominant heterotrophic bacterioplankton lineage SAR11, as well as the less abundant picophytoplankton, Synechococcus. In contrast, virioplankton abundance was positively correlated to the dominant picophytoplankton lineage Prochlorococcus, and the less abundant alpha-proteobacteria, Rhodobacteraceae. Seasonally, virioplankton abundances were highly synchronous with Prochlorococcus distributions and the virioplankton to Prochlorococcus ratio remained remarkably constant during periods of water column stratification. The data suggest that a significant fraction of viruses in the mid-euphotic zone of the subtropical gyres may be cyanophages and patterns in their abundance are largely determined by Prochlorococcus dynamics in response to water column stability. This high-resolution, decadal survey of virioplankton abundance provides insight into the possible controls of virioplankton dynamics in the open ocean. PMID

  4. Dynamic Adipocyte Phosphoproteome Reveals that Akt Directly Regulates mTORC2

    PubMed Central

    Humphrey, Sean J.; Yang, Guang; Yang, Pengyi; Fazakerley, Daniel J.; Stöckli, Jacqueline; Yang, Jean Y.; James, David E.

    2013-01-01

    Summary A major challenge of the post-genomics era is to define the connectivity of protein phosphorylation networks. Here, we quantitatively delineate the insulin signaling network in adipocytes by high-resolution mass spectrometry-based proteomics. These data reveal the complexity of intracellular protein phosphorylation. We identified 37,248 phosphorylation sites on 5,705 proteins in this single-cell type, with approximately 15% responding to insulin. We integrated these large-scale phosphoproteomics data using a machine learning approach to predict physiological substrates of several diverse insulin-regulated kinases. This led to the identification of an Akt substrate, SIN1, a core component of the mTORC2 complex. The phosphorylation of SIN1 by Akt was found to regulate mTORC2 activity in response to growth factors, revealing topological insights into the Akt/mTOR signaling network. The dynamic phosphoproteome described here contains numerous phosphorylation sites on proteins involved in diverse molecular functions and should serve as a useful functional resource for cell biologists. PMID:23684622

  5. Revealing nonergodic dynamics in living cells from a single particle trajectory

    NASA Astrophysics Data System (ADS)

    Lanoiselée, Yann; Grebenkov, Denis S.

    2016-05-01

    We propose the improved ergodicity and mixing estimators to identify nonergodic dynamics from a single particle trajectory. The estimators are based on the time-averaged characteristic function of the increments and can thus capture additional information on the process as compared to the conventional time-averaged mean-square displacement. The estimators are first investigated and validated for several models of anomalous diffusion, such as ergodic fractional Brownian motion and diffusion on percolating clusters, and nonergodic continuous-time random walks and scaled Brownian motion. The estimators are then applied to two sets of earlier published trajectories of mRNA molecules inside live Escherichia coli cells and of Kv2.1 potassium channels in the plasma membrane. These statistical tests did not reveal nonergodic features in the former set, while some trajectories of the latter set could be classified as nonergodic. Time averages along such trajectories are thus not representative and may be strongly misleading. Since the estimators do not rely on ensemble averages, the nonergodic features can be revealed separately for each trajectory, providing a more flexible and reliable analysis of single-particle tracking experiments in microbiology.

  6. Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq

    PubMed Central

    Shepard, Peter J.; Choi, Eun-A; Lu, Jente; Flanagan, Lisa A.; Hertel, Klemens J.; Shi, Yongsheng

    2011-01-01

    Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3′ untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation. PMID:21343387

  7. Transcription closed and open complex dynamics studies reveal balance between genetic determinants and co-factors

    NASA Astrophysics Data System (ADS)

    Sala, Adrien; Shoaib, Muhammad; Anufrieva, Olga; Mutharasu, Gnanavel; Jahan Hoque, Rawnak; Yli-Harja, Olli; Kandhavelu, Meenakshisundaram

    2015-05-01

    In E. coli, promoter closed and open complexes are key steps in transcription initiation, where magnesium-dependent RNA polymerase catalyzes RNA synthesis. However, the exact mechanism of initiation remains to be fully elucidated. Here, using single mRNA detection and dual reporter studies, we show that increased intracellular magnesium concentration affects Plac initiation complex formation resulting in a highly dynamic process over the cell growth phases. Mg2+ regulates transcription transition, which modulates bimodality of mRNA distribution in the exponential phase. We reveal that Mg2+ regulates the size and frequency of the mRNA burst by changing the open complex duration. Moreover, increasing magnesium concentration leads to higher intrinsic and extrinsic noise in the exponential phase. RNAP-Mg2+ interaction simulation reveals critical movements creating a shorter contact distance between aspartic acid residues and Nucleotide Triphosphate residues and increasing electrostatic charges in the active site. Our findings provide unique biophysical insights into the balanced mechanism of genetic determinants and magnesium ion in transcription initiation regulation during cell growth.

  8. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

    PubMed Central

    Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard

    2016-01-01

    Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility. DOI: http://dx.doi.org/10.7554/eLife.12765.001 PMID:27228154

  9. Live Imaging of Calcium Dynamics during Axon Degeneration Reveals Two Functionally Distinct Phases of Calcium Influx

    PubMed Central

    Yamagishi, Yuya; Tessier-Lavigne, Marc

    2015-01-01

    Calcium is a key regulator of axon degeneration caused by trauma and disease, but its specific spatial and temporal dynamics in injured axons remain unclear. To clarify the function of calcium in axon degeneration, we observed calcium dynamics in single injured neurons in live zebrafish larvae and tested the temporal requirement for calcium in zebrafish neurons and cultured mouse DRG neurons. Using laser axotomy to induce Wallerian degeneration (WD) in zebrafish peripheral sensory axons, we monitored calcium dynamics from injury to fragmentation, revealing two stereotyped phases of axonal calcium influx. First, axotomy triggered a transient local calcium wave originating at the injury site. This initial calcium wave only disrupted mitochondria near the injury site and was not altered by expression of the protective WD slow (WldS) protein. Inducing multiple waves with additional axotomies did not change the kinetics of degeneration. In contrast, a second phase of calcium influx occurring minutes before fragmentation spread as a wave throughout the axon, entered mitochondria, and was abolished by WldS expression. In live zebrafish, chelating calcium after the first wave, but before the second wave, delayed the progress of fragmentation. In cultured DRG neurons, chelating calcium early in the process of WD did not alter degeneration, but chelating calcium late in WD delayed fragmentation. We propose that a terminal calcium wave is a key instructive component of the axon degeneration program. SIGNIFICANCE STATEMENT Axon degeneration resulting from trauma or neurodegenerative disease can cause devastating deficits in neural function. Understanding the molecular and cellular events that execute axon degeneration is essential for developing treatments to address these conditions. Calcium is known to contribute to axon degeneration, but its temporal requirements in this process have been unclear. Live calcium imaging in severed zebrafish neurons and temporally controlled

  10. Event-Scale Morphodynamics and Sediment Sorting in a Dynamic Braided River Revealed by TLS

    NASA Astrophysics Data System (ADS)

    Vericat, D.; Brasington, J.

    2008-12-01

    In the last decade, advances in topographic survey and digital elevation modelling have enabled a revolution in the study of fluvial morphodynamics. Despite this recent progress, our understanding of braided river dynamics remains limited by the time-space scale of studies. Hindered by high labour and flight costs, together with slow ground-based survey methods, studies to date have focused either on event-scale dynamics of morphological units (Ferguson and Ashworth, 1992; Lane et al., 1995; Milan et al., 2007) or seasonal-annual dynamics of larger system-scale reaches (sensu Lane, 2006; e.g., Brasington et al., 2003; Lane et al., 2003). Terrestrial Laser Scanning technology offers the potential to acquire rapidly, reach-scale datasets which record topographic information at the resolution of bed grain-scale upwards. However, as yet, no detailed 3d datasets exist that reveal the system-scale evolution of a braided river through a continuous sequence of floods. Such data are urgently required to address unresolved and fundamental questions concerning the controls and behaviour of braided rivers and are also needed to validate morphodynamic simulation models (Brasington and Richards, 2007). Our recent wok has demonstrated that TLS can be applied to recover centimetre-scale channel morphology, maps of particle size, sorting, packing and floodplain roughness (Brasington et al., 2007, 2008; Antonarakis, 2008a,b; Hodge et al., in review). This potential is illustrated by the results obtained in a field study conducted in January 2008. This used TLS to monitor the evolution of channel morphology and develop methods to derive models of bed roughness and facies in a small 500 x 300 m reach of the actively braided Rees River, New Zealand. Fieldwork comprised repeat surveys before and after 3 competent events, combining laser scans from eight positions with bathymetric data obtained by RTK GPS. The resulting point clouds incorporated between 48-110 million survey points, with

  11. Gas Dynamics and Outflow in the Barred Starburst Galaxy NGC 1808 Revealed with ALMA

    NASA Astrophysics Data System (ADS)

    Salak, Dragan; Nakai, Naomasa; Hatakeyama, Takuya; Miyamoto, Yusuke

    2016-05-01

    NGC 1808 is a nearby barred starburst galaxy with an outflow from the nuclear region. To study the inflow and outflow processes related to star formation and dynamical evolution of the galaxy, we have carried out 12CO (J=1-0) mapping observations of the central r ∼ 4 kpc of NGC 1808 using the Atacama Large Millimeter/submillimeter Array. Four distinct components of molecular gas are revealed at high spatial resolution of 2″ (∼100 pc): (1) a compact (r < 200 pc) circumnuclear disk (CND), (2) r ∼ 500 pc ring, (3) gas-rich galactic bar, and (4) spiral arms. Basic geometric and kinematic parameters are derived for the central 1 kpc region using tilted-ring modeling. The derived rotation curve reveals multiple mass components that include (1) a stellar bulge, (2) a nuclear bar and molecular CND, and (3) an unresolved massive (∼107 M ⊙) core. Two systemic velocities, 998 km s‑1 for the CND and 964 km s‑1 for the 500 pc ring, are revealed, indicating a kinematic offset. The pattern speed of the primary bar, derived by using a cloud-orbit model, is 56 ± 11 km s‑1 kpc‑1. Noncircular motions are detected associated with a nuclear spiral pattern and outflow in the central 1 kpc region. The ratio of the mass outflow rate to the star formation rate is {\\dot{M}}{out}/{SFR}∼ 0.2 in the case of optically thin CO (1–0) emission in the outflow, suggesting low efficiency of star formation quenching.

  12. Mechanically Untying a Protein Slipknot: Multiple Pathways Revealed by Force Spectroscopy and Steered Molecular Dynamics Simulations

    PubMed Central

    He, Chengzhi; Genchev, Georgi Z.; Lu, Hui; Li, Hongbin

    2013-01-01

    Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechancial unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie, and the polypeptide chain to completely extend. These distinct unfolding pathways proceed either via a two-state or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus provding a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechancial unfolding pathway of AFV3-109, and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding. PMID:22626004

  13. Fluctuation Analysis of Centrosomes Reveals a Cortical Function of Kinesin-1.

    PubMed

    Winkler, Franziska; Gummalla, Maheshwar; Künneke, Lutz; Lv, Zhiyi; Zippelius, Annette; Aspelmeier, Timo; Grosshans, Jörg

    2015-09-01

    The actin and microtubule networks form the dynamic cytoskeleton. Network dynamics is driven by molecular motors applying force onto the networks and the interactions between the networks. Here we assay the dynamics of centrosomes in the scale of seconds as a proxy for the movement of microtubule asters. With this assay we want to detect the role of specific motors and of network interaction. During interphase of syncytial embryos of Drosophila, cortical actin and the microtubule network depend on each other. Centrosomes induce cortical actin to form caps, whereas F-actin anchors microtubules to the cortex. In addition, lateral interactions between microtubule asters are assumed to be important for regular spatial organization of the syncytial embryo. The functional interaction between the microtubule asters and cortical actin has been largely analyzed in a static manner, so far. We recorded the movement of centrosomes at 1 Hz and analyzed their fluctuations for two processes—pair separation and individual movement. We found that F-actin is required for directional movements during initial centrosome pair separation, because separation proceeds in a diffusive manner in latrunculin-injected embryos. For assaying individual movement, we established a fluctuation parameter as the deviation from temporally and spatially slowly varying drift movements. By analysis of mutant and drug-injected embryos, we found that the fluctuations were suppressed by both cortical actin and microtubules. Surprisingly, the microtubule motor Kinesin-1 also suppressed fluctuations to a similar degree as F-actin. Kinesin-1 may mediate linkage of the microtubule (+)-ends to the actin cortex. Consistent with this model is our finding that Kinesin-1-GFP accumulates at the cortical actin caps. PMID:26331244

  14. Reduction in Dynamic Visual Acuity Reveals Gaze Control Changes Following Spaceflight

    NASA Technical Reports Server (NTRS)

    Peters, Brian T.; Brady, Rachel A.; Miller, Chris; Lawrence, Emily L.; Mulavara Ajitkumar P.; Bloomberg, Jacob J.

    2010-01-01

    INTRODUCTION: Exposure to microgravity causes adaptive changes in eye-head coordination that can lead to altered gaze control. This could affect postflight visual acuity during head and body motion. The goal of this study was to characterize changes in dynamic visual acuity after long-duration spaceflight. METHODS: Dynamic Visual Acuity (DVA) data from 14 astro/cosmonauts were collected after long-duration (6 months) spaceflight. The difference in acuity between seated and walking conditions provided a metric of change in the subjects ability to maintain gaze fixation during self-motion. In each condition, a psychophysical threshold detection algorithm was used to display Landolt ring optotypes at a size that was near each subject s acuity threshold. Verbal responses regarding the orientation of the gap were recorded as the optotypes appeared sequentially on a computer display 4 meters away. During the walking trials, subjects walked at 6.4 km/h on a motorized treadmill. RESULTS: A decrement in mean postflight DVA was found, with mean values returning to baseline within 1 week. The population mean showed a consistent improvement in DVA performance, but it was accompanied by high variability. A closer examination of the individual subject s recovery curves revealed that many did not follow a pattern of continuous improvement with each passing day. When adjusted on the basis of previous long-duration flight experience, the population mean shows a "bounce" in the re-adaptation curve. CONCLUSION: Gaze control during self-motion is altered following long-duration spaceflight and changes in postflight DVA performance indicate that vestibular re-adaptation may be more complex than a gradual return to normal.

  15. Electrocorticography reveals the temporal dynamics of posterior parietal cortical activity during recognition memory decisions

    PubMed Central

    Gonzalez, Alex; Hutchinson, J. Benjamin; Uncapher, Melina R.; Chen, Janice; LaRocque, Karen F.; Foster, Brett L.; Rangarajan, Vinitha; Parvizi, Josef; Wagner, Anthony D.

    2015-01-01

    Theories of the neurobiology of episodic memory predominantly focus on the contributions of medial temporal lobe structures, based on extensive lesion, electrophysiological, and imaging evidence. Against this backdrop, functional neuroimaging data have unexpectedly implicated left posterior parietal cortex (PPC) in episodic retrieval, revealing distinct activation patterns in PPC subregions as humans make memory-related decisions. To date, theorizing about the functional contributions of PPC has been hampered by the absence of information about the temporal dynamics of PPC activity as retrieval unfolds. Here, we leveraged electrocorticography to examine the temporal profile of high gamma power (HGP) in dorsal PPC subregions as participants made old/new recognition memory decisions. A double dissociation in memory-related HGP was observed, with activity in left intraparietal sulcus (IPS) and left superior parietal lobule (SPL) differing in time and sign for recognized old items (Hits) and correctly rejected novel items (CRs). Specifically, HGP in left IPS increased for Hits 300–700 ms poststimulus onset, and decayed to baseline ∼200 ms preresponse. By contrast, HGP in left SPL increased for CRs early after stimulus onset (200−300 ms) and late in the memory decision (from 700 ms to response). These memory-related effects were unique to left PPC, as they were not observed in right PPC. Finally, memory-related HGP in left IPS and SPL was sufficiently reliable to enable brain-based decoding of the participant’s memory state at the single-trial level, using multivariate pattern classification. Collectively, these data provide insights into left PPC temporal dynamics as humans make recognition memory decisions. PMID:26283375

  16. Structure-dynamics relationships in bursting neuronal networks revealed using a prediction framework.

    PubMed

    Mäki-Marttunen, Tuomo; Aćimović, Jugoslava; Ruohonen, Keijo; Linne, Marja-Leena

    2013-01-01

    The question of how the structure of a neuronal network affects its functionality has gained a lot of attention in neuroscience. However, the vast majority of the studies on structure-dynamics relationships consider few types of network structures and assess limited numbers of structural measures. In this in silico study, we employ a wide diversity of network topologies and search among many possibilities the aspects of structure that have the greatest effect on the network excitability. The network activity is simulated using two point-neuron models, where the neurons are activated by noisy fluctuation of the membrane potential and their connections are described by chemical synapse models, and statistics on the number and quality of the emergent network bursts are collected for each network type. We apply a prediction framework to the obtained data in order to find out the most relevant aspects of network structure. In this framework, predictors that use different sets of graph-theoretic measures are trained to estimate the activity properties, such as burst count or burst length, of the networks. The performances of these predictors are compared with each other. We show that the best performance in prediction of activity properties for networks with sharp in-degree distribution is obtained when the prediction is based on clustering coefficient. By contrast, for networks with broad in-degree distribution, the maximum eigenvalue of the connectivity graph gives the most accurate prediction. The results shown for small ([Formula: see text]) networks hold with few exceptions when different neuron models, different choices of neuron population and different average degrees are applied. We confirm our conclusions using larger ([Formula: see text]) networks as well. Our findings reveal the relevance of different aspects of network structure from the viewpoint of network excitability, and our integrative method could serve as a general framework for structure-dynamics

  17. Clonal expansion during Staphylococcus aureus infection dynamics reveals the effect of antibiotic intervention.

    PubMed

    McVicker, Gareth; Prajsnar, Tomasz K; Williams, Alexander; Wagner, Nelly L; Boots, Michael; Renshaw, Stephen A; Foster, Simon J

    2014-02-01

    To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population "bottleneck" whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where

  18. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy.

    PubMed

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  19. The Dynamics of Sea Straits Reveals Large-Scale Modes of Variability

    NASA Astrophysics Data System (ADS)

    Rubino, Angelo; Androsov, Alexey; Zanchettin, Davide; Voltzinger, Naum

    2016-04-01

    Using a very high resolution 3D numerical model we investigate the tidal dynamics in the Strait of Messina. We show that different stratifications at the southern boundaries, consistent with observed stratifications in the Ionian approaches to the Strait, yield different mean sea level heights. On this basis we search for long-term variations in sea level heights measured in the tidal stations of Catania, Messina and Marseille, and associate them with the concomitant phase of dominant modes of interannual-to-decadal climate variability in the Euro-Mediterranean sector. We focus on the atmospheric North Atlantic Oscillation (NAO) and on the Adriatic-Ionian Bimodal Oscillating System (BiOS) to illustrate the grand variability in sea level teleconnections during the last four decades. In particular, observations reveal a strong imprint of both NAO and BiOS on all sea level records in the 21st century, when NAO and BiOS are inversely correlated. In the 1990s, a well known period of persistent positive NAO anomalies, the NAO imprint on sea level becomes weaker compared to the most recent period, although it remains clear on decadal trends, while the BiOS shows very weak positive variability. In the 1970s and early 1980s, when the NAO was on a neutral phase with weak variability, the NAO imprint on sea levels is weakest, and sea levels in Marseille and Sicily anticorrelate with each other, in contrast to the positive correlations found during the later periods. Based on these observational evidence, we discuss how our modeling results provide a basis to understand the local dynamics that contributed to determine such observed decadal variability.

  20. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

    PubMed Central

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  1. Particle Motion Analysis Reveals Nanoscale Bond Characteristics and Enhances Dynamic Range for Biosensing.

    PubMed

    Visser, Emiel W A; van IJzendoorn, Leo J; Prins, Menno W J

    2016-03-22

    Biofunctionalized colloidal particles are widely used as labels in bioanalytical assays, lab-on-chip devices, biophysical research, and in studies on live biological systems. With detection resolution going down to the level of single particles and single molecules, understanding the nature of the interaction of the particles with surfaces and substrates becomes of paramount importance. Here, we present a comprehensive study of motion patterns of colloidal particles maintained in close proximity to a substrate by short molecular tethers (40 nm). The motion of the particles (500-1000 nm) was optically tracked with a very high localization accuracy (below 3 nm). A surprisingly large variation in motion patterns was observed, which can be attributed to properties of the particle-molecule-substrate system, namely the bond number, the nature of the bond, particle protrusions, and substrate nonuniformities. Experimentally observed motion patterns were compared to numerical Monte Carlo simulations, revealing a close correspondence between the observed motion patterns and properties of the molecular system. Particles bound via single tethers show distinct disc-, ring-, and bell-shaped motion patterns, where the ring- and bell-shaped patterns are caused by protrusions on the particle in the direct vicinity of the molecular attachment point. Double and triple tethered particles exhibit stripe-shaped and triangular-shaped motion patterns, respectively. The developed motion pattern analysis allows for discrimination between particles bound by different bond types, which opens the possibility to improve the limit of detection and the dynamic range of bioanalytical assays, with a projected increase of dynamic range by nearly 2 orders of magnitude. PMID:26913834

  2. Structure-Dynamics Relationships in Bursting Neuronal Networks Revealed Using a Prediction Framework

    PubMed Central

    Mäki-Marttunen, Tuomo; Aćimović, Jugoslava; Ruohonen, Keijo; Linne, Marja-Leena

    2013-01-01

    The question of how the structure of a neuronal network affects its functionality has gained a lot of attention in neuroscience. However, the vast majority of the studies on structure-dynamics relationships consider few types of network structures and assess limited numbers of structural measures. In this in silico study, we employ a wide diversity of network topologies and search among many possibilities the aspects of structure that have the greatest effect on the network excitability. The network activity is simulated using two point-neuron models, where the neurons are activated by noisy fluctuation of the membrane potential and their connections are described by chemical synapse models, and statistics on the number and quality of the emergent network bursts are collected for each network type. We apply a prediction framework to the obtained data in order to find out the most relevant aspects of network structure. In this framework, predictors that use different sets of graph-theoretic measures are trained to estimate the activity properties, such as burst count or burst length, of the networks. The performances of these predictors are compared with each other. We show that the best performance in prediction of activity properties for networks with sharp in-degree distribution is obtained when the prediction is based on clustering coefficient. By contrast, for networks with broad in-degree distribution, the maximum eigenvalue of the connectivity graph gives the most accurate prediction. The results shown for small () networks hold with few exceptions when different neuron models, different choices of neuron population and different average degrees are applied. We confirm our conclusions using larger () networks as well. Our findings reveal the relevance of different aspects of network structure from the viewpoint of network excitability, and our integrative method could serve as a general framework for structure-dynamics studies in biosciences. PMID:23935998

  3. Seasonal-Resolution δ18O in Speleothems by Ion Microprobe: Revealing Asian Monsoon Dynamics

    NASA Astrophysics Data System (ADS)

    Orland, I. J.; Edwards, R. L.; Cheng, H.; Kozdon, R.; Valley, J. W.

    2014-12-01

    Over the last decade, ion microprobe analysis of speleothems (cave carbonates) has increased the temporal resolution of their oxygen isotope (δ18O) paleoclimate proxy records. Recent improvements in methodology, standardization, and imaging at the WiscSIMS lab make it possible to examine sub-annual patterns of δ18O variability at 10-µm-scale, revealing new seasonal paleoenvironmental information. We applied this technique to an important suite of Chinese stalagmites with conventional drill-sampled δ18O records that reflect changes in Asian Monsoon dynamics across the last deglaciation. Seasonal-resolution δ18O analyses in the Chinese stalagmites reveal regular patterns of annual δ18O variability. Quantitative assessment of the patterns identifies two important components in the δ18O records. First, the source and rainout histories of water vapors that ultimately yield rainfall over China play a primary role in determining the δ18O value of speleothem calcite year-round. Second, intra-annual patterns of calcite δ18O variability indicate that the annual proportion of monsoon precipitation changes systematically during the last deglaciation; the annual proportion of monsoon rainfall is greater during the Holocene and Bølling-Allerød than during the Younger Dryas. This is the first time these components have been characterized in any speleothem δ18O record of monsoon dynamics because seasonal δ18O variability is lost by conventional drill-sampling. Ion microprobe analysis of speleothems can also produce year-by-year records of δ18O across abrupt climate change events. At the Younger Dryas-Holocene transition in a Kulishu Cave stalagmite, which spanned 16 years at 11.53 ky BP, there is a relatively smooth decrease in year-round δ18O(calcite). In contrast, the intra-annual δ18O patterns indicate that the increase in the annual proportion of monsoon rainfall across this transition is stochastic, implying that this record can distinguish the regional

  4. Molecular Dynamic Simulations Reveal the Structural Determinants of Fatty Acid Binding to Oxy-Myoglobin

    PubMed Central

    Chintapalli, Sree V.; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L.; van Rossum, Damian B.; Anishkin, Andriy; Adams, Sean H.

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  5. The neural dynamics of face detection in the wild revealed by MVPA.

    PubMed

    Cauchoix, Maxime; Barragan-Jason, Gladys; Serre, Thomas; Barbeau, Emmanuel J

    2014-01-15

    Previous magnetoencephalography/electroencephalography (M/EEG) studies have suggested that face processing is extremely rapid, indeed faster than any other object category. Most studies, however, have been performed using centered, cropped stimuli presented on a blank background resulting in artificially low interstimulus variability. In contrast, the aim of the present study was to assess the underlying temporal dynamics of face detection presented in complex natural scenes. We recorded EEG activity while participants performed a rapid go/no-go categorization task in which they had to detect the presence of a human face. Subjects performed at ceiling (94.8% accuracy), and traditional event-related potential analyses revealed only modest modulations of the two main components classically associated with face processing (P100 and N170). A multivariate pattern analysis conducted across all EEG channels revealed that face category could, however, be readout very early, under 100 ms poststimulus onset. Decoding was linked to reaction time as early as 125 ms. Decoding accuracy did not increase monotonically; we report an increase during an initial 95-140 ms period followed by a plateau ∼140-185 ms-perhaps reflecting a transitory stabilization of the face information available-and a strong increase afterward. Further analyses conducted on individual images confirmed these phases, further suggesting that decoding accuracy may be initially driven by low-level stimulus properties. Such latencies appear to be surprisingly short given the complexity of the natural scenes and the large intraclass variability of the face stimuli used, suggesting that the visual system is highly optimized for the processing of natural scenes. PMID:24431443

  6. Molecular dynamic simulations reveal the structural determinants of Fatty Acid binding to oxy-myoglobin.

    PubMed

    Chintapalli, Sree V; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L; van Rossum, Damian B; Anishkin, Andriy; Adams, Sean H

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a "U" shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  7. Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2.

    PubMed

    Kumar, Atul; Paeger, Lars; Kosmas, Kosmas; Kloppenburg, Peter; Noegel, Angelika A; Peche, Vivek S

    2016-01-01

    Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2(gt/gt) mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2(gt/gt) with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2(gt/gt) neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics. PMID:27507934

  8. Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2

    PubMed Central

    Kumar, Atul; Paeger, Lars; Kosmas, Kosmas; Kloppenburg, Peter; Noegel, Angelika A.; Peche, Vivek S.

    2016-01-01

    Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2gt/gt mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2gt/gt with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2gt/gt neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics. PMID:27507934

  9. Revealing spatio-spectral electroencephalographic dynamics of musical mode and tempo perception by independent component analysis

    PubMed Central

    2014-01-01

    Background Music conveys emotion by manipulating musical structures, particularly musical mode- and tempo-impact. The neural correlates of musical mode and tempo perception revealed by electroencephalography (EEG) have not been adequately addressed in the literature. Method This study used independent component analysis (ICA) to systematically assess spatio-spectral EEG dynamics associated with the changes of musical mode and tempo. Results Empirical results showed that music with major mode augmented delta-band activity over the right sensorimotor cortex, suppressed theta activity over the superior parietal cortex, and moderately suppressed beta activity over the medial frontal cortex, compared to minor-mode music, whereas fast-tempo music engaged significant alpha suppression over the right sensorimotor cortex. Conclusion The resultant EEG brain sources were comparable with previous studies obtained by other neuroimaging modalities, such as functional magnetic resonance imaging (fMRI) and positron emission tomography (PET). In conjunction with advanced dry and mobile EEG technology, the EEG results might facilitate the translation from laboratory-oriented research to real-life applications for music therapy, training and entertainment in naturalistic environments. PMID:24581119

  10. Unfolding mechanism of thrombin-binding aptamer revealed by molecular dynamics simulation and Markov State Model

    PubMed Central

    Zeng, Xiaojun; Zhang, Liyun; Xiao, Xiuchan; Jiang, Yuanyuan; Guo, Yanzhi; Yu, Xinyan; Pu, Xuemei; Li, Menglong

    2016-01-01

    Thrombin-binding aptamer (TBA) with the sequence 5′GGTTGGTGTGGTTGG3′ could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation. PMID:27045335

  11. Dynamic Allostery of the Catabolite Activator Protein Revealed by Interatomic Forces

    PubMed Central

    Louet, Maxime; Seifert, Christian; Hensen, Ulf; Gräter, Frauke

    2015-01-01

    The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals. PMID:26244893

  12. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation.

    PubMed

    Shalek, Alex K; Satija, Rahul; Shuga, Joe; Trombetta, John J; Gennert, Dave; Lu, Diana; Chen, Peilin; Gertner, Rona S; Gaublomme, Jellert T; Yosef, Nir; Schwartz, Schraga; Fowler, Brian; Weaver, Suzanne; Wang, Jing; Wang, Xiaohui; Ding, Ruihua; Raychowdhury, Raktima; Friedman, Nir; Hacohen, Nir; Park, Hongkun; May, Andrew P; Regev, Aviv

    2014-06-19

    High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a 'core' module of antiviral genes is expressed very early by a few 'precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced 'peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses. PMID:24919153

  13. Intravital Placenta Imaging Reveals Microcirculatory Dynamics Impact on Sequestration and Phagocytosis of Plasmodium-Infected Erythrocytes

    PubMed Central

    de Moraes, Luciana Vieira; Tadokoro, Carlos Eduardo; Gómez-Conde, Iván; Olivieri, David N.; Penha-Gonçalves, Carlos

    2013-01-01

    Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE. PMID:23382682

  14. Diversity of sharp-wave–ripple LFP signatures reveals differentiated brain-wide dynamical events

    PubMed Central

    Ramirez-Villegas, Juan F.; Logothetis, Nikos K.; Besserve, Michel

    2015-01-01

    Sharp-wave–ripple (SPW-R) complexes are believed to mediate memory reactivation, transfer, and consolidation. However, their underlying neuronal dynamics at multiple scales remains poorly understood. Using concurrent hippocampal local field potential (LFP) recordings and functional MRI (fMRI), we study local changes in neuronal activity during SPW-R episodes and their brain-wide correlates. Analysis of the temporal alignment between SPW and ripple components reveals well-differentiated SPW-R subtypes in the CA1 LFP. SPW-R–triggered fMRI maps show that ripples aligned to the positive peak of their SPWs have enhanced neocortical metabolic up-regulation. In contrast, ripples occurring at the trough of their SPWs relate to weaker neocortical up-regulation and absent subcortical down-regulation, indicating differentiated involvement of neuromodulatory pathways in the ripple phenomenon mediated by long-range interactions. To our knowledge, this study provides the first evidence for the existence of SPW-R subtypes with differentiated CA1 activity and metabolic correlates in related brain areas, possibly serving different memory functions. PMID:26540729

  15. Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy

    PubMed Central

    Andrecka, Joanna; Ortega Arroyo, Jaime; Takagi, Yasuharu; de Wit, Gabrielle; Fineberg, Adam; MacKinnon, Lachlan; Young, Gavin; Sellers, James R; Kukura, Philipp

    2015-01-01

    Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds in a controlled manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an ångstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head–head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps. DOI: http://dx.doi.org/10.7554/eLife.05413.001 PMID:25748137

  16. Single Centrosome Manipulation Reveals Its Electric Charge and Associated Dynamic Structure

    PubMed Central

    Hormeño, S.; Ibarra, B.; Chichón, F.J.; Habermann, K.; Lange, B.M.H.; Valpuesta, J.M.; Carrascosa, J.L.; Arias-Gonzalez, J.R.

    2009-01-01

    Abstract The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 ± 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 103 electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca2+, over the thermally induced dynamic structure of the centrosome. PMID:19686649

  17. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer.

    PubMed

    Bindea, Gabriela; Mlecnik, Bernhard; Tosolini, Marie; Kirilovsky, Amos; Waldner, Maximilian; Obenauf, Anna C; Angell, Helen; Fredriksen, Tessa; Lafontaine, Lucie; Berger, Anne; Bruneval, Patrick; Fridman, Wolf Herman; Becker, Christoph; Pagès, Franck; Speicher, Michael R; Trajanoski, Zlatko; Galon, Jérôme

    2013-10-17

    The complex interactions between tumors and their microenvironment remain to be elucidated. Combining large-scale approaches, we examined the spatio-temporal dynamics of 28 different immune cell types (immunome) infiltrating tumors. We found that the immune infiltrate composition changed at each tumor stage and that particular cells had a major impact on survival. Densities of T follicular helper (Tfh) cells and innate cells increased, whereas most T cell densities decreased along with tumor progression. The number of B cells, which are key players in the core immune network and are associated with prolonged survival, increased at a late stage and showed a dual effect on recurrence and tumor progression. The immune control relevance was demonstrated in three endoscopic orthotopic colon-cancer mouse models. Genomic instability of the chemokine CXCL13 was a mechanism associated with Tfh and B cell infiltration. CXCL13 and IL21 were pivotal factors for the Tfh/B cell axis correlating with survival. This integrative study reveals the immune landscape in human colorectal cancer and the major hallmarks of the microenvironment associated with tumor progression and recurrence. PMID:24138885

  18. Dynamics of oscillatory phenotypes in S. cerevisiae reveal a network of genome-wide transcriptional oscillators

    PubMed Central

    Chin, Shwe L.; Marcus, Ian M.; Klevecz, Robert R.; Li, Caroline M.

    2012-01-01

    Genetic and environmental factors are well-studied influences on phenotype; however, time is a variable that is rarely considered when studying changes in cellular phenotype. Time-resolved microarray data revealed genome-wide transcriptional oscillation in a yeast continuous culture system with ~2 and ~4 h periods. We mapped the global patterns of transcriptional oscillations into a 3D map to represent different cellular phenotypes of redox cycles. This map shows the dynamic nature of gene expression in that transcripts are ordered and coupled to each other through time and concentration space. Although cells differed in oscillation periods, transcripts involved in certain processes were conserved in a deterministic way. When oscillation period lengthened, the peak to trough ratio of transcripts increased and the fraction of cells in the unbudded (G0/G1) phase of the cell division cycle increased. Decreasing the glucose level in the culture media was one way to increase the redox cycle, possibly from changes in metabolic flux. The period may be responding to lower glucose levels by increasing the fraction of cells in G1 and reducing S-phase gating so that cells can spend more time in catabolic processes. Our results support that gene transcripts are coordinated with metabolic functions and the cell division cycle. PMID:22289124

  19. Molecular markers reveal infestation dynamics of the bed bug (Hemiptera: Cimicidae) within apartment buildings.

    PubMed

    Booth, Warren; Saenz, Virna L; Santangelo, Richard G; Wang, Changlu; Schal, Coby; Vargo, Edward L

    2012-05-01

    The bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), has experienced an extraordinary global resurgence in recent years, the reasons for which remain poorly understood. Once considered a pest of lower socioeconomic classes, bed bugs are now found extensively across all residential settings, with widespread infestations established in multiapartment buildings. Within such buildings, understanding the population genetic structure and patterns of dispersal may prove critical to the development of effective control strategies. Here, we describe the development of 24 high-resolution microsatellite markers through next generation 454 pyrosequencing and their application to elucidate infestation dynamics within three multistory apartment buildings in the United States. Results reveal contrasting characteristics potentially representative of geographic or locale differences. In Raleigh, NC, an infestation within an apartment building seemed to have started from a single introduction followed by extensive spread. In Jersey City, NJ, two or more introductions followed by spread are evident in two buildings. Populations within single apartments in all buildings were characterized by high levels of relatedness and low levels of diversity, indicative of foundation from small, genetically depauperate propagules. Regardless of the number of unique introductions, genetic data indicate that spread within buildings is extensive, supporting both active and human-mediated dispersal within and between adjacent rooms or apartments spanning multiple floors. PMID:22679860

  20. Dynamic imaging reveals that BDNF can independently regulate motility and direction of RMS neuroblast migration

    PubMed Central

    Bagley, Joshua A.; Belluscio, Leonardo

    2010-01-01

    Neuronal precursors generated in the subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB). Although, the mechanisms regulating this migration remain largely unknown, studies have suggested that molecular factors, such as Brain-Derived Neurotrophic Factor (BDNF) emanating from the OB, may function as chemoattractants drawing neuroblasts toward their target. To better understand the role of BDNF in RMS migration, we used an acute slice preparation from early postnatal mice to track the tangential migration of GAD65-GFP labeled RMS neuroblasts with confocal time-lapse imaging. By quantifying the cell dynamics using specific directional and motility criteria, our results showed that removal of the OB did not alter the overall directional trajectory of neuroblasts, but did reduce their motility. This suggested that additional guidance factors may be present locally within the RMS. Thus, we next demonstrated that BDNF and its high affinity receptor, TrkB, are indeed heterogeneously expressed within the RMS at postnatal day 7, and by altering BDNF levels within the entire pathway, showed that reduced BDNF signaling changes both neuroblast motility and direction, while increased BDNF levels changes only motility. Together these data reveal that during this early postnatal period BDNF plays a complex role in regulating both the motility and direction of RMS flow, and that it arises from within the RMS itself, as well as from the olfactory bulb. PMID:20538046

  1. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation

    NASA Astrophysics Data System (ADS)

    Shalek, Alex K.; Satija, Rahul; Shuga, Joe; Trombetta, John J.; Gennert, Dave; Lu, Diana; Chen, Peilin; Gertner, Rona S.; Gaublomme, Jellert T.; Yosef, Nir; Schwartz, Schraga; Fowler, Brian; Weaver, Suzanne; Wang, Jing; Wang, Xiaohui; Ding, Ruihua; Raychowdhury, Raktima; Friedman, Nir; Hacohen, Nir; Park, Hongkun; May, Andrew P.; Regev, Aviv

    2014-06-01

    High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a `core' module of antiviral genes is expressed very early by a few `precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced `peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.

  2. Adiponectin fine-tuning of liver regeneration dynamics revealed through cellular network modelling.

    PubMed

    Correnti, Jason M; Cook, Daniel; Aksamitiene, Edita; Swarup, Aditi; Ogunnaike, Babatunde; Vadigepalli, Rajanikanth; Hoek, Jan B

    2015-01-15

    Following partial hepatectomy, the liver initiates a regenerative programme involving hepatocyte priming and replication driven by the coordinated actions of cytokine and growth factors. We investigated the mechanisms underlying adiponectin's (Adn) regulation of liver regeneration through modulation of these mediators. Adn(-/-) mice showed delayed onset of hepatocyte replication, but accelerated cell cycle progression relative to wild-type mice, suggesting Adn has multiple effects fine-tuning the kinetics of liver regeneration. We developed a computational model describing the molecular and physiological kinetics of liver regeneration in Adn(-/-) mice. We employed this computational model to evaluate the underlying regulatory mechanisms. Our analysis predicted that Adn is required for an efficient early cytokine response to partial hepatectomy, but is inhibitory to later growth factor actions. Consistent with this prediction, Adn knockout reduced hepatocyte responses to interleukin-6 during the priming phase, but enhanced growth factor levels through peak hepatocyte replication. By contrast, supraphysiological concentrations of Adn resulting from rosiglitazone treatment suppressed regeneration by reducing growth factor levels during S phase, consistent with computational predictions. Together, these results revealed that Adn fine-tunes the progression of liver regeneration through dynamically modulating molecular mediator networks and cellular interactions within the liver. PMID:25630259

  3. Adiponectin fine-tuning of liver regeneration dynamics revealed through cellular network modelling

    PubMed Central

    Correnti, Jason M; Cook, Daniel; Aksamitiene, Edita; Swarup, Aditi; Ogunnaike, Babatunde; Vadigepalli, Rajanikanth; Hoek, Jan B

    2015-01-01

    Following partial hepatectomy, the liver initiates a regenerative programme involving hepatocyte priming and replication driven by the coordinated actions of cytokine and growth factors. We investigated the mechanisms underlying adiponectin's (Adn) regulation of liver regeneration through modulation of these mediators. Adn–/– mice showed delayed onset of hepatocyte replication, but accelerated cell cycle progression relative to wild-type mice, suggesting Adn has multiple effects fine-tuning the kinetics of liver regeneration. We developed a computational model describing the molecular and physiological kinetics of liver regeneration in Adn–/– mice. We employed this computational model to evaluate the underlying regulatory mechanisms. Our analysis predicted that Adn is required for an efficient early cytokine response to partial hepatectomy, but is inhibitory to later growth factor actions. Consistent with this prediction, Adn knockout reduced hepatocyte responses to interleukin-6 during the priming phase, but enhanced growth factor levels through peak hepatocyte replication. By contrast, supraphysiological concentrations of Adn resulting from rosiglitazone treatment suppressed regeneration by reducing growth factor levels during S phase, consistent with computational predictions. Together, these results revealed that Adn fine-tunes the progression of liver regeneration through dynamically modulating molecular mediator networks and cellular interactions within the liver. PMID:25630259

  4. Unfolding mechanism of thrombin-binding aptamer revealed by molecular dynamics simulation and Markov State Model

    NASA Astrophysics Data System (ADS)

    Zeng, Xiaojun; Zhang, Liyun; Xiao, Xiuchan; Jiang, Yuanyuan; Guo, Yanzhi; Yu, Xinyan; Pu, Xuemei; Li, Menglong

    2016-04-01

    Thrombin-binding aptamer (TBA) with the sequence 5‧GGTTGGTGTGGTTGG3‧ could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation.

  5. Dynamic Allostery of the Catabolite Activator Protein Revealed by Interatomic Forces.

    PubMed

    Louet, Maxime; Seifert, Christian; Hensen, Ulf; Gräter, Frauke

    2015-08-01

    The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals. PMID:26244893

  6. Size distribution dynamics reveal particle-phase chemistry in organic aerosol formation.

    PubMed

    Shiraiwa, Manabu; Yee, Lindsay D; Schilling, Katherine A; Loza, Christine L; Craven, Jill S; Zuend, Andreas; Ziemann, Paul J; Seinfeld, John H

    2013-07-16

    Organic aerosols are ubiquitous in the atmosphere and play a central role in climate, air quality, and public health. The aerosol size distribution is key in determining its optical properties and cloud condensation nucleus activity. The dominant portion of organic aerosol is formed through gas-phase oxidation of volatile organic compounds, so-called secondary organic aerosols (SOAs). Typical experimental measurements of SOA formation include total SOA mass and atomic oxygen-to-carbon ratio. These measurements, alone, are generally insufficient to reveal the extent to which condensed-phase reactions occur in conjunction with the multigeneration gas-phase photooxidation. Combining laboratory chamber experiments and kinetic gas-particle modeling for the dodecane SOA system, here we show that the presence of particle-phase chemistry is reflected in the evolution of the SOA size distribution as well as its mass concentration. Particle-phase reactions are predicted to occur mainly at the particle surface, and the reaction products contribute more than half of the SOA mass. Chamber photooxidation with a midexperiment aldehyde injection confirms that heterogeneous reaction of aldehydes with organic hydroperoxides forming peroxyhemiacetals can lead to a large increase in SOA mass. Although experiments need to be conducted with other SOA precursor hydrocarbons, current results demonstrate coupling between particle-phase chemistry and size distribution dynamics in the formation of SOAs, thereby opening up an avenue for analysis of the SOA formation process. PMID:23818634

  7. The Gating Mechanism of the Human Aquaporin 5 Revealed by Molecular Dynamics Simulations

    PubMed Central

    Janosi, Lorant; Ceccarelli, Matteo

    2013-01-01

    Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5) shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value. PMID:23565173

  8. Diversity of sharp-wave-ripple LFP signatures reveals differentiated brain-wide dynamical events.

    PubMed

    Ramirez-Villegas, Juan F; Logothetis, Nikos K; Besserve, Michel

    2015-11-17

    Sharp-wave-ripple (SPW-R) complexes are believed to mediate memory reactivation, transfer, and consolidation. However, their underlying neuronal dynamics at multiple scales remains poorly understood. Using concurrent hippocampal local field potential (LFP) recordings and functional MRI (fMRI), we study local changes in neuronal activity during SPW-R episodes and their brain-wide correlates. Analysis of the temporal alignment between SPW and ripple components reveals well-differentiated SPW-R subtypes in the CA1 LFP. SPW-R-triggered fMRI maps show that ripples aligned to the positive peak of their SPWs have enhanced neocortical metabolic up-regulation. In contrast, ripples occurring at the trough of their SPWs relate to weaker neocortical up-regulation and absent subcortical down-regulation, indicating differentiated involvement of neuromodulatory pathways in the ripple phenomenon mediated by long-range interactions. To our knowledge, this study provides the first evidence for the existence of SPW-R subtypes with differentiated CA1 activity and metabolic correlates in related brain areas, possibly serving different memory functions. PMID:26540729

  9. Optical tweezers reveal a dynamic mechanical response of cationic peptide-DNA complexes

    NASA Astrophysics Data System (ADS)

    Lee, Amy; Zheng, Tai; Sucayan, Sarah; Chou, Szu-Ting; Tricoli, Lucas; Hustedt, Jason; Kahn, Jason; Mixson, A. James; Seog, Joonil

    2013-03-01

    Nonviral carriers have been developed to deliver nucleic acids by forming nanoscale complexes; however, there has been limited success in achieving high transfection efficiency. Our hypothesis is that a factor affecting gene delivery efficiency is the mechanical response of the condensed complex. To begin to test this hypothesis, we directly measured the mechanical properties of DNA-carrier complexes using optical tweezers. Histidine-lysine (HK) polymer, Asparagine-lysine (NK) polymer and poly-L-lysine were used to form complexes with a single DNA molecule. As carriers were introduced, a sudden decrease in DNA extension occurrs at a force level which is defined as critical force (Fc). Fc is carrier and concentration dependent. Pulling revealed reduction in DNA extension length for HK-DNA complexes. The characteristics of force profiles vary by agent and can be dynamically manipulated by changes in environmental conditions such as ionic strength of the buffer as well as pH. Heparin can remove cationic reagents which are otherwise irreversibly bound to DNA. The implications for optimizing molecular interactions to enhance transfection efficiency will be discussed.

  10. Low-level chemiluminescent analysis of nondiluted human blood reveals its dynamic system properties

    NASA Astrophysics Data System (ADS)

    Voeikov, Vladimir L.; Novikov, Cyril N.; Vilenskaya, Natalia D.

    1999-01-01

    Lucigenin- and luminol-dependent chemiluminescence [(LC- CL) and (LM-CL)] in nondiluted human blood was studied. LM-CL was low in fresh blood and disappeared after its storage for 3 h, though the respiratory burst (RB) stimulated in blood was followed by high intensity and long- lasting LM-CL. LC-CL was high in fresh blood and was steadily increasing with blood storage. Blood dilution with saline resulted in LC-CL attenuation and LM-CL elevation. LC-CL did not depend on air supply to blood, while LM-CL elevation during RB needed constant blood aeration. The results suggest that besides a well-known mechanism of reactive oxygen species production by neutrophils during RB, another process of electron excited state generation reflected by LC-CL operates in blood. It needs blood integrity for its manifestation and uses oxygen supplied by erythrocytes. Dynamic system properties of blood were revealed also in experiments with blood transfer from one sample to another in the course of RB. Highly nonlinear changes of CL intensity both in a `donor' and in a `recipient' sample resulted in strong differences in CL levels in two samples, one of which was prepared by blood subtraction, and another by blood addition. We suggest that CL data from measurements on nondiluted blood may be informative of integrative properties of blood tissue in addition to its being a measure of some sort of oxidative metabolism in it.

  11. The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences

    PubMed Central

    Kensche, Philip Reiner; Hoeijmakers, Wieteke Anna Maria; Toenhake, Christa Geeke; Bras, Maaike; Chappell, Lia; Berriman, Matthew; Bártfai, Richárd

    2016-01-01

    In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen. PMID:26578577

  12. iRED analysis of TAR RNA reveals motional coupling, long-range correlations, and a dynamical hinge.

    PubMed

    Musselman, Catherine; Al-Hashimi, Hashim M; Andricioaei, Ioan

    2007-07-15

    The HIV-1 transactivation response RNA element (TAR), which is essential to the lifecycle of the virus, has been suggested, based on NMR and hydrodynamic measurements, to undergo substantial, collective, structural dynamics that are important for its function. To deal with the significant coupling between overall diffusional rotation and internal motion expected to exist in TAR, here we utilize an isotropic reorientational eigenmode dynamics analysis of simulated molecular trajectories to obtain a detailed description of TAR dynamics and an accurately quantified pattern of dynamical correlations. The analysis demonstrates the inseparability of internal and overall motional modes, confirms the existence and reveals the nature of collective domain dynamics, and additionally reveals that the hinge for these motions is centered on residues U23, C24, and C41. Results also indicate the existence of long-range communication between the loop and the core of the RNA, and between the loop and the bulge. Additionally, the isotropic reorientational eigenmode dynamics analysis explains, from a dynamical perspective, several existing biochemical mutational studies and suggests new mutations for future structural dynamics studies. PMID:17449677

  13. Distributed Modeling Reveals the Ecohydrological Dynamics Linked with Woody Plant Encroachment in the Sonoran Desert

    NASA Astrophysics Data System (ADS)

    Pierini, N. A.; Vivoni, E. R.; Anderson, C.; Saripalli, S.; Robles-Morua, A.

    2012-12-01

    moisture and temperature distributions through comparisons of canopy and intercanopy sites. The field and remote sensing observations are then used in simulations using the TIN-based Real-time Integrated Basin Simulator (tRIBS) at high spatiotemporal resolutions over the two study years (2011-2012). Numerical experiments are designed to reveal the influence of the mesquite encroachment patterns on the watershed dynamics. Through the spatiotemporal analysis of model outputs, we identify how and when mesquite trees affect the spatial patterns of energy and water fluxes and their linkage to runoff production. As a result, the distributed model application provides a more complete understanding of the impact of woody encroachment on watershed-scale hydrologic patterns.

  14. Horizontal Structure of Dynamical Instability at Marine Stratocumulus Cloud Top Revealed in Polarized Light

    NASA Astrophysics Data System (ADS)

    Davis, A. B.; Diner, D. J.; Matheou, G.; Teixeira, J.; Qu, Z.; Emde, C.

    2014-12-01

    Marine stratocumulus (Sc) layers cover vast regions and, due to their high opacities, they play a major role in the Earth's solar radiation budget. They also have remarkably flat upper boundaries due to strong gradients in relative humidity at the top of the boundary layer (BL). However, those very gradients are unstable at scales as small as meters depending on fluctuations of temperature and liquid water content, hence radiative cooling in the thermal IR. The ensuing turbulent mixing of moist and dry air at cloud top due to such small-scale dynamical processes is not benign. It controls the structure of the entire marine BL, hence the Sc life-cycle, hence large-scale subsidence, hence global circulation and, ultimately, climate. This physical connection across many orders of magnitude in scale makes the prognosis and microphysical parameterization of marine Sc particularly challenging for climate modelers. It also makes these clouds high-value targets for remote sensing, both space-based and airborne. Airborne sensors can easily achieve the resolution required to image cloud-top instabilities but natural sunlight is so highly scattered that the finest spatial features are all but erased by the "radiative smoothing" process. However, we will show that JPL's Airborne Multi-angle Spectro-Polarimetric Imager (AirMSPI), which flies on NASA's ER-2 aircraft at 20 km altitude, reveals in near-backscattered polarized light the previously unseen horizontal structure of the marine Sc cloud top physics and dynamics at 10 m resolution across a 10 km swath. It appears as a complex network of meandering filaments. Large-Eddy Simulation modeling of these oceanic clouds with bin microphysics and state-of-the-art polarized 3D radiative transfer have been harnessed to model AirMSPI observations of the first three Stokes vector components in the relevant observational geometry for a 2.5x2.5 km^2 region. Synthetic imagery obtained at JPL's High-Performance Computing facility shows

  15. Change in Magma Dynamics at Okataina Rhyolite Caldera revealed by Plagioclase Textures and Geochemistry

    NASA Astrophysics Data System (ADS)

    Shane, P. A. R.

    2015-12-01

    A fundamental reorganization of magma dynamics at Okataina volcano, New Zealand, occurred at 26 ka involving a change from smaller volume, high-temperature rhyodacite magmas to a lower eruptive tempo of larger volume, low-temperature, rhyolite magmas. Zircon studies demonstrate the presence of a periodically active, long-lived (100,000 yr) magmatic reservoir. However, there is little correlation between periods of zircon crystallization and eruption events. In contrast, the changing magmatic dynamics is revealed in plagioclase growth histories. Crystals from the ~0.7 ka Kaharoa eruption are characterized by resorbed cores displaying a cellular-texture of high-An (>40) zones partially replaced by low-An (<30) zones, surrounded by a resorption surface and a prominent normal-zoned rim (An50-20). Elevated An, Fe, Mg, Sr and Ti follow the resorption surface and display rimward depletion trends, accompanied by Ba and REE enrichment. The zonation is consistent with fractional crystallization and cooling. The cores display wide trace element diversity, pointing to crystallization in a variety of melts, before transport and mixing into a common magma where the rims grew. Plagioclase from the ~36 ka Hauparu eruption display several regrowth zones separated by resorption surfaces, which surround small resorbed cores with a spongy cellular texture of variable An content (An 40-50). The crystals display step-wise re-growth of successively higher An, Fe, Mg and Ti content, consistent with progressive mafic recharge. Two crystal groups are distinguished by trace element chemistry indicating growth in separate melts and co-occurrence via magma-mingling. The contrasting zoning patterns in plagioclase correspond to the evolutionary history of magmatism at Okataina. Emptying of the magma reservoir following caldera eruption at 46 ka reduced barriers to mafic magma ascent. This is recorded by the frequent resorption and recharge episodes in Hauparu crystals. Subsequent re

  16. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn

    PubMed Central

    Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

    2015-01-01

    The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize. PMID:26587848

  17. Temporal Dynamics of Avian Populations during Pleistocene Revealed by Whole-Genome Sequences.

    PubMed

    Nadachowska-Brzyska, Krystyna; Li, Cai; Smeds, Linnea; Zhang, Guojie; Ellegren, Hans

    2015-05-18

    Global climate fluctuations have significantly influenced the distribution and abundance of biodiversity. During unfavorable glacial periods, many species experienced range contraction and fragmentation, expanding again during interglacials. An understanding of the evolutionary consequences of both historical and ongoing climate changes requires knowledge of the temporal dynamics of population numbers during such climate cycles. Variation in abundance should have left clear signatures in the patterns of intraspecific genetic variation in extant species, from which historical effective population sizes (N(e)) can be estimated. We analyzed whole-genome sequences of 38 avian species in a pairwise sequentially Markovian coalescent (PSMC, [5]) framework to quantitatively reveal changes in N(e) from approximately 10 million to 10 thousand years ago. Significant fluctuations in N(e) over time were evident for most species. The most pronounced pattern observed in many species was a severe reduction in N(e) coinciding with the beginning of the last glacial period (LGP). Among species, N(e) varied by at least three orders of magnitude, exceeding 1 million in the most abundant species. Several species on the IUCN Red List of Threatened Species showed long-term reduction in population size, predating recent declines. We conclude that cycles of population expansions and contractions have been a common feature of many bird species during the Quaternary period, likely coinciding with climate cycles. Population size reduction should have increased the risk of extinction but may also have promoted speciation. Species that have experienced long-term declines may be especially vulnerable to recent anthropogenic threats. PMID:25891404

  18. Temporal Dynamics of Avian Populations during Pleistocene Revealed by Whole-Genome Sequences

    PubMed Central

    Nadachowska-Brzyska, Krystyna; Li, Cai; Smeds, Linnea; Zhang, Guojie; Ellegren, Hans

    2015-01-01

    Summary Global climate fluctuations have significantly influenced the distribution and abundance of biodiversity [1]. During unfavorable glacial periods, many species experienced range contraction and fragmentation, expanding again during interglacials [2–4]. An understanding of the evolutionary consequences of both historical and ongoing climate changes requires knowledge of the temporal dynamics of population numbers during such climate cycles. Variation in abundance should have left clear signatures in the patterns of intraspecific genetic variation in extant species, from which historical effective population sizes (Ne) can be estimated [3]. We analyzed whole-genome sequences of 38 avian species in a pairwise sequentially Markovian coalescent (PSMC, [5]) framework to quantitatively reveal changes in Ne from approximately 10 million to 10 thousand years ago. Significant fluctuations in Ne over time were evident for most species. The most pronounced pattern observed in many species was a severe reduction in Ne coinciding with the beginning of the last glacial period (LGP). Among species, Ne varied by at least three orders of magnitude, exceeding 1 million in the most abundant species. Several species on the IUCN Red List of Threatened Species showed long-term reduction in population size, predating recent declines. We conclude that cycles of population expansions and contractions have been a common feature of many bird species during the Quaternary period, likely coinciding with climate cycles. Population size reduction should have increased the risk of extinction but may also have promoted speciation. Species that have experienced long-term declines may be especially vulnerable to recent anthropogenic threats. PMID:25891404

  19. Metabolomics Reveals Dynamic Metabolic Changes Associated with Age in Early Childhood

    PubMed Central

    Chiu, Chih-Yung; Yeh, Kuo-Wei; Lin, Gigin; Chiang, Meng-Han; Yang, Shu-Chen; Chao, Wei-Ju; Yao, Tsung-Chieh; Tsai, Ming-Han; Hua, Man-Chin; Liao, Sui-Ling; Lai, Shen-Hao

    2016-01-01

    Objectives A detailed understanding of the metabolic processes governing rapid growth in early life is still lacking. The aim of this study was to investigate the age-related metabolic changes in healthy children throughout early childhood. Methods Healthy children from a birth cohort were enrolled in this study from birth through 4 years of age. Urinary metabolites were assessed at 6 months, and 1, 2, 3, and 4 yr of age by using 1H-nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate statistical analysis including principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). Metabolic pathway analysis was performed using the MetPA web tool. Results A total of 105 urine samples from 30 healthy children were collected and analyzed. Metabolites contributing to the discrimination between age groups were identified by using supervised PLS-DA (Q2 = 0.60; R2 = 0.66). A significantly higher urinary trimethylamine N-oxide (TMAO) and betaine level was found in children aged 6 months. Urinary glycine and glutamine levels declined significantly after 6 months of age and there was a concomitant compensatory increase in urinary creatine and creatinine. Metabolic pathway analysis using MetPA revealed similar nitrogen metabolism associated energy production across all ages assessed. Pathways associated with amino acid metabolism were significantly different between infants aged 6 months and 1 year, whereas pathways associated with carbohydrate metabolism were significantly different between children at ages 2 and 3 years. Conclusions Urine metabolomics ideally represents dynamic metabolic changes across age. Urinary metabolic profiles change significantly within the first year of life, which can potentially provide crucial information about infant nutrition and growth. PMID:26914934

  20. Dynamic habitat suitability modelling reveals rapid poleward distribution shift in a mobile apex predator.

    PubMed

    Hill, Nicholas J; Tobin, Andrew J; Reside, April E; Pepperell, Julian G; Bridge, Tom C L

    2016-03-01

    Many taxa are undergoing distribution shifts in response to anthropogenic climate change. However, detecting a climate signal in mobile species is difficult due to their wide-ranging, patchy distributions, often driven by natural climate variability. For example, difficulties associated with assessing pelagic fish distributions have rendered fisheries management ill-equipped to adapt to the challenges posed by climate change, leaving pelagic species and ecosystems vulnerable. Here, we demonstrate the value of citizen science data for modelling the dynamic habitat suitability of a mobile pelagic predator (black marlin, Istiompax indica) within the south-west Pacific Ocean. The extensive spatial and temporal coverage of our occurrence data set (n = 18 717), collected at high resolution (~1.85 km(2) ), enabled identification of suitable habitat at monthly time steps over a 16-year period (1998-2013). We identified considerable monthly, seasonal and interannual variability in the extent and distribution of suitable habitat, predominately driven by chlorophyll a and sea surface height. Interannual variability correlated with El Nino Southern Oscillation (ENSO) events, with suitable habitat extending up to ~300 km further south during La Nina events. Despite the strong influence of ENSO, our model revealed a rapid poleward shift in the geometric mean of black marlin habitat, occurring at 88.2 km decade(-1) . By incorporating multiple environmental factors at monthly time steps, we were able to demonstrate a rapid distribution shift in a mobile pelagic species. Our findings suggest that the rapid velocity of climate change in the south-west Pacific Ocean is likely affecting mobile pelagic species, indicating that they may be more vulnerable to climate change than previously thought. PMID:26464050

  1. Longitudinal Alterations in the Dynamic Autoregulation of Optic Nerve Head Blood Flow Revealed in Experimental Glaucoma

    PubMed Central

    Wang, Lin; Cull, Grant; Burgoyne, Claude F.; Thompson, Simon; Fortune, Brad

    2014-01-01

    Purpose. To use a novel dynamic autoregulation analysis (dAR) to test the hypothesis that the optic nerve head (ONH) blood flow (BF) autoregulation is disrupted during early stages of experimental glaucoma (EG) in nonhuman primates. Methods. Retinal nerve fiber layer thickness (RNFLT, assessed by optical coherence tomography) and ONH BF (assessed by laser speckle imaging technique) were measured biweekly before and after unilateral laser treatment to the trabecular meshwork. Each nonhuman primate was followed until reaching either an early stage of damage (RNFLT loss < 20%, n = 6) or moderate to advanced stages of damage (RNFLT loss > 20%, n = 9). At each test, dAR was assessed by characterizing ONH BF changes during the first minute of rapid manometrical intraocular pressure (IOP) elevation from 10 to 40 mm Hg. The dAR analysis extracted the following parameters: baseline BF, average BF 10 seconds before IOP elevation; BFΔmax, maximum BF change from baseline BF; Tr, time from baseline BF to the BFΔmax; Kr, average descending BF rate. Results. Mean postlaser IOP was 20.2 ± 5.9 and 12.3 ± 2.6 mm Hg in EG and control eyes, respectively (P < 0.0001). Compared with prelaser values, baseline BF was higher in early EG, but lower in moderate to advanced EG (P = 0.01). Tr was increased and Kr was reduced in both stages (P < 0.01). BFΔmax was smaller in the early EG (P = 0.05) and remained low in the moderate to advanced EG (P = 0.15). No changes in the parameters were observed in control eyes. Conclusions. Chronic IOP elevation causes ONH autoregulation dysfunction in the early stage of EG, characterized by a disrupted BF response and delayed Tr, revealed by dAR analysis. PMID:24812551

  2. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    PubMed

    Dong, Yongbin; Wang, Qilei; Zhang, Long; Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

    2015-01-01

    The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize. PMID:26587848

  3. Dynamic regulation of alternative splicing and chromatin structure in Drosophila gonads revealed by RNA-seq

    PubMed Central

    Gan, Qiang; Chepelev, Iouri; Wei, Gang; Tarayrah, Lama; Cui, Kairong; Zhao, Keji; Chen, Xin

    2010-01-01

    Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understandings of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles from Drosophila testes and ovaries using RNA-seq. We identified a set of genes that have sex-specific isoforms in wild-type (wt) gonads, including several transcription factors. We found that differentiation of sperms from undifferentiated germ cells induced a dramatic down-regulation of RNA splicing factors. Our data confirmed that RNA splicing events are significantly more frequent in the undifferentiated-cell enriched bag of marbles (bam) mutant testis, but down-regulated upon differentiation in wt testis. Consistent with this, we showed that genes required for meiosis and terminal differentiation in wt testis were mainly regulated at the transcriptional level, but not by alternative splicing. Unexpectedly, we observed an increase in expression of all families of chromatin remodeling factors and histone modifying enzymes in the undifferentiated cell-enriched bam testis. More interestingly, chromatin regulators and histone modifying enzymes with opposite enzymatic activities are co-enriched in undifferentiated cells in testis, suggesting these cells may possess dynamic chromatin architecture. Finally, our data revealed many new features of the Drosophila gonadal transcriptomes, and will lead to a more comprehensive understanding of how differential gene expression and splicing regulate gametogenesis in Drosophila. Our data provided a foundation for the systematic study of gene expression and alternative splicing in many interesting areas of germ cell biology in Drosophila, such as the molecular basis for sexual

  4. Competitive Semantic Memory Retrieval: Temporal Dynamics Revealed by Event-Related Potentials

    PubMed Central

    Hellerstedt, Robin; Johansson, Mikael

    2016-01-01

    Memories compete for retrieval when they are related to a common retrieval cue. Previous research has shown that retrieval of a target memory may lead to subsequent retrieval-induced forgetting (RIF) of currently irrelevant competing memories. In the present study, we investigated the time course of competitive semantic retrieval and examined the neurocognitive mechanisms underlying RIF. We contrasted two theoretical accounts of RIF by examining a critical aspect of this memory phenomenon, namely the extent to which it depends on successful retrieval of the target memory. Participants first studied category-exemplar word-pairs (e.g. Fruit—Apple). Next, we recorded electrophysiological measures of brain activity while the participants performed a competitive semantic cued-recall task. In this task, the participants were provided with the studied categories but they were instructed to retrieve other unstudied exemplars (e.g. Fruit—Ma__?). We investigated the event-related potential (ERP) correlates of retrieval success by comparing ERPs from successful and failed retrieval trials. To isolate the ERP correlates of continuous retrieval attempts from the ERP correlates of retrieval success, we included an impossible retrieval condition, with incompletable word-stem cues (Drinks—Wy__) and compared it with a non-retrieval presentation baseline condition (Occupation—Dentist). The participants’ memory for all the studied exemplars was tested in the final phase of the experiment. Taken together, the behavioural results suggest that RIF is independent of target retrieval. Beyond investigating the mechanisms underlying RIF, the present study also elucidates the temporal dynamics of semantic cued-recall by isolating the ERP correlates of retrieval attempt and retrieval success. The ERP results revealed that retrieval attempt is reflected in a late posterior negativity, possibly indicating construction of candidates for completing the word-stem cue and retrieval

  5. Dynamic full field optical coherence tomography: subcellular metabolic contrast revealed in tissues by interferometric signals temporal analysis

    PubMed Central

    Apelian, Clement; Harms, Fabrice; Thouvenin, Olivier; Boccara, A. Claude

    2016-01-01

    We developed a new endogenous approach to reveal subcellular metabolic contrast in fresh ex vivo tissues taking advantage of the time dependence of the full field optical coherence tomography interferometric signals. This method reveals signals linked with local activity of the endogenous scattering elements which can reveal cells where other OCT-based techniques fail or need exogenous contrast agents. We benefit from the micrometric transverse resolution of full field OCT to image intracellular features. We used this time dependence to identify different dynamics at the millisecond scale on a wide range of organs in normal or pathological conditions. PMID:27446672

  6. Typification of Natural Seasonal Dynamics of Vegetation to Reveal Impact of Land Surface Change on Environment (by Satellite Data)

    NASA Astrophysics Data System (ADS)

    Shevyrnogov, A.; Vysotskaya, G.; Sidko, A.; Dunaev, K.

    Deep insight into types of vegetation variability provided by AVHRR space scanner images of vegetation index spatial distribution helps reveal impact of land surface changes on environment.The Institute of Computational Modeling SB RAS has developed nonparametric algorithms of automatic to classify and recognize patterns of these images which helped to reveal: (1) major variability types (generally connected); (2) areas belonging to small classes, which can be used to reveal deviations from ``normal'' (e.g., forest fires, etc.); (3) deviation from a certain type of dynamics indicative of changes in condition of plants, which can be used to diagnose pathology at early stages; (4) impact of economical activities on vegetation in Norilsk area. The authors provide biological interpretation of the satellite data. Computer-animated dynamics and color maps are presented. Nonparametric algorithms of an automatic classification and pattern recognition were provided by the Institute of Computational Modeling SB RAS

  7. Dynamic ordering of nuclei in syncytial embryos: a quantitative analysis of the role of cytoskeletal networks.

    PubMed

    Kanesaki, Takuma; Edwards, Carina M; Schwarz, Ulrich S; Grosshans, Jörg

    2011-11-01

    In syncytial embryos nuclei undergo cycles of division and rearrangement within a common cytoplasm. It is presently unclear to what degree and how the nuclear array maintains positional order in the face of rapid cell divisions. Here we establish a quantitative assay, based on image processing, for analysing the dynamics of the nuclear array. By tracking nuclear trajectories in Drosophila melanogaster embryos, we are able to define and evaluate local and time-dependent measures for the level of geometrical order in the array. We find that after division, order is re-established in a biphasic manner, indicating the competition of different ordering processes. Using mutants and drug injections, we show that the order of the nuclear array depends on cytoskeletal networks organised by centrosomes. While both f-actin and microtubules are required for re-establishing order after mitosis, only f-actin is required to maintain the stability of this arrangement. Furthermore, f-actin function relies on myosin-independent non-contractile filaments that suppress individual nuclear mobility, whereas microtubules promote mobility and attract adjacent nuclei. Actin caps are shown to act to prevent nuclear incorporation into adjacent microtubule baskets. Our data demonstrate that two principal ordering mechanisms thus simultaneously contribute: (1) a passive crowding mechanism in which nuclei and actin caps act as spacers and (2) an active self-organisation mechanism based on a microtubule network. PMID:22001900

  8. Dynamical regime shifts in the North Atlantic climate variability during the last 2 ka as revealed by terrestrial proxies

    NASA Astrophysics Data System (ADS)

    Franke, Jasper G.; Donner, Reik V.

    2016-04-01

    The climate during the last two millennia is in general considered to be exceptionally stable compared to prior times. Nevertheless, there have been different episodes of distinguishable climate dynamics, most prominently the Medieval Climate Anomaly (MCA) and the Little Ice Age (LIA). In this study, we test a set of terrestrial paleoclimate records from Northern Europe for indications of temporary time-reversal asymmetry implying that during the thus identified periods of time, the data cannot be described by a linear Gaussian process and thus exhibit marked (possibly nonlinear) dynamics. Our analysis reveals that the onsets of both the MCA and the LIA are characterized by such complex dynamics indicating possible dynamical regime shifts in the regional climate system. Furthermore, the end of the Roman Warm Period as well as the 1.4k event are accompanied by similar signatures of time-reversal asymmetry.

  9. Revealing the pure confinement effect in glass-forming liquids by dynamic mechanical analysis

    NASA Astrophysics Data System (ADS)

    Koppensteiner, J.; Schranz, W.; Carpenter, M. A.

    2010-01-01

    The dynamic mechanical response of mesoporous silica with coated inner surfaces confining the glass-forming liquid salol is measured as a function of temperature and frequency (1-100 Hz) for various pore sizes (2.4-7.3 nm). Compared to former results on natural pores, a distinct acceleration of dynamics due to the removal of surface-related retardation of molecular dynamics is found now, which can be fitted by a homogeneous relaxation using an unmodified Vogel-Fulcher-Tammann relation. This lubrication effect leads to a stronger decrease in the glass transition temperature Tg with decreasing pore size. The present data allow to quantify and separate competing side effects as surface bondings and negative pressure from the pure confinement induced acceleration of molecular dynamics with decreasing pore size. We analyze the dynamic elastic susceptibility data in terms of a recently proposed procedure [C. Dalle-Ferrier , Phys. Rev. E 76, 041510 (2007)], which relates the number Ncorr,T of molecules, whose dynamics is correlated with a local enthalpy fluctuation, to the three-point dynamic susceptibility χT . The observed increase of Ncorr,T with decreasing temperature strongly indicates that the size ξ of dynamic heterogeneities increases when approaching the glass transition.

  10. Will a Category Cue Attract You? Motor Output Reveals Dynamic Competition across Person Construal

    ERIC Educational Resources Information Center

    Freeman, Jonathan B.; Ambady, Nalini; Rule, Nicholas O.; Johnson, Kerri L.

    2008-01-01

    People use social categories to perceive others, extracting category cues to glean membership. Growing evidence for continuous dynamics in real-time cognition suggests, contrary to prevailing social psychological accounts, that person construal may involve dynamic competition between simultaneously active representations. To test this, the authors…

  11. Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

    PubMed Central

    Doshi, Urmi; Holliday, Michael J.; Eisenmesser, Elan Z.; Hamelberg, Donald

    2016-01-01

    Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue–residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general. PMID:27071107

  12. Do Dynamic Compared to Static Facial Expressions of Happiness and Anger Reveal Enhanced Facial Mimicry?

    PubMed Central

    Rymarczyk, Krystyna; Żurawski, Łukasz; Jankowiak-Siuda, Kamila; Szatkowska, Iwona

    2016-01-01

    Facial mimicry is the spontaneous response to others’ facial expressions by mirroring or matching the interaction partner. Recent evidence suggested that mimicry may not be only an automatic reaction but could be dependent on many factors, including social context, type of task in which the participant is engaged, or stimulus properties (dynamic vs static presentation). In the present study, we investigated the impact of dynamic facial expression and sex differences on facial mimicry and judgment of emotional intensity. Electromyography recordings were recorded from the corrugator supercilii, zygomaticus major, and orbicularis oculi muscles during passive observation of static and dynamic images of happiness and anger. The ratings of the emotional intensity of facial expressions were also analysed. As predicted, dynamic expressions were rated as more intense than static ones. Compared to static images, dynamic displays of happiness also evoked stronger activity in the zygomaticus major and orbicularis oculi, suggesting that subjects experienced positive emotion. No muscles showed mimicry activity in response to angry faces. Moreover, we found that women exhibited greater zygomaticus major muscle activity in response to dynamic happiness stimuli than static stimuli. Our data support the hypothesis that people mimic positive emotions and confirm the importance of dynamic stimuli in some emotional processing. PMID:27390867

  13. Do Dynamic Compared to Static Facial Expressions of Happiness and Anger Reveal Enhanced Facial Mimicry?

    PubMed

    Rymarczyk, Krystyna; Żurawski, Łukasz; Jankowiak-Siuda, Kamila; Szatkowska, Iwona

    2016-01-01

    Facial mimicry is the spontaneous response to others' facial expressions by mirroring or matching the interaction partner. Recent evidence suggested that mimicry may not be only an automatic reaction but could be dependent on many factors, including social context, type of task in which the participant is engaged, or stimulus properties (dynamic vs static presentation). In the present study, we investigated the impact of dynamic facial expression and sex differences on facial mimicry and judgment of emotional intensity. Electromyography recordings were recorded from the corrugator supercilii, zygomaticus major, and orbicularis oculi muscles during passive observation of static and dynamic images of happiness and anger. The ratings of the emotional intensity of facial expressions were also analysed. As predicted, dynamic expressions were rated as more intense than static ones. Compared to static images, dynamic displays of happiness also evoked stronger activity in the zygomaticus major and orbicularis oculi, suggesting that subjects experienced positive emotion. No muscles showed mimicry activity in response to angry faces. Moreover, we found that women exhibited greater zygomaticus major muscle activity in response to dynamic happiness stimuli than static stimuli. Our data support the hypothesis that people mimic positive emotions and confirm the importance of dynamic stimuli in some emotional processing. PMID:27390867

  14. X-Ray Photon Correlation Spectroscopy Reveals Intermittent Aging Dynamics in a Metallic Glass.

    PubMed

    Evenson, Zach; Ruta, Beatrice; Hechler, Simon; Stolpe, Moritz; Pineda, Eloi; Gallino, Isabella; Busch, Ralf

    2015-10-23

    We use coherent x rays to probe the aging dynamics of a metallic glass directly on the atomic level. Contrary to the common assumption of a steady slowing down of the dynamics usually observed in macroscopic studies, we show that the structural relaxation processes underlying aging in this metallic glass are intermittent and highly heterogeneous at the atomic scale. Moreover, physical aging is triggered by cooperative atomic rearrangements, driven by the relaxation of internal stresses. The rich diversity of this behavior reflects a complex energy landscape, giving rise to a unique type of glassy-state dynamics. PMID:26551125

  15. X-Ray Photon Correlation Spectroscopy Reveals Intermittent Aging Dynamics in a Metallic Glass

    NASA Astrophysics Data System (ADS)

    Evenson, Zach; Ruta, Beatrice; Hechler, Simon; Stolpe, Moritz; Pineda, Eloi; Gallino, Isabella; Busch, Ralf

    2015-10-01

    We use coherent x rays to probe the aging dynamics of a metallic glass directly on the atomic level. Contrary to the common assumption of a steady slowing down of the dynamics usually observed in macroscopic studies, we show that the structural relaxation processes underlying aging in this metallic glass are intermittent and highly heterogeneous at the atomic scale. Moreover, physical aging is triggered by cooperative atomic rearrangements, driven by the relaxation of internal stresses. The rich diversity of this behavior reflects a complex energy landscape, giving rise to a unique type of glassy-state dynamics.

  16. Slow dynamics of a protein backbone in molecular dynamics simulation revealed by time-structure based independent component analysis

    SciTech Connect

    Naritomi, Yusuke; Fuchigami, Sotaro

    2013-12-07

    We recently proposed the method of time-structure based independent component analysis (tICA) to examine the slow dynamics involved in conformational fluctuations of a protein as estimated by molecular dynamics (MD) simulation [Y. Naritomi and S. Fuchigami, J. Chem. Phys. 134, 065101 (2011)]. Our previous study focused on domain motions of the protein and examined its dynamics by using rigid-body domain analysis and tICA. However, the protein changes its conformation not only through domain motions but also by various types of motions involving its backbone and side chains. Some of these motions might occur on a slow time scale: we hypothesize that if so, we could effectively detect and characterize them using tICA. In the present study, we investigated slow dynamics of the protein backbone using MD simulation and tICA. The selected target protein was lysine-, arginine-, ornithine-binding protein (LAO), which comprises two domains and undergoes large domain motions. MD simulation of LAO in explicit water was performed for 1 μs, and the obtained trajectory of C{sub α} atoms in the backbone was analyzed by tICA. This analysis successfully provided us with slow modes for LAO that represented either domain motions or local movements of the backbone. Further analysis elucidated the atomic details of the suggested local motions and confirmed that these motions truly occurred on the expected slow time scale.

  17. Slow dynamics of a protein backbone in molecular dynamics simulation revealed by time-structure based independent component analysis

    NASA Astrophysics Data System (ADS)

    Naritomi, Yusuke; Fuchigami, Sotaro

    2013-12-01

    We recently proposed the method of time-structure based independent component analysis (tICA) to examine the slow dynamics involved in conformational fluctuations of a protein as estimated by molecular dynamics (MD) simulation [Y. Naritomi and S. Fuchigami, J. Chem. Phys. 134, 065101 (2011)]. Our previous study focused on domain motions of the protein and examined its dynamics by using rigid-body domain analysis and tICA. However, the protein changes its conformation not only through domain motions but also by various types of motions involving its backbone and side chains. Some of these motions might occur on a slow time scale: we hypothesize that if so, we could effectively detect and characterize them using tICA. In the present study, we investigated slow dynamics of the protein backbone using MD simulation and tICA. The selected target protein was lysine-, arginine-, ornithine-binding protein (LAO), which comprises two domains and undergoes large domain motions. MD simulation of LAO in explicit water was performed for 1 μs, and the obtained trajectory of Cα atoms in the backbone was analyzed by tICA. This analysis successfully provided us with slow modes for LAO that represented either domain motions or local movements of the backbone. Further analysis elucidated the atomic details of the suggested local motions and confirmed that these motions truly occurred on the expected slow time scale.

  18. Concerted dynamic motions of an FABP4 model and its ligands revealed by microsecond molecular dynamics simulations.

    PubMed

    Li, Yan; Li, Xiang; Dong, Zigang

    2014-10-14

    In this work, we investigate the dynamic motions of fatty acid binding protein 4 (FABP4) in the absence and presence of a ligand by explicitly solvated all-atom molecular dynamics simulations. The dynamics of one ligand-free FABP4 and four ligand-bound FABP4s is compared via multiple 1.2 μs simulations. In our simulations, the protein interconverts between the open and closed states. Ligand-free FABP4 prefers the closed state, whereas ligand binding induces a conformational transition to the open state. Coupled with opening and closing of FABP4, the ligand adopts distinct binding modes, which are identified and compared with crystal structures. The concerted dynamics of protein and ligand suggests that there may exist multiple FABP4-ligand binding conformations. Thus, this work provides details about how ligand binding affects the conformational preference of FABP4 and how ligand binding is coupled with a conformational change of FABP4 at an atomic level. PMID:25231537

  19. Dynamical phase transitions reveal amyloid-like states on protein folding landscapes.

    PubMed

    Weber, Jeffrey K; Jack, Robert L; Schwantes, Christian R; Pande, Vijay S

    2014-08-19

    Developing an understanding of protein misfolding processes presents a crucial challenge for unlocking the mysteries of human disease. In this article, we present our observations of β-sheet-rich misfolded states on a number of protein dynamical landscapes investigated through molecular dynamics simulation and Markov state models. We employ a nonequilibrium statistical mechanical theory to identify the glassy states in a protein's dynamics, and we discuss the nonnative, β-sheet-rich states that play a distinct role in the slowest dynamics within seven protein folding systems. We highlight the fundamental similarity between these states and the amyloid structures responsible for many neurodegenerative diseases, and we discuss potential consequences for mechanisms of protein aggregation and intermolecular amyloid formation. PMID:25140433

  20. Noise Response Data Reveal Novel Controllability Gramian for Nonlinear Network Dynamics.

    PubMed

    Kashima, Kenji

    2016-01-01

    Control of nonlinear large-scale dynamical networks, e.g., collective behavior of agents interacting via a scale-free connection topology, is a central problem in many scientific and engineering fields. For the linear version of this problem, the so-called controllability Gramian has played an important role to quantify how effectively the dynamical states are reachable by a suitable driving input. In this paper, we first extend the notion of the controllability Gramian to nonlinear dynamics in terms of the Gibbs distribution. Next, we show that, when the networks are open to environmental noise, the newly defined Gramian is equal to the covariance matrix associated with randomly excited, but uncontrolled, dynamical state trajectories. This fact theoretically justifies a simple Monte Carlo simulation that can extract effectively controllable subdynamics in nonlinear complex networks. In addition, the result provides a novel insight into the relationship between controllability and statistical mechanics. PMID:27264780

  1. Noise Response Data Reveal Novel Controllability Gramian for Nonlinear Network Dynamics

    NASA Astrophysics Data System (ADS)

    Kashima, Kenji

    2016-06-01

    Control of nonlinear large-scale dynamical networks, e.g., collective behavior of agents interacting via a scale-free connection topology, is a central problem in many scientific and engineering fields. For the linear version of this problem, the so-called controllability Gramian has played an important role to quantify how effectively the dynamical states are reachable by a suitable driving input. In this paper, we first extend the notion of the controllability Gramian to nonlinear dynamics in terms of the Gibbs distribution. Next, we show that, when the networks are open to environmental noise, the newly defined Gramian is equal to the covariance matrix associated with randomly excited, but uncontrolled, dynamical state trajectories. This fact theoretically justifies a simple Monte Carlo simulation that can extract effectively controllable subdynamics in nonlinear complex networks. In addition, the result provides a novel insight into the relationship between controllability and statistical mechanics.

  2. Noise Response Data Reveal Novel Controllability Gramian for Nonlinear Network Dynamics

    PubMed Central

    Kashima, Kenji

    2016-01-01

    Control of nonlinear large-scale dynamical networks, e.g., collective behavior of agents interacting via a scale-free connection topology, is a central problem in many scientific and engineering fields. For the linear version of this problem, the so-called controllability Gramian has played an important role to quantify how effectively the dynamical states are reachable by a suitable driving input. In this paper, we first extend the notion of the controllability Gramian to nonlinear dynamics in terms of the Gibbs distribution. Next, we show that, when the networks are open to environmental noise, the newly defined Gramian is equal to the covariance matrix associated with randomly excited, but uncontrolled, dynamical state trajectories. This fact theoretically justifies a simple Monte Carlo simulation that can extract effectively controllable subdynamics in nonlinear complex networks. In addition, the result provides a novel insight into the relationship between controllability and statistical mechanics. PMID:27264780

  3. Dynamical age differences among coeval star clusters as revealed by blue stragglers.

    PubMed

    Ferraro, F R; Lanzoni, B; Dalessandro, E; Beccari, G; Pasquato, M; Miocchi, P; Rood, R T; Sigurdsson, S; Sills, A; Vesperini, E; Mapelli, M; Contreras, R; Sanna, N; Mucciarelli, A

    2012-12-20

    Globular star clusters that formed at the same cosmic time may have evolved rather differently from the dynamical point of view (because that evolution depends on the internal environment) through a variety of processes that tend progressively to segregate stars more massive than the average towards the cluster centre. Therefore clusters with the same chronological age may have reached quite different stages of their dynamical history (that is, they may have different 'dynamical ages'). Blue straggler stars have masses greater than those at the turn-off point on the main sequence and therefore must be the result of either a collision or a mass-transfer event. Because they are among the most massive and luminous objects in old clusters, they can be used as test particles with which to probe dynamical evolution. Here we report that globular clusters can be grouped into a few distinct families on the basis of the radial distribution of blue stragglers. This grouping corresponds well to an effective ranking of the dynamical stage reached by stellar systems, thereby permitting a direct measure of the cluster dynamical age purely from observed properties. PMID:23257880

  4. Asymmetric collapse in biomimetic complex coacervates revealed by local polymer and water dynamics.

    PubMed

    Ortony, Julia H; Hwang, Dong Soo; Franck, John M; Waite, J Herbert; Han, Songi

    2013-05-13

    Complex coacervation is a phenomenon characterized by the association of oppositely charged polyelectrolytes into micrometer-scale liquid condensates. This process is the purported first step in the formation of underwater adhesives by sessile marine organisms, as well as the process harnessed for the formation of new synthetic and protein-based contemporary materials. Efforts to understand the physical nature of complex coacervates are important for developing robust adhesives, injectable materials, or novel drug delivery vehicles for biomedical applications; however, their internal fluidity necessitates the use of in situ characterization strategies of their local dynamic properties, capabilities not offered by conventional techniques such as X-ray scattering, microscopy, or bulk rheological measurements. Herein, we employ the novel magnetic resonance technique Overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP), together with electron paramagnetic resonance (EPR) line shape analysis, to concurrently quantify local molecular and hydration dynamics, with species- and site-specificity. We observe striking differences in the structure and dynamics of the protein-based biomimetic complex coacervates from their synthetic analogues, which is an asymmetric collapse of the polyelectrolyte constituents. From this study we suggest charge heterogeneity within a given polyelectrolyte chain to be an important parameter by which the internal structure of complex coacervates may be tuned. Acquiring molecular-level insight to the internal structure and dynamics of dynamic polymer complexes in water through the in situ characterization of site- and species-specific local polymer and hydration dynamics should be a promising general approach that has not been widely employed for materials characterization. PMID:23540713

  5. The dynamics of attentional sampling during visual search revealed by Fourier analysis of periodic noise interference.

    PubMed

    Dugué, Laura; Vanrullen, Rufin

    2014-01-01

    What are the temporal dynamics of perceptual sampling during visual search tasks, and how do they differ between a difficult (or inefficient) and an easy (or efficient) task? Does attention focus intermittently on the stimuli, or are the stimuli processed continuously over time? We addressed these questions by way of a new paradigm using periodic fluctuations of stimulus information during a difficult (color-orientation conjunction) and an easy (+ among Ls) search task. On each stimulus, we applied a dynamic visual noise that oscillated at a given frequency (2-20 Hz, 2-Hz steps) and phase (four cardinal phase angles) for 500 ms. We estimated the dynamics of attentional sampling by computing an inverse Fourier transform on subjects' d-primes. In both tasks, the sampling function presented a significant peak at 2 Hz; we showed that this peak could be explained by nonperiodic search strategies such as increased sensitivity to stimulus onset and offset. Specifically in the difficult task, however, a second, higher-frequency peak was observed at 9 to 10 Hz, with a similar phase for all subjects; this isolated frequency component necessarily entails oscillatory attentional dynamics. In a second experiment, we presented difficult search arrays with dynamic noise that was modulated by the previously obtained grand-average attention sampling function or by its converse function (in both cases omitting the 2 Hz component to focus on genuine oscillatory dynamics). We verified that performance was higher in the latter than in the former case, even for subjects who had not participated in the first experiment. This study supports the idea of a periodic sampling of attention during a difficult search task. Although further experiments will be needed to extend these findings to other search tasks, the present report validates the usefulness of this novel paradigm for measuring the temporal dynamics of attention. PMID:24525262

  6. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    NASA Astrophysics Data System (ADS)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  7. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    PubMed Central

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-01-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. PMID:27032695

  8. Revealing Dissociative Electron Attachment Dynamics in Polyatomic Molecules Using Momentum Imaging Experiments and Electron Scattering Calculations

    NASA Astrophysics Data System (ADS)

    Belkacem, Ali; Slaughter, Daniel

    2015-05-01

    Understanding electron-driven chemical reactions is important for improving a variety of technological applications such as materials processing and the important role they play in the radiation damage in bulk matter. Furthermore, dissociative electron attachment often exhibits site-selective bond cleavage, which holds promise for prediction and precise control of electron-driven chemical reactions. Recent dynamical studies of these reactions have demonstrated that an understanding of anion dissociation dynamics beyond simple one-dimensional models is crucial in interpreting the measured fragment angular distributions. We combine ion fragment momentum imaging experiments with electron attachment entrance amplitude calculations to interrogate the non-Born-Oppenheimer dynamics of dissociative electron attachment in polyatomic molecules. We will report recent experimental developments in molecules of technological interest including methanol, methane and uracil. Work supported by Chemical Sciences, Geosciences and Biosciences division of BES/DOE.

  9. Coupled surface-subsurface hydrologic measurements reveal infiltration, recharge, and discharge dynamics across the swash zone of a sandy beach

    NASA Astrophysics Data System (ADS)

    Heiss, James W.; Puleo, Jack A.; Ullman, William J.; Michael, Holly A.

    2015-11-01

    Swash-groundwater interactions affect the biogeochemistry of beach aquifers and the transport of solutes and sediment across the beachface. Improved understanding of the complex, coupled dynamics of surface and subsurface flow processes in the swash zone is required to better estimate chemical fluxes to the sea and predict the morphological evolution of beaches. Simultaneous high-frequency measurements of saturation, water table elevation, and the cross-shore locations of runup and the boundary between the saturated and unsaturated beachface (surface saturation boundary) were collected on a sandy beach to link groundwater flow dynamics with swash zone forcing. Saturation and lysimeter measurements showed the dynamic response of subsurface saturation to swash events and permitted estimation of infiltration rates. Surface and subsurface observations revealed a decoupling of the surface saturation boundary and the intersection between the water table and the beachface. Surface measurements alone were insufficient to delineate the infiltration and discharge zones, which moved independently of the surface saturation boundary. Results show for the first time the motion and areal extent of infiltration and recharge zones, and constrain the maximum size of the subaerial discharge zone over swash and tidal time scales. The width of the infiltration zone was controlled by swash processes, and subaerial discharge was controlled primarily by tidal processes. These dynamics reveal the tightly coupled nature of surface and subsurface processes over multiple time scales, with implications for sediment transport and fluid and solute fluxes through the hydrologically and biogeochemically active intertidal zone of sandy beaches.

  10. Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

    PubMed

    Sato, Yuko; Hliscs, Marion; Dunst, Josefine; Goosmann, Christian; Brinkmann, Volker; Montagna, Georgina N; Matuschewski, Kai

    2016-07-15

    Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin-binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping. PMID:27226484

  11. Profiling the Dynamics of a Human Phosphorylome Reveals New Components in HGF/c-Met Signaling

    PubMed Central

    Wan, Jun; Xia, Shuli; Newman, Robert; Hu, Jianfei; Zhang, Jin; Hayward, S. Diane; Qian, Jiang; Laterra, John; Zhu, Heng

    2013-01-01

    Protein phosphorylation is a dynamic and reversible event that greatly influences cellular function. Identifying the key regulatory elements that determine cellular phenotypes during development and oncogenesis requires the ability to dynamically monitor proteome-wide events. Here, we report the development of a new strategy to monitor dynamic changes of protein phosphorylation in cells and tissues using functional protein microarrays as the readout. To demonstrate this technology's ability to identify condition-dependent phosphorylation events, human protein microarrays were incubated with lysates from cells or tissues under activation or inhibition of c-Met, a receptor tyrosine kinase involved in tissue morphogenesis and malignancy. By comparing the differences between the protein phosphorylation profiles obtained using the protein microarrays, we were able to recover many of the proteins that are known to be specifically activated (i.e., phosphorylated) upon c-Met activation by the hepatocyte growth factor (HGF). Most importantly, we discovered many proteins that were differentially phosphorylated by lysates from cells or tissues when the c-Met pathway was active. Using phosphorylation-specific antibodies, we were able to validate several candidate proteins as new downstream components of the c-Met signaling pathway in cells. We envision that this new approach, like its DNA microarray counterpart, can be further extended toward profiling dynamics of global protein phosphorylation under many different physiological conditions both in cellulo and in vivo in a high-throughput and cost-effective fashion. PMID:24023761

  12. Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imaging.

    PubMed

    Kuznetsov, Andrey V; Hermann, Martin; Troppmair, Jakob; Margreiter, Raimund; Hengster, Paul

    2010-01-01

    Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission-fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were (i) fast fission and fusion; (ii) small oscillating movements of the mitochondrial network; (iii) larger movements, including filament extension, retraction, fast (0.1-0.3 mum/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; (iv) as well as combinations of these actions and (v) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 mum/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems. PMID:19382913

  13. STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics

    PubMed Central

    2015-01-01

    Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality. PMID:26235350

  14. Stable metal isotopes reveal copper accumulation and loss dynamics in the freshwater bivalve Corbucula

    USGS Publications Warehouse

    Croteau, M.-N.; Luoma, S.N.; Topping, B.R.; Lopez, C.B.

    2004-01-01

    Characterization of uptake and loss dynamics is critical to understanding risks associated with contaminant exposure in aquatic animals. Dynamics are especially important in addressing questions such as why coexisting species in nature accumulate different levels of a contaminant. Here we manipulated copper (Cu) stable isotopic ratios (as an alternative to radioisotopes) to describe for the first time Cu dynamics in a freshwater invertebrate, the bivalve Corbicula fluminea. In the laboratory, Corbicula uptake and loss rate constants were determined from an environmentally realistic waterborne exposure to 65Cu (5.7 ??g L-1). That is, we spiked deionized water with Cu that was 99.4% 65Cu. Net tracer uptake was detectable after 1 day and strongly evident after 4 days. Thus, short-term exposures necessary to determine uptake dynamics are feasible with stable isotopes of Cu. In Corbicula, 65Cu depuration was biphasic. An unusually low rate constant of loss (0.0038 d-1) characterized the slow component of efflux, explaining why Corbicula strongly accumulates copper in nature. We incorporated our estimates of rate constants for dissolved 65Cu uptake and physiological efflux into a bioaccumulation model and showed that dietary exposure to Cu is likely an important bioaccumulation pathway for Corbicula.

  15. Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, MethylRO.

    PubMed

    Ueda, Jun; Maehara, Kazumitsu; Mashiko, Daisuke; Ichinose, Takako; Yao, Tatsuma; Hori, Mayuko; Sato, Yuko; Kimura, Hiroshi; Ohkawa, Yasuyuki; Yamagata, Kazuo

    2014-06-01

    In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states. PMID:24936475

  16. Heterochromatin Dynamics during the Differentiation Process Revealed by the DNA Methylation Reporter Mouse, MethylRO

    PubMed Central

    Ueda, Jun; Maehara, Kazumitsu; Mashiko, Daisuke; Ichinose, Takako; Yao, Tatsuma; Hori, Mayuko; Sato, Yuko; Kimura, Hiroshi; Ohkawa, Yasuyuki; Yamagata, Kazuo

    2014-01-01

    Summary In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states. PMID:24936475

  17. Application of dynamical systems theory to global weather phenomena revealed by satellite imagery

    NASA Technical Reports Server (NTRS)

    Saltzman, Barry; Ebisuzaki, Wesley; Maasch, Kirk A.; Oglesby, Robert; Pandolfo, Lionel; Tang, Chung-Muh

    1989-01-01

    Theoretical studies of low frequency and seasonal weather variability; dynamical properties of observational and general circulation model (GCM)-generated records; effects of the hydrologic cycle and latent heat release on extratropical weather; and Earth-system science studies are summarized.

  18. STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics.

    PubMed

    Vicidomini, Giuseppe; Ta, Haisen; Honigmann, Alf; Mueller, Veronika; Clausen, Mathias P; Waithe, Dominic; Galiani, Silvia; Sezgin, Erdinc; Diaspro, Alberto; Hell, Stefan W; Eggeling, Christian

    2015-09-01

    Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality. PMID:26235350

  19. Vinculin Binding Angle in Podosomes Revealed by High Resolution Microscopy

    PubMed Central

    Walde, Marie; Monypenny, James; Heintzmann, Rainer; Jones, Gareth E.; Cox, Susan

    2014-01-01

    Podosomes are highly dynamic actin-rich adhesive structures formed predominantly by cells of the monocytic lineage, which degrade the extracellular matrix. They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin. We have characterised the structure of podosomes in macrophages, particularly the structure of the ring, using three super-resolution fluorescence microscopy techniques: stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy. Rather than being round, as previously assumed, we found the vinculin ring to be created from relatively straight strands of vinculin, resulting in a distinctly polygonal shape. The strands bind preferentially at angles between 116° and 135°. Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth. PMID:24523880

  20. The Dynamic Conformational Cycle of the Group I Chaperonin C-Termini Revealed via Molecular Dynamics Simulation

    PubMed Central

    Dalton, Kevin M.; Frydman, Judith; Pande, Vijay S.

    2015-01-01

    Chaperonins are large ring shaped oligomers that facilitate protein folding by encapsulation within a central cavity. All chaperonins possess flexible C-termini which protrude from the equatorial domain of each subunit into the central cavity. Biochemical evidence suggests that the termini play an important role in the allosteric regulation of the ATPase cycle, in substrate folding and in complex assembly and stability. Despite the tremendous wealth of structural data available for numerous orthologous chaperonins, little structural information is available regarding the residues within the C-terminus. Herein, molecular dynamics simulations are presented which localize the termini throughout the nucleotide cycle of the group I chaperonin, GroE, from Escherichia coli. The simulation results predict that the termini undergo a heretofore unappreciated conformational cycle which is coupled to the nucleotide state of the enzyme. As such, these results have profound implications for the mechanism by which GroE utilizes nucleotide and folds client proteins. PMID:25822285

  1. The dynamic conformational cycle of the group I chaperonin C-termini revealed via molecular dynamics simulation.

    PubMed

    Dalton, Kevin M; Frydman, Judith; Pande, Vijay S

    2015-01-01

    Chaperonins are large ring shaped oligomers that facilitate protein folding by encapsulation within a central cavity. All chaperonins possess flexible C-termini which protrude from the equatorial domain of each subunit into the central cavity. Biochemical evidence suggests that the termini play an important role in the allosteric regulation of the ATPase cycle, in substrate folding and in complex assembly and stability. Despite the tremendous wealth of structural data available for numerous orthologous chaperonins, little structural information is available regarding the residues within the C-terminus. Herein, molecular dynamics simulations are presented which localize the termini throughout the nucleotide cycle of the group I chaperonin, GroE, from Escherichia coli. The simulation results predict that the termini undergo a heretofore unappreciated conformational cycle which is coupled to the nucleotide state of the enzyme. As such, these results have profound implications for the mechanism by which GroE utilizes nucleotide and folds client proteins. PMID:25822285

  2. Chain Dynamics in Solid Polymers and Polymerizing Systems as Revealed by Broadband Dielectric Spectroscopy

    NASA Astrophysics Data System (ADS)

    Williams, Graham

    2008-08-01

    A number of techniques are used to study the chain-dynamics of solid polymers, including those of dielectric relaxation [1-4], dynamic mechanical thermal analysis (DMTA) [1, 5], multinuclear NMR relaxations [6], quasi-elastic dynamic light scattering [7] and neutron scattering [8] (QELS & QENS) and transient fluorescence depolarization (TFD) [9]. Each technique has its own particular probe of the dynamics in a material. e.g. dielectric relaxation gives information on the angular motions of molecular chain-dipoles (for dipole relaxation) and the translational motions of ions (for f-dependent electrical conduction); NMR relaxations relate to the angular motions of chemical bonds; QELS relates to fluctuations in local refractive index; QENS to the time-dependent van Hove correlation function (suitably-defined) for proton-containing groups; TFD to the angular motions of fluorescent groups in a chain. Due to its relevance to practical applications of materials, DMTA is pre-eminent among the many physical techniques applied to solid polymers, but interpretations of behaviour in terms of molecular properties remain difficult since the direct link between an applied macroscopic stress and the molecular response of polymer chains in a bulk material remains an unsolved problem. Of the above techniques, Broadband Dielectric Spectroscopy (BDS) offers several advantages. (a) Materials may be studied in the frequency range 10-6 to 1010 Hz, over wide ranges of temperature and applied pressure, using commercially-available instrumentation. (b) Since the electrical capacitance of a film is inversely proportional its thickness, free-standing and supported films may be studied down to nm-thicknesses, giving e.g. information on the behaviour of the dynamic Tg as sample thickness approaches molecular dimensions. (c) Theoretical interpretations of dielectric relaxation and a.c. conduction are well-established in terms of Fourier transforms of molecular time correlation functions (TCFs

  3. A model of gene expression based on random dynamical systems reveals modularity properties of gene regulatory networks.

    PubMed

    Antoneli, Fernando; Ferreira, Renata C; Briones, Marcelo R S

    2016-06-01

    Here we propose a new approach to modeling gene expression based on the theory of random dynamical systems (RDS) that provides a general coupling prescription between the nodes of any given regulatory network given the dynamics of each node is modeled by a RDS. The main virtues of this approach are the following: (i) it provides a natural way to obtain arbitrarily large networks by coupling together simple basic pieces, thus revealing the modularity of regulatory networks; (ii) the assumptions about the stochastic processes used in the modeling are fairly general, in the sense that the only requirement is stationarity; (iii) there is a well developed mathematical theory, which is a blend of smooth dynamical systems theory, ergodic theory and stochastic analysis that allows one to extract relevant dynamical and statistical information without solving the system; (iv) one may obtain the classical rate equations form the corresponding stochastic version by averaging the dynamic random variables (small noise limit). It is important to emphasize that unlike the deterministic case, where coupling two equations is a trivial matter, coupling two RDS is non-trivial, specially in our case, where the coupling is performed between a state variable of one gene and the switching stochastic process of another gene and, hence, it is not a priori true that the resulting coupled system will satisfy the definition of a random dynamical system. We shall provide the necessary arguments that ensure that our coupling prescription does indeed furnish a coupled regulatory network of random dynamical systems. Finally, the fact that classical rate equations are the small noise limit of our stochastic model ensures that any validation or prediction made on the basis of the classical theory is also a validation or prediction of our model. We illustrate our framework with some simple examples of single-gene system and network motifs. PMID:27036626

  4. Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock

    USGS Publications Warehouse

    Kamath, Pauline L.; Foster, Jeffrey T.; Drees, Kevin P.; Luikart, Gordon; Quance, Christine; Anderson, Neil J.; Clarke, P. Ryan; Cole, Eric K.; Drew, Mark L.; Edwards, William H.; Rhyan, Jack C.; Treanor, John J.; Wallen, Rick L.; White, Patrick J.; Robbe-Austerman, Suelee; Cross, Paul C.

    2016-01-01

    Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (B3 to 8 km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations.

  5. A distributed cell division counter reveals growth dynamics in the gut microbiota

    PubMed Central

    Myhrvold, Cameron; Kotula, Jonathan W.; Hicks, Wade M.; Conway, Nicholas J.; Silver, Pamela A.

    2015-01-01

    Microbial population growth is typically measured when cells can be directly observed, or when death is rare. However, neither of these conditions hold for the mammalian gut microbiota, and, therefore, standard approaches cannot accurately measure the growth dynamics of this community. Here we introduce a new method (distributed cell division counting, DCDC) that uses the accurate segregation at cell division of genetically encoded fluorescent particles to measure microbial growth rates. Using DCDC, we can measure the growth rate of Escherichia coli for >10 consecutive generations. We demonstrate experimentally and theoretically that DCDC is robust to error across a wide range of temperatures and conditions, including in the mammalian gut. Furthermore, our experimental observations inform a mathematical model of the population dynamics of the gut microbiota. DCDC can enable the study of microbial growth during infection, gut dysbiosis, antibiotic therapy or other situations relevant to human health. PMID:26615910

  6. Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

    PubMed Central

    Loucaides, Eva M.; von Kirchbach, Johann C.; Foeglein, Ágnes; Sharps, Jane; Fodor, Ervin; Digard, Paul

    2009-01-01

    The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity. PMID:19744689

  7. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    SciTech Connect

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U.; Elsen, A.; Baumann, Ph.; Nuzzo, R. G.; Stach, E. A.; Frenkel, A. I.

    2015-06-29

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction—ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. Lastly, this method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.

  8. Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock.

    PubMed

    Kamath, Pauline L; Foster, Jeffrey T; Drees, Kevin P; Luikart, Gordon; Quance, Christine; Anderson, Neil J; Clarke, P Ryan; Cole, Eric K; Drew, Mark L; Edwards, William H; Rhyan, Jack C; Treanor, John J; Wallen, Rick L; White, Patrick J; Robbe-Austerman, Suelee; Cross, Paul C

    2016-01-01

    Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (∼3 to 8 km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations. PMID:27165544

  9. Revealing the signature of dipolar interactions in dynamic spectra of polydisperse magnetic nanoparticles.

    PubMed

    Ivanov, Alexey O; Zverev, Vladimir S; Kantorovich, Sofia S

    2016-04-21

    We investigate, via a modified mean field approach, the dynamic magnetic response of a polydisperse dipolar suspension to a weak, linearly polarised, AC field. We introduce an additional term into the Fokker-Planck equation, which takes into account dipole-dipole interaction in the form of the first order perturbation, and allows for particle polydispersity. The analytical expressions, obtained for the real and imaginary dynamic susceptibilities, predict three measurable effects: the increase of the real part low-frequency plateaux; the enhanced growth of the imaginary part in the low-frequency range; and the shift of the imaginary part maximum. Our theoretical predictions find an experimental confirmation and explain the changes in the spectrum. PMID:26890415

  10. Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons.

    PubMed

    Habib, Naomi; Li, Yinqing; Heidenreich, Matthias; Swiech, Lukasz; Avraham-Davidi, Inbal; Trombetta, John J; Hession, Cynthia; Zhang, Feng; Regev, Aviv

    2016-08-26

    Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2'-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues. PMID:27471252

  11. Pupil dilation deconvolution reveals the dynamics of attention at high temporal resolution.

    PubMed

    Wierda, Stefan M; van Rijn, Hedderik; Taatgen, Niels A; Martens, Sander

    2012-05-29

    The size of the human pupil increases as a function of mental effort. However, this response is slow, and therefore its use is thought to be limited to measurements of slow tasks or tasks in which meaningful events are temporally well separated. Here we show that high-temporal-resolution tracking of attention and cognitive processes can be obtained from the slow pupillary response. Using automated dilation deconvolution, we isolated and tracked the dynamics of attention in a fast-paced temporal attention task, allowing us to uncover the amount of mental activity that is critical for conscious perception of relevant stimuli. We thus found evidence for specific temporal expectancy effects in attention that have eluded detection using neuroimaging methods such as EEG. Combining this approach with other neuroimaging techniques can open many research opportunities to study the temporal dynamics of the mind's inner eye in great detail. PMID:22586101

  12. Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

    SciTech Connect

    Loucaides, Eva M.; Kirchbach, Johann C. von; Foeglein, Agnes; Sharps, Jane; Fodor, Ervin; Digard, Paul

    2009-11-10

    The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity.

  13. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes.

    PubMed

    Li, Y; Zakharov, D; Zhao, S; Tappero, R; Jung, U; Elsen, A; Baumann, Ph; Nuzzo, R G; Stach, E A; Frenkel, A I

    2015-01-01

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction-ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. This method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes. PMID:26119246

  14. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    NASA Astrophysics Data System (ADS)

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U.; Elsen, A.; Baumann, Ph.; Nuzzo, R. G.; Stach, E. A.; Frenkel, A. I.

    2015-06-01

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction--ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. This method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.

  15. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    PubMed Central

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U.; Elsen, A.; Baumann, Ph.; Nuzzo, R.G.; Stach, E.A.; Frenkel, A.I.

    2015-01-01

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction—ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. This method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes. PMID:26119246

  16. Revealing the Complex Dynamics of the Atmospheres of Red Supergiants with the Very Large Telescope Interferometer

    NASA Astrophysics Data System (ADS)

    Ohnaka, K.; Weigelt, G.; Hofmann, K.-H.; Schertl, D.

    2015-12-01

    Massive stars lose a significant fraction of their initial mass when they evolve to red supergiants before they end their life in supernova explosions. The mass loss greatly affects their final fate. However, the mass loss from these dying supergiants is not yet understood well. Here we present our efforts to spatially resolve the dynamics of the atmospheres of red supergiants with the Very Large Telescope Interferometer (VLTI) and the AMBER instrument to clarify the physical mechanism behind the mass loss. The VLTI/AMBER's combination of milliarcsecond spatial resolution and high spectral resolution allows us to spatially resolve stellar atmospheres and extract the dynamical information at each position over the star and the atmosphere — just like observations of the Sun.

  17. Complex structural dynamics of nanocatalysts revealed in Operando conditions by correlated imaging and spectroscopy probes

    DOE PAGESBeta

    Li, Y.; Zakharov, D.; Zhao, S.; Tappero, R.; Jung, U.; Elsen, A.; Baumann, Ph.; Nuzzo, R. G.; Stach, E. A.; Frenkel, A. I.

    2015-06-29

    Understanding how heterogeneous catalysts change size, shape and structure during chemical reactions is limited by the paucity of methods for studying catalytic ensembles in working state, that is, in operando conditions. Here by a correlated use of synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy in operando conditions, we quantitatively describe the complex structural dynamics of supported Pt catalysts exhibited during an exemplary catalytic reaction—ethylene hydrogenation. This work exploits a microfabricated catalytic reactor compatible with both probes. The results demonstrate dynamic transformations of the ensemble of Pt clusters that spans a broad size range throughout changing reaction conditions. Lastly,more » this method is generalizable to quantitative operando studies of complex systems using a wide variety of X-ray and electron-based experimental probes.« less

  18. Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock

    PubMed Central

    Kamath, Pauline L.; Foster, Jeffrey T.; Drees, Kevin P.; Luikart, Gordon; Quance, Christine; Anderson, Neil J.; Clarke, P. Ryan; Cole, Eric K.; Drew, Mark L.; Edwards, William H.; Rhyan, Jack C.; Treanor, John J.; Wallen, Rick L.; White, Patrick J.; Robbe-Austerman, Suelee; Cross, Paul C.

    2016-01-01

    Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (∼3 to 8 km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations. PMID:27165544

  19. Stochastic expression dynamics of a transcription factor revealed by single-molecule noise analysis.

    PubMed

    Hensel, Zach; Feng, Haidong; Han, Bo; Hatem, Christine; Wang, Jin; Xiao, Jie

    2012-08-01

    Gene expression is inherently stochastic; precise gene regulation by transcription factors is important for cell-fate determination. Many transcription factors regulate their own expression, suggesting that autoregulation counters intrinsic stochasticity in gene expression. Using a new strategy, cotranslational activation by cleavage (CoTrAC), we probed the stochastic expression dynamics of cI, which encodes the bacteriophage λ repressor CI, a fate-determining transcription factor. CI concentration fluctuations influence both lysogenic stability and induction of bacteriophage λ. We found that the intrinsic stochasticity in cI expression was largely determined by CI expression level irrespective of autoregulation. Furthermore, extrinsic, cell-to-cell variation was primarily responsible for CI concentration fluctuations, and negative autoregulation minimized CI concentration heterogeneity by counteracting extrinsic noise and introducing memory. This quantitative study of transcription factor expression dynamics sheds light on the mechanisms cells use to control noise in gene regulatory networks. PMID:22751020

  20. Dynamic views of living cell fine structure revealed by birefringence imaging

    NASA Astrophysics Data System (ADS)

    Oldenbourg, Rudolf

    2001-11-01

    We have been developing and applying a new type of polarized light microscope, the new Pol-Scope, which dramatically enhances the unique capabilities of the traditional polarizing microscope. In living cells, without applying exogenous dyes or florescent labels, we have studied the dynamic organization of filamentous actin in neuronal growth cones and improved the efficiency of spindle imaging for in-vitro fertilization and enucleation procedures.

  1. Dynamics inside the cancer cell attractor reveal cell heterogeneity, limits of stability, and escape

    PubMed Central

    Li, Qin; Wennborg, Anders; Aurell, Erik; Dekel, Erez; Zou, Jie-Zhi; Xu, Yuting; Huang, Sui; Ernberg, Ingemar

    2016-01-01

    The observed intercellular heterogeneity within a clonal cell population can be mapped as dynamical states clustered around an attractor point in gene expression space, owing to a balance between homeostatic forces and stochastic fluctuations. These dynamics have led to the cancer cell attractor conceptual model, with implications for both carcinogenesis and new therapeutic concepts. Immortalized and malignant EBV-carrying B-cell lines were used to explore this model and characterize the detailed structure of cell attractors. Any subpopulation selected from a population of cells repopulated the whole original basin of attraction within days to weeks. Cells at the basin edges were unstable and prone to apoptosis. Cells continuously changed states within their own attractor, thus driving the repopulation, as shown by fluorescent dye tracing. Perturbations of key regulatory genes induced a jump to a nearby attractor. Using the Fokker–Planck equation, this cell population behavior could be described as two virtual, opposing influences on the cells: one attracting toward the center and the other promoting diffusion in state space (noise). Transcriptome analysis suggests that these forces result from high-dimensional dynamics of the gene regulatory network. We propose that they can be generalized to all cancer cell populations and represent intrinsic behaviors of tumors, offering a previously unidentified characteristic for studying cancer. PMID:26929366

  2. Functional connectivity dynamics during film viewing reveal common networks for different emotional experiences.

    PubMed

    Raz, Gal; Touroutoglou, Alexandra; Wilson-Mendenhall, Christine; Gilam, Gadi; Lin, Tamar; Gonen, Tal; Jacob, Yael; Atzil, Shir; Admon, Roee; Bleich-Cohen, Maya; Maron-Katz, Adi; Hendler, Talma; Barrett, Lisa Feldman

    2016-08-01

    Recent theoretical and empirical work has highlighted the role of domain-general, large-scale brain networks in generating emotional experiences. These networks are hypothesized to process aspects of emotional experiences that are not unique to a specific emotional category (e.g., "sadness," "happiness"), but rather that generalize across categories. In this article, we examined the dynamic interactions (i.e., changing cohesiveness) between specific domain-general networks across time while participants experienced various instances of sadness, fear, and anger. We used a novel method for probing the network connectivity dynamics between two salience networks and three amygdala-based networks. We hypothesized, and found, that the functional connectivity between these networks covaried with the intensity of different emotional experiences. Stronger connectivity between the dorsal salience network and the medial amygdala network was associated with more intense ratings of emotional experience across six different instances of the three emotion categories examined. Also, stronger connectivity between the dorsal salience network and the ventrolateral amygdala network was associated with more intense ratings of emotional experience across five out of the six different instances. Our findings demonstrate that a variety of emotional experiences are associated with dynamic interactions of domain-general neural systems. PMID:27142636

  3. Multidimensional infrared spectroscopy reveals the vibrational and solvation dynamics of isoniazid

    NASA Astrophysics Data System (ADS)

    Shaw, Daniel J.; Adamczyk, Katrin; Frederix, Pim W. J. M.; Simpson, Niall; Robb, Kirsty; Greetham, Gregory M.; Towrie, Michael; Parker, Anthony W.; Hoskisson, Paul A.; Hunt, Neil T.

    2015-06-01

    The results of infrared spectroscopic investigations into the band assignments, vibrational relaxation, and solvation dynamics of the common anti-tuberculosis treatment Isoniazid (INH) are reported. INH is known to inhibit InhA, a 2-trans-enoyl-acyl carrier protein reductase enzyme responsible for the maintenance of cell walls in Mycobacterium tuberculosis but as new drug-resistant strains of the bacterium appear, next-generation therapeutics will be essential to combat the rise of the disease. Small molecules such as INH offer the potential for use as a biomolecular marker through which ultrafast multidimensional spectroscopies can probe drug binding and so inform design strategies but a complete characterization of the spectroscopy and dynamics of INH in solution is required to inform such activity. Infrared absorption spectroscopy, in combination with density functional theory calculations, is used to assign the vibrational modes of INH in the 1400-1700 cm-1 region of the infrared spectrum while ultrafast multidimensional spectroscopy measurements determine the vibrational relaxation dynamics and the effects of solvation via spectral diffusion of the carbonyl stretching vibrational mode. These results are discussed in the context of previous linear spectroscopy studies on solid-phase INH and its usefulness as a biomolecular probe.

  4. Multidimensional infrared spectroscopy reveals the vibrational and solvation dynamics of isoniazid.

    PubMed

    Shaw, Daniel J; Adamczyk, Katrin; Frederix, Pim W J M; Simpson, Niall; Robb, Kirsty; Greetham, Gregory M; Towrie, Michael; Parker, Anthony W; Hoskisson, Paul A; Hunt, Neil T

    2015-06-01

    The results of infrared spectroscopic investigations into the band assignments, vibrational relaxation, and solvation dynamics of the common anti-tuberculosis treatment Isoniazid (INH) are reported. INH is known to inhibit InhA, a 2-trans-enoyl-acyl carrier protein reductase enzyme responsible for the maintenance of cell walls in Mycobacterium tuberculosis but as new drug-resistant strains of the bacterium appear, next-generation therapeutics will be essential to combat the rise of the disease. Small molecules such as INH offer the potential for use as a biomolecular marker through which ultrafast multidimensional spectroscopies can probe drug binding and so inform design strategies but a complete characterization of the spectroscopy and dynamics of INH in solution is required to inform such activity. Infrared absorption spectroscopy, in combination with density functional theory calculations, is used to assign the vibrational modes of INH in the 1400-1700 cm(-1) region of the infrared spectrum while ultrafast multidim