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Sample records for microtubule doublet interactions

  1. Structural insights into microtubule doublet interactions inaxonemes

    SciTech Connect

    Downing, Kenneth H.; Sui, Haixin

    2007-06-06

    Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of the axoneme. Recent structural studies of the axoneme have used cryo-electron tomography to reveal new details of the interactions among some of the multitude of proteins that form the axoneme and regulate its movement. Connections among the several sets of dyneins, in particular, suggest ways in which their actions may be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding the doublet's mechanical properties that are related to the bending of the axoneme, and has also offered insight into its potential role in the mechanism of dynein activity regulation.

  2. Microtubule doublets are double-track railways for intraflagellar transport trains.

    PubMed

    Stepanek, Ludek; Pigino, Gaia

    2016-05-06

    The cilium is a large macromolecular machine that is vital for motility, signaling, and sensing in most eukaryotic cells. Its conserved core structure, the axoneme, contains nine microtubule doublets, each comprising a full A-microtubule and an incomplete B-microtubule. However, thus far, the function of this doublet geometry has not been understood. We developed a time-resolved correlative fluorescence and three-dimensional electron microscopy approach to investigate the dynamics of intraflagellar transport (IFT) trains, which carry ciliary building blocks along microtubules during the assembly and disassembly of the cilium. Using this method, we showed that each microtubule doublet is used as a bidirectional double-track railway: Anterograde IFT trains move along B-microtubules, and retrograde trains move along A-microtubules. Thus, the microtubule doublet geometry provides direction-specific rails to coordinate bidirectional transport of ciliary components.

  3. Molecular architecture of axonemal microtubule doublets revealedby cryo-electron tomography

    SciTech Connect

    Sui, Haixin; Downing, Kenneth H.

    2006-05-22

    The axoneme, which forms the core of eukaryotic flagella and cilia, is one of the largest macromolecular machines with a structure that is largely conserved from protists to mammals. Microtubule doublets are structural components of axonemes containing a number of proteins besides tubulin, and are usually found in arrays of nine doublets arranged around two singlet microtubules. Coordinated sliding of adjacent doublets, which involves a host of other proteins in the axoneme, produces periodic beating movements of the axoneme. We have obtained a 3D density map of intact microtubule doublets using cryo-electron tomography and image averaging. Our map, with a resolution of about 3 nm, provides insights into locations of particular proteins within the doublets and the structural features of the doublets that define their mechanical properties. We identify likely candidates for several of these non-tubulin components of the doublets. This work offers novel insight on how tubulin protofilaments and accessory proteins attach together to form the doublets and provides a structural basis for understanding doublet function in axonemes.

  4. Biochemical characterization of tektins from sperm flagellar doublet microtubules.

    PubMed

    Linck, R W; Stephens, R E

    1987-04-01

    Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little obvious similarity to either alpha- or beta-tubulin. With reverse-phase HPLC on a C18 column, using 6 M guanidine-HCl solubilization and a 0.1% trifluoroacetic acid/CH3CN gradient system (Stephens, R. E., 1984, J. Cell Biol. 90:37a [Abstr.]), the relatively less hydrophobic 51-kD tektin elutes at greater than 45% CH3CN, immediately followed by the 55-kD chain. The 47-kD tektin is substantially more hydrophobic, eluting between the two tubulins. The amino acid compositions of the tektins are very similar to each other but totally distinct from tubulin chains, being characterized by a greater than 50% higher arginine plus lysine content (in good agreement with the number of tryptic peptides) and about half the content of glycine, histidine, proline, and tyrosine. The proline content correlates well with the fact that tektin filaments have twice as much alpha-helical content as tubulin. Total hydrophobic amino acid content correlates with HPLC elution times for the tektins but not tubulins. The average amino acid composition of the tektins indicates that they resemble intermediate filament proteins, as originally postulated from structural, solubility, and electrophoretic properties. Tektins have higher cysteine and tryptophan contents than desmin and vimentin, which characteristically have only one residue of each, more closely resembling certain keratins in these amino acids.

  5. Biochemical characterization of tektins from sperm flagellar doublet microtubules

    PubMed Central

    1987-01-01

    Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little obvious similarity to either alpha- or beta- tubulin. With reverse-phase HPLC on a C18 column, using 6 M guanidine- HCl solubilization and a 0.1% trifluoroacetic acid/CH3CN gradient system (Stephens, R. E., 1984, J. Cell Biol. 90:37a [Abstr.]), the relatively less hydrophobic 51-kD tektin elutes at greater than 45% CH3CN, immediately followed by the 55-kD chain. The 47-kD tektin is substantially more hydrophobic, eluting between the two tubulins. The amino acid compositions of the tektins are very similar to each other but totally distinct from tubulin chains, being characterized by a greater than 50% higher arginine plus lysine content (in good agreement with the number of tryptic peptides) and about half the content of glycine, histidine, proline, and tyrosine. The proline content correlates well with the fact that tektin filaments have twice as much alpha-helical content as tubulin. Total hydrophobic amino acid content correlates with HPLC elution times for the tektins but not tubulins. The average amino acid composition of the tektins indicates that they resemble intermediate filament proteins, as originally postulated from structural, solubility, and electrophoretic properties. Tektins have higher cysteine and tryptophan contents than desmin and vimentin, which characteristically have only one residue of each, more closely resembling certain keratins in these amino acids. PMID:3558479

  6. Insights into the Structure and Function of Ciliary and Flagellar Doublet Microtubules

    PubMed Central

    Linck, Richard; Fu, Xiaofeng; Lin, Jianfeng; Ouch, Christna; Schefter, Alexandra; Steffen, Walter; Warren, Peter; Nicastro, Daniela

    2014-01-01

    Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems. PMID:24794867

  7. Coordination of outer arm dynein activity along axonemal doublet microtubules.

    PubMed

    Seetharam, Raviraja N; Satir, Peter

    2008-07-01

    This study considers the mechanism by which ODA based sliding is produced and the relationship of that mechanism to the determination of beat frequency. Two models of activity have been examined: a stochastic model, where ODA activity is random and a metachronal model, where activity is sequentially triggered along a doublet. Inactivation of a few ODAs would have virtually no effect on stochastic activity, but would completely block metachronal activity. We (Seetharam and Satir [2005]: Cell Motil Cytoskeleton 60:96-103) previously demonstrated that ODAs produce high speed sliding of about 200 mum/s, followed by a pause. IDAs produce slow, 5 mum/s, continuous sliding. We have examined the effects of nM concentrations of vanadate on sliding, measuring velocity and extent of high speed sliding and pause distribution or sliding cessation. In 5 nM vanadate, where photocleavage experiments show about 16/270 ODAs per doublet are affected, no differences from control are seen, but at 10 and 25 nM vanadate, high speed velocity is greatly reduced and pause distribution changes. The results support a model, in which high speed sliding is produced by metachronal activity. Blockage of two or more heavy chains of one ODA or a small group of adjacent ODAs produces cessation of sliding, but cessation is only temporary, probably because IDA activity continues, allowing ODA activity re-initiation beyond the block. These conclusions are consistent with Sugino and Naitoh's [1982; Nature 295:609-611] proposal, whereby during each beat, every ODA along a doublet becomes activated in succession, with repetitive activation determining beat frequency.

  8. Extrusion of microtubule doublet outer dense fibers 5-6 associating with fibrous sheath sliding in mouse sperm flagella.

    PubMed

    Si, Y; Okuno, M

    1995-11-01

    Our previous experiments (Si and Okuno [1993a] Exp. Cell Res., 208:170-174) provided evidence that the fibrous sheath (FS) slid headward to middle piece in the activated mouse sperm flagellum when doublet microtubules together with their outer dense fibers (ODFs) extruded from the axoneme. Of the extruded doublet-ODFs, however, which one was responsible for the FS sliding remained unresolved. The present study demonstrated that the FS sliding and the order of doublet-ODFs extrusion in mouse sperm flagella were trypsin concentration dependent. Under the condition of mild trypsinization (0.1 micrograms/ml), only doublet-ODFs 4, 5-6 (doublet-ODFs 5 and 6 were always paired), and 7 extruded from the axoneme. Furthermore, the extrusion of doublet-ODFs 5-6 was identified to precede doublet-ODFs 4 and 7, and was considered the candidate responsible for FS sliding. In contrast, the high-concentration trypsinization (4 micrograms/ml) led to extrusion of doublet-ODFs 1, 2, and 9 following doublet-ODFs 4, 5-6, and 7. FS sliding, however, did not occur.

  9. Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable protofilaments.

    PubMed

    Linck, Richard; Fu, Xiaofeng; Lin, Jianfeng; Ouch, Christna; Schefter, Alexandra; Steffen, Walter; Warren, Peter; Nicastro, Daniela

    2014-06-20

    Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems.

  10. Kinetochore-microtubule interactions during cell division.

    PubMed

    Maiato, Helder; Sunkel, Claudio E

    2004-01-01

    Proper segregation of chromosomes during cell division is essential for the maintenance of genetic stability. During this process chromosomes must establish stable functional interactions with microtubules through the kinetochore, a specialized protein structure located on the surface of the centromeric heterochromatin. Stable attachment of kinetochores to a number of microtubules results in the formation of a kinetochore fibre that mediates chromosome movement. How the kinetochore fibre is formed and how chromosome motion is produced and regulated remain major questions in cell biology. Here we look at some of the history of research devoted to the study of kinetochore-microtubule interaction and attempt to identify significant advances in the knowledge of the basic processes. Ultrastructural work has provided substantial insights into the structure of the kinetochore and associated microtubules during different stages of mitosis. Also, recent in-vivo studies have probed deep into the dynamics of kinetochore-attached microtubules suggesting possible models for the way in which kinetochores harness the capacity of microtubules to do work and turn it into chromosome motion. Much of the research in recent years suggests that indeed multiple mechanisms are involved in both formation of the k-fibre and chromosome motion. Thus, rather than moving to a unified theory, it has become apparent that most cell types have the capacity to build the spindle using multiple and probably redundant mechanisms.

  11. End-on microtubule-dynein interactions and pulling-based positioning of microtubule organizing centers

    PubMed Central

    Laan, Liedewij; Roth, Sophie; Dogterom, Marileen

    2012-01-01

    During important cellular processes such as centrosome and spindle positioning, dynein at the cortex interacts with dynamic microtubules in an apparent “end-on” fashion. It is well-established that dynein can generate forces by moving laterally along the microtubule lattice, but much less is known about dynein’s interaction with dynamic microtubule ends. In this paper, we review recent in vitro experiments that show that dynein, attached to an artificial cortex, is able to capture microtubule ends, regulate microtubule dynamics and mediate the generation of pulling forces on shrinking microtubules. We further review existing ideas on the involvement of dynein-mediated cortical pulling forces in the positioning of microtubule organizing centers such as centrosomes. Recent in vitro experiments have demonstrated that cortical pulling forces in combination with pushing forces can lead to reliable centering of microtubule asters in quasi two-dimensional microfabricated chambers. In these experiments, pushing leads to slipping of microtubule ends along the chamber boundaries, resulting in an anisotropic distribution of cortical microtubule contacts that favors centering, once pulling force generators become engaged. This effect is predicted to be strongly geometry-dependent, and we therefore finally discuss ongoing efforts to repeat these experiments in three-dimensional, spherical and deformable geometries. PMID:22895049

  12. The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes

    PubMed Central

    Bower, Raqual; Tritschler, Douglas; VanderWaal, Kristyn; Perrone, Catherine A.; Mueller, Joshua; Fox, Laura; Sale, Winfield S.; Porter, M. E.

    2013-01-01

    The nexin–dynein regulatory complex (N-DRC) is proposed to coordinate dynein arm activity and interconnect doublet microtubules. Here we identify a conserved region in DRC4 critical for assembly of the N-DRC into the axoneme. At least 10 subunits associate with DRC4 to form a discrete complex distinct from other axonemal substructures. Transformation of drc4 mutants with epitope-tagged DRC4 rescues the motility defects and restores assembly of missing DRC subunits and associated inner-arm dyneins. Four new DRC subunits contain calcium-signaling motifs and/or AAA domains and are nearly ubiquitous in species with motile cilia. However, drc mutants are motile and maintain the 9 + 2 organization of the axoneme. To evaluate the function of the N-DRC, we analyzed ATP-induced reactivation of isolated axonemes. Rather than the reactivated bending observed with wild-type axonemes, ATP addition to drc-mutant axonemes resulted in splaying of doublets in the distal region, followed by oscillatory bending between pairs of doublets. Thus the N-DRC provides some but not all of the resistance to microtubule sliding and helps to maintain optimal alignment of doublets for productive flagellar motility. These findings provide new insights into the mechanisms that regulate motility and further highlight the importance of the proximal region of the axoneme in generating flagellar bending. PMID:23427265

  13. The smallest active fragment of microtubule-associated protein 4 and its interaction with microtubules in phosphate buffer.

    PubMed

    Hashi, Yurika; Nagase, Lisa; Matsushima, Kazuyuki; Kotani, Susumu

    2012-01-01

    To analyze the interaction between microtubule-associated protein (MAP) 4 and microtubules physicochemically, a MAP4 active site fragment was designed for nuclear magnetic resonance (NMR) use. The fragment was bacterially expressed and purified to homogeneity. The buffer conditions for NMR were optimized to support microtubule assembly. The fragment was found to bind to microtubules under the optimized buffer conditions.

  14. Molecular architecture of the Dam1 complex–microtubule interaction

    PubMed Central

    Legal, Thibault; Zou, Juan; Sochaj, Alicja; Rappsilber, Juri

    2016-01-01

    Mitosis is a highly regulated process that allows the equal distribution of the genetic material to the daughter cells. Chromosome segregation requires the formation of a bipolar mitotic spindle and assembly of a multi-protein structure termed the kinetochore to mediate attachments between condensed chromosomes and spindle microtubules. In budding yeast, a single microtubule attaches to each kinetochore, necessitating robustness and processivity of this kinetochore–microtubule attachment. The yeast kinetochore-localized Dam1 complex forms a direct interaction with the spindle microtubule. In vitro, the Dam1 complex assembles as a ring around microtubules and couples microtubule depolymerization with cargo movement. However, the subunit organization within the Dam1 complex, its higher-order oligomerization and how it interacts with microtubules remain under debate. Here, we used chemical cross-linking and mass spectrometry to define the architecture and subunit organization of the Dam1 complex. This work reveals that both the C termini of Duo1 and Dam1 subunits interact with the microtubule and are critical for microtubule binding of the Dam1 complex, placing Duo1 and Dam1 on the inside of the ring structure. Integrating this information with available structural data, we provide a coherent model for how the Dam1 complex self-assembles around microtubules. PMID:26962051

  15. Modulation of Microtubule Interprotofilament Interactions by Modified Taxanes

    PubMed Central

    Matesanz, Ruth; Rodríguez-Salarichs, Javier; Pera, Benet; Canales, Ángeles; Andreu, José Manuel; Jiménez-Barbero, Jesús; Bras, Wim; Nogales, Aurora; Fang, Wei-Shuo; Díaz, José Fernando

    2011-01-01

    Microtubules assembled with paclitaxel and docetaxel differ in their numbers of protofilaments, reflecting modification of the lateral association between αβ-tubulin molecules in the microtubule wall. These modifications of microtubule structure, through a not-yet-characterized mechanism, are most likely related to the changes in tubulin-tubulin interactions responsible for microtubule stabilization by these antitumor compounds. We have used a set of modified taxanes to study the structural mechanism of microtubule stabilization by these ligands. Using small-angle x-ray scattering, we have determined how modifications in the shape and size of the taxane substituents result in changes in the interprotofilament angles and in their number. The observed effects have been explained using NMR-aided docking and molecular dynamic simulations of taxane binding at the microtubule pore and luminal sites. Modeling results indicate that modification of the size of substituents at positions C7 and C10 of the taxane core influence the conformation of three key elements in microtubule lateral interactions (the M-loop, the S3 β-strand, and the H3 helix) that modulate the contacts between adjacent protofilaments. In addition, modifications of the substituents at position C2 slightly rearrange the ligand in the binding site, modifying the interaction of the C7 substituent with the M-loop. PMID:22208196

  16. Direct interaction of microtubule- and actin-based transport motors

    NASA Technical Reports Server (NTRS)

    Huang, J. D.; Brady, S. T.; Richards, B. W.; Stenolen, D.; Resau, J. H.; Copeland, N. G.; Jenkins, N. A.

    1999-01-01

    The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

  17. FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas.

    PubMed

    Yanagisawa, Haru-aki; Mathis, Garrison; Oda, Toshiyuki; Hirono, Masafumi; Richey, Elizabeth A; Ishikawa, Hiroaki; Marshall, Wallace F; Kikkawa, Masahide; Qin, Hongmin

    2014-05-01

    The axoneme-the conserved core of eukaryotic cilia and flagella-contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy shows that the mutant axonemes lack the IJ, and cryo-electron tomography combined with a structural labeling method reveals that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of the axoneme and may be involved in planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component, because uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beak's localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra-B-tubular substructures required for a planar asymmetrical waveform.

  18. Interaction of chicken gizzard smooth muscle calponin with brain microtubules.

    PubMed

    Fujii, T; Hiromori, T; Hamamoto, M; Suzuki, T

    1997-08-01

    Calponin, a major actin-, tropomyosin-, and calmodulin-binding protein in smooth muscle, interacted with tubulin, a main constituent of microtubules, in a concentration-dependent fashion in vitro. The apparent K(d) value of calponin to tubulin was calculated to be 5.2 microM with 2 mol of calponin maximally bound per 1 mol of tubulin. At low ionic strength, tubulin bound to calponin immobilized on Sepharose 4B, and the bound protein was released at about 270 mM NaCl. Chemical cross-linking experiments showed that a 1:1 molar covalent complex of calponin and tubulin was produced. The amount of calponin bound to microtubules decreased with increasing ionic strength or Ca2+ concentration. The addition of calmodulin or S100 to the mixture of calponin and microtubule proteins caused the removal of calponin from microtubules in the presence of Ca2+, but not in the presence of EGTA. Calponin-related proteins including tropomyosin, SM22, and caldesmon had little effect on the calponin binding to microtubules, whereas MAP2 inhibited the binding. Interestingly, there was little, if any, effect of mycalolide B-treated actin on the binding of calponin to microtubules. Furthermore, only about 20% of calponin-F-actin interaction was inhibited in the presence of an excess amount of tubulin (4 mol per mol of calponin), indicating that tubulin binds to calponin at a different site from that of actin. Compared with MAP2, calponin had little effect on microtubule polymerization.

  19. A mathematical model of stress generation in microtubule pair interactions

    NASA Astrophysics Data System (ADS)

    Fang, Fang; Betterton, Meredith; Shelley, Michael

    2014-11-01

    Microtubules and motor proteins are basic ingredients in many cellular structures and of new biosynthetic ``active'' suspensions. The interaction of microtubules with their surrounding fluid medium depends fundamentally upon the force generation afforded them through cross-linking motile motor proteins. Here we develop a simple mathematical model, based on the statistical mechanics, motor proteins binding and unbinding, to study the generation of active fluid stresses. We study the role and contributions of ``polarity sorting'' and ``tether'' relaxation on the generation of intrinsic, destabilizing stresses.

  20. Computer simulations reveal motor properties generating stable antiparallel microtubule interactions.

    PubMed

    Nédélec, François

    2002-09-16

    An aster of microtubules is a set of flexible polar filaments with dynamic plus ends that irradiate from a common location at which the minus ends of the filaments are found. Processive soluble oligomeric motor complexes can bind simultaneously to two microtubules, and thus exert forces between two asters. Using computer simulations, I have explored systematically the possible steady-state regimes reached by two asters under the action of various kinds of oligomeric motors. As expected, motor complexes can induce the asters to fuse, for example when the complexes consist only of minus end-directed motors, or to fully separate, when the motors are plus end directed. More surprisingly, complexes made of two motors of opposite directionalities can also lead to antiparallel interactions between overlapping microtubules that are stable and sustained, like those seen in mitotic spindle structures. This suggests that such heterocomplexes could have a significant biological role, if they exist in the cell.

  1. Hydrophobic interaction of organic chemicals with microtubule assembly in vitro.

    PubMed

    Stoiber, Thomas; Unger, Eberhard; Dorn, Susanne B; Degen, Gisela H; Bolt, Hermann M

    2008-09-01

    A recent concept connecting the lipophilicity of organic chemicals with their genotoxicity on a chromosomal level implies that the lipophilic character of organic chemicals determines a certain background of chromosomal genotoxicity that can be addressed as "non-specific". This is opposed to compounds with more "specific" modes of action. Such mechanisms influence the processes of karyokinesis and cytokinesis. A critical partial process for the chromosomal segregation is the dynamics of assembly and disassembly of microtubules. To broaden the present database for such interactions, chemicals were selected based on their lipophilicity (log P between -1.5 and +1.0) and on hints from the literature pointing to possibilities of interaction with the tubulin-microtubule system. Thus, acetamide, acrylamide, methylmethane sulfonate, acetonitrile, acrylonitrile and cyclohexanone were assessed as to their potencies to influence the dynamic processes of microtubule assembly and disassembly in a cell-free system in vitro. These compounds covered a range of log P between -1.5 and 1.0, complementary to compounds investigated earlier. The entire body of data supports the general concept that hydrophobic interactions are connected with non-specific processes, which contribute to a background genotoxicity on a chromosomal level. It also points to the dynamics of microtubule assembly and disassembly as a decisive partial process involved.

  2. Kinetochore-Dependent Microtubule Rescue Ensures Their Efficient and Sustained Interactions in Early Mitosis

    PubMed Central

    Gandhi, Sapan R.; Gierliński, Marek; Mino, Akihisa; Tanaka, Kozo; Kitamura, Etsushi; Clayton, Lesley; Tanaka, Tomoyuki U.

    2011-01-01

    Summary How kinetochores regulate microtubule dynamics to ensure proper kinetochore-microtubule interactions is unknown. Here, we studied this during early mitosis in Saccharomyces cerevisiae. When a microtubule shrinks and its plus end reaches a kinetochore bound to its lateral surface, the microtubule end attempts to tether the kinetochore. This process often fails and, responding to this failure, microtubule rescue (conversion from shrinkage to growth) occurs, preventing kinetochore detachment from the microtubule end. This rescue is promoted by Stu2 transfer (ortholog of vertebrate XMAP215/ch-TOG) from the kinetochore to the microtubule end. Meanwhile, microtubule rescue distal to the kinetochore is also promoted by Stu2, which is transported by a kinesin-8 motor Kip3 along the microtubule from the kinetochore. Microtubule extension following rescue facilitates interaction with other widely scattered kinetochores, diminishing long delays in collecting the complete set of kinetochores by microtubules. Thus, kinetochore-dependent microtubule rescue ensures efficient and sustained kinetochore-microtubule interactions in early mitosis. PMID:22075150

  3. PACSIN1, a Tau-interacting protein, regulates axonal elongation and branching by facilitating microtubule instability.

    PubMed

    Liu, Yingying; Lv, Kaosheng; Li, Zenglong; Yu, Albert C H; Chen, Jianguo; Teng, Junlin

    2012-11-16

    Tau is a major member of the neuronal microtubule-associated proteins. It promotes tubulin assembly and stabilizes axonal microtubules. Previous studies have demonstrated that Tau forms cross-bridges between microtubules, with some particles located on cross-bridges, suggesting that some proteins interact with Tau and might be involved in regulating Tau-related microtubule dynamics. This study reports that PACSIN1 interacts with Tau in axon. PACSIN1 blockade results in impaired axonal elongation and a higher number of primary axonal branches in mouse dorsal root ganglia neurons, which is induced by increasing the binding ability of Tau to microtubules. In PACSIN1-blocked dorsal root ganglia neurons, a greater amount of Tau is inclined to accumulate in the central domain of growth cones, and it promotes the stability of the microtubule network. Taken together, these results suggest that PACSIN1 is an important Tau binding partner in regulating microtubule dynamics and forming axonal plasticity.

  4. Xenopus oocyte wound healing as a model system for analysis of microtubule-actin interactions.

    PubMed

    Zhang, Tong; Mandato, Craig A

    2007-01-01

    Microtubule-actin interactions are fundamental to many cellular processes such as cytokinesis and cellular locomotion. Investigating the mechanism of microtubule-actin interactions is the key to understand the cellular morphogenesis and related pathological processes. The abundance and highly dynamic nature of microtubules and F-actin raise a serious challenge when trying to distinguish between the real and fortuitous interactions within a cell. Xenopus oocyte wound model represents an ideal system to study microtubule-actin interactions as well as microtubule-dependent control of the actin polymerization. Here, we describe a series of cytoskeleton specific treatments in Xenopus oocyte wound healing experiments and use confocal fluorescence microscopy to analyze fixed oocytes to examine microtubule-actin interactions.

  5. The von Hippel-Lindau tumour suppressor interacts with microtubules through kinesin-2.

    PubMed

    Lolkema, Martijn P; Mans, Dorus A; Snijckers, Cristel M; van Noort, Mascha; van Beest, Moniek; Voest, Emile E; Giles, Rachel H

    2007-10-02

    Synthesis and maintenance of primary cilia are regulated by the von Hippel-Lindau (VHL) tumour suppressor protein. Recent studies indicate that this regulation is linked to microtubule-dependent functions of pVHL such as orienting microtubule growth and increasing plus-end microtubule stability, however little is known how this occurs. We have identified the kinesin-2 motor complex, known to regulate cilia, as a novel and endogenous pVHL binding partner. The interaction with kinesin-2 facilitates pVHL binding to microtubules. These data suggest that microtubule-dependent functions of pVHL are influenced by kinesin-2.

  6. Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling

    PubMed Central

    DeBonis, Salvatore; Neumann, Emmanuelle; Skoufias, Dimitrios A.

    2015-01-01

    TPPP/p25 is a microtubule-associated protein, detected in protein inclusions associated with various neurodegenerative diseases. Deletion analysis data show that TPPP/p25 has two microtubule binding sites, both located in intrinsically disordered domains, one at the N-terminal and the other in the C-terminal domain. In copolymerization assays the full-length protein exhibits microtubule stimulation and bundling activity. In contrast, at the same ratio relative to tubulin, truncated forms of TPPP/p25 exhibit either lower or no microtubule stimulation and no bundling activity, suggesting a cooperative phenomenon which is enhanced by the presence of the two binding sites. The binding characteristics of the N- and C-terminally truncated proteins to taxol-stabilized microtubules are similar to the full-length protein. However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules. Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors. The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules. PMID:26289831

  7. Dynamic regulation of cortical microtubule organization through prefoldin-DELLA interaction.

    PubMed

    Locascio, Antonella; Blázquez, Miguel A; Alabadí, David

    2013-05-06

    Plant morphogenesis relies on specific patterns of cell division and expansion. It is well established that cortical microtubules influence the direction of cell expansion, but less is known about the molecular mechanisms that regulate microtubule arrangement. Here we show that the phytohormones gibberellins (GAs) regulate microtubule orientation through physical interaction between the nuclear-localized DELLA proteins and the prefoldin complex, a cochaperone required for tubulin folding. In the presence of GA, DELLA proteins are degraded, and the prefoldin complex stays in the cytoplasm and is functional. In the absence of GA, the prefoldin complex is localized to the nucleus, which severely compromises α/β-tubulin heterodimer availability, affecting microtubule organization. The physiological relevance of this molecular mechanism was confirmed by the observation that the daily rhythm of plant growth was accompanied by coordinated oscillation of DELLA accumulation, prefoldin subcellular localization, and cortical microtubule reorientation.

  8. Microtubule bundling and shape transitions: Mechanics, interactions, and self-assemblies

    NASA Astrophysics Data System (ADS)

    Needleman, Daniel Joseph

    two arrays of hierarchically structured nanotubules occurs through a novel phase transition driven by a discrete conformational change in the constituent tubulin subunit. The microtubule associated protein tau may cause microtubules to bundle and the binding of tau seems to affect the microtubule structure. The interaction of tau with microtubules is strongly dependent on the particular tau isoform.

  9. Interaction of an Overexpressed gamma-Tubulin with Microtubules In Vivo and In Vitro.

    PubMed

    Kofron, M; Nadezdina, E; Vassilev, A; Matuliene, J; Essner, R; Kato, J; Kuriyama, R

    1998-08-01

    gamma-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for gamma-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus gamma-tubulin. When CHO cells were transiently transfected with the gamma-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such gamma-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that gamma-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas gamma-tubulin purified from insect Sf9 cells (), interaction between gamma-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with gamma-tubulin monomers in vitro were associated with more gold particles conjugated with gamma-tubulin than in controls where no exogenous gamma-tubulin was added. However, binding of gamma-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although gamma-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (; ), which might be ascribed to the difference in the level of protein expression in transfected cells.

  10. Probing interactions between CLIP-170, EB1, and microtubules

    PubMed Central

    Gupta, Kamlesh K.; Joyce, Michelle V.; Slabbekoorn, Aranda R.; Zhu, Zhiqing; Paulson, Benjamin A.; Boggess, Bill; Goodson, Holly V.

    2010-01-01

    CLIP-170 is a microtubule (MT) plus-end tracking protein (+TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other +TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of β-tubulin. However, the observation that CLIP-170 has two-CAP-Gly motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α- and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α- and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to EB1 via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and the MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the +TIP network. PMID:19913027

  11. Microtubule Dynamics Interacting with Stabilizing Agents in a Cell-like Environment

    NASA Astrophysics Data System (ADS)

    Shojania Feizabadi, Mitra

    2010-03-01

    Microtubules, key components of the cytoskeleton, are involved in several biological functions. They are highly dynamic polymers that stochastically switch between growing and shrinking phases. Due to their critical role in the process of cell division, they are the target of anti-cancer drugs. Antimitotic drugs usually suppress the dynamic instability of microtubules and, therefore, affect the process of cell division. In this work, the dynamic of microtubules interacting with catastrophe suppressing drugs as stabilizing agents, introduced by Mishra et al. (Phys. Rev. E. 72, 51914, 2005), is modified in a confined geometry and associated with the obtained and analyzed steady state solutions.

  12. Models of Yukawa interaction in the two Higgs doublet model, and their collider phenomenology

    SciTech Connect

    Aoki, Mayumi; Kanemura, Shinya; Yagyu, Kei; Tsumura, Koji

    2009-07-01

    Possible models of Yukawa interaction are discussed in the two Higgs doublet model (THDM) under the discrete symmetry imposed to avoid the flavor changing neutral current at the leading order. It is known that there are four types of such models corresponding to the possible different assignment of charges for the discrete symmetry on quarks and leptons. We first examine the decay properties of Higgs bosons in each type model, and summarize constraints on the models from current experimental data. We then shed light on the differences among these models in collider phenomenology. In particular, we mainly discuss the so-called type-II THDM and type-X THDM. The type-II THDM corresponds to the model with the same Yukawa interaction as the minimal supersymmetric standard model. On the other hand, in the type-X THDM, additional Higgs bosons can predominantly decay into leptons. This scenario may be interesting because of the motivation for a light charged Higgs boson scenario such as in the TeV-scale model of neutrinos, dark matter, and baryogenesis. We study how we can distinguish the type-X THDM from the minimal supersymmetric standard model at the Large Hadron Collider and the International Linear Collider.

  13. Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

    PubMed Central

    Bouchet, Benjamin P; Gough, Rosemarie E; Ammon, York-Christoph; van de Willige, Dieudonnée; Post, Harm; Jacquemet, Guillaume; Altelaar, AF Maarten; Heck, Albert JR; Goult, Benjamin T; Akhmanova, Anna

    2016-01-01

    The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules. DOI: http://dx.doi.org/10.7554/eLife.18124.001 PMID:27410476

  14. Probing protein interactions in living mammalian cells on a microtubule bench

    PubMed Central

    Boca, Mirela; Kretov, Dmitry A.; Desforges, Bénédicte; Mephon-Gaspard, Alix; Curmi, Patrick A.; Pastré, David

    2015-01-01

    Microtubules are μm-long cylinders of about 25 nm in diameter which are present in the cytoplasm of eukaryotic cells. Here, we have developed a new method which uses these cylindrical structures as platforms to detect protein interactions in cells. The principle is simple: a protein of interest used as bait is brought to microtubules by fusing it to Tau, a microtubule-associated protein. The presence of a protein prey on microtubules then reveals an interaction between bait and prey. This method requires only a conventional optical microscope and straightforward fluorescence image analysis for detection and quantification of protein interactions. To test the reliability of this detection scheme, we used it to probe the interactions among three mRNA-binding proteins in both fixed and living cells and compared the results to those obtained by pull-down assays. We also tested whether the molecular interactions of Cx43, a membrane protein, can be investigated with this system. Altogether, the results indicate that microtubules can be used as platforms to detect protein interactions in mammalian cells, which should provide a basis for investigating pathogenic protein interactions involved in human diseases. PMID:26610591

  15. Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons.

    PubMed

    Janning, Dennis; Igaev, Maxim; Sündermann, Frederik; Brühmann, Jörg; Beutel, Oliver; Heinisch, Jürgen J; Bakota, Lidia; Piehler, Jacob; Junge, Wolfgang; Brandt, Roland

    2014-11-05

    The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP-tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin-microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport.

  16. The femtomolar-acting NAP interacts with microtubules: Novel aspects of astrocyte protection.

    PubMed

    Gozes, Illana; Divinski, Inna

    2004-12-01

    Activity-dependent neuroprotective protein (ADNP), a gene product essential for brain formation, contains a short octapeptide sequence NAPVSIPQ (NAP) that protects neurons against a wide variety of insults. At the pico-molar concentration range, NAP provides neuroprotection by direct interaction with neurons. At the femtomolar concentration range, NAP requires the presence of glial cells to provide neuroprotection. To further understand the mechanism of neuroprotection afforded by NAP, specific binding proteins were searched for. Tubulin, the major subunit protein of microtubules, was identified as a NAP binding molecule. NAP structure allows membrane penetration, followed by tubulin binding and facilitation of microtubule assembly toward cellular protection in astrocytes. NAP (10(-15) M) promoted microtubule assembly in vitro and protected astrocytes against zinc intoxication which is associated with microtubule disruption. A two hour incubation period of astrocytes with femtomolar concentrations of NAP resulted in microtubule re-organization and transient increases in immunoreactive non-phosphorylated tau. Microtubules are the key component of the neuronal and glial cytoskeleton that regulates cell division, differentiation and protection, while tau pathology is a major contributor to Alzheimer's disease and other dementias. The findings described here may open up new horizons in research and development of neuroprotective compounds.

  17. Tobacco mosaic virus movement protein interacts with green fluorescent protein-tagged microtubule end-binding protein 1.

    PubMed

    Brandner, Katrin; Sambade, Adrian; Boutant, Emmanuel; Didier, Pascal; Mély, Yves; Ritzenthaler, Christophe; Heinlein, Manfred

    2008-06-01

    The targeting of the movement protein (MP) of Tobacco mosaic virus to plasmodesmata involves the actin/endoplasmic reticulum network and does not require an intact microtubule cytoskeleton. Nevertheless, the ability of MP to facilitate the cell-to-cell spread of infection is tightly correlated with interactions of the protein with microtubules, indicating that the microtubule system is involved in the transport of viral RNA. While the MP acts like a microtubule-associated protein able to stabilize microtubules during late infection stages, the protein was also shown to cause the inactivation of the centrosome upon expression in mammalian cells, thus suggesting that MP may interact with factors involved in microtubule attachment, nucleation, or polymerization. To further investigate the interactions of MP with the microtubule system in planta, we expressed the MP in the presence of green fluorescent protein (GFP)-fused microtubule end-binding protein 1a (EB1a) of Arabidopsis (Arabidopsis thaliana; AtEB1a:GFP). The two proteins colocalize and interact in vivo as well as in vitro and exhibit mutual functional interference. These findings suggest that MP interacts with EB1 and that this interaction may play a role in the associations of MP with the microtubule system during infection.

  18. Arabidopsis phospholipase D alpha 1-derived phosphatidic acid regulates microtubule organization and cell development under microtubule-interacting drugs treatment.

    PubMed

    Zhang, Qun; Qu, Yana; Wang, Qing; Song, Ping; Wang, Peipei; Jia, Qianru; Guo, Jinhe

    2017-01-01

    Phospholipase D (PLD) and its product phosphatidic acid (PA) are emerging as essential regulators of cytoskeleton organization in plants. However, the underlying molecular mechanisms of PA-mediated microtubule reorganization in plants remain largely unknown. In this study, we used pharmacological and genetic approaches to analyze the function of Arabidopsis thaliana PLDα1 in the regulation of microtubule organization and cell development in response to microtubule-affecting drugs. Treatment with the microtubule-stabilizing drug paclitaxel resulted in less growth inhibition and decreased rightward slant of roots, longitudinal alignment of microtubules, and enhanced length of hypocotyl epidermal cells in the pldα1 mutant, the phenotype of which was rescued by exogenous application of PA. Moreover, the pldα1 mutant was sensitive to the microtubule-disrupting drugs oryzalin and propyzamide in terms of seedling survival ratio, left-skewing angle of roots and microtubule organization. In addition, both disruption and stabilization of microtubules induced by drugs activated PLDα1 activity. Our findings demonstrate that in A. thaliana, PLDα1/PA might regulate cell development by modulating microtubule organization in an activity-dependent manner.

  19. Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster.

    PubMed

    Henríquez, J P; Cambiazo, V; Maccioni, R B

    1996-05-24

    The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequential affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behaviour of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.

  20. Meiosis I chromosome segregation is established through regulation of microtubule-kinetochore interactions.

    PubMed

    Miller, Matthew P; Unal, Elçin; Brar, Gloria A; Amon, Angelika

    2012-12-18

    During meiosis, a single round of DNA replication is followed by two consecutive rounds of nuclear divisions called meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate, while sister chromatids remain together. Determining how this unusual chromosome segregation behavior is established is central to understanding germ cell development. Here we show that preventing microtubule-kinetochore interactions during premeiotic S phase and prophase I is essential for establishing the meiosis I chromosome segregation pattern. Premature interactions of kinetochores with microtubules transform meiosis I into a mitosis-like division by disrupting two key meiosis I events: coorientation of sister kinetochores and protection of centromeric cohesin removal from chromosomes. Furthermore we find that restricting outer kinetochore assembly contributes to preventing premature engagement of microtubules with kinetochores. We propose that inhibition of microtubule-kinetochore interactions during premeiotic S phase and prophase I is central to establishing the unique meiosis I chromosome segregation pattern.DOI:http://dx.doi.org/10.7554/eLife.00117.001.

  1. Structural basis of inter-protofilament interaction and lateral deformation of microtubules

    PubMed Central

    Sui, Haixin; Downing, Kenneth H.

    2010-01-01

    Summary The diverse functions of microtubules require stiff structures possessing sufficient lateral flexibility to enable bending with high curvature. We used cryo-electron microscopy to investigate the molecular basis for these critical mechanical properties. High-quality structural maps were used to build pseudo-atomic models of microtubules containing 11 to 16 protofilaments, representing a wide range of lateral curvature. Protofilaments in all these microtubules were connected primarily via inter-protofilament interactions between the M loops, and the H1′-S2 and H2-S3 loops. We postulate that the tolerance of the loop-loop interactions to lateral deformation provides the capacity for high-curvature bending without breaking. On the other hand, the local molecular architecture that surrounds these connecting loops contributes to the overall rigidity. Inter-protofilament interactions in the seam region are similar to those in the normal helical regions, suggesting that the existence of the seam does not significantly affect the mechanical properties of microtubules. PMID:20696402

  2. Signaling Scaffold Protein IQGAP1 Interacts with Microtubule Plus-end Tracking Protein SKAP and Links Dynamic Microtubule Plus-end to Steer Cell Migration*

    PubMed Central

    Cao, Dan; Su, Zeqi; Wang, Wenwen; Wu, Huihui; Liu, Xing; Akram, Saima; Qin, Bo; Zhou, Jiajia; Zhuang, Xiaoxuan; Adams, Gregory; Jin, Changjiang; Wang, Xiwei; Liu, Lifang; Hill, Donald L.; Wang, Dongmei; Ding, Xia; Yao, Xuebiao

    2015-01-01

    Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex. PMID:26242911

  3. DAPK interacts with Patronin and the microtubule cytoskeleton in epidermal development and wound repair.

    PubMed

    Chuang, Marian; Hsiao, Tiffany I; Tong, Amy; Xu, Suhong; Chisholm, Andrew D

    2016-09-23

    Epidermal barrier epithelia form a first line of defense against the environment, protecting animals against infection and repairing physical damage. In C. elegans, death-associated protein kinase (DAPK-1) regulates epidermal morphogenesis, innate immunity and wound repair. Combining genetic suppressor screens and pharmacological tests, we find that DAPK-1 maintains epidermal tissue integrity through regulation of the microtubule (MT) cytoskeleton. dapk-1 epidermal phenotypes are suppressed by treatment with microtubule-destabilizing drugs and mimicked or enhanced by microtubule-stabilizing drugs. Loss of function in ptrn-1, the C. elegans member of the Patronin/Nezha/CAMSAP family of MT minus-end binding proteins, suppresses dapk-1 epidermal and innate immunity phenotypes. Over-expression of the MT-binding CKK domain of PTRN-1 triggers epidermal and immunity defects resembling those of dapk-1 mutants, and PTRN-1 localization is regulated by DAPK-1. DAPK-1 and PTRN-1 physically interact in co-immunoprecipitation experiments, and DAPK-1 itself undergoes MT-dependent transport. Our results uncover an unexpected interdependence of DAPK-1 and the microtubule cytoskeleton in maintenance of epidermal integrity.

  4. DAPK interacts with Patronin and the microtubule cytoskeleton in epidermal development and wound repair

    PubMed Central

    Chuang, Marian; Hsiao, Tiffany I; Tong, Amy; Xu, Suhong; Chisholm, Andrew D

    2016-01-01

    Epidermal barrier epithelia form a first line of defense against the environment, protecting animals against infection and repairing physical damage. In C. elegans, death-associated protein kinase (DAPK-1) regulates epidermal morphogenesis, innate immunity and wound repair. Combining genetic suppressor screens and pharmacological tests, we find that DAPK-1 maintains epidermal tissue integrity through regulation of the microtubule (MT) cytoskeleton. dapk-1 epidermal phenotypes are suppressed by treatment with microtubule-destabilizing drugs and mimicked or enhanced by microtubule-stabilizing drugs. Loss of function in ptrn-1, the C. elegans member of the Patronin/Nezha/CAMSAP family of MT minus-end binding proteins, suppresses dapk-1 epidermal and innate immunity phenotypes. Over-expression of the MT-binding CKK domain of PTRN-1 triggers epidermal and immunity defects resembling those of dapk-1 mutants, and PTRN-1 localization is regulated by DAPK-1. DAPK-1 and PTRN-1 physically interact in co-immunoprecipitation experiments, and DAPK-1 itself undergoes MT-dependent transport. Our results uncover an unexpected interdependence of DAPK-1 and the microtubule cytoskeleton in maintenance of epidermal integrity. DOI: http://dx.doi.org/10.7554/eLife.15833.001 PMID:27661253

  5. Kinetochore-microtubule error correction is driven by differentially regulated interaction modes.

    PubMed

    Kalantzaki, Maria; Kitamura, Etsushi; Zhang, Tongli; Mino, Akihisa; Novák, Béla; Tanaka, Tomoyuki U

    2015-04-01

    For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore-microtubule attachments are disrupted (error correction) by aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still formed efficiently to enable fresh attempts at bi-orientation. How this is possible remains an enigma. Here we show that kinetochore attachment to the microtubule lattice (lateral attachment) is impervious to aurora B regulation, but attachment to the microtubule plus end (end-on attachment) is disrupted by this kinase. Thus, a new lateral attachment is formed without interference, then converted to end-on attachment and released if incorrect. This process continues until bi-orientation is established and stabilized by tension across sister kinetochores. We reveal how aurora B specifically promotes disruption of the end-on attachment through phospho-regulation of kinetochore components Dam1 and Ndc80. Our results reveal fundamental mechanisms for promoting error correction for bi-orientation.

  6. The spindle matrix protein, Chromator, is a novel tubulin binding protein that can interact with both microtubules and free tubulin.

    PubMed

    Yao, Changfu; Wang, Chao; Li, Yeran; Ding, Yun; Rath, Uttama; Sengupta, Saheli; Girton, Jack; Johansen, Kristen M; Johansen, Jørgen

    2014-01-01

    The chromodomain protein, Chromator, is localized to chromosomes during interphase; however, during cell division together with other nuclear proteins Chromator redistributes to form a macro molecular spindle matrix complex that embeds the microtubule spindle apparatus. It has been demonstrated that the CTD of Chromator is sufficient for localization to the spindle matrix and that expression of this domain alone could partially rescue Chro mutant microtubule spindle defects. Furthermore, the presence of frayed and unstable microtubule spindles during mitosis after Chromator RNAi depletion in S2 cells indicated that Chromator may interact with microtubules. In this study using a variety of biochemical assays we have tested this hypothesis and show that Chromator not only has binding activity to microtubules with a Kd of 0.23 µM but also to free tubulin. Furthermore, we have mapped the interaction with microtubules to a relatively small stretch of 139 amino acids in the carboxy-terminal region of Chromator. This sequence is likely to contain a novel microtubule binding interface since database searches did not find any sequence matches with known microtubule binding motifs.

  7. Growth, interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells.

    PubMed

    Mitchison, Timothy; Wühr, Martin; Nguyen, Phuong; Ishihara, Keisuke; Groen, Aaron; Field, Christine M

    2012-10-01

    Ray Rappaport spent many years studying microtubule asters, and how they induce cleavage furrows. Here, we review recent progress on aster structure and dynamics in zygotes and early blastomeres of Xenopus laevis and Zebrafish, where cells are extremely large. Mitotic and interphase asters differ markedly in size, and only interphase asters span the cell. Growth of interphase asters occurs by a mechanism that allows microtubule density at the aster periphery to remain approximately constant as radius increases. We discuss models for aster growth, and favor a branching nucleation process. Neighboring asters that grow into each other interact to block further growth at the shared boundary. We compare the morphology of interaction zones formed between pairs of asters that grow out from the poles of the same mitotic spindle (sister asters) and between pairs not related by mitosis (non-sister asters) that meet following polyspermic fertilization. We argue growing asters recognize each other by interaction between antiparallel microtubules at the mutual boundary, and discuss models for molecular organization of interaction zones. Finally, we discuss models for how asters, and the centrosomes within them, are positioned by dynein-mediated pulling forces so as to generate stereotyped cleavage patterns. Studying these problems in extremely large cells is starting to reveal how general principles of cell organization scale with cell size.

  8. Microtubule-associated proteins and tubulin interaction by isothermal titration calorimetry.

    PubMed

    Tsvetkov, P O; Barbier, P; Breuzard, G; Peyrot, V; Devred, F

    2013-01-01

    Microtubules play an important role in a number of vital cell processes such as cell division, intracellular transport, and cell architecture. The highly dynamic structure of microtubules is tightly regulated by a number of stabilizing and destabilizing microtubule-associated proteins (MAPs), such as tau and stathmin. Because of their importance, tubulin-MAPs interactions have been extensively studied using various methods that provide researchers with complementary but sometimes contradictory thermodynamic data. Isothermal titration calorimetry (ITC) is the only direct thermodynamic method that enables a full thermodynamic characterization (stoichiometry, enthalpy, entropy of binding, and association constant) of the interaction after a single titration experiment. This method has been recently applied to study tubulin-MAPs interactions in order to bring new insights into molecular mechanisms of tubulin regulation. In this chapter, we review the technical specificity of this method and then focus on the use of ITC in the investigation of tubulin-MAPs binding. We describe technical issues which could arise during planning and carrying out the ITC experiments, in particular with fragile proteins such as tubulin. Using examples of stathmin and tau, we demonstrate how ITC can be used to gain major insights into tubulin-MAP interaction.

  9. Centaurin-α₂ interacts with β-tubulin and stabilizes microtubules.

    PubMed

    Zuccotti, Paola; Cartelli, Daniele; Stroppi, Michela; Pandini, Vittorio; Venturin, Marco; Aliverti, Alessandro; Battaglioli, Elena; Cappelletti, Graziella; Riva, Paola

    2012-01-01

    Centaurin-α₂ is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α₂ can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α₂ interacting protein, β-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of β-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α₂ overexpression in HeLa cells and extraction of soluble (αβ dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α₂ mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α₂ remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α₂ and tubulin confirmed that Centaurin-α₂ promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α₂ overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α₂ role on MT stabilization. Centaurin-α₂ interacts with β-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α₂ as a new microtubule-associated protein (MAP) increasing MT stability.

  10. The hepatitis E virus open reading frame 3 product interacts with microtubules and interferes with their dynamics.

    PubMed

    Kannan, Harilakshmi; Fan, Sumin; Patel, Deendayal; Bossis, Ioannis; Zhang, Yan-Jin

    2009-07-01

    Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated alpha-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.

  11. Precise and Reversible Protein-Microtubule-Like Structure with Helicity Driven by Dual Supramolecular Interactions.

    PubMed

    Yang, Guang; Zhang, Xiang; Kochovski, Zdravko; Zhang, Yufei; Dai, Bin; Sakai, Fuji; Jiang, Lin; Lu, Yan; Ballauff, Matthias; Li, Xueming; Liu, Cong; Chen, Guosong; Jiang, Ming

    2016-02-17

    Protein microtubule is a significant self-assembled architecture found in nature with crucial biological functions. However, mimicking protein microtubules with precise structure and controllable self-assembly behavior remains highly challenging. In this work, we demonstrate that by using dual supramolecular interactions from a series of well-designed ligands, i.e., protein-sugar interaction and π-π stacking, highly homogeneous protein microtubes were achieved from tetrameric soybean agglutinin without any chemical or biological modification. Using combined cryo-EM single-particle reconstruction and computational modeling, the accurate structure of protein microtube was determined. The helical protein microtube is consisted of three protofilaments, each of which features an array of soybean agglutinin tetramer linked by the designed ligands. Notably, the microtubes resemble the natural microtubules in their structural and dynamic features such as the shape and diameter and the controllable and reversible assembly behavior, among others. Furthermore, the protein microtubes showed an ability to enhance immune response, demonstrating its great potential for biological applications.

  12. Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells

    PubMed Central

    Dugina, Vera; Alieva, Irina; Khromova, Natalya; Kireev, Igor; Gunning, Peter W.; Kopnin, Pavel

    2016-01-01

    Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells. PMID:27683037

  13. Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells.

    PubMed

    Dugina, Vera; Alieva, Irina; Khromova, Natalya; Kireev, Igor; Gunning, Peter W; Kopnin, Pavel

    2016-11-08

    Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells.

  14. Pattern formation of microtubules and motors: Inelastic interaction of polar rods

    NASA Astrophysics Data System (ADS)

    Aranson, Igor S.; Tsimring, Lev S.

    2005-05-01

    We derive a model describing spatiotemporal organization of an array of microtubules interacting via molecular motors. Starting from a stochastic model of inelastic polar rods with a generic anisotropic interaction kernel we obtain a set of equations for the local rods concentration and orientation. At large enough mean density of rods and concentration of motors, the model describes orientational instability. We demonstrate that the orientational instability leads to the formation of vortices and (for large density and/or kernel anisotropy) asters seen in recent experiments.

  15. Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis[OPEN

    PubMed Central

    Buschmann, Henrik; Lloyd, Clive W.

    2015-01-01

    Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric 14N/15N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning. PMID:26432860

  16. The 1997 Kagoshima (Japan) Earthquake Doublet: A Quantitative Analysis of Stress Interaction

    NASA Astrophysics Data System (ADS)

    Woessner, J.; Hauksson, E.; Wiemer, S.; Neukomm, S.

    2003-12-01

    Understanding how the nucleation of earthquakes is affected by sudden changes in the state of stress in their immediate vicinity may provide insight into the elusive relationship between static stress changes and earthquake occurrence. As working hypothesis, we assume that if aftershocks are in part caused by stress changes from their mainshock, changes in their decay rate may reflect changes in the state of stress induced by nearby large earthquakes. The 1997 Kagoshima (Japan) earthquake doublet provides a unique opportunity to analyze this hypothesis for two moderate M6 events that occurred on adjacent faults with the epicenters located in a distance of about 5 km from each other. We map the Omori law parameters on an equally spaced grid for the time period between the two mainshocks (the learning period of 47.8 days) using four Omori law type models with increasing complexity. The best fitting model is found using the corrected Akaike Criterion Information. We then forecast the number of aftershocks for the next 50 days and compare it to the actual observed number. Uncertainties in the rate forecasts are obtained by a bootstrap approach, allowing us to compute a detailed map of the significance of the relative rate changes. We find four regions with highly significant relative rate changes, three negative and one positive, which are a consequence of the activation and deactivation of aftershock activity due to the second mainshock, respectively. While our results confirm a relative rate drop in the Western part of the aftershock sequence that is in agreement with the stress triggering hypothesis (Stein, 2003), the other changes cannot be readily explained. Because of the importance of rate changes for the evaluation of the stress triggering hypothesis as well as for rate and state friction models, we consider our quantitative analysis technique introduced here important and a step forward in the process of understanding the behavior of aftershocks.

  17. Mapping the bound conformation and protein interactions of microtubule destabilizing peptides by STD-NMR spectroscopy.

    PubMed

    Milton, Mark J; Thomas Williamson, R; Koehn, Frank E

    2006-08-15

    Using the hemiasterlin analogs taltobulin (I, HTI-286), II, and III as model compounds, we demonstrate that relaxation-compensated STD-NMR can be used as an effective tool to efficiently provide a qualitative epitope map for microtubule destabilizing peptides. Due to the disparate relaxation behavior of the protons in these model compounds, it was essential to collect STD with very short saturation times to render an accurate picture of the binding interaction. The conformation of HTI-286 (I) in complex with the protein was determined from TRNOESY/ROESY experiments and is similar to the X-ray crystal structure conformation observed for hemiasterlin methyl ester in the absence of protein.

  18. EB1 acetylation by P300/CBP-associated factor (PCAF) ensures accurate kinetochore–microtubule interactions in mitosis

    PubMed Central

    Xia, Peng; Wang, Zhikai; Liu, Xing; Wu, Bing; Wang, Juncheng; Ward, Tarsha; Zhang, Liangyu; Ding, Xia; Gibbons, Gary; Shi, Yunyu; Yao, Xuebiao

    2012-01-01

    In eukaryotes, microtubules are essential for cellular plasticity and dynamics. Here we show that P300/CBP-associated factor (PCAF), a kinetochore-associated acetyltransferase, acts as a negative modulator of microtubule stability through acetylation of EB1, a protein that controls the plus ends of microtubules. PCAF acetylates EB1 on K220 and disrupts the stability of a hydrophobic cavity on the dimerized EB1 C terminus, which was previously reported to interact with plus-end tracking proteins (TIPs) containing the SxIP motif. As determined with an EB1 acetyl-K220–specific antibody, K220 acetylation is dramatically increased in mitosis and localized to the spindle microtubule plus ends. Surprisingly, persistent acetylation of EB1 delays metaphase alignment, resulting in impaired checkpoint silencing. Consequently, suppression of Mad2 overrides mitotic arrest induced by persistent EB1 acetylation. Thus, our findings identify dynamic acetylation of EB1 as a molecular mechanism to orchestrate accurate kinetochore–microtubule interactions in mitosis. These results establish a previously uncharacterized regulatory mechanism governing localization of microtubule plus-end tracking proteins and thereby the plasticity and dynamics of cells. PMID:23001180

  19. Mechanism of action of antitumor drugs that interact with microtubules and tubulin.

    PubMed

    Jordan, M A

    2002-01-01

    Microtubules, major structural components in cells, are the target of a large and diverse group of natural product anticancer drugs. Given the success of this class of drugs in cancer treatment, it can be argued that microtubules represent the single best cancer target identified to date. Microtubules are highly dynamic assemblies of the protein tubulin. They readily polymerize and depolymerize in cells, and they undergo two interesting kinds of dynamics called dynamic instability and treadmilling. These dynamic behaviors are crucial to mitosis, the process of chromosomal division to form new cells. Microtubule dynamics are highly regulated during the cell cycle by endogenous cellular regulators. In addition, many antitumor drugs and natural compounds alter the polymerization dynamics of microtubules, blocking mitosis, and consequently, inducing cell death by apoptosis. These drugs include several that inhibit microtubule polymerization at high drug concentrations, namely, the Vinca alkaloids, cryptophycins, halichondrins, estramustine, and colchicine. Another group of these compounds stimulates microtubule polymerization and stabilizes microtubules at high concentrations. These include Taxol, Taxotere, eleutherobins, epothilones, laulimalide, sarcodictyins, and discodermolide. Importantly, considerable evidence indicates that, at lower concentrations, these drugs have a common mechanism of action; they suppress the dynamics of microtubules without appreciably changing the mass of microtubules in the cell. The drugs bind to diverse sites on tubulin and at different positions within the microtubule, and they have diverse effects on microtubule dynamics. However, by their common mechanism of suppression microtubule dynamics, they all block mitosis at the metaphase/anaphase transition, and induce cell death.

  20. Effect of repulsive interactions on the rate of doublet formation of colloidal nanoparticles in the presence of convective transport.

    PubMed

    Lattuada, Marco; Morbidelli, Massimo

    2011-03-01

    In this work, we have performed a systematic investigation of the effect of electrostatic repulsive interactions on the aggregation rate of colloidal nanoparticles to from doublets in the presence of a convective transport mechanism. The aggregation rate has been computed by solving numerically the Fuchs-Smoluchowski diffusion-convection equation. Two convective transport mechanisms have been considered: extensional flow field and gravity-induced relative sedimentation. A broad range of conditions commonly encountered in the applications of colloidal dispersions has been analyzed. The relative importance of convective to diffusive contributions has been quantified by using the Peclet number Pe. The simulation results indicate that, in the presence of repulsive interactions, the evolution of the aggregation rate as a function of Pe can always be divided into three distinct regimes, no matter which convective mechanism is considered. At low Pe values the rate of aggregation is independent of convection and is dominated by repulsive interactions. At high Pe values, the rate of aggregation is dominated by convection, and independent of repulsive interactions. At intermediate Pe values, a sharp transition between these two regimes occurs. During this transition, which occurs usually over a 10-100-fold increase in Pe values, the aggregation rate can change by several orders of magnitude. The interval of Pe values where this transition occurs depends upon the nature of the convective transport mechanism, as well as on the height and characteristic lengthscale of the repulsive barrier. A simplified model has been proposed that is capable of quantitatively accounting for the simulations results. The obtained results reveal unexpected features of the effect of ionic strength and particle size on the stability of colloidal suspensions under shear or sedimentation, which have relevant consequences in industrial applications.

  1. TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana

    PubMed Central

    Tian, Juan; Wang, Xiaohong; Mao, Tonglin; Yuan, Ming; Li, Yunhai

    2016-01-01

    How cell shape is controlled is a fundamental question in developmental biology, but the genetic and molecular mechanisms that determine cell shape are largely unknown. Arabidopsis trichomes have been used as a good model system to investigate cell shape at the single-cell level. Here we describe the trichome cell shape 1 (tcs1) mutants with the reduced trichome branch number in Arabidopsis. TCS1 encodes a coiled-coil domain-containing protein. Pharmacological analyses and observations of microtubule dynamics show that TCS1 influences the stability of microtubules. Biochemical analyses and live-cell imaging indicate that TCS1 binds to microtubules and promotes the assembly of microtubules. Further results reveal that TCS1 physically associates with KCBP/ZWICHEL, a microtubule motor involved in the regulation of trichome branch number. Genetic analyses indicate that kcbp/zwi is epistatic to tcs1 with respect to trichome branch number. Thus, our findings define a novel genetic and molecular mechanism by which TCS1 interacts with KCBP to regulate trichome cell shape by influencing the stability of microtubules. PMID:27768706

  2. Large-scale pattern formation in active particles suspensions: from interacting microtubules to swimming bacteria

    NASA Astrophysics Data System (ADS)

    Aranson, Igor

    2006-03-01

    We consider two biological systems of active particles exhibiting large-scale collective behavior: microtubules interacting with molecular motors and hydrodynamically entrained swimming bacteria. Starting from a generic stochastic microscopic model of inelastically colliding polar rods with an anisotropic interaction kernel, we derive set of equations for the local rods concentration and orientation. Above certain critical density of rods the model exhibits orientational instability and onset of large-scale coherence. For the microtubules and molecular motors system we demonstrate that the orientational instability leads to the formation of vortices and asters seen in recent experiments. Similar approach is applied to colonies of swimming bacteria Bacillus subtilis confined in thin fluid film. The model is formulated in term of two-dimensional equations for local density and orientation of bacteria coupled to the low Reynolds number Navier-Stokes equation for the fluid flow velocity. The collective swimming of bacteria is represented by additional source term in the Navier-Stokes equation. We demonstrate that this system exhibits formation of dynamic large-scale patterns with the typical scale determined by the density of bacteria.

  3. Valley spin-orbit interaction for the triplet and doublet 1sground states of lithium donor center in monoisotopic {sup 28}Si

    SciTech Connect

    Ezhevskii, Alexander A.; Popkov, Sergey A.; Soukhorukov, Andrey V.; Guseinov, Davud V.; Konakov, Anton A.; Abrosimov, Nikolai V.; Riemann, Helge

    2013-12-04

    Valley spin-orbit interaction for the triplet and doublet 1s-ground states of lithium donor center in monoisotopic {sup 28}Si was studied in order to determine its contribution to the electron spin relaxation rate. We observed new electron paramagnetic resonance spectra of lithium in monoisotopic silicon with g<2.000 and found the spin Hamiltonian parameters for it. Using our experimental results and taking into account spin-orbit coupling between the triplet states and the triplet and doublet states we found that the lithium donor electron spectrum and g-factors for its states strongly depend on both the internal strains in the crystal and the intervalley spin-orbit interactions.

  4. Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance

    PubMed Central

    Tischfield, Max A.; Baris, Hagit N.; Wu, Chen; Rudolph, Guenther; Van Maldergem, Lionel; He, Wei; Chan, Wai-Man; Andrews, Caroline; Demer, Joseph L.; Robertson, Richard L.; Mackey, David A.; Ruddle, Jonathan B.; Bird, Thomas D.; Gottlob, Irene; Pieh, Christina; Traboulsi, Elias I.; Pomeroy, Scott L.; Hunter, David G.; Soul, Janet S.; Newlin, Anna; Sabol, Louise J.; Doherty, Edward J.; de Uzcátegui, Clara E.; de Uzcátegui, Nicolas; Collins, Mary Louise Z.; Sener, Emin C.; Wabbels, Bettina; Hellebrand, Heide; Meitinger, Thomas; de Berardinis, Teresa; Magli, Adriano; Schiavi, Costantino; Pastore-Trossello, Marco; Koc, Feray; Wong, Agnes M.; Levin, Alex V.; Geraghty, Michael T.; Descartes, Maria; Flaherty, Maree; Jamieson, Robyn V.; Møller, H. U.; Meuthen, Ingo; Callen, David F.; Kerwin, Janet; Lindsay, Susan; Meindl, Alfons; Gupta, Mohan L.; Pellman, David; Engle, Elizabeth C.

    2011-01-01

    We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific β-tubulin isotype III, result in a spectrum of human nervous system disorders we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves, and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate normal TUBB3 is required for axon guidance and maintenance in mammals. PMID:20074521

  5. Genetic Interactions among Chlamydomonas Reinhardtii Mutations That Confer Resistance to anti-Microtubule Herbicides

    PubMed Central

    James, S. W.; Lefebvre, P. A.

    1992-01-01

    We previously described two types of genetic interactions among recessive mutations in the APM1 and APM2 loci of Chlamydomonas reinhardtii that may reflect a physical association of the gene products or their involvement in a common structure/process: (1) allele-specific synthetic lethality, and (2) unlinked noncomplementation, or dominant enhancement. To further investigate these interactions, we isolated revertants in which the heat sensitivity caused by the apm2-1 mutation is lost. The heat-insensitive revertants were either fully or partially suppressed for the drug-resistance caused by the apm2-1 allele. In recombination tests the revertants behaved as if the suppressing mutation mapped within the APM2 locus; the partial suppressors of apm2-1 herbicide resistance failed to complement apm2-1, leading to the conclusion that they were likely to be intragenic pseudorevertants. The apm2-1 partial suppressor mutations reversed apm1(-)apm2-1 synthetic lethality in an allele-specific manner with respect both to apm1(-) alleles and apm2-1 suppressor mutations. Those apm1(-) apm2-1(rev) strains that regained viability also regained heat sensitivity characteristic of the original apm2-1 mutation, even though the apm2-1 suppressor strains were fully heat-insensitive. The Hs(+) phenotypes of apm2-1 partial suppressors were also reversed by treatment with the microtubule-stabilizing agent deuterium oxide (D(2)O). In addition to the above interactions, we observed interallelic complementation and phenotypic enhancement of temperature conditionality among apm1(-) alleles. Evidence of a role for the products of the two genes in microtubule-based processes was obtained from studying flagellar assembly in apm1(-) and apm2(-) mutants. PMID:1311696

  6. Effects of mutual interaction of Laccaria laccata with Trichoderma harzianum and T. virens on the morphology of microtubules and mitochondria.

    PubMed

    Zadworny, M; Tuszyńska, S; Samardakiewicz, S; Werner, A

    2007-01-01

    Organelles are known to respond to challenges caused by many stress factors. The morphology of the microtubular cytoskeleton and mitochondria during mutual interaction in coculture of Laccaria laccata with Trichoderma harzianum and T. virens were examined. Hyphae from the interaction region were sampled between 4 and 12 days of growth. Microtubules were labelled with a specific antibody and mitochondria with 3,3'-dihexyloxacarbocyanine iodide, and the organelles were examined microscopically. The morphology of microtubules and mitochondria were similar in all three fungi. Microtubules were arranged in long arrays parallel to the hyphal axis and mitochondria formed an interconnected network. In hyphae growing within the interaction zone, microtubules became wavy and eventually fragmented or depolymerised, and mitochondria also became fragmented. The effects were time-dependent. In general, the organelles of all three fungi were affected during the interaction, but L. laccata was affected the least and to the same extent by each of the saprotrophic fungi. The saprotrophic fungi were affected by L. laccata to a similar extent at 4 and 8 days of interaction. Our results suggest that the studied fungi antagonistically affect each other at the cellular level, although the mechanisms involved remain to be elucidated.

  7. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration.

    PubMed Central

    Billingsley, M L; Kincaid, R L

    1997-01-01

    This review attempts to summarize what is known about tau phosphorylation in the context of both normal cellular function and dysfunction. However, conceptions of tau function continue to evolve, and it is likely that the regulation of tau distribution and metabolism is complex. The roles of microtubule-associated kinases and phosphatases have yet to be fully described, but may afford insight into how tau phosphorylation at the distal end of the axon regulates cytoskeletal-membrane interactions. Finally, lipid and glycosaminoglycan modification of tau structure affords yet more complexity for regulation and aggregation. Continued work will help to determine what is causal and what is coincidental in Alzheimer's disease, and may lead to identification of therapeutic targets for halting the progression of paired helical filament formation. PMID:9169588

  8. Dynamic microtubule-dependent interactions position homotypic neurones in regular monolayered arrays during retinal development.

    PubMed

    Galli-Resta, Lucia; Novelli, Elena; Viegi, Alessandro

    2002-08-01

    In the vertebrate retina cell layers support serial processing, while monolayered arrays of homotypic neurones tile each layer to allow parallel processing. How neurones form layers and arrays is still largely unknown. We show that monolayered retinal arrays are dynamic structures based on dendritic interactions between the array cells. The analysis of three developing retinal arrays shows that these become regular as a net of dendritic processes links neighbouring array cells. Molecular or pharmacological perturbations of microtubules within dendrites lead to a stereotyped and reversible disruption of array organization: array cells lose their regular spacing and the arrangement in a monolayer. This leads to a micro-mechanical explanation of how monolayers of regularly spaced 'like-cells' are formed.

  9. Mechanics of kinetochore microtubules and their interactions with chromosomes during cell division

    NASA Astrophysics Data System (ADS)

    Nazockdast, Ehssan; Fürthauer, Sebastian; Redemann, Stephanie; Baumgart, Johannes; Lindow, Norbert; Kratz, Andrea; Prohaska, Steffen; Müller-Reichert, Thomas; Shelley, Michael

    2016-11-01

    The accurate segregation of chromosomes, and subsequent cell division, in Eukaryotic cells is achieved by the interactions of an assembly of microtubules (MTs) and motor-proteins, known as the mitotic spindle. We use a combination of our computational platform for simulating cytoskeletal assemblies and our structural data from high-resolution electron tomography of the mitotic spindle, to study the kinetics and mechanics of MTs in the spindle, and their interactions with chromosomes during chromosome segregation in the first cell division in C.elegans embryo. We focus on kinetochore MTs, or KMTs, which have one end attached to a chromosome. KMTs are thought to be a key mechanical component in chromosome segregation. Using exploratory simulations of MT growth, bending, hydrodynamic interactions, and attachment to chromosomes, we propose a mechanical model for KMT-chromosome interactions that reproduces observed KMT length and shape distributions from electron tomography. We find that including detailed hydrodynamic interactions between KMTs is essential for agreement with the experimental observations.

  10. The Interaction of Microtubules with Stabilizers Characterized at Biochemical and Structural Levels

    NASA Astrophysics Data System (ADS)

    Díaz, J. F.; Andreu, J. M.; Jiménez-Barbero, J.

    Since the discovery of paclitaxel and its peculiar mechanism of cytotoxicity, which has made it and its analogues widely used antitumour drugs, great effort has been made to understand the way they produce their effect in microtubules and to find other products that share this effect without the undesired side effects of low solubility and development of multidrug resistance by tumour cells. This chapter reviews the actual knowledge about the biochemical and structural mechanisms of microtubule stabilization by microtubule stabilizing agents, and illustrates the way paclitaxel and its biomimetics induce microtubule assembly, the thermodynamics of their binding, the way they reach their binding site and the conformation they have when bound.

  11. The role of host microfilaments and microtubules during opsonin-independent interactions of Cryptococcus neoformans with mammalian lung cells.

    PubMed

    Choo, K K; Chong, P P; Ho, A S H; Yong, P V C

    2015-12-01

    The purpose of this investigation was to characterise the interactions of Cryptococcus neoformans with mammalian host alveolar epithelial cells and alveolar macrophages, with emphasis on the roles of the cryptococcal capsule and the host cell cytoskeletons. The adherence and internalisation of C. neoformans into mammalian lung cells and the roles of host cell cytoskeletons in host-pathogen interactions were studied using in vitro models coupled with a differential fluorescence assay, fluorescence staining, immunofluorescence and drug inhibition of actin and microtubule polymerisation. Under conditions devoid of opsonin and macrophage activation, C. neoformans has a high affinity towards MH-S alveolar macrophages, yet associated poorly to A549 alveolar epithelial cells. Acapsular C. neoformans adhered to and internalised into the mammalian cells more effectively compared to encapsulated cryptococci. Acapsular C. neoformans induced prominent actin reorganisation at the host-pathogen interface in MH-S alveolar macrophages, but minimally affected actin reorganisation in A549 alveolar epithelial cells. Acapsular C. neoformans also induced localisation of microtubules to internalised cryptococci in MH-S cells. Drug inhibition of actin and microtubule polymerisation both reduced the association of acapsular C. neoformans to alveolar macrophages. The current study visualises and confirms the interactions of C. neoformans with mammalian alveolar cells during the establishment of infection in the lungs. The acapsular form of C. neoformans effectively adhered to and internalised into alveolar macrophages by inducing localised actin reorganisation, relying on the host's actin and microtubule activities.

  12. Fragile X mental retardation protein interactions with the microtubule associated protein 1B RNA.

    PubMed

    Menon, Lakshmi; Mader, Samantha Ann; Mihailescu, Mihaela-Rita

    2008-08-01

    Fragile X mental retardation syndrome, the most common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine (RGG) box to bind to a subset of RNA targets that form a G quadruplex structure. We performed a detailed analysis of the interactions between the FMRP RGG box and the microtubule associated protein 1B (MAP1B) mRNA, a relevant in vivo FMRP target. We show that MAP1B RNA forms an intramolecular G quadruplex structure, which is bound with high affinity and specificity by the FMRP RGG box. We determined that hydrophobic interactions are important in the FMRP RGG box-MAP1B RNA association, with minor contributions from electrostatic interactions. Our findings that at low protein:RNA ratios the RNA G quadruplex structure is slightly stabilized, whereas at high ratios is unfolded, suggest a mechanism by which the FMRP concentration variation in response to a neurotransmitter stimulation event could act as a regulatory switch for the protein function, from translation repressor to translation activator.

  13. Interaction between the microtubule inhibitor tubulozole and gamma-irradiation in murine tumors in vivo

    SciTech Connect

    Distelmans, W.; Van Ginckel, R.; Vanherck, W.; Willebrords, R.; De Brabander, M.; Wouters, L.; Van den Winkel, P.; De Backer, G.

    1989-01-01

    The combined effect of the microtubule inhibitor tubulozole and gamma-irradiation has been investigated in vivo in subcutaneous MO4 fibrosarcomas and Lewis Lung carcinomas. A marked interactive effect on tumor growth was observed when 160 mg/kg tubulozole was orally administered before the tumors were treated with 10 Gy radiation. Dose dependency and optimal effect were obtained on tumor growth of MO4 tumor bearing animals when the drug treatment was given 6 hr prior to the irradiation. The optimal pretreatment time coincided with the time at which a peak mitotic index in the tumor tissue was observed. An enhancing effect is also noticed at other doses of radiation in MO4 tumors pretreated 6 hr before with 160 mg/kg tubulozole. The interactive effect is maintained in a clinically relevant dose fractionation schedule whereby 8 fractions of 2 Gy each were pretreated 6 hr before with 80 mg/kg tubulozole. Tubulozole-T, the stereo-isomer of tubulozole, neither exhibits any antimicrotubular action nor exerts an antitumoral effect on its own or in combination with gamma-irradiation. The possible mechanisms of interaction between tubulozole and gamma-irradiation in tumor tissue are discussed.

  14. Interaction of Antiparallel Microtubules in the Phragmoplast Is Mediated by the Microtubule-Associated Protein MAP65-3 in Arabidopsis[W

    PubMed Central

    Ho, Chin-Min Kimmy; Hotta, Takashi; Guo, Fengli; Roberson, Robert W.; Lee, Yuh-Ru Julie; Liu, Bo

    2011-01-01

    In plant cells, microtubules (MTs) in the cytokinetic apparatus phragmoplast exhibit an antiparallel array and transport Golgi-derived vesicles toward MT plus ends located at or near the division site. By transmission electron microscopy, we observed that certain antiparallel phragmoplast MTs overlapped and were bridged by electron-dense materials in Arabidopsis thaliana. Robust MT polymerization, reported by fluorescently tagged End Binding1c (EB1c), took place in the phragmoplast midline. The engagement of antiparallel MTs in the central spindle and phragmoplast was largely abolished in mutant cells lacking the MT-associated protein, MAP65-3. We found that endogenous MAP65-3 was selectively detected on the middle segments of the central spindle MTs at late anaphase. When MTs exhibited a bipolar appearance with their plus ends placed in the middle, MAP65-3 exclusively decorated the phragmoplast midline. A bacterially expressed MAP65-3 protein was able to establish the interdigitation of MTs in vitro. MAP65-3 interacted with antiparallel microtubules before motor Kinesin-12 did during the establishment of the phragmoplast MT array. Thus, MAP65-3 selectively cross-linked interdigitating MTs (IMTs) to allow antiparallel MTs to be closely engaged in the phragmoplast. Although the presence of IMTs was not essential for vesicle trafficking, they were required for the phragmoplast-specific motors Kinesin-12 and Phragmoplast-Associated Kinesin-Related Protein2 to interact with MT plus ends. In conclusion, we suggest that the phragmoplast contains IMTs and highly dynamic noninterdigitating MTs, which work in concert to bring about cytokinesis in plant cells. PMID:21873565

  15. Accurate phosphoregulation of kinetochore–microtubule affinity requires unconstrained molecular interactions

    PubMed Central

    Zaytsev, Anatoly V.; Sundin, Lynsie J.R.; DeLuca, Keith F.

    2014-01-01

    Accurate chromosome segregation relies on dynamic interactions between microtubules (MTs) and the NDC80 complex, a major kinetochore MT-binding component. Phosphorylation at multiple residues of its Hec1 subunit may tune kinetochore–MT binding affinity for diverse mitotic functions, but molecular details of such phosphoregulation remain elusive. Using quantitative analyses of mitotic progression in mammalian cells, we show that Hec1 phosphorylation provides graded control of kinetochore–MT affinity. In contrast, modeling the kinetochore interface with repetitive MT binding sites predicts a switchlike response. To reconcile these findings, we hypothesize that interactions between NDC80 complexes and MTs are not constrained, i.e., the NDC80 complexes can alternate their binding between adjacent kinetochore MTs. Experiments using cells with phosphomimetic Hec1 mutants corroborate predictions of such a model but not of the repetitive sites model. We propose that accurate regulation of kinetochore–MT affinity is driven by incremental phosphorylation of an NDC80 molecular “lawn,” in which the NDC80–MT bonds reorganize dynamically in response to the number and stability of MT attachments. PMID:24982430

  16. CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions

    PubMed Central

    Shahbazi, Marta N.; Megias, Diego; Epifano, Carolina; Akhmanova, Anna; Gundersen, Gregg G.; Fuchs, Elaine

    2013-01-01

    Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. In this paper, we report that the MT plus end–binding protein CLASP2 localizes to adherens junctions (AJs) via direct interaction with p120-catenin (p120) in primary basal mouse keratinocytes. Reductions in the levels of p120 or CLASP2 decreased the localization of the other protein to cell–cell contacts and altered AJ dynamics and stability. These features were accompanied by decreased MT density and altered MT dynamics at intercellular junction sites. Interestingly, CLASP2 was enriched at the cortex of basal progenitor keratinocytes, in close localization to p120. Our findings suggest the existence of a new mechanism of MT targeting to AJs with potential functional implications in the maintenance of proper cell–cell adhesion in epidermal stem cells. PMID:24368809

  17. Proteome analysis of microtubule-associated proteins and their interacting partners from mammalian brain.

    PubMed

    Kozielski, Frank; Riaz, Tahira; DeBonis, Salvatore; Koehler, Christian J; Kroening, Mario; Panse, Isabel; Strozynski, Margarita; Donaldson, Ian M; Thiede, Bernd

    2011-07-01

    The microtubule (MT) cytoskeleton is essential for a variety of cellular processes. MTs are finely regulated by distinct classes of MT-associated proteins (MAPs), which themselves bind to and are regulated by a large number of additional proteins. We have carried out proteome analyses of tubulin-rich and tubulin-depleted MAPs and their interacting partners isolated from bovine brain. In total, 573 proteins were identified giving us unprecedented access to brain-specific MT-associated proteins from mammalian brain. Most of the standard MAPs were identified and at least 500 proteins have been reported as being associated with MTs. We identified protein complexes with a large number of subunits such as brain-specific motor/adaptor/cargo complexes for kinesins, dynein, and dynactin, and proteins of an RNA-transporting granule. About 25% of the identified proteins were also found in the synaptic vesicle proteome. Analysis of the MS/MS data revealed many posttranslational modifications, amino acid changes, and alternative splice variants, particularly in tau, a key protein implicated in Alzheimer's disease. Bioinformatic analysis of known protein-protein interactions of the identified proteins indicated that the number of MAPs and their associated proteins is larger than previously anticipated and that our database will be a useful resource to identify novel binding partners.

  18. Cell cycle regulators interact with pathways that modulate microtubule stability in Saccharomyces cerevisiae.

    PubMed

    Shohat-Tal, Aya; Eshel, Dan

    2011-12-01

    The integrity of mitosis is dependent upon strict regulation of microtubule stability and dynamics. Although much information has been accumulated on regulators of the microtubule cytoskeleton, our knowledge of the specific pathways involved is still limited. Here we designed genetic screens to identify regulators of microtubule stability that are dispensable in the wild type yet become essential under microtubule-disrupting conditions. We found that the transcriptional cofactor Swi6p and activator Swi4p, as well as the G(2)/M-specific cyclin Clb2p, are required in a microtubule-destabilizing environment. Swi6p and Swi4p can combine as a transcriptional complex, called the SBF complex (SBF for Swi4/6 cell cycle box [SCB]-binding factor) that is functionally homologous to the metazoan DP1/2-E2F complex and that controls the G(1)/S transition through the genes it regulates. We show that Swi6p's contribution to microtubule stability can be either dependent or independent of the SBF complex. The SBF-dependent pathway requires downregulation of SBF complex levels and may thereby reroute the transcriptional program in favor of greater microtubule stability. This pathway can be triggered by overexpression of Fcp1p, a phosphatase in the general transcription machinery, or by expression of an allele of SWI6 that is associated with reduced transcription from SBF-controlled promoters. The SBF-independent pathway is activated by a constitutively nuclear allele of Swi6p. Our results introduce novel roles in microtubule stability for genes whose participation in the process may be masked under normal conditions yet nonetheless acquire a dominant role when microtubule stability is compromised.

  19. Evaluating the microtubule cytoskeleton and its interacting proteins in monocots by mining the rice genome

    PubMed Central

    Guo, Longbiao; Ho, Chin-Min Kimmy; Kong, Zhaosheng; Lee, Yuh-Ru Julie; Qian, Qian; Liu, Bo

    2009-01-01

    Background Microtubules (MTs) are assembled by heterodimers of α- and β-tubulins, which provide tracks for directional transport and frameworks for the spindle apparatus and the phragmoplast. MT nucleation and dynamics are regulated by components such as the γ-tubulin complex which are conserved among eukaryotes, and other components which are unique to plants. Following remarkable progress made in the model plant Arabidopsis thaliana toward revealing key components regulating MT activities, the completed rice (Oryza sativa) genome has prompted a survey of the MT cytoskeleton in this important crop as a model for monocots. Scope The rice genome contains three α-tubulin genes, eight β-tubulin genes and a single γ-tubulin gene. A functional γ-tubulin ring complex is expected to form in rice as genes encoding all components of the complex are present. Among proteins that interact with MTs, compared with A. thaliana, rice has more genes encoding some members such as the MAP65/Ase1p/PRC1 family, but fewer for the motor kinesins, the end-binding protein EB1 and the mitotic kinase Aurora. Although most known MT-interacting factors have apparent orthologues in rice, no orthologues of arabidopsis RIC1 and MAP18 have been identified in rice. Among all proteins surveyed here, only a few have had their functions characterized by genetic means in rice. Elucidating functions of proteins of the rice MT cytoskeleton, aided by recent technical advances made in this model monocot, will greatly advance our knowledge of how monocots employ their MTs to regulate their growth and form. PMID:19106179

  20. Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells

    PubMed Central

    1984-01-01

    Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h. PMID:6547721

  1. Krit 1 interactions with microtubules and membranes are regulated by Rap1 and integrin cytoplasmic domain associated protein-1.

    PubMed

    Béraud-Dufour, Sophie; Gautier, Romain; Albiges-Rizo, Corinne; Chardin, Pierre; Faurobert, Eva

    2007-11-01

    The small G protein Rap1 regulates diverse cellular processes such as integrin activation, cell adhesion, cell-cell junction formation and cell polarity. It is crucial to identify Rap1 effectors to better understand the signalling pathways controlling these processes. Krev interaction trapped 1 (Krit1), a protein with FERM (band four-point-one/ezrin/radixin/moesin) domain, was identified as a Rap1 partner in a yeast two-hybrid screen, but this interaction was not confirmed in subsequent studies. As the evidence suggests a role for Krit1 in Rap1-dependent pathways, we readdressed this question. In the present study, we demonstrate by biochemical assays that Krit1 interacts with Rap1A, preferentially its GTP-bound form. We show that, like other FERM proteins, Krit1 adopts two conformations: a closed conformation in which its N-terminal NPAY motif interacts with its C-terminus and an opened conformation bound to integrin cytoplasmic domain associated protein (ICAP)-1, a negative regulator of focal adhesion assembly. We show that a ternary complex can form in vitro between Krit1, Rap1 and ICAP-1 and that Rap1 binds the Krit1 FERM domain in both closed and opened conformations. Unlike ICAP-1, Rap1 does not open Krit1. Using sedimentation assays, we show that Krit1 binds in vitro to microtubules through its N- and C-termini and that Rap1 and ICAP-1 inhibit Krit1 binding to microtubules. Consistently, YFP-Krit1 localizes on cyan fluorescent protein-labelled microtubules in baby hamster kidney cells and is delocalized from microtubules upon coexpression with activated Rap1V12. Finally, we show that Krit1 binds to phosphatidylinositol 4,5-P(2)-containing liposomes and that Rap1 enhances this binding. Based on these results, we propose a model in which Krit1 would be delivered by microtubules to the plasma membrane where it would be captured by Rap1 and ICAP-1.

  2. Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton

    PubMed Central

    1996-01-01

    By immunogold labeling, we demonstrate that "millipede-like" structures seen previously in mammalian cell cytoskeletons after removal of actin by treatment with gelsolin are composed of the cores of vimentin IFs with sidearms containing plectin. These plectin sidearms connect IFs to microtubules, the actin-based cytoskeleton and possibly membrane components. Plectin binding to microtubules was significantly increased in cells from transgenic mice lacking IFs and was reversed by microinjection of exogenous vimentin. These results suggest the existence of a pool of plectin which preferentially associates with IFs but may also be competed for by microtubules. The association of IFs with microtubules did not show a preference for Glu-tubulin. Nor did it depend upon the presence of MAP4 since plectin links were retained after specific immunodepletion of MAP4. The association of IFs with stress fibers survived actin depletion by gelsolin suggesting that myosin II minifilaments or components closely associated with them may play a role as plectin targets. Our results provide direct structural evidence for the hypothesis that plectin cross-links elements of the cytoskeleton thus leading to integration of the cytoplasm. PMID:8922382

  3. Doublet craters on Venus

    NASA Astrophysics Data System (ADS)

    Cook, Cheryl M.; Melosh, H. Jay; Bottke, William F.

    2003-09-01

    Of the impact craters on Earth larger than 20 km in diameter, 10-15% (3 out of 28) are doublets, having been formed by the simultaneous impact of two well-separated projectiles. The most likely scenario for their formation is the impact of well-separated binary asteroids. If a population of binary asteroids is capable of striking the Earth, it should also be able to hit the other terrestrial planets as well. Venus is a promising planet to search for doublet craters because its surface is young, erosion is nearly nonexistent, and its crater population is significantly larger than the Earth's. After a detailed investigation of single craters separated by less than 150 km and "multiple" craters having diameters greater than 10 km, we found that the proportion of doublet craters on Venus is at most 2.2%, significantly smaller than Earth's, although several nearly incontrovertible doublets were recognized. We believe this apparent deficit relative to the Earth's doublet population is a consequence of atmospheric screening of small projectiles on Venus rather than a real difference in the population of impacting bodies. We also examined "splotches," circular radar reflectance features in the Magellan data. Projectiles that are too small to form craters probably formed these features. After a careful study of these patterns, we believe that the proportion of doublet splotches on Venus (14%) is comparable to the proportion of doublet craters found on Earth (10-15%). Thus, given the uncertainties of interpretation and the statistics of small numbers, it appears that the doublet crater population on Venus is consistent with that of the Earth.

  4. The Microtubule Plus-End Tracking Proteins SPR1 and EB1b Interact to Maintain Polar Cell Elongation and Directional Organ Growth in Arabidopsis[W

    PubMed Central

    Galva, Charitha; Kirik, Viktor; Lindeboom, Jelmer J.; Kaloriti, Despoina; Rancour, David M.; Hussey, Patrick J.; Bednarek, Sebastian Y.; Ehrhardt, David W.; Sedbrook, John C.

    2014-01-01

    The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues. PMID:25415978

  5. The microtubule plus-end tracking proteins SPR1 and EB1b interact to maintain polar cell elongation and directional organ growth in Arabidopsis.

    PubMed

    Galva, Charitha; Kirik, Viktor; Lindeboom, Jelmer J; Kaloriti, Despoina; Rancour, David M; Hussey, Patrick J; Bednarek, Sebastian Y; Ehrhardt, David W; Sedbrook, John C

    2014-11-01

    The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.

  6. Microtubule-associated protein/microtubule affinity-regulating kinase (p110mark). A novel protein kinase that regulates tau-microtubule interactions and dynamic instability by phosphorylation at the Alzheimer-specific site serine 262.

    PubMed

    Drewes, G; Trinczek, B; Illenberger, S; Biernat, J; Schmitt-Ulms, G; Meyer, H E; Mandelkow, E M; Mandelkow, E

    1995-03-31

    Aberrant phosphorylation of the microtubule-associated protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1992) J. Biol. Chem. 26, 17047-17054) and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro (Biernat, J., Gustke, N., Drewes, G., Mandelkow, E.-M., and Mandelkow, E. (1993) Neuron 11, 153-163). Here we describe the purification and characterization of a protein-serine kinase from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes them dynamically unstable, in contrast to other protein kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel kinase in cellular events involving rearrangement of the microtuble-associated proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease.

  7. Plant microtubule cytoskeleton complexity: microtubule arrays as fractals.

    PubMed

    Gardiner, John; Overall, Robyn; Marc, Jan

    2012-01-01

    Biological systems are by nature complex and this complexity has been shown to be important in maintaining homeostasis. The plant microtubule cytoskeleton is a highly complex system, with contributing factors through interactions with microtubule-associated proteins (MAPs), expression of multiple tubulin isoforms, and post-translational modification of tubulin and MAPs. Some of this complexity is specific to microtubules, such as a redundancy in factors that regulate microtubule depolymerization. Plant microtubules form partial helical fractals that play a key role in development. It is suggested that, under certain cellular conditions, other categories of microtubule fractals may form including isotropic fractals, triangular fractals, and branched fractals. Helical fractal proteins including coiled-coil and armadillo/beta-catenin repeat proteins and the actin cytoskeleton are important here too. Either alone, or in combination, these fractals may drive much of plant development.

  8. Cortical microtubule rearrangements and cell wall patterning

    PubMed Central

    Oda, Yoshihisa

    2015-01-01

    Plant cortical microtubules, which form a highly ordered array beneath the plasma membrane, play essential roles in determining cell shape and function by directing the arrangement of cellulosic and non-cellulosic compounds on the cell surface. Interphase transverse arrays of cortical microtubules self-organize through their dynamic instability and inter-microtubule interactions, and by branch-form microtubule nucleation and severing. Recent studies revealed that distinct spatial signals including ROP GTPase, cellular geometry, and mechanical stress regulate the behavior of cortical microtubules at the subcellular and supercellular levels, giving rise to dramatic rearrangements in the cortical microtubule array in response to internal and external cues. Increasing evidence indicates that negative regulators of microtubules also contribute to the rearrangement of the cortical microtubule array. In this review, I summarize recent insights into how the rearrangement of the cortical microtubule array leads to proper, flexible cell wall patterning. PMID:25904930

  9. CYTOPLASMIC MICROTUBULES

    PubMed Central

    Slautterback, David B.

    1963-01-01

    Small cytoplasmic tubules are present in the interstitial cells and cnidoblasts of hydra. They are referred to here as "microtubules." These tubular elements have an outside diameter of 180 A and an inside diameter of 80 A. By difference, the membranous wall is estimated to be 50 A thick. The maximum length of the microtubules cannot be determined from thin sections but is known to exceed 1.5 µ. In the interstitial cells the microtubules are found in the intercellular bridges, free in the cytoplasm and in association with the centrioles. In the cnidoblast they form a framework around the developing nematocyst and in late stages are related to the cnidocil forming a tight skein in the basal part of the cell. Especially in this cell, confluence of microtubules with small spherical vesicles of the Golgi complex has been observed. It is proposed that these tubules function in the transport of water, ions, or small molecules. PMID:14079495

  10. On and around microtubules: an overview.

    PubMed

    Wade, Richard H

    2009-10-01

    Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from alphabeta-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the beta-tubulin subunit facing the microtubule plus end conferring a structural polarity. The alpha- and beta-tubulins are highly conserved. A third member of the tubulin family, gamma-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with beta-tubulin so that microtubules consist principally of 'GDP-tubulin' stabilized at the plus end by a short 'cap'. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins.

  11. The Oligomeric Outer Dynein Arm Assembly Factor CCDC103 Is Tightly Integrated within the Ciliary Axoneme and Exhibits Periodic Binding to Microtubules*

    PubMed Central

    King, Stephen M.; Patel-King, Ramila S.

    2015-01-01

    CCDC103 is an ∼29-kDa protein consisting of a central RPAP3_C domain flanked by N- and C-terminal coiled coils. Defects in CCDC103 lead to primary ciliary dyskinesia caused by the loss of outer dynein arms. This protein is present along the entire length of the ciliary axoneme and does not require other dynein or docking complex components for its integration. Unlike other known dynein assembly factors within the axoneme, CCDC103 is not solubilized by 0.6 m NaCl and requires more chaotropic conditions, such as 0.5 m KI. Alternatively, it can be extracted using 0.3% sarkosyl. CCDC103 forms stable dimers and other oligomers in solution through interactions involving the central domain. The smallest particle observed by dynamic light scattering has a hydrodynamic diameter of ∼25 nm. Furthermore, CCDC103 binds microtubules directly, forming ∼9-nm diameter particles that exhibit a 12-nm spacing on the microtubule lattice, suggesting that there may be two CCDC103 units per outer arm dynein repeat. Although the outer dynein arm docking complex is necessary to form arrays of dyneins along microtubules, it is not sufficient to set up a single array in a precise location on each axonemal doublet. We propose that CCDC103 helps generate a high-affinity site on the doublets for outer arm assembly, either through direct interactions or indirectly, perhaps by modifying the underlying microtubule lattice. PMID:25572396

  12. USP8, a regulator of endosomal sorting, is involved in mouse acrosome biogenesis through interaction with the spermatid ESCRT-0 complex and microtubules.

    PubMed

    Berruti, Giovanna; Ripolone, Michela; Ceriani, Michela

    2010-05-01

    Ubiquitin-specific peptidase 8 (USP8) is a deubiquitinating enzyme that works as a regulator of endosomal sorting and vesicle morphology in cultured cells. Its function in vivo is, however, unknown as USP8 gene deletion leads to embryonic lethality. Previously, we have shown that USP8 is highly expressed in male germ cells. These cells develop a peculiar acidic vesicle that is indispensable for fertilization, the acrosome; USP8 might be involved in vivo in acrosomogenesis. The objective of this study was to test this hypothesis by determining if selective components of the early endosomal machinery interact functionally with USP8 during acrosomogenesis using protein-protein interaction assays and double/triple immunolabeling. Moreover, by exploiting the characteristic of USP8 that exhibits a microtubule interacting and trafficking/transport (MIT) domain, we verified whether USP8 effectively associates with spermatid microtubules by microtubule cosedimentation and binding assays. USP8 was able to interact with spermatid ESCRT-0 (endosomal-sorting complex required for transport-0) and microtubule structures; USP8/ESCRT-0-labeled vesicles, monitored by fluorescence microscopy, were found to contribute to acrosome formation while USP8 can directly link, via its MIT domain, the labeled vesicles/developing acrosome to microtubules, which could favor both acrosome assembly and shaping. VPS54, the vacuolar-sorting protein responsible for early endocytic retrograde transport, was here detected for the first time in male germ cells; VPS54 followed the intracellular route of USP8/ESCRT-0-labeled vesicles during acrosomogenesis. We concluded that in vivo USP8 has a role strongly associated with acrosome biogenesis and that the early endosome pathway is significantly involved in the process, which suggests that the acrosome could be a novel lysosome-related organelle.

  13. Quantitative Analysis of the Microtubule Interaction of Rabies Virus P3 Protein: Roles in Immune Evasion and Pathogenesis

    PubMed Central

    Brice, Aaron; Whelan, Donna R.; Ito, Naoto; Shimizu, Kenta; Wiltzer-Bach, Linda; Lo, Camden Y.; Blondel, Danielle; Jans, David A.; Bell, Toby D. M.; Moseley, Gregory W.

    2016-01-01

    Although microtubules (MTs) are known to have important roles in intracellular transport of many viruses, a number of reports suggest that specific viral MT-associated proteins (MAPs) target MTs to subvert distinct MT-dependent cellular processes. The precise functional importance of these interactions and their roles in pathogenesis, however, remain largely unresolved. To assess the association with disease of the rabies virus (RABV) MAP, P3, we quantitatively compared the phenotypes of P3 from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derivative strain, Ni-CE. Using confocal/live-cell imaging and dSTORM super-resolution microscopy to quantify protein interactions with the MT network and with individual MT filaments, we found that the interaction by Ni-CE-P3 is significantly impaired compared with Ni-P3. This correlated with an impaired capacity to effect association of the transcription factor STAT1 with MTs and to antagonize interferon (IFN)/STAT1-dependent antiviral signaling. Importantly, we identified a single mutation in Ni-CE-P3 that is sufficient to inhibit MT-association and IFN-antagonist function of Ni-P3, and showed that this mutation alone attenuates the pathogenicity of RABV. These data provide evidence that the viral protein-MT interface has important roles in pathogenesis, suggesting that this interface could provide targets for vaccine/antiviral drug development. PMID:27649849

  14. Quantitative Analysis of the Microtubule Interaction of Rabies Virus P3 Protein: Roles in Immune Evasion and Pathogenesis.

    PubMed

    Brice, Aaron; Whelan, Donna R; Ito, Naoto; Shimizu, Kenta; Wiltzer-Bach, Linda; Lo, Camden Y; Blondel, Danielle; Jans, David A; Bell, Toby D M; Moseley, Gregory W

    2016-09-21

    Although microtubules (MTs) are known to have important roles in intracellular transport of many viruses, a number of reports suggest that specific viral MT-associated proteins (MAPs) target MTs to subvert distinct MT-dependent cellular processes. The precise functional importance of these interactions and their roles in pathogenesis, however, remain largely unresolved. To assess the association with disease of the rabies virus (RABV) MAP, P3, we quantitatively compared the phenotypes of P3 from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derivative strain, Ni-CE. Using confocal/live-cell imaging and dSTORM super-resolution microscopy to quantify protein interactions with the MT network and with individual MT filaments, we found that the interaction by Ni-CE-P3 is significantly impaired compared with Ni-P3. This correlated with an impaired capacity to effect association of the transcription factor STAT1 with MTs and to antagonize interferon (IFN)/STAT1-dependent antiviral signaling. Importantly, we identified a single mutation in Ni-CE-P3 that is sufficient to inhibit MT-association and IFN-antagonist function of Ni-P3, and showed that this mutation alone attenuates the pathogenicity of RABV. These data provide evidence that the viral protein-MT interface has important roles in pathogenesis, suggesting that this interface could provide targets for vaccine/antiviral drug development.

  15. βI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug-tubulin interactions and microtubule stability.

    PubMed

    Kanakkanthara, Arun; Rowe, Matthew R; Field, Jessica J; Northcote, Peter T; Teesdale-Spittle, Paul H; Miller, John H

    2015-09-01

    Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective against a broad spectrum of cancer cells. The interactions of PLA and LAU with tubulin have attracted a great deal of attention, mainly because they bind to β-tubulin at a site that is different from the classical taxoid site. Multiple βI-tubulin amino acid residues have been predicted by computer modelling studies and more recently by protein crystallography to participate in the binding of PLA and LAU to tubulin. The relevance of these residues in determining cellular sensitivity to the compounds, however, remains largely uncertain. To determine the role of four binding site residues, Q291, D295, V333, and N337 on PLA and LAU activity, we introduced single mutations to these sites by site-directed mutagenesis and transfected each mutant tubulin separately into HEK and/or HeLa cells. We found that a Q291M βI-tubulin mutation increased sensitivity of the cells to PLA, but not to LAU, paclitaxel (PTX), or vinblastine (VBL). In contrast, V333W and N337L mutations led to less stable microtubules, with the V333W causing resistance to PLA and PTX, but not LAU, and the N337L causing resistance to PLA, LAU, and PTX. Moreover, cells expressing either W333 or L337 were hypersensitive to the microtubule-destabilizing agent, VBL. The D295I mutation conferred resistance to both PLA and LAU without affecting microtubule stability or sensitivity to PTX or ixabepilone (IXB). This study identifies the first mammalian βI-tubulin mutation that specifically increases sensitivity to PLA, and reports mutations at PLA and LAU binding site residues that can either reduce microtubule stability or impair drug-tubulin binding, conferring resistance to these microtubule-stabilizing agents. This information provides insights on β-tubulin residues important for maintaining microtubule structural integrity and for sensitivity to microtubule-targeting agents, and suggests novel

  16. ADNP/NAP dramatically increase microtubule end-binding protein-Tau interaction: a novel avenue for protection against tauopathy.

    PubMed

    Ivashko-Pachima, Y; Sayas, C Laura; Malishkevich, A; Gozes, I

    2017-01-24

    Activity-dependent neuroprotective protein (ADNP), vital for brain formation and cognitive function, is mutated in autism and linked to neurodegenerative/psychiatric diseases. An eight-amino-acid peptide snippet of ADNP, NAP (NAPVSIPQ), identified as a smallest active fragment, includes the SxIP microtubule (MT) end-binding protein (EB) association motif, and enhances ADNP-EB3 interaction. Depletion of EB1 or EB3 abolishes NAP protection against zinc intoxication. Furthermore, NAP enhances Tau-MT interaction, and Tau regulates the localization and function of EB1 and EB3 in developing neuronal cells. Here, we asked how NAP (ADNP) enhances Tau-MT interactions and whether this is mediated by EBs. We showed, for we believe the first time, that NAP augmented endogenous EB1 comet density in the N1E-115 neuroblastoma neuronal model. This finding was substantiated by cell transfection with fluorescent EB1 and live cell imaging. NAP increased comet amounts, length and speed. At the molecular level, NAP enhanced EB3 homodimer formation, while decreasing EB1-EB3 heterodimer content and driving EB1- and EB3-Tau interactions (dramatic 20-fold increases), leading to recruitment of EB1/EB3 and Tau to MTs under zinc intoxication. Our previous results showed that while NAP protected neuronal-like cells against oxidative stress, it did not protect NIH3T3 fibroblasts. Here, NAP did not protect NIH3T3 cells against zinc intoxication, unless these cells were transfected with Tau. Interestingly, other MT associated proteins (MAPs) may replace Tau, thus, EB-Tau (MAPs) interaction is identified as a novel target for endogenous ADNP neuroprotection, and a future target for drug development, with NAP as a prototype.Molecular Psychiatry advance online publication, 24 January 2017; doi:10.1038/mp.2016.255.

  17. NuMA-microtubule interactions are critical for spindle orientation and the morphogenesis of diverse epidermal structures

    PubMed Central

    Seldin, Lindsey; Muroyama, Andrew; Lechler, Terry

    2016-01-01

    Mitotic spindle orientation is used to generate cell fate diversity and drive proper tissue morphogenesis. A complex of NuMA and dynein/dynactin is required for robust spindle orientation in a number of cell types. Previous research proposed that cortical dynein/dynactin was sufficient to generate forces on astral microtubules (MTs) to orient the spindle, with NuMA acting as a passive tether. In this study, we demonstrate that dynein/dynactin is insufficient for spindle orientation establishment in keratinocytes and that NuMA’s MT-binding domain, which targets MT tips, is also required. Loss of NuMA-MT interactions in skin caused defects in spindle orientation and epidermal differentiation, leading to neonatal lethality. In addition, we show that NuMA-MT interactions are also required in adult mice for hair follicle morphogenesis and spindle orientation within the transit-amplifying cells of the matrix. Loss of spindle orientation in matrix cells results in defective differentiation of matrix-derived lineages. Our results reveal an additional and direct function of NuMA during mitotic spindle positioning, as well as a reiterative use of spindle orientation in the skin to build diverse structures. DOI: http://dx.doi.org/10.7554/eLife.12504.001 PMID:26765568

  18. Selective extraction of isolated mitotic apparatus. Evidence that typical microtubule protein is extracted by organic mercurial.

    PubMed

    Bibring, T; Baxandall, J

    1971-02-01

    Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.

  19. Centriolar CPAP/SAS-4 Imparts Slow Processive Microtubule Growth.

    PubMed

    Sharma, Ashwani; Aher, Amol; Dynes, Nicola J; Frey, Daniel; Katrukha, Eugene A; Jaussi, Rolf; Grigoriev, Ilya; Croisier, Marie; Kammerer, Richard A; Akhmanova, Anna; Gönczy, Pierre; Steinmetz, Michel O

    2016-05-23

    Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and "caps" microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control.

  20. Adhesive interactions of N-cadherin limit the recruitment of microtubules to cell-cell contacts through organization of actomyosin.

    PubMed

    Plestant, Charlotte; Strale, Pierre-Olivier; Seddiki, Rima; Nguyen, Emmanuelle; Ladoux, Benoit; Mège, René-Marc

    2014-04-15

    Adhesive interactions of cadherins induce crosstalk between adhesion complexes and the actin cytoskeleton, allowing strengthening of adhesions and cytoskeletal organization. The underlying mechanisms are not completely understood, and microtubules (MTs) might be involved, as for integrin-mediated cell-extracellular-matrix adhesions. Therefore, we investigated the relationship between N-cadherin and MTs by analyzing the influence of N-cadherin engagement on MT distribution and dynamics. MTs progressed less, with a lower elongation rate, towards cadherin adhesions than towards focal adhesions. Increased actin treadmilling and the presence of an actomyosin contractile belt, suggested that actin relays inhibitory signals from cadherin adhesions to MTs. The reduced rate of MT elongation, associated with reduced recruitment of end-binding (EB) proteins to plus ends, was alleviated by expression of truncated N-cadherin, but was only moderately affected when actomyosin was disrupted. By contrast, destabilizing actomyosin fibers allowed MTs to enter the adhesion area, suggesting that tangential actin bundles impede MT growth independently of MT dynamics. Blocking MT penetration into the adhesion area strengthened cadherin adhesions. Taken together, these results establish a crosstalk between N-cadherin, F-actin and MTs. The opposing effects of cadherin and integrin engagement on actin organization and MT distribution might induce bias of the MT network during cell polarization.

  1. Effective theory for electroweak doublet dark matter

    NASA Astrophysics Data System (ADS)

    Dedes, A.; Karamitros, D.; Spanos, V. C.

    2016-11-01

    We perform a detailed study of an effective field theory which includes the standard model particle content extended by a pair of Weyl fermionic SU(2) doublets with opposite hypercharges. A discrete symmetry guarantees that a linear combination of the doublet components is stable and can act as a candidate particle for dark matter. The dark sector fermions interact with the Higgs and gauge bosons through renormalizable d =4 operators, and nonrenormalizable d =5 operators that appear after integrating out extra degrees of freedom above the TeV scale. We study collider, cosmological and astrophysical probes for this effective theory of dark matter. We find that a weakly interacting dark matter particle with a mass nearby the electroweak scale, and thus observable at the LHC, is consistent with collider and astrophysical data only when fairly large magnetic dipole moment transition operators with the gauge bosons exist, together with moderate Yukawa interactions.

  2. Protein-Protein Interactions between Intermediate Chains and the Docking Complex of Chlamydomonas Flagellar Outer Arm Dynein

    PubMed Central

    Ide, Takahiro; Owa, Mikito; King, Stephen M.; Kamiya, Ritsu; Wakabayashi, Ken-ichi

    2013-01-01

    Outer arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association. PMID:23747306

  3. End-binding proteins sensitize microtubules to the action of microtubule-targeting agents.

    PubMed

    Mohan, Renu; Katrukha, Eugene A; Doodhi, Harinath; Smal, Ihor; Meijering, Erik; Kapitein, Lukas C; Steinmetz, Michel O; Akhmanova, Anna

    2013-05-28

    Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert on microtubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process.

  4. Microtubule alignment and manipulation using AC electrokinetics.

    PubMed

    Uppalapati, Maruti; Huang, Ying-Ming; Jackson, Thomas N; Hancock, William O

    2008-09-01

    The kinesin-microtubule system plays an important role in intracellular transport and is a model system for integrating biomotor-driven transport into microengineered devices. AC electrokinetics provides a novel tool for manipulating and organizing microtubules in solution, enabling new experimental geometries for investigating and controlling the interactions of microtubules and microtubule motors in vitro. By fabricating microelectrodes on glass substrates and generating AC electric fields across solutions of microtubules in low-ionic-strength buffers, bundles of microtubules are collected and aligned and the electrical properties of microtubules in solution are measured. The AC electric fields result in electro-osmotic flow, electrothermal flow, and dielectrophoresis of microtubules, which can be controlled by varying the solution conductivity, AC frequency, and electrode geometry. By mapping the solution conductivity and AC frequency over which positive dielectrophoresis occurs, the apparent conductivity of taxol-stabilized bovine-brain microtubules in PIPES buffer is measured to be 250 mS m(-1). By maximizing dielectrophoretic forces and minimizing electro-osmotic and electrothermal flow, microtubules are assembled into opposed asters. These experiments demonstrate that AC electrokinetics provides a powerful new tool for kinesin-driven transport applications and for investigating the role of microtubule motors in development and maintenance of the mitotic spindle.

  5. Interacting genes that affect microtubule function in Drosophila melanogaster: Two classes of mutation revert the failure to complement between hay sup nc2 and mutations in tubulin genes

    SciTech Connect

    Regan, C.L.; Fuller, M.T. )

    1990-05-01

    The recessive male sterile mutation hay{sup nc2} of Drosophila melanogaster fails to complement certain {beta}{sub 2}-tubulin and {alpha}-tubulin mutations, suggesting that the haywire product plays a role in microtubule function, perhaps as a structural component of microtubules. The genetic interaction appears to require the presence of the aberrant product encoded by hay{sup nc2}, which may act as a structural poison. Based on this observation, the authors have isolated ten new mutations with EMS that revert the failure to complement between hay{sup nc2} and B2t{sup n}. The revertants tested behaved as intragenic mutations of hay in recombination tests, and feel into two phenotypic classes, suggesting two functional domains of the hay gene product. Some revertants were hemizygous viable and less severe than hay{sup nc2} in their recessive phenotype. These mutations might revert the poison by restoring the aberrant product encoded by the hay{sup nc2} allele to more wild-type function. Most of the revertants were recessive lethal mutations, indicating that the hay gene product is essential for viability. These more extreme mutations could revert the poison by destroying the ability of the aberrant haywire{sup nc2} product to interact structurally with microtubules. Flies heterozygous for the original hay{sup nc2} allele and an extreme revertant show defects in both the structure and the function of the male meiotic spindle.

  6. Interacting Genes That Affect Microtubule Function in Drosophila Melanogaster: Two Classes of Mutation Revert the Failure to Complement between Hay(nc2) and Mutations in Tubulin Genes

    PubMed Central

    Regan, C. L.; Fuller, M. T.

    1990-01-01

    The recessive male sterile mutation hay(nc2) of Drosophila melanogaster fails to complement certain β(2)-tubulin and α-tubulin mutations, suggesting that the haywire product plays a role in microtubule function, perhaps as a structural component of microtubules. The genetic interaction appears to require the presence of the aberrant product encoded by hay(nc2), which may act as a structural poison. Based on this observation, we have isolated ten new mutations that revert the failure to complement between hay(nc2) and B2t(n). The revertants tested behaved as intragenic mutations of hay in recombination tests, and fell into two phenotypic classes, suggesting two functional domains of the hay gene product. Some revertants were hemizygous viable and less severe than hay(nc2) in their recessive phenotype. These mutations might revert the poison by restoring the aberrant product encoded by the hay(nc2) allele to more wild-type function. Most of the revertants were recessive lethal mutations, indicating that the hay gene product is essential for viability. These more extreme mutations could revert the poison by destroying the ability of the aberrant haywire(nc2) product to interact structurally with microtubules. Flies heterozygous for the original hay(nc2) allele and an extreme revertant show defects in both the structure and the function of the male meiotic spindle. PMID:2111265

  7. Real-time monitoring of changes in microtubule mechanical properties in response to microtubule-destabilizing drug treatment.

    PubMed

    Han, Sung-Woong; Simona, Patriche; Banu, Mihaela; Adachi, Taiji

    2013-03-01

    Microtubules are cylindrical protein polymers that play important roles in a number of cellular functions. The properties of microtubules are dynamically changed by interacting with many microtubule-related proteins and drugs. In this study, we used atomic force microscopy to evaluate the changes in microtubule mechanical properties induced by treatment with nocodazole, which is a microtubule-destabilizing drug. The average spring constant of the microtubules, which was used as a measure of microtubule lateral stiffness, was drastically decreased by treatment with nocodazole within 30 min from 0.052 +/- 0.014 N/m to 0.029 +/- 0.015 N/m. Our findings will aid in the understanding of microtubule dynamics, protein interactions in response to drug treatment, microtubule-related diseases, and drug development.

  8. Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length

    PubMed Central

    Zheng, Xiangdong; Ramani, Anand; Soni, Komal; Gottardo, Marco; Zheng, Shuangping; Ming Gooi, Li; Li, Wenjing; Feng, Shan; Mariappan, Aruljothi; Wason, Arpit; Widlund, Per; Pozniakovsky, Andrei; Poser, Ina; Deng, Haiteng; Ou, Guangshuo; Riparbelli, Maria; Giuliano, Callaini; Hyman, Anthony A.; Sattler, Michael; Gopalakrishnan, Jay; Li, Haitao

    2016-01-01

    Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAPF375A, with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAPEE343RR that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a ‘clutch-like' mechanism. PMID:27306797

  9. Automation design of cemented doublet

    NASA Astrophysics Data System (ADS)

    Romanova, Galina; Ivanova, Tatiana; Korotkova, Natalia

    2015-09-01

    Algorithm and software for cemented doublet synthesis by Slusarev's methodology are presented. Slusarev's methodology is based on lookup tables that allow calculating doublet radii by given value of third-order coma, spherical aberration and chromatic aberration by specific algorithm. This calculation is automated in this work. The input parameters for algorithm are desired values of third-order coma, spherical aberration and chromatic aberration of cemented doublet. The software looks up few pairs of optical glasses corresponding to specified value of chromatic aberration and then calculates radii of surfaces for each pair of glasses corresponding to specified third-order coma and spherical aberration. The resulted third-order aberrations and real aberrations on the edge of the pupil are calculated for obtained radiuses. Several doublets can be analyzed in result table and the chosen one can be imported into Zemax. The calculated cemented doublet parameters can be analyzed and optimized in optical system design software. The software allows to make the first step of optical system design fast and simple. It allows to design not only the system which is free of the third-order spherical aberration, coma and axial color, but obtain necessary value of aberration for compensation of aberrations in another part of optical system. Possibility to look up optical glasses automatically, what affects the chromatic aberration correction and aberration correction in general, is especially important. Examples of automatic calculation of cemented doublet and compensation of aberrations in another part of optical system are presented in the paper.

  10. Comparison of the Microtubule Proteins of Neuroblastoma Cells, Brain, and Chlamydomonas Flagella

    PubMed Central

    Olmsted, J. B.; Witman, G. B.; Carlson, K.; Rosenbaum, Joel L.

    1971-01-01

    Intact A microtubules isolated from outer doublet microtubules of Chlamydomonas flagella contain two separable proteins (tubulins) that differ in molecular weight and in amino-acid composition. The microtubule protein isolated from brain or neuroblastoma cells also has two electrophoretically distinct tubulins. Although the two tubulins of brain and neuroblastoma cells are electrophoretically similar to each other, only one of these tubulins migrates with the flagellar tubulins. This is the first evidence that (a) isolated, morphologically intact, single microtubules from flagella contain at least two different tubulins, and (b) at least one of these tubulins differs from tubulins that are isolated from other sources. Images PMID:5289385

  11. Identification of novel microtubule-binding proteins by taxol-mediated microtubule stabilization and mass spectrometry analysis

    PubMed Central

    He, Xianfei; Liu, Zhu; He, Qianqian; Qin, Juan; Liu, Ningning; Zhang, Linlin; Li, Dengwen; Zhou, Jun; Shui, Wenqing; Liu, Min

    2015-01-01

    Microtubule-binding proteins (MBPs) are structurally and functionally diverse regulators of microtubule-mediated cellular processes. Alteration of MBPs has been implicated in the pathogenesis of human diseases, including cancer. MBPs can stabilize or destabilize microtubules or move along microtubules to transport various cargoes. In addition, MBPs can control microtubule dynamics through direct interaction with microtubules or coordination with other proteins. To better understand microtubule structure and function, it is necessary to identify additional MBPs. In this study, we isolated microtubules and MBPs from mammalian cells by a taxol-based method and then profiled a panel of MBPs by mass spectrometry. We discovered a number of previously uncharacterized MBPs, including several membrane-associated proteins and proteins involved in post-translational modifications, in addition to several structural components. These results support the notion that microtubules have a wide range of functions and may undergo more exquisite regulation than previously recognized. PMID:26445615

  12. Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose Synthesis in Arabidopsis.

    PubMed

    Liu, Zengyu; Schneider, Rene; Kesten, Christopher; Zhang, Yi; Somssich, Marc; Zhang, Youjun; Fernie, Alisdair R; Persson, Staffan

    2016-08-08

    Cellulose is the most abundant biopolymer on Earth and is the major contributor to plant morphogenesis. Cellulose is synthesized by plasma membrane-localized cellulose synthase complexes (CSCs). Nascent cellulose microfibrils become entangled in the cell wall, and further catalysis therefore drives the CSC forward through the membrane: a process guided by cortical microtubules via the protein CSI1/POM2. Still, it is unclear how the microtubules can withstand the forces generated by the motile CSCs to effectively direct CSC movement. Here, we identified a family of microtubule-associated proteins, the cellulose synthase-microtubule uncouplings (CMUs), that located as static puncta along cortical microtubules. Functional disruption of the CMUs caused lateral microtubule displacement and compromised microtubule-based guidance of CSC movement. CSCs that traversed the microtubules interacted with the microtubules via CSI1/POM2, which prompted the lateral microtubule displacement. Hence, we have revealed how microtubules can withstand the propulsion of the CSCs during cellulose biosynthesis and thus sustain anisotropic plant cell growth.

  13. Polyribosome targeting to microtubules: enrichment of specific mRNAs in a reconstituted microtubule preparation from sea urchin embryos

    PubMed Central

    1994-01-01

    A subset of mRNAs, polyribosomes, and poly(A)-binding proteins copurify with microtubules from sea urchin embryos. Several lines of evidence indicate that the interaction of microtubules with ribosomes is specific: a distinct stalk-like structure appears to mediate their association; ribosomes bind to microtubules with a constant stoichiometry through several purification cycles; and the presence of ribosomes in these preparations depends on the presence of intact microtubules. Five specific mRNAs are enriched with the microtubule- bound ribosomes, indicating that translation of specific proteins may occur on the microtubule scaffolding in vivo. PMID:7962079

  14. Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning

    PubMed Central

    Peng, Yutian; Moritz, Michelle; Han, Xuemei; Giddings, Thomas H.; Lyon, Andrew; Kollman, Justin; Winey, Mark; Yates, John; Agard, David A.; Drubin, David G.; Barnes, Georjana

    2015-01-01

    Casein kinase 1δ (CK1δ) family members associate with microtubule-organizing centers (MTOCs) from yeast to humans, but their mitotic roles and targets have yet to be identified. We show here that budding yeast CK1δ, Hrr25, is a γ-tubulin small complex (γTuSC) binding factor. Moreover, Hrr25's association with γTuSC depends on its kinase activity and its noncatalytic central domain. Loss of Hrr25 kinase activity resulted in assembly of unusually long cytoplasmic microtubules and defects in spindle positioning, consistent with roles in regulation of γTuSC-mediated microtubule nucleation and the Kar9 spindle-positioning pathway, respectively. Hrr25 directly phosphorylated γTuSC proteins in vivo and in vitro, and this phosphorylation promoted γTuSC integrity and activity. Because CK1δ and γTuSC are highly conserved and present at MTOCs in diverse eukaryotes, similar regulatory mechanisms are expected to apply generally in eukaryotes. PMID:25971801

  15. Binding of microtubule protein to DNA and chromatin: possibility of simultaneous linkage of microtubule to nucleic and assembly of the microtubule structure.

    PubMed Central

    Villasante, A; Corces, V G; Manso-Martínez, R; Avila, J

    1981-01-01

    Microtubule protein binds to DNA through microtubule associated polypeptides (MAPs). Among MAPs there is one high molecular weight polypeptide (MAP2) which interacts with DNA fundamentally through certain polynucleotide sequences. This interaction is not affected by the presence of histones and other chromosomal proteins. DNA can associate to assembled microtubules and when a determinate DNA/protein ratio is reached the nucleic acid behaves as a microtubule associated molecule. The nucleic acid fragments which preferentially bind to microtubules have been isolated and characterized. These fragments contain DNA regions enriched in repetitive sequences that hybridizes preferentially to the pericentromeric zone of metaphase chromosomes. These results give further support to the model of interaction microtubule-chromosome based upon the mediator function of the microtubule associated proteins. Images PMID:7232207

  16. A novel model of interaction between high frequency electromagnetic non-ionizing fields and microtubules viewed as coupled two-degrees of freedom harmonic oscillators.

    PubMed

    Caligiuri, Luigi Maxmilian

    2015-01-01

    The question regarding the potential biological and adverse health effects of non-ionizing electromagnetic fields on living organisms is of primary importance in biophysics and medicine. Despite the several experimental evidences showing such occurrence in a wide frequency range from extremely low frequency to microwaves, a definitive theoretical model able to explain a possible mechanism of interaction between electromagnetic fields and living matter, especially in the case of weak and very weak intensities, is still missing. In this paper it has been suggested a possible mechanism of interaction involving the resonant absorption of electromagnetic radiation by microtubules. To this aim these have been modeled as non-dissipative forced harmonic oscillators characterized by two coupled "macroscopic" degrees of freedom, respectively describing longitudinal and transversal vibrations induced by the electromagnetic field. We have shown that the proposed model, although at a preliminary stage, is able to explain the ability of even weak electromagnetic radiating electromagnetic fields to transfer high quantities of energy to living systems by means of a resonant mechanism, so capable to easily damage microtubules structure.

  17. Expansion and Polarity Sorting in Microtubule-Dynein Bundles

    NASA Astrophysics Data System (ADS)

    Zemel, A.; Mogilner, A.

    Interactions of multiple molecular motors with dynamicpolymers, such as actin and microtubules, form the basis for many processes in the cell cytoskeleton. One example is the active `sorting' of microtubule bundles by dynein molecular motors into aster-like arrays of microtubules; in these bundles dynein motors cross-link and slide neighboring microtubules apart. A number of models have been suggested to quantify the active dynamics of cross-linked bundles of polar filaments. In the case of densely packed bundles, however, a major complication arises from the fact that each microtubule interacts with multiple neighboring filaments. To explicitly take these interactions into account we performed detailed computer simulations in which the equations of motion for all microtubules in the bundle were iteratively solved. Our simulations demonstrate the phenomenon of polarity sorting and reveal the variable-rate of the concurrent bundle expansion and its dependence on the nature of the microtubule-motor interactions.

  18. Compressing the Inert Doublet Model

    SciTech Connect

    Blinov, Nikita; Kozaczuk, Jonathan; Morrissey, David E.; de la Puente, Alejandro

    2016-02-16

    The Inert Doublet Model relies on a discrete symmetry to prevent couplings of the new scalars to Standard Model fermions. We found that this stabilizes the lightest inert state, which can then contribute to the observed dark matter density. In the presence of additional approximate symmetries, the resulting spectrum of exotic scalars can be compressed. Here, we study the phenomenological and cosmological implications of this scenario. In conclusion, we derive new limits on the compressed Inert Doublet Model from LEP, and outline the prospects for exclusion and discovery of this model at dark matter experiments, the LHC, and future colliders.

  19. Microtubule dynamics and organization

    NASA Astrophysics Data System (ADS)

    Dogterom, Marileen

    2000-03-01

    Microtubules are rigid biopolymers found in all higher order cells. They are a mayor part of the cytoskeleton, the network of protein polymers that gives the cell its shape and rigidity and allows for various forms of (intra)cellular motility. The intracellular spatial organization of the microtubule network is constantly changing as the microtubules adapt to their different functions. In part, this spatial organization depends on the assembly dynamics (including microtubule nucleation) and forces generated by the microtubules themselves. To understand these mechanisms, we study the physical aspects connected with the assembly, force generation and spatial organization of microtubules in simplified model systems, in the absence of other cellular components. We measure the forces generated by individual microtubules by making them grow against a microfabricated barrier. These experiments show that a single microtubule can generate at least several picoNewton of force, comparable to what is known for motor proteins. Theoretical modeling of force-generation by multi-protofilament polymers is used to predict force-velocity relations that can be compared to experimental data. We study the self-organization of microtubules by confining them to microfabricated chambers that mimic the geometry of living cells. The distribution of microtubule nucleation sites in these chambers is controlled to study its effect on the organization of the microtubule network. We find that so-called microtubule asters position themselves in response to forces generated by dynamic microtubules. Experiments aimed at measuring the forces acting on these asters using optical trapping techniques will be described.

  20. Micropattern-Guided Assembly of Overlapping Pairs of Dynamic Microtubules

    PubMed Central

    Fourniol, Franck J.; Li, Tai-De; Bieling, Peter; Mullins, R. Dyche; Fletcher, Daniel A.; Surrey, Thomas

    2014-01-01

    Interactions between antiparallel microtubules are essential for the organization of spindles in dividing cells. The ability to form immobilized antiparallel microtubule pairs in vitro, combined with the ability to image them via TIRF microscopy, permits detailed biochemical characterization of microtubule cross-linking proteins and their effects on microtubule dynamics. Here, we describe methods for chemical micropatterning of microtubule seeds on glass surfaces in configurations that specifically promote the formation of antiparallel microtubule overlaps in vitro. We demonstrate that this assay is especially well suited for reconstitution of minimal midzone overlaps stabilized by the antiparallel microtubule cross-linking protein PRC1 and its binding partners. The micropatterning method is suitable for use with a broad range of proteins, and the assay is generally applicable to any microtubule cross-linking protein. PMID:24630116

  1. Valosin-containing Protein-interacting Membrane Protein (VIMP) Links the Endoplasmic Reticulum with Microtubules in Concert with Cytoskeleton-linking Membrane Protein (CLIMP)-63*

    PubMed Central

    Noda, Chikano; Kimura, Hana; Arasaki, Kohei; Matsushita, Mitsuru; Yamamoto, Akitsugu; Wakana, Yuichi; Inoue, Hiroki; Tagaya, Mitsuo

    2014-01-01

    The distribution and morphology of the endoplasmic reticulum (ER) in mammalian cells depend on both dynamic and static interactions of ER membrane proteins with microtubules (MTs). Cytoskeleton-linking membrane protein (CLIMP)-63 is exclusively localized in sheet-like ER membranes, typical structures of the rough ER, and plays a pivotal role in the static interaction with MTs. Our previous study showed that the 42-kDa ER-residing form of syntaxin 5 (Syn5L) regulates ER structure through the interactions with both CLIMP-63 and MTs. Here, we extend our previous study and show that the valosin-containing protein/p97-interacting membrane protein (VIMP)/SelS is also a member of the family of proteins that shape the ER by interacting with MTs. Depletion of VIMP causes the spreading of the ER to the cell periphery and affects an MT-dependent process on the ER. Although VIMP can interact with CLIMP-63 and Syn5L, it does not interact with MT-binding ER proteins (such as Reep1) that shape the tubular smooth ER, suggesting that different sets of MT-binding ER proteins are used to organize different ER subdomains. PMID:25008318

  2. Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly

    PubMed Central

    1981-01-01

    B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication- derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components. PMID:7251656

  3. Movement of chromosomes with severed kinetochore microtubules.

    PubMed

    Forer, Arthur; Johansen, Kristen M; Johansen, Jørgen

    2015-05-01

    Experiments dating from 1966 and thereafter showed that anaphase chromosomes continued to move poleward after their kinetochore microtubules were severed by ultraviolet microbeam irradiation. These observations were initially met with scepticism as they contradicted the prevailing view that kinetochore fibre microtubules pulled chromosomes to the pole. However, recent experiments using visible light laser microbeam irradiations have corroborated these earlier experiments as anaphase chromosomes again were shown to move poleward after their kinetochore microtubules were severed. Thus, multiple independent studies using different techniques have shown that chromosomes can indeed move poleward without direct microtubule connections to the pole, with only a kinetochore 'stub' of microtubules. An issue not yet settled is: what propels the disconnected chromosome? There are two not necessarily mutually exclusive proposals in the literature: (1) chromosome movement is propelled by the kinetochore stub interacting with non-kinetochore microtubules and (2) chromosome movement is propelled by a spindle matrix acting on the stub. In this review, we summarise the data indicating that chromosomes can move with severed kinetochore microtubules and we discuss proposed mechanisms for chromosome movement with severed kinetochore microtubules.

  4. The b1 isoform of protocadherin-gamma (Pcdhgamma) interacts with the microtubule-destabilizing protein SCG10.

    PubMed

    Gayet, Odile; Labella, Vincenzo; Henderson, Christopher E; Kallenbach, Sacha

    2004-12-03

    Due to their structural characteristics and their diversity, the 22 members of the protocadherin-gamma (Pcdhgamma) family have been suggested to contribute to the establishment of specific connections in the nervous system. Here, we focus on a single isoform, Pcdhgamma-b1. Its expression is found in different brain regions and in developing spinal cord it is restricted to scattered cells, whereas all cells are labeled using an antibody that recognizes all Pcdhgamma isoforms. As a first step to understanding the signaling mechanisms downstream of Pcdhgamma, we identify the microtubule-destabilizing protein SCG10 as a cytoplasmic interactor for Pcdhgamma-b1 and other isoforms of the Pcdhgamma-b subfamily, and show that SCG10 and Pcdhgamma-b1 are found together in certain neuronal growth cones.

  5. Microtubule-Destabilizing Agents: Structural and Mechanistic Insights from the Interaction of Colchicine and Vinblastine with Tubulin

    NASA Astrophysics Data System (ADS)

    Gigant, B.; Cormier, A.; Dorléans, A.; Ravelli, R. B. G.; Knossow, M.

    Microtubules (MTs) are dynamic structures of the eukaryotic cytoskeleton that, during cell division, form the mitotic spindle. Perturbing them leads to mitotic arrest and ultimately to cell death. Consistently, MTs and their building block, αβ tubulin, are one of the best characterized targets in anti-cancer chemotherapy. Drugs that interfere with MTs either stabilize or destabilize them. The latter class is the subject of this review. These ligands bind to the colchicine site or to the vinca domain, two distinct sites located at a distance from each other on tubulin. Nevertheless the effects of both classes of ligands share a common theme, they prevent the formation of MT specific contacts, therefore triggering their disassembly.

  6. Cortical microtubule contacts position the spindle in C. elegans embryos.

    PubMed

    Kozlowski, Cleopatra; Srayko, Martin; Nedelec, Francois

    2007-05-04

    Interactions between microtubules and the cell cortex play a critical role in positioning organelles in a variety of biological contexts. Here we used Caenorhabditis elegans as a model system to study how cortex-microtubule interactions position the mitotic spindle in response to polarity cues. Imaging EBP-2::GFP and YFP::alpha-tubulin revealed that microtubules shrink soon after cortical contact, from which we propose that cortical adaptors mediate microtubule depolymerization energy into pulling forces. We also observe association of dynamic microtubules to form astral fibers that persist, despite the catastrophe events of individual microtubules. Computer simulations show that these effects, which are crucially determined by microtubule dynamics, can explain anaphase spindle oscillations and posterior displacement in 3D.

  7. Evolving tip structures can explain age-dependent microtubule catastrophe.

    PubMed

    Coombes, Courtney E; Yamamoto, Ami; Kenzie, Madeline R; Odde, David J; Gardner, Melissa K

    2013-07-22

    Microtubules are key structural and transport elements in cells. The dynamics at microtubule ends are characterized by periods of slow growth, followed by stochastic switching events termed "catastrophes," in which microtubules suddenly undergo rapid shortening. Growing microtubules are thought to be protected from catastrophe by a GTP-tubulin "cap": GTP-tubulin subunits add to the tips of growing microtubules but are subsequently hydrolyzed to GDP-tubulin subunits once they are incorporated into the microtubule lattice. Loss of the GTP-tubulin cap exposes GDP-tubulin subunits at the microtubule tip, resulting in a catastrophe event. However, the mechanistic basis for sudden loss of the GTP cap, leading to catastrophe, is not known. To investigate microtubule catastrophe events, we performed 3D mechanochemical simulations that account for interactions between neighboring protofilaments. We found that there are two separate factors that contribute to catastrophe events in the 3D simulation: the GTP-tubulin cap size, which settles into a steady-state value that depends on the free tubulin concentration during microtubule growth, and the structure of the microtubule tip. Importantly, 3D simulations predict, and both fluorescence and electron microscopy experiments confirm, that microtubule tips become more tapered as the microtubule grows. This effect destabilizes the tip and ultimately contributes to microtubule catastrophe. Thus, the likelihood of a catastrophe event may be intimately linked to the aging physical structure of the growing microtubule tip. These results have important consequences for catastrophe regulation in cells, as microtubule-associated proteins could promote catastrophe events in part by modifying microtubule tip structures.

  8. Discodermolide interferes with the binding of tau protein to microtubules.

    PubMed

    Kar, Santwana; Florence, Gordon J; Paterson, Ian; Amos, Linda A

    2003-03-27

    We investigated whether discodermolide, a novel antimitotic agent, affects the binding to microtubules of tau protein repeat motifs. Like taxol, the new drug reduces the proportion of tau that pellets with microtubules. Despite their differing structures, discodermolide, taxol and tau repeats all bind to a site on beta-tubulin that lies within the microtubule lumen and is crucial in controlling microtubule assembly. Low concentrations of tau still bind strongly to the outer surfaces of preformed microtubules when the acidic C-terminal regions of at least six tubulin dimers are available for interaction with each tau molecule; otherwise binding is very weak.

  9. Vfa1 Binds to the N-terminal Microtubule-interacting and Trafficking (MIT) Domain of Vps4 and Stimulates Its ATPase Activity*

    PubMed Central

    Vild, Cody J.; Xu, Zhaohui

    2014-01-01

    The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function. PMID:24567329

  10. Colchicine-binding activity distinguishes sea urchin egg and outer doublet tubulins

    PubMed Central

    1984-01-01

    The colchicine-binding activity of tubulin has been utilized to distinguish the tubulins from two distinct microtubule systems of the same species, the sea urchin Strongylocentrotus purpuratus. We have analyzed the colchicine-binding affinities of highly purified tubulins from the unfertilized eggs and from the flagellar outer doublet microtubules by van't Hoff analysis, and have found significant differences in the free energy, enthalpy, and entropy changes characterizing the binding of colchicine to the two tubulins. The data indicate that significant chemical differences in the tubulins from the two functionally distinct microtubule systems exist, and that the differences are expressed in the native forms of the tubulins. Our findings are discussed in terms of the possibility that the colchicine- binding site may be an important regulatory site on the tubulin molecule. PMID:6539784

  11. Presence of a carboxy-terminal pseudorepeat and disease-like pseudohyperphosphorylation critically influence tau’s interaction with microtubules in axon-like processes

    PubMed Central

    Niewidok, Benedikt; Igaev, Maxim; Sündermann, Frederik; Janning, Dennis; Bakota, Lidia; Brandt, Roland

    2016-01-01

    A current challenge of cell biology is to investigate molecular interactions in subcellular compartments of living cells to overcome the artificial character of in vitro studies. To dissect the interaction of the neuronal microtubule (MT)-associated protein tau with MTs in axon-like processes, we used a refined fluorescence decay after photoactivation approach and single-molecule tracking. We found that isoform variation had only a minor influence on the tau–MT interaction, whereas the presence of a C-terminal pseudorepeat region (PRR) greatly increased MT binding by a greater-than-sixfold reduction of the dissociation rate. Bioinformatic analysis revealed that the PRR contained a highly conserved motif of 18 amino acids. Disease-associated tau mutations in the PRR (K369I, G389R) did not influence apparent MT binding but increased its dynamicity. Simulation of disease-like tau hyperphosphorylation dramatically diminished the tau–MT interaction by a greater-than-fivefold decrease of the association rate with no major change in the dissociation rate. Apparent binding of tau to MTs was similar in axons and dendrites but more sensitive to increased phosphorylation in axons. Our data indicate that under the conditions of high MT density that prevail in the axon, tau’s MT binding and localization are crucially affected by the presence of the PRR and tau hyperphosphorylation. PMID:27582388

  12. Microtubules regulate disassembly of epithelial apical junctions

    PubMed Central

    Ivanov, Andrei I; McCall, Ingrid C; Babbin, Brian; Samarin, Stanislav N; Nusrat, Asma; Parkos, Charles A

    2006-01-01

    Background Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion. Results Calcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex. Conclusion Our data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins. PMID:16509970

  13. Tubulin acetylation protects long-lived microtubules against mechanical ageing.

    PubMed

    Portran, Didier; Schaedel, Laura; Xu, Zhenjie; Théry, Manuel; Nachury, Maxence V

    2017-04-01

    Long-lived microtubules endow the eukaryotic cell with long-range transport abilities. While long-lived microtubules are acetylated on Lys40 of α-tubulin (αK40), acetylation takes place after stabilization and does not protect against depolymerization. Instead, αK40 acetylation has been proposed to mechanically stabilize microtubules. Yet how modification of αK40, a residue exposed to the microtubule lumen and inaccessible to microtubule-associated proteins and motors, could affect microtubule mechanics remains an open question. Here we develop FRET-based assays that report on the lateral interactions between protofilaments and find that αK40 acetylation directly weakens inter-protofilament interactions. Congruently, αK40 acetylation affects two processes largely governed by inter-protofilament interactions, reducing the nucleation frequency and accelerating the shrinkage rate. Most relevant to the biological function of acetylation, microfluidics manipulations demonstrate that αK40 acetylation enhances flexibility and confers resilience against repeated mechanical stresses. Thus, unlike deacetylated microtubules that accumulate damage when subjected to repeated stresses, long-lived microtubules are protected from mechanical ageing through their acquisition of αK40 acetylation. In contrast to other tubulin post-translational modifications that act through microtubule-associated proteins, motors and severing enzymes, intraluminal acetylation directly tunes the compliance and resilience of microtubules.

  14. Microtubules, Tubulins and Associated Proteins.

    ERIC Educational Resources Information Center

    Raxworthy, Michael J.

    1988-01-01

    Reviews much of what is known about microtubules, which are biopolymers consisting predominantly of subunits of the globular protein, tubulin. Describes the functions of microtubules, their structure and assembly, microtube associated proteins, and microtubule-disrupting agents. (TW)

  15. Effects of the fluorescence dye DAPI on microtubule structure in vitro: formation of novel types of tubulin assembly products.

    PubMed

    Vater, W; Böhm, K J; Unger, E

    1993-02-01

    It has been found that the DNA fluorescence dye 4',6-diamidino-2-phenylindole (DAPI) is able to stain also microtubules. However, electron microscopy revealed that DAPI changed microtubule structure and induced the formation of a broad spectrum of polymorphic tubulin assembly products. Upon addition of DAPI to microtubules assembled from 10 to 15 mumol tubulin (molar DAPI/tubulin ratios of 10 to 40) in the presence of microtubule-associated proteins, most of the microtubules were decorated with additional protofilaments usually running parallel to the protofilaments of the microtubule wall (microtubule-protofilament complexes). When DAPI was already present during assembly, curved C- and S-shaped protofilament ribbons and microtubule-ribbon complexes with 6-shaped profiles were the most prominent products, beside microtubules. Additionally, protofilament bundles, some flat sheets, and hoops occurred. Electrophoresis revealed that DAPI lowered the amount of associated proteins, especially of tau-proteins, bound to the assembly products. Nevertheless, DAPI stimulated the assembly, enabled pure tubulin to assemble even at concentrations as low as 10 mumol, and stabilized the assembly products against cold. The microtubule-protofilament complexes, observed for the first time, are interpreted as the result of DAPI-induced protofilament linking as well as of activation of an additional tubulin-tubulin binding site which is possibly identical to that involved in the formation of microtubule doublets.

  16. Elevated Estrogen Receptor-α in VHL-Deficient Condition Induces Microtubule Organizing Center Amplification via Disruption of BRCA1/Rad51 Interaction

    PubMed Central

    Jung, Youn-Sang; Chun, Ho-Young; Yoon, Min-Ho; Park, Bum-Joon

    2014-01-01

    Since loss of VHL is frequently detected early phase genetic event in human renal cell carcinoma, pVHL is assumed to be indispensable for suppression of tumor initiation step. However, induction of HIF-1α, target of pVHL E3 ligase, is more adequate to angiogenesis step after tumor mass formation. Concerning this, it has been reported that pVHL is involved in centrosome location during metaphase and regulates ER-α signaling. Here, we provide the evidences that pVHL-mediated ER-α suppression is critical for microtubule organizing center (MTOC) maintaining and elevated ER-α promotes MTOC amplification through disruption of BRCA1-Rad51 interaction. In fact, numerous MTOC in VHL- or BRCA1-deficient cells are reduced by Fulvestrant, inhibitor of ER-α expression as well as antagonist. In addition, we reveal that activation of ER signaling can increase γ-tubulin, core factor of TuRC and render the resistance to Taxol. Thus, Fulvestrant but not Tamoxifen, antagonist against ER-α, can restore the Taxol sensitivity in VHL- or BRCA1-deficient cells. Our results suggest that pVHL-mediated ER-α suppression is important for regulation of MTOC as well as drug resistance. PMID:25499220

  17. Microtubules Growth Rate Alteration in Human Endothelial Cells

    PubMed Central

    Alieva, Irina B.; Zemskov, Evgeny A.; Kireev, Igor I.; Gorshkov, Boris A.; Wiseman, Dean A.; Black, Stephen M.; Verin, Alexander D.

    2010-01-01

    To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC) and “fast” (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules. PMID:20445745

  18. Microtubules growth rate alteration in human endothelial cells.

    PubMed

    Alieva, Irina B; Zemskov, Evgeny A; Kireev, Igor I; Gorshkov, Boris A; Wiseman, Dean A; Black, Stephen M; Verin, Alexander D

    2010-01-01

    To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with "normal" (similar to those in monolayer EC) and "fast" (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.

  19. Singlet-Doublet Dark Matter

    SciTech Connect

    Cohen, Timothy; Kearney, John; Pierce, Aaron; Tucker-Smith, David; /Williams Coll.

    2012-02-15

    In light of recent data from direct detection experiments and the Large Hadron Collider, we explore models of dark matter in which an SU(2){sub L} doublet is mixed with a Standard Model singlet. We impose a thermal history. If the new particles are fermions, this model is already constrained due to null results from XENON100. We comment on remaining regions of parameter space and assess prospects for future discovery. We do the same for the model where the new particles are scalars, which at present is less constrained. Much of the remaining parameter space for both models will be probed by the next generation of direct detection experiments. For the fermion model, DeepCore may also play an important role.

  20. A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei

    PubMed Central

    1992-01-01

    The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule- associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins. PMID:1348252

  1. A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei.

    PubMed

    Hemphill, A; Affolter, M; Seebeck, T

    1992-04-01

    The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule-associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins.

  2. General theory for the mechanics of confined microtubule asters

    NASA Astrophysics Data System (ADS)

    Ma, Rui; Laan, Liedewij; Dogterom, Marileen; Pavin, Nenad; Jülicher, Frank

    2014-01-01

    In cells, dynamic microtubules organize into asters or spindles to assist positioning of organelles. Two types of forces are suggested to contribute to the positioning process: (i) microtubule-growth based pushing forces; and (ii) motor protein mediated pulling forces. In this paper, we present a general theory to account for aster positioning in a confinement of arbitrary shape. The theory takes account of microtubule nucleation, growth, catastrophe, slipping, as well as interaction with cortical force generators. We calculate microtubule distributions and forces acting on microtubule organizing centers in a sphere and in an ellipsoid. Positioning mechanisms based on both pushing forces and pulling forces can be distinguished in our theory for different parameter regimes or in different geometries. In addition, we investigate positioning of microtubule asters in the case of asymmetric distribution of motors. This analysis enables us to characterize situations relevant for Caenorrhabditis elegans embryos.

  3. Achromatic doublets using group indices of refraction

    NASA Astrophysics Data System (ADS)

    Rosete-Aguilar, M.; Estrada-Silva, F. C.; Román-Moreno, C. J.; Ortega-Martínez, R.

    2008-03-01

    One main function of short pulses is to concentrate energy in time and space [1]. The use of refractive lenses allows us to concentrate energy in a small volume of focusing around the focal point of the lens. When using refractive lenses, there are three effects that affect the concentration of energy around the focal point of the lens. These are the group velocity dispersion (GVD), the propagation time difference (PTD), and the aberrations of the lens. In this paper, we study lenses which are diffraction limited so that the monochromatic aberrations are negligible; the group velocity dispersion and the propagation time difference are the main effects affecting the spreading of the pulse at the focus. We will show that for 100-fs pulses the spatial spreading is larger than the temporal spreading of the pulse. It is already known that the effect of spatial spreading of the pulse due to PTD can be reduced by using achromatic optics. We use the theory proposed by A. Vaughan to analyze simple lenses and normal achromatic doublets, where normal means doublets that we can buy from catalogs. We then use the Vaughan theory to design achromatic doublets in phase and group, which produce no spatial spreading of the pulse, i.e., PTD = 0, when the doublet is designed for the carrier of the pulse. We compare these phase and group achromatic doublets with normal achromatic doublets. Finally, we show that apochromatic optics can give a much better correction of PTD than using normal achromatic doublets.

  4. Multimodal microtubule binding by the Ndc80 kinetochore complex.

    PubMed

    Alushin, Gregory M; Musinipally, Vivek; Matson, Daniel; Tooley, John; Stukenberg, P Todd; Nogales, Eva

    2012-11-01

    The Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular 'head' formed by tandem calponin-homology domains and an 80-amino-acid unstructured 'tail' that contains sites of phosphoregulation by the Aurora B kinase. Using biochemical, cell biological and electron microscopy analyses, we dissected the roles of the tail in binding of microtubules and mediation of cooperative interactions between Ndc80 complexes. Two segments of the tail that contain Aurora B phosphorylation sites become ordered at interfaces; one with tubulin and the second with an adjacent Ndc80 head on the microtubule surface, forming interactions that are disrupted by phosphorylation. We propose a model in which Ndc80's interaction with either growing or shrinking microtubule ends can be tuned by the phosphorylation state of its tail.

  5. Association between microtubules and Golgi vesicles isolated from rat parotid glands.

    PubMed

    Coffe, G; Raymond, M N

    1990-01-01

    We report an isolation procedure of trans-Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans-Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol-induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV-bound microtubules co-migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue-stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 micrograms of microtubule protein bound to 500 micrograms of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 micrograms/ml. Electron microscopy of negatively stained re-floated material showed numerous microtubule-vesicle complexes. Coating of microtubules with an excess of brain microtubule-associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold-stable microtubules represents 10-20% of the total tubulin content of the parotid cell.

  6. Microtubules Remodel Actomyosin Networks in Xenopus Egg Extracts via Two Mechanisms of F-Actin Transport

    PubMed Central

    Waterman-Storer, Clare; Duey, Devin Y.; Weber, Kari L.; Keech, John; Cheney, Richard E.; Salmon, E.D.; Bement, William M.

    2000-01-01

    Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid (∼250–300 nm/s) jerking and slow (∼50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings. PMID:10908578

  7. Producing Conditional Mutants for Studying Plant Microtubule Function

    SciTech Connect

    Richard Cyr

    2009-09-29

    The cytoskeleton, and in particular its microtubule component, participates in several processes that directly affect growth and development in higher plants. Normal cytoskeletal function requires the precise and orderly arrangement of microtubules into several cell cycle and developmentally specific arrays. One of these, the cortical array, is notable for its role in directing the deposition of cellulose (the most prominent polymer in the biosphere). An understanding of how these arrays form, and the molecular interactions that contribute to their function, is incomplete. To gain a better understanding of how microtubules work, we have been working to characterize mutants in critical cytoskeletal genes. This characterization is being carried out at the subcellular level using vital microtubule gene constructs. In the last year of funding colleagues have discovered that gamma-tubulin complexes form along the lengths of cortical microtubules where they act to spawn new microtubules at a characteristic 40 deg angle. This finding complements nicely the finding from our lab (which was funded by the DOE) showing that microtubule encounters are angle dependent; high angles encounters results in catastrophic collisions while low angle encounters result in favorable zippering. The finding of a 40 deg spawn of new microtubules from extant microtubule, together with aforementioned rules of encounters, insures favorable co-alignment in the array. I was invited to write a New and Views essay on this topic and a PDF is attached (News and Views policy does not permit funding acknowledgments and so I was not allowed to acknowledge support from the DOE).

  8. Theoretical Description of Microtubule Dynamics in Fission Yeast During Interphase

    NASA Astrophysics Data System (ADS)

    Oei, Yung-Chin; Jiménez-Dalmaroni, Andrea; Vilfan, Andrej; Duke, Thomas

    2009-03-01

    Fission yeast (S. pombe) is a unicellular organism with a characteristic cylindrical shape. Cell growth during interphase is strongly influenced by microtubule self-organization - a process that has been experimentally well characterised. The microtubules are organized in 3 to 4 bundles, called ``interphase microtubule assemblies'' (IMAs). Each IMA is composed of several microtubules, arranged with their dynamic ``plus'' ends facing the cell tips and their ``minus'' ends overlapping at the cell middle. Although the main protein factors involved in interphase microtubule organization have been identified, an understanding of how their collective interaction with microtubules leads to the organization and structures observed in vivo is lacking. We present a physical model of microtubule dynamics that aims to provide a quantitative description of the self-organization process. First, we solve equations for the microtubule length distribution in steady-state, taking into account the way that a limited tubulin pool affects the nucleation, growth and shrinkage of microtubules. Then we incorporate passive and active crosslinkers (the bundling factor Ase1 and molecular motor Klp2) and investigate the formation of IMA structures. Analytical results are complemented by a 3D stochastic simulation.

  9. Inert doublet model and LEP II limits

    SciTech Connect

    Lundstroem, Erik; Gustafsson, Michael; Edsjoe, Joakim

    2009-02-01

    The inert doublet model is a minimal extension of the standard model introducing an additional SU(2) doublet with new scalar particles that could be produced at accelerators. While there exists no LEP II analysis dedicated for these inert scalars, the absence of a signal within searches for supersymmetric neutralinos can be used to constrain the inert doublet model. This translation however requires some care because of the different properties of the inert scalars and the neutralinos. We investigate what restrictions an existing DELPHI Collaboration study of neutralino pair production can put on the inert scalars and discuss the result in connection with dark matter. We find that although an important part of the inert doublet model parameter space can be excluded by the LEP II data, the lightest inert particle still constitutes a valid dark matter candidate.

  10. The Ndc80 kinetochore complex forms oligomeric arrays along microtubules

    PubMed Central

    Alushin, Gregory M.; Ramey, Vincent H.; Pasqualato, Sebastiano; Ball, David A.; Grigorieff, Nikolaus; Musacchio, Andrea; Nogales, Eva

    2010-01-01

    The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment, but the molecular mechanism underlying its function remains unknown. Here we present a subnanometer resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that Ndc80 binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments via interactions mediated by the amino-terminal tail of the Ndc80 protein, the site of phospho-regulation by the Aurora B kinase. Ndc80's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing Ndc80-microtubule attachments. PMID:20944740

  11. Human SAS-6 C-Terminus Nucleates and Promotes Microtubule Assembly in Vitro by Binding to Microtubules.

    PubMed

    Gupta, Hindol; Badarudeen, Binshad; George, Athira; Thomas, Geethu Emily; Gireesh, K K; Manna, Tapas K

    2015-10-20

    Centrioles are essential components of the animal centrosome and play crucial roles in the formation of cilia and flagella. They are cylindrical structures composed of nine triplet microtubules organized around a central cartwheel. Recent studies have identified spindle assembly abnormal protein SAS-6 as a critical component necessary for formation of the cartwheel. However, the molecular details of how the cartwheel participates in centriolar microtubule assembly have not been clearly understood. In this report, we show that the C-terminal tail (residues 470-657) of human SAS-6, HsSAS-6 C, the region that has been shown to extend toward the centriolar wall where the microtubule triplets are organized, nucleated and induced microtubule polymerization in vitro. The N-terminus (residues 1-166) of HsSAS-6, the domain known to be involved in formation of the central hub of the cartwheel, did not, however, exert any effect on microtubule polymerization. HsSAS-6 C bound to the microtubules and localized along the lengths of the microtubules in vitro. Microtubule pull-down and coimmunoprecipitation (Co-IP) experiments with S-phase synchronized HeLa cell lysates showed that the endogenous HsSAS-6 coprecipitated with the microtubules, and it mediated interaction with tubulin. Isothermal calorimetry titration and size exclusion chromatography showed that HsSAS-6 C bound to the αβ-tubulin dimer in vitro. The results demonstrate that HsSAS-6 possesses an intrinsic microtubule assembly promoting activity and further implicate that its outer exposed C-terminal tail may play critical roles in microtubule assembly and stabilizing microtubule attachment with the centriolar cartwheel.

  12. Do prokaryotes contain microtubules?

    PubMed Central

    Bermudes, D; Hinkle, G; Margulis, L

    1994-01-01

    In eukaryotic cells, microtubules are 24-nm-diameter tubular structures composed of a class of conserved proteins called tubulin. They are involved in numerous cell functions including ciliary motility, nerve cell elongation, pigment migration, centrosome formation, and chromosome movement. Although cytoplasmic tubules and fibers have been observed in bacteria, some with diameters similar to those of eukaryotes, no homologies to eukaryotic microtubules have been established. Certain groups of bacteria including azotobacters, cyanobacteria, enteric bacteria, and spirochetes have been frequently observed to possess microtubule-like structures, and others, including archaebacteria, have been shown to be sensitive to drugs that inhibit the polymerization of microtubules. Although little biochemical or molecular biological information is available, the differences observed among these prokaryotic structures suggest that their composition generally differs among themselves as well as from that of eukaryotes. We review the distribution of cytoplasmic tubules in prokaryotes, even though, in all cases, their functions remain unknown. At least some tend to occur in cells that are large, elongate, and motile, suggesting that they may be involved in cytoskeletal functions, intracellular motility, or transport activities comparable to those performed by eukaryotic microtubules. In Escherichia coli, the FtsZ protein is associated with the formation of a ring in the division zone between the newly forming offspring cells. Like tubulin, FtsZ is a GTPase and shares with tubulin a 7-amino-acid motif, making it a promising candidate in which to seek the origin of tubulins. Images PMID:7968920

  13. Do prokaryotes contain microtubules?

    NASA Technical Reports Server (NTRS)

    Bermudes, D.; Hinkle, G.; Margulis, L.

    1994-01-01

    In eukaryotic cells, microtubules are 24-nm-diameter tubular structures composed of a class of conserved proteins called tubulin. They are involved in numerous cell functions including ciliary motility, nerve cell elongation, pigment migration, centrosome formation, and chromosome movement. Although cytoplasmic tubules and fibers have been observed in bacteria, some with diameters similar to those of eukaryotes, no homologies to eukaryotic microtubules have been established. Certain groups of bacteria including azotobacters, cyanobacteria, enteric bacteria, and spirochetes have been frequently observed to possess microtubule-like structures, and others, including archaebacteria, have been shown to be sensitive to drugs that inhibit the polymerization of microtubules. Although little biochemical or molecular biological information is available, the differences observed among these prokaryotic structures suggest that their composition generally differs among themselves as well as from that of eukaryotes. We review the distribution of cytoplasmic tubules in prokaryotes, even though, in all cases, their functions remain unknown. At least some tend to occur in cells that are large, elongate, and motile, suggesting that they may be involved in cytoskeletal functions, intracellular motility, or transport activities comparable to those performed by eukaryotic microtubules. In Escherichia coli, the FtsZ protein is associated with the formation of a ring in the division zone between the newly forming offspring cells. Like tubulin, FtsZ is a GTPase and shares with tubulin a 7-amino-acid motif, making it a promising candidate in which to seek the origin of tubulins.

  14. A study of microtubule dipole lattices

    NASA Astrophysics Data System (ADS)

    Nandi, Shubhendu

    Microtubules are cytoskeletal protein polymers orchestrating a host of important cellular functions including, but not limited to, cell support, cell division, cell motility and cell transport. In this thesis, we construct a toy-model of the microtubule lattice composed of vector Ising spins representing tubulin molecules, the building block of microtubules. Nearest-neighbor and next-to-nearest neighbor interactions are considered within an anisotropic dielectric medium. As a consequence of the helical topology, we observe that certain spin orientations render the lattice frustrated with nearest neighbor ferroelectric and next-to-nearest neighbor antiferroelectric bonds. Under these conditions, the lattice displays the remarkable property of stabilizing certain spin patterns that are robust to thermal fluctuations. We model this behavior in the framework of a generalized Ising model known as the J1 - J2 model and theoretically determine the set of stable patterns. Employing Monte-Carlo methods, we demonstrate the stability of such patterns in the microtubule lattice at human physiological temperatures. This suggests a novel biological mechanism for storing information in living organisms, whereby the tubulin spin (dipole moment) states become information bits and information gets stored in microtubules in a way that is robust to thermal fluctuations.

  15. Buckling of microtubules on elastic media via breakable bonds.

    PubMed

    Afrin, Tanjina; Kabir, Arif Md Rashedul; Sada, Kazuki; Kakugo, Akira; Nitta, Takahiro

    2016-11-04

    Buckling of microtubules observed in cells has been reconstructed on a two-dimensional elastic medium consisting of kinesins grafted over compressible substrates, enabling precise control of experimental conditions and quantitative analysis. However, interpretations of the observations have ambiguities due to inevitable experimental difficulties. In this study, with computer simulations, we investigated importance of the mode of interaction of microtubule with elastic medium in the buckling behavior of microtubule. By taking into consideration of forced-induced detachments of kinesins from microtubules, our simulations reproduced the previous experimental results, and showed deviations from predictions of the elastic foundation model. On the other hand, with hypothetical linkers permanently bound to microtubules, our simulation reproduced the predictions of the elastic foundation model. By analyzing the results of the simulations, we investigated as to why the difference arose. These findings indicate the importance of the mode of interaction of microtubule with the medium in the buckling behavior of microtubule. Our findings would bring new insights on buckling of microtubules in living cells.

  16. BRIDGES BETWEEN MICROTUBULES

    PubMed Central

    McIntosh, J. R.

    1974-01-01

    Bridges between microtubules have been studied with the electron microscope in the axostyle of Saccinobaculus and in various tubule systems of chicken testis, including the helix of tubules surrounding the elongating spermatid nucleus and the flagellum of the sperm tail. In addition to the previously described periodic bridges, evidence is presented that nonperiodic bridges exist between certain tubules. An analysis of axial spacing between adjacent nonperiodic bridges suggests that these structures are attached to periodic binding sites on the microtubule wall, but that not all the binding sites are filled. The bridges appear nonperiodic as a result of random occupancy of some fraction of the periodic sites. The distribution of these binding sites is related to the substructure of the microtubule wall as seen with negative staining and optical diffraction. PMID:4132065

  17. Expression of developmentally regulated plasma membrane polypeptide (DREPP2) in rice root tip and interaction with Ca(2+)/CaM complex and microtubule.

    PubMed

    Yamada, Nana; Theerawitaya, Cattarin; Kageyama, Hakuto; Cha-Um, Suriyan; Takabe, Teruhiro

    2015-11-01

    The cytoplasmic free Ca(2+) could play an important role for salt tolerance in rice root (Oryza sativa L.). Here, we compared the expression profiles of two putative developmentally regulated plasma membrane polypeptides (DREPP1 and DREPP2) in rice roots of salt-tolerant cv. Pokkali and salt-sensitive cv. IR29. The messenger RNA (mRNA) for OsDREPP1 could be detected in all parts of root and did not change upon salt stress, whereas the mRNA for OsDREPP2 was detected only in root tips. The transcript level of OsDREPP2 first disappeared upon salt stress, then recovered in Pokkali, but not recovered in IR29. The gene-encoding OsDREPP2 was cloned from cv. Pokkali and expressed in Escherichia coli, and its biochemical properties were studied. It was found that OsDREPP2 is a Ca(2+)-binding protein and binds also to calmodulin (CaM) as well as microtubules. The mutation of Trp4 and Phe16 in OsDREPP2 to Ala decreased the binding of DREPP2 to Ca(2+)/CaM complex, indicating the N-terminal basic domain is involved for the binding. The binding of OsDREPP2 to microtubules was inhibited by Ca(2+)/CaM complex, while the binding of double-mutant OsDREPP2 protein to microtubules was not inhibited by Ca(2+)/CaM complex. We propose that CaM inhibits the binding of DREPP2 to cortical microtubules, causes the inhibition of microtubule depolymerization, and enhances the cell elongation.

  18. Mechanical model of kinesin moving on microtubule

    NASA Astrophysics Data System (ADS)

    To, Kiwing; Chou, Ya-Chang; Hsiao, Yi-Feng; Chen, Kuan-Hua

    Kinesins are biomolecules that serve as intercellular motors for carrying cellular cargos along microtubules. Although the mechanism of converting the chemical energy of ATP to mechanical work is not fully understood, the motion of a kinesin on a microtubule has been measured and two different mechanisms, namely the ``hand-over-hand'' and ``inchworm'', has been proposed. The particular shape of kinesin and microtubules suggest a possible mechanism for force generation similar to Brownian ratchet. Using a bead chain connected to two heads that are attracted to a vibrated ratchet plate as a scaled up analog of the kinesinmicrotubule system, we manage to simulate both ``handoverhand'' and ``inchworm'' motion [Chou, et. al., Physica A443, 66 (2015)]. In addition, we find that chain, which play the role of the stalk in a kinesin molecule, can also generate force by interacting with the ratchet plate [Chen, et. al. Phys. Rev. E87, 012711 (2013)].

  19. Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy

    PubMed Central

    Wang, Yan; Zheng, Xiyin; Yu, Bingjie; Han, Shaojie; Guo, Jiangbo; Tang, Haiping; Yu, Alice Yunzi L; Deng, Haiteng; Hong, Yiguo; Liu, Yule

    2015-01-01

    Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation. PMID:26566764

  20. CYLD Regulates Noscapine Activity in Acute Lymphoblastic Leukemia via a Microtubule-Dependent Mechanism.

    PubMed

    Yang, Yunfan; Ran, Jie; Sun, Lei; Sun, Xiaodong; Luo, Youguang; Yan, Bing; Tala; Liu, Min; Li, Dengwen; Zhang, Lei; Bao, Gang; Zhou, Jun

    2015-01-01

    Noscapine is an orally administrable drug used worldwide for cough suppression and has recently been demonstrated to disrupt microtubule dynamics and possess anticancer activity. However, the molecular mechanisms regulating noscapine activity remain poorly defined. Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL). Flow cytometry and immunofluorescence microscopy reveal that CYLD increases the ability of noscapine to induce mitotic arrest and apoptosis. Examination of cellular microtubules as well as in vitro assembled microtubules shows that CYLD enhances the effect of noscapine on microtubule polymerization. Microtubule cosedimentation and fluorescence titration assays further reveal that CYLD interacts with microtubule outer surface and promotes noscapine binding to microtubules. These findings thus demonstrate CYLD as a critical regulator of noscapine activity and have important implications for ALL treatment.

  1. Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

    PubMed Central

    Howes, Stuart C.; Alushin, Gregory M.; Shida, Toshinobu; Nachury, Maxence V.; Nogales, Eva

    2014-01-01

    Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments. PMID:24227885

  2. Tensile stress stimulates microtubule outgrowth in living cells

    NASA Technical Reports Server (NTRS)

    Kaverina, Irina; Krylyshkina, Olga; Beningo, Karen; Anderson, Kurt; Wang, Yu-Li; Small, J. Victor

    2002-01-01

    Cell motility is driven by the sum of asymmetric traction forces exerted on the substrate through adhesion foci that interface with the actin cytoskeleton. Establishment of this asymmetry involves microtubules, which exert a destabilising effect on adhesion foci via targeting events. Here, we demonstrate the existence of a mechano-sensing mechanism that signals microtubule polymerisation and guidance of the microtubules towards adhesion sites under increased stress. Stress was applied either by manipulating the body of cells moving on glass with a microneedle or by stretching a flexible substrate that cells were migrating on. We propose a model for this mechano-sensing phenomenon whereby microtubule polymerisation is stimulated and guided through the interaction of a microtubule tip complex with actin filaments under tension.

  3. Microtubule stabilising peptides rescue tau phenotypes in-vivo

    PubMed Central

    Quraishe, Shmma; Sealey, Megan; Cranfield, Louise; Mudher, Amritpal

    2016-01-01

    The microtubule cytoskeleton is a highly dynamic, filamentous network underpinning cellular structure and function. In Alzheimer’s disease, the microtubule cytoskeleton is compromised, leading to neuronal dysfunction and eventually cell death. There are currently no disease-modifying therapies to slow down or halt disease progression. However, microtubule stabilisation is a promising therapeutic strategy that is being explored. We previously investigated the disease-modifying potential of a microtubule-stabilising peptide NAP (NAPVSIPQ) in a well-established Drosophila model of tauopathy characterised by microtubule breakdown and axonal transport deficits. NAP prevented as well as reversed these phenotypes even after they had become established. In this study, we investigate the neuroprotective capabilities of an analogous peptide SAL (SALLRSIPA). We found that SAL mimicked NAP’s protective effects, by preventing axonal transport disruption and improving behavioural deficits, suggesting both NAP and SAL may act via a common mechanism. Both peptides contain a putative ‘SIP’ (Ser-Ile-Pro) domain that is important for interactions with microtubule end-binding proteins. Our data suggests this domain may be central to the microtubule stabilising function of both peptides and the mechanism by which they rescue phenotypes in this model of tauopathy. Our observations support microtubule stabilisation as a promising disease-modifying therapeutic strategy for tauopathies like Alzheimer’s disease. PMID:27910888

  4. Multiscale modeling and simulation of microtubule-motor-protein assemblies

    NASA Astrophysics Data System (ADS)

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, M. D.; Shelley, Michael J.

    2015-12-01

    Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.

  5. Multiscale modeling and simulation of microtubule-motor-protein assemblies.

    PubMed

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A; Betterton, M D; Shelley, Michael J

    2015-01-01

    Microtubules and motor proteins self-organize into biologically important assemblies including the mitotic spindle and the centrosomal microtubule array. Outside of cells, microtubule-motor mixtures can form novel active liquid-crystalline materials driven out of equilibrium by adenosine triphosphate-consuming motor proteins. Microscopic motor activity causes polarity-dependent interactions between motor proteins and microtubules, but how these interactions yield larger-scale dynamical behavior such as complex flows and defect dynamics is not well understood. We develop a multiscale theory for microtubule-motor systems in which Brownian dynamics simulations of polar microtubules driven by motors are used to study microscopic organization and stresses created by motor-mediated microtubule interactions. We identify polarity-sorting and crosslink tether relaxation as two polar-specific sources of active destabilizing stress. We then develop a continuum Doi-Onsager model that captures polarity sorting and the hydrodynamic flows generated by these polar-specific active stresses. In simulations of active nematic flows on immersed surfaces, the active stresses drive turbulent flow dynamics and continuous generation and annihilation of disclination defects. The dynamics follow from two instabilities, and accounting for the immersed nature of the experiment yields unambiguous characteristic length and time scales. When turning off the hydrodynamics in the Doi-Onsager model, we capture formation of polar lanes as observed in the Brownian dynamics simulation.

  6. α-Synuclein is a Novel Microtubule Dynamase.

    PubMed

    Cartelli, Daniele; Aliverti, Alessandro; Barbiroli, Alberto; Santambrogio, Carlo; Ragg, Enzio M; Casagrande, Francesca V M; Cantele, Francesca; Beltramone, Silvia; Marangon, Jacopo; De Gregorio, Carmelita; Pandini, Vittorio; Emanuele, Marco; Chieregatti, Evelina; Pieraccini, Stefano; Holmqvist, Staffan; Bubacco, Luigi; Roybon, Laurent; Pezzoli, Gianni; Grandori, Rita; Arnal, Isabelle; Cappelletti, Graziella

    2016-09-15

    α-Synuclein is a presynaptic protein associated to Parkinson's disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson's disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics.

  7. α-Synuclein is a Novel Microtubule Dynamase

    PubMed Central

    Cartelli, Daniele; Aliverti, Alessandro; Barbiroli, Alberto; Santambrogio, Carlo; Ragg, Enzio M.; Casagrande, Francesca V.M.; Cantele, Francesca; Beltramone, Silvia; Marangon, Jacopo; De Gregorio, Carmelita; Pandini, Vittorio; Emanuele, Marco; Chieregatti, Evelina; Pieraccini, Stefano; Holmqvist, Staffan; Bubacco, Luigi; Roybon, Laurent; Pezzoli, Gianni; Grandori, Rita; Arnal, Isabelle; Cappelletti, Graziella

    2016-01-01

    α-Synuclein is a presynaptic protein associated to Parkinson’s disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson’s disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics. PMID:27628239

  8. Idiotypic mimicry and the assembly of a supramolecular structure: an anti-idiotypic antibody that mimics taxol in its tubulin-microtubule interactions.

    PubMed Central

    Leu, J G; Chen, B X; Diamanduros, A W; Erlanger, B F

    1994-01-01

    Taxol, originally extracted from the bark of the western yew, Taxus brevifolia, is reportedly the first of a new class of anti-cancer agents. It acts by promoting and irreversibly stabilizing microtubule assembly, thus interfering with the dynamic processes required for cell viability and multiplication. With the aim of using immunological techniques to study the mechanism of action of taxol, a monoclonal anti-idiotypic antibody that mimics taxol was prepared, using an auto-anti-idiotypic strategy. It and its Fab fragment inhibited the binding of [3H]taxol to microtubules. Moreover, like taxol, both promoted the assembly of tubulin into microtubules. These findings provide an example of an anti-idiotypic antibody capable of assembling an organized supramolecular structure from soluble cellular components. In addition, it further establishes the ability of anti-idiotypic antibodies to be functional mimics of ligand molecules bearing no structural similarity to immunoglobulins. The variable regions of the antibody have been sequenced. With the exception of the complementarity-determining region 3, the sequence of the heavy chain variable region is strikingly similar to that of an anti-idiotypic antibody raised to anti-insulin. The finding that a polypeptide can mimic taxol raises the possibility that taxol acts as a peptidomimetic compound that interferes with the function of an endogenous polypeptide. Images PMID:7840821

  9. The Dynamic Behavior of Individual Microtubules Associated with Chromosomes In Vitro

    PubMed Central

    Hunt, Alan J.; McIntosh, J. Richard

    1998-01-01

    Mitotic movements of chromosomes are usually coupled to the elongation and shortening of the microtubules to which they are bound. The lengths of kinetochore-associated microtubules change by incorporation or loss of tubulin subunits, principally at their chromosome-bound ends. We have reproduced aspects of this phenomenon in vitro, using a real-time assay that displays directly the movements of individual chromosome-associated microtubules as they elongate and shorten. Chromosomes isolated from cultured Chinese hamster ovary cells were adhered to coverslips and then allowed to bind labeled microtubules. In the presence of tubulin and GTP, these microtubules could grow at their chromosome-bound ends, causing the labeled segments to move away from the chromosomes, even in the absence of ATP. Sometimes a microtubule would switch to shortening, causing the direction of movement to change abruptly. The link between a microtubule and a chromosome was mechanically strong; 15 pN of tension was generally insufficient to detach a microtubule, even though it could add subunits at the kinetochore–microtubule junction. The behavior of the microtubules in vitro was regulated by the chromosomes to which they were bound; the frequency of transitions from polymerization to depolymerization was decreased, and the speed of depolymerization-coupled movement toward chromosomes was only one-fifth the rate of shortening for microtubules free in solution. Our results are consistent with a model in which each microtubule interacts with an increasing number of chromosome-associated binding sites as it approaches the kinetochore. PMID:9763448

  10. Structural Analysis of Mutations in the Drosophila β2-Tubulin Isoform Reveals Regions in the β-Tubulin Molecule Required for General and for Tissue-Specific Microtubule Functions

    PubMed Central

    Fackenthal, J. D.; Hutchens, J. A.; Turner, F. R.; Raff, E. C.

    1995-01-01

    We have determined the lesions in a number of mutant alleles of βTub85D, the gene that encodes the testis-specific β2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the β2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all β-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t(6) contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate β-tubulins. Correspondingly, B2t(6) disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that β3, a developmentally regulated Drosophila β-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type β2-tubulin. We show here by complementation analysis that β3 and the B2t(6) product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the β2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the β2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the β2 variant lacking the carboxy terminus and the B2t(6) variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from

  11. Doublet Tracer Testing in Klamath Falls, Oregon

    SciTech Connect

    Gudmundsson, J.S.; Johnson, S.E.; Horne, R.N.; Jackson, P.B.; Culver, G.G.

    1983-12-15

    A tracer test was carried out in a geothermal doublet system to study the injection behavior of a developed reservoir known to be fractured. The doublet produces about 320 gpm of 160 F water that is used for space heating and then injected; the wells are spaced 250 ft apart. Tracer breakthrough was observed in 2 hours and 45 minutes in the production well, indicating fracture flow. However, the tracer concentrations were low and indicated porous media flow; the tracers mixed with a reservoir volume much larger than a fracture.

  12. Doublet-doublet fluorescence of benzyl, p-methylbenzyl and p-chlorobenzyl radicals in solution

    NASA Astrophysics Data System (ADS)

    Tokumura, Kunihiro; Udagawa, Masahiro; Ozaki, Tomomi; Itoh, Michiya

    1987-11-01

    The doublet-doublet fluorescence spectra of benzyl, p-methylbenzyl and p-chlorobenzyl radicals were detected in the 450-650 nm region upon the double-pulse (248 nm→308 nm) excitation of corresponding benzyl chlorides in hexane at room temperature. The respective fluorescence lifetimes were determined to be ≈ 1 ns or less (benzyl), 14 ns ( p-methylbenzyl) and 81 ns ( p-chlorobenzyl). Extensively temperature-dependent non-radiative relaxation was confirmed for these benzyl radicals with close-lying lowest doublet excited states.

  13. Depletion force induced collective motion of microtubules driven by kinesin.

    PubMed

    Inoue, Daisuke; Mahmot, Bulbul; Kabir, Arif Md Rashedul; Farhana, Tamanna Ishrat; Tokuraku, Kiyotaka; Sada, Kazuki; Konagaya, Akihiko; Kakugo, Akira

    2015-11-21

    Collective motion is a fascinating example of coordinated behavior of self-propelled objects, which is often associated with the formation of large scale patterns. Nowadays, the in vitro gliding assay is being considered a model system to experimentally investigate various aspects of group behavior and pattern formation by self-propelled objects. In the in vitro gliding assay, cytoskeletal filaments F-actin or microtubules are driven by the surface immobilized associated biomolecular motors myosin or dynein respectively. Although the F-actin/myosin or microtubule/dynein system was found to be promising in understanding the collective motion and pattern formation by self-propelled objects, the most widely used biomolecular motor system microtubule/kinesin could not be successfully employed so far in this regard. Failure in exhibiting collective motion by kinesin driven microtubules is attributed to the intrinsic properties of kinesin, which was speculated to affect the behavior of individual gliding microtubules and mutual interactions among them. In this work, for the first time, we have demonstrated the collective motion of kinesin driven microtubules by regulating the mutual interaction among the gliding microtubules, by employing a depletion force among them. Proper regulation of the mutual interaction among the gliding microtubules through the employment of the depletion force was found to allow the exhibition of collective motion and stream pattern formation by the microtubules. This work offers a universal means for demonstrating the collective motion using the in vitro gliding assay of biomolecular motor systems and will help obtain a meticulous understanding of the fascinating coordinated behavior and pattern formation by self-propelled objects.

  14. Microtubule dynamics in fish melanophores

    PubMed Central

    1994-01-01

    We have studied the dynamics of microtubules in black tetra (Gymnocorymbus ternetzi) melanophores to test the possible correlation of microtubule stability and intracellular particle transport. X- rhodamine-or caged fluorescein-conjugated tubulin were microinjected and visualized by fluorescence digital imaging using a cooled charge coupled device and videomicroscopy. Microtubule dynamics were evaluated by determining the time course of tubulin incorporation after pulse injection, by time lapse observation, and by quantitation of fluorescence redistribution after photobleaching and photoactivation. The time course experiments showed that the kinetics of incorporation of labeled tubulin into microtubules were similar for cells with aggregated or dispersed pigment with most microtubules becoming fully labeled within 15-20 min after injection. Quantitation by fluorescence redistribution after photobleaching and photoactivation confirmed that microtubule turnover was rapid in both states, t1/2 = 3.5 +/- 1.5 and 6.1 +/- 3.0 min for cells with aggregated and dispersed pigment, respectively. In addition, immunostaining with antibodies specific to posttranslationally modified alpha-tubulin, which is usually enriched in stable microtubules, showed that microtubules composed exclusively of detyrosinated tubulin were absent and microtubules containing acetylated tubulin were sparse. We conclude that the microtubules of melanophores are very dynamic, that their dynamic properties do not depend critically on the state of pigment distribution, and that their stabilization is not a prerequisite for intracellular transport. PMID:8089178

  15. Three doublet lepton-specific model

    NASA Astrophysics Data System (ADS)

    Merchand, Marco; Sher, Marc

    2017-03-01

    In the lepton-specific version of two Higgs doublet models, a discrete symmetry is used to couple one Higgs boson, Φ2, to quarks and the other, Φ1, to leptons. The symmetry eliminates tree level flavor changing neutral currents. Motivated by strong constraints on such currents in the quark sector from meson-antimeson mixing, and by hints of h →μ τ in the lepton sector, we study a simple three Higgs doublet model in which one doublet couples to quarks and the other two to leptons. Unlike most other studies of three Higgs doublet models, we impose no flavor symmetry and just use a Z2 symmetry to constrain the Yukawa couplings. We present the model and discuss the various mixing angles. Constraining the parameters to be consistent with observations of the Higgs boson at the LHC, we study the properties of the charged Higgs boson(s) in the model, focusing on the case in which the charged Higgs boson is above the top threshold. It is found that one can have the branching fraction of the charged Higgs boson into τ ντ comparable to t b ¯ decay without needing very large values for the ratios of vacuum expectation values. One can also get a large branching fraction for the much more easily observable μ ντ decay.

  16. Cell Biology: Microtubule Collisions to the Rescue.

    PubMed

    Gardner, Melissa K

    2016-12-19

    The proper regulation of microtubule lengths is fundamental to their cellular function. New work now reports that the collision of a growing microtubule end with another object, such as a microtubule, can contribute to the regulation of microtubule lengths by leaving behind damage that ultimately acts to stabilize the microtubule network.

  17. The physical basis of microtubule structure and stability.

    PubMed

    Sept, David; Baker, Nathan A; McCammon, J Andrew

    2003-10-01

    Microtubules are cylindrical polymers found in every eukaryotic cell. They have a unique helical structure that has implications at both the cellular level, in terms of the functions they perform, and at the multicellular level, such as determining the left-right symmetry in plants. Through the combination of an atomically detailed model for a microtubule and large-scale computational techniques for computing electrostatic interactions, we are able to explain the observed microtubule structure. On the basis of the lateral interactions between protofilaments, we have determined that B lattice is the most favorable configuration. Further, we find that these lateral bonds are significantly weaker than the longitudinal bonds along protofilaments. This explains observations of microtubule disassembly and may serve as another step toward understanding the basis for dynamic instability.

  18. A theory of microtubule catastrophes and their regulation.

    PubMed

    Brun, Ludovic; Rupp, Beat; Ward, Jonathan J; Nédélec, François

    2009-12-15

    Dynamic instability, in which abrupt transitions occur between growing and shrinking states, is an intrinsic property of microtubules that is regulated by both mechanics and specialized proteins. We discuss a model of dynamic instability based on the popular idea that growth is maintained by a cap at the tip of the fiber. The loss of this cap is thought to trigger the transition from growth to shrinkage, called a catastrophe. The model includes longitudinal interactions between the terminal tubulins of each protofilament and "gating rescues" between neighboring protofilaments. These interactions allow individual protofilaments to transiently shorten during a phase of overall microtubule growth. The model reproduces the reported dependency of the catastrophe rate on tubulin concentration, the time between tubulin dilution and catastrophe, and the induction of microtubule catastrophes by walking depolymerases. The model also reproduces the comet tail distribution that is characteristic of proteins that bind to the tips of growing microtubules.

  19. Microtubules Accelerate the Kinase Activity of Aurora-B by a Reduction in Dimensionality

    PubMed Central

    Noujaim, Michael; Bechstedt, Susanne; Wieczorek, Michal; Brouhard, Gary J.

    2014-01-01

    Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a “reduction in dimensionality.” We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates. PMID:24498282

  20. Physical Modeling of Microtubules Network

    NASA Astrophysics Data System (ADS)

    Allain, Pierre; Kervrann, Charles

    2014-10-01

    Microtubules (MT) are highly dynamic tubulin polymers that are involved in many cellular processes such as mitosis, intracellular cell organization and vesicular transport. Nevertheless, the modeling of cytoskeleton and MT dynamics based on physical properties is difficult to achieve. Using the Euler-Bernoulli beam theory, we propose to model the rigidity of microtubules on a physical basis using forces, mass and acceleration. In addition, we link microtubules growth and shrinkage to the presence of molecules (e.g. GTP-tubulin) in the cytosol. The overall model enables linking cytosol to microtubules dynamics in a constant state space thus allowing usage of data assimilation techniques.

  1. Microtubules (tau) as an emerging therapeutic target: NAP (davunetide).

    PubMed

    Gozes, Illana

    2011-01-01

    This review focuses on the discovery of activity-dependent neuroprotective protein (ADNP) and the ensuing discovery of NAP (davunetide) toward clinical development with emphasis on microtubule protection. ADNP immunoreactivity was shown to occasionally decorate microtubules and ADNP silencing inhibited neurite outgrowth as measured by microtubule associated protein 2 (MAP2) labeling. ADNP knockout is lethal, while 50% reduction in ADNP (ADNP haploinsufficiency) resulted in the microtubule associated protein tau pathology coupled to cognitive dysfunction and neurodegeneration. NAP (davunetide), an eight amino acid peptide derived from ADNP partly ameliorated deficits associated with ADNP deficiency. NAP (davunetide) interacted with microtubules, protected against microtubule toxicity associated with zinc, nocodazole and oxidative stress in vitro and against tau pathology and MAP6 (stable tubuleonly polypeptide - STOP) pathology in vivo. NAP (davunetide) provided neurotrophic functions promoting neurite outgrowth as measured by increases in MAP2 immunoreactivity and synapse formation by increasing synaptophysin expression. NAP (davunetide) protection against neurodegeneration has recently been shown to extend to katanin-related microtubule disruption under conditions of tau deficiencies. In conclusion, NAP (davunetide) provided potent neuroprotection in a broad range of neurodegenerative models, protecting the neuroglial cytoskeleton in vitro and inhibiting tau pathology (tauopathy) in vivo. Based on these extensive preclinical results, davunetide (NAP) is now being evaluated in a Phase II/III study of the tauopathy, progressive supranuclear palsy (PSP); (Allon Therapeutics Inc.).

  2. Impact of C24:0 on actin-microtubule interaction in human neuronal SK-N-BE cells: evaluation by FRET confocal spectral imaging microscopy after dual staining with rhodamine-phalloidin and tubulin tracker green.

    PubMed

    Zarrouk, Amira; Nury, Thomas; Dauphin, Aurélien; Frère, Perrine; Riedinger, Jean-Marc; Bachelet, Claude-Marie; Frouin, Frédérique; Moreau, Thibault; Hammami, Mohamed; Kahn, Edmond; Lizard, Gérard

    2015-01-01

    Disorganization of the cytoskeleton of neurons has major consequences on the transport of neurotransmitters via the microtubule network. The interaction of cytoskeleton proteins (actin and tubulin) was studied in neuronal SK-N-BE cells treated with tetracosanoic acid (C24:0), which is cytotoxic and increased in Alzheimer's disease patients. When SK-N-BE cells were treated with C24:0, mitochondrial dysfunctions and a non-apoptotic mode of cell death were observed. Fluorescence microscopy revealed shrunken cells with perinuclear condensation of actin and tubulin. Impact of C24:0 on actin-microtubule interaction in human neuronal SK-N-BE cells: evaluation by FRET confocal spectral imaging microscopy after dual staining with rhodamine-phalloidin and tubulin tracker green After staining with rhodamine-phalloidin and with an antibody raised against α-/β-tubulin, modifications of F-actin and α-/β-tubulin levels were detected by flow cytometry. Lower levels of α-tubulin were found by Western blotting. In C24:0-treated cells, spectral analysis and fluorescence recovery after photobleaching (FRAP) measured by confocal microscopy proved the existence of fluorescence resonance energy transfer (FRET) when actin and tubulin were stained with tubulin tracker and rhodamine-phalloidin demonstrating actin and tubulin co-localization/interaction. In control cells, no FRET was observed. Our data demonstrate quantitative changes in actin and tubulin, and modified interactions between actin and tubulin in SK-N-BE cells treated with C24:0. They also show that FRET confocal imaging microscopy is an interesting method for specifying the impact of cytotoxic compounds on cytoskeleton proteins.

  3. TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types

    PubMed Central

    Nwagbara, Belinda U.; Faris, Anna E.; Bearce, Elizabeth A.; Erdogan, Burcu; Ebbert, Patrick T.; Evans, Matthew F.; Rutherford, Erin L.; Enzenbacher, Tiffany B.; Lowery, Laura Anne

    2014-01-01

    Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end–tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics. PMID:25187649

  4. Localization of tektin filaments in microtubules of sea urchin sperm flagella by immunoelectron microscopy

    PubMed Central

    1985-01-01

    Extraction of doublet microtubules from the sperm flagella of the sea urchin Strongylocentrotus purpuratus with sarkosyl (0.5%)-urea (2.5 M) yields a highly pure preparation of "tektin" filaments that we have previously shown to resemble intermediate filament proteins. They form filaments 2-3 nm in diameter as seen by negative stain electron microscopy and are composed of approximately equal amounts of three polypeptide bands with apparent molecular weights of 47,000, 51,000, and 55,000, as determined by SDS PAGE. We prepared antibodies to this set of proteins to localize them in the doublet microtubules of S. purpuratus and other species. Tektins and tubulin were antigenically distinct when tested by immunoblotting with affinity-purified antitektin and antitubulin antibodies. Fixed sperm or axonemes from several different species of sea urchin showed immunofluorescent staining with antitektin antibodies. We also used antibodies coupled to gold spheres to localize the proteins by electron microscopy. Whereas a monoclonal antitubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol. 93:576-582) decorates intact microtubules along their lengths, antitektins labeled only the ends of intact microtubules and sarkosyl-insoluble ribbons. However, if microtubules and ribbons attached to electron microscope grids were first extracted with sarkosyl-urea, the tektin filaments that remain were decorated by antitektin antibodies throughout their length. These results suggest that tektins form integral filaments of flagellar microtubule walls, whose antigenic sites are normally masked, perhaps by the presence of tubulin around them. PMID:3880749

  5. The Ndc80 complex uses a tripartite attachment point to couple microtubule depolymerization to chromosome movement.

    PubMed

    Tooley, John G; Miller, Stephanie A; Stukenberg, P Todd

    2011-04-15

    In kinetochores, the Ndc80 complex couples the energy in a depolymerizing microtubule to perform the work of moving chromosomes. The complex directly binds microtubules using an unstructured, positively charged N-terminal tail located on Hec1/Ndc80. Hec1/Ndc80 also contains a calponin homology domain (CHD) that increases its affinity for microtubules in vitro, yet whether it is required in cells and how the tail and CHD work together are critical unanswered questions. Human kinetochores containing Hec1/Ndc80 with point mutations in the CHD fail to align chromosomes or form productive microtubule attachments. Kinetochore architecture and spindle checkpoint protein recruitment are unaffected in these mutants, and the loss of CHD function cannot be rescued by removing Aurora B sites from the tail. The interaction between the Hec1/Ndc80 CHD and a microtubule is facilitated by positively charged amino acids on two separate regions of the CHD, and both are required for kinetochores to make stable attachments to microtubules. Chromosome congression in cells also requires positive charge on the Hec1 tail to facilitate microtubule contact. In vitro binding data suggest that charge on the tail regulates attachment by directly increasing microtubule affinity as well as driving cooperative binding of the CHD. These data argue that in vertebrates there is a tripartite attachment point facilitating the interaction between Hec1/Ndc80 and microtubules. We discuss how such a complex microtubule-binding interface may facilitate the coupling of depolymerization to chromosome movement.

  6. Kinesin-4 KIF21B is a potent microtubule pausing factor

    PubMed Central

    van Riel, Wilhelmina E; Rai, Ankit; Bianchi, Sarah; Katrukha, Eugene A; Liu, Qingyang; Heck, Albert JR; Hoogenraad, Casper C; Steinmetz, Michel O; Kapitein, Lukas C; Akhmanova, Anna

    2017-01-01

    Microtubules are dynamic polymers that in cells can grow, shrink or pause, but the factors that promote pausing are poorly understood. Here, we show that the mammalian kinesin-4 KIF21B is a processive motor that can accumulate at microtubule plus ends and induce pausing. A few KIF21B molecules are sufficient to induce strong growth inhibition of a microtubule plus end in vitro. This property depends on non-motor microtubule-binding domains located in the stalk region and the C-terminal WD40 domain. The WD40-containing KIF21B tail displays preference for a GTP-type over a GDP-type microtubule lattice and contributes to the interaction of KIF21B with microtubule plus ends. KIF21B also contains a motor-inhibiting domain that does not fully block the interaction of the protein with microtubules, but rather enhances its pause-inducing activity by preventing KIF21B detachment from microtubule tips. Thus, KIF21B combines microtubule-binding and regulatory activities that together constitute an autonomous microtubule pausing factor. DOI: http://dx.doi.org/10.7554/eLife.24746.001 PMID:28290984

  7. Analysis of Soybean Microtubule Persistence Length; New Evidence on the Correlation between Structural Composition and Mechanical Properties

    NASA Astrophysics Data System (ADS)

    Shojania Feizabadi, Mitra; Winton, Carly; Barrientos, Jimmy

    2012-02-01

    Recent studies on microtubules composed of different β tubulin isotypes indicate their different functionality in terms of their dynamical behavior or the mechanism of their interaction with chemotherapeutic drugs. Along these lines, the result of our recent study measuring the rigidity of neural and non-neural samples of microtubules with different β tubulin isotype compositions suggests that the distinguished mechanical properties of microtubules, such as rigidity, may also be associated with the different distribution of their β tubulin isotypes. In our current study, we have reported the persistence length of a single soybean microtubule. This plant microtubule has a structural composition different from that of mammalian microtubules. Under the same experimental methods of measurement, the soybean microtubules showed a different persistence length as compared to the value of the persistence length that we estimated in the study of both single Bovine Brain and MCF7 microtubules.

  8. Doublet III: status and future plans

    SciTech Connect

    Rawls, J.M.

    1980-04-01

    A synopsis is presented of the experimental results from the ohmic heating phase of Doublet III, with emphasis on the production of good target plasmas for the upcoming neutral beam injection phase. The program plan for the device over the life of the US-Japan cooperative program is discussed, as is the status of the preliminary investigation into replacing the present vacuum vessel by one better suited for ETF simulation.

  9. Active sliding between cytoplasmic microtubules.

    PubMed

    Koonce, M P; Tong, J; Euteneuer, U; Schliwa, M

    Microtubules are versatile cellular polymers that play a role in cell shape determination and mediate various motile processes such as ciliary and flagellar bending, chromosome movements and organelle transport. That a sliding microtubule mechanism can generate force has been demonstrated in highly ordered structures such as axonemes, and microtubule-based force generation almost certainly contributes to the function of mitotic and meiotic spindles. Most cytoplasmic microtubule arrays, however, do not exhibit the structural regularity of axonemes and some spindles, and often appear disorganized. Yet many cellular activities (such as shape changes during morphogenesis, axonal extension and spindle assembly) involve highly coordinated microtubule behaviour and possibly require force generated by an intermicrotubule sliding mechanism, or perhaps use sliding to move microtubules rapidly into a protrusion for stabilization. Here we show that active sliding between cytoplasmic microtubules can occur in microtubule bundles of the amoeba Reticulomyxa. A force-producing mechanism of this sort could be used by this organism to facilitate the extension of cell processes and to generate the dynamic movements of the cytoplasmic network.

  10. Anomalous Flexural Behaviors of Microtubules

    PubMed Central

    Liu, Xiaojing; Zhou, Youhe; Gao, Huajian; Wang, Jizeng

    2012-01-01

    Apparent controversies exist on whether the persistence length of microtubules depends on its contour length. This issue is particularly challenging from a theoretical point of view due to the tubular structure and strongly anisotropic material property of microtubules. Here we adopt a higher order continuum orthotropic thin shell model to study the flexural behavior of microtubules. Our model overcomes some key limitations of a recent study based on a simplified anisotropic shell model and results in a closed-form solution for the contour-length-dependent persistence length of microtubules, with predictions in excellent agreement with experimental measurements. By studying the ratio between their contour and persistence lengths, we find that microtubules with length at ∼1.5 μm show the lowest flexural rigidity, whereas those with length at ∼15 μm show the highest flexural rigidity. This finding may provide an important theoretical basis for understanding the mechanical structure of mitotic spindles during cell division. Further analysis on the buckling of microtubules indicates that the critical buckling load becomes insensitive to the tube length for relatively short microtubules, in drastic contrast to the classical Euler buckling. These rich flexural behaviors of microtubules are of profound implication for many biological functions and biomimetic molecular devices. PMID:22768935

  11. Microtubule dynamics in plant cells.

    PubMed

    Buschmann, Henrik; Sambade, Adrian; Pesquet, Edouard; Calder, Grant; Lloyd, Clive W

    2010-01-01

    This chapter describes some of the choices and unavoidable compromises to be made when studying microtubule dynamics in plant cells. The choice of species still depends very much on the ability to produce transgenic plants and most work has been done in the relatively small cells of Arabidopsis plants or in tobacco BY-2 suspension cells. Fluorescence-tagged microtubule proteins have been used to label entire microtubules, or their plus ends, but there are still few minus-end markers for these acentrosomal cells. Pragmatic decisions have to be made about probes, balancing the efficacy of microtubule labeling against a tendency to overstabilize and bundle the microtubules and even induce helical plant growth. A key limitation in visualizing plant microtubules is the ability to keep plants alive for long periods under the microscope and we describe a biochamber that allows for plant cell growth and development while allowing gas exchange and reducing evaporation. Another major difficulty is the limited fluorescence lifetime and we describe imaging strategies to reduce photobleaching in long-term imaging. We also discuss methods of measuring microtubule dynamics, with emphasis on the behavior of plant-specific microtubule arrays.

  12. Simulation of Pake doublet with classical spins and correspondence between the quantum and classical approaches

    NASA Astrophysics Data System (ADS)

    Henner, Victor K.; Klots, Andrey; Belozerova, Tatyana

    2016-12-01

    Problems of interacting quantum magnetic moments become exponentially complex with increasing number of particles. As a result, classical equations are often used to model spin systems. In this paper we show that a classical spins based approach can be used to describe the phenomena essentially quantum in nature such as of the Pake doublet.

  13. Multiscale polar theory of microtubule and motor-protein assemblies.

    PubMed

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A; Betterton, M D; Shelley, Michael J

    2015-01-30

    Microtubules and motor proteins are building blocks of self-organized subcellular biological structures such as the mitotic spindle and the centrosomal microtubule array. These same ingredients can form new "bioactive" liquid-crystalline fluids that are intrinsically out of equilibrium and which display complex flows and defect dynamics. It is not yet well understood how microscopic activity, which involves polarity-dependent interactions between motor proteins and microtubules, yields such larger-scale dynamical structures. In our multiscale theory, Brownian dynamics simulations of polar microtubule ensembles driven by cross-linking motors allow us to study microscopic organization and stresses. Polarity sorting and cross-link relaxation emerge as two polar-specific sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. The results connect local polar structure to flow structures and defect dynamics.

  14. Multiscale Polar Theory of Microtubule and Motor-Protein Assemblies

    NASA Astrophysics Data System (ADS)

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, M. D.; Shelley, Michael J.

    2015-01-01

    Microtubules and motor proteins are building blocks of self-organized subcellular biological structures such as the mitotic spindle and the centrosomal microtubule array. These same ingredients can form new "bioactive" liquid-crystalline fluids that are intrinsically out of equilibrium and which display complex flows and defect dynamics. It is not yet well understood how microscopic activity, which involves polarity-dependent interactions between motor proteins and microtubules, yields such larger-scale dynamical structures. In our multiscale theory, Brownian dynamics simulations of polar microtubule ensembles driven by cross-linking motors allow us to study microscopic organization and stresses. Polarity sorting and cross-link relaxation emerge as two polar-specific sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. The results connect local polar structure to flow structures and defect dynamics.

  15. Mechanism and Dynamics of Breakage of Fluorescent Microtubules

    PubMed Central

    Guo, Honglian; Xu, Chunhua; Liu, Chunxiang; Qu, E.; Yuan, Ming; Li, Zhaolin; Cheng, Bingying; Zhang, Daozhong

    2006-01-01

    The breakage of fluorescence-labeled microtubules under irradiation of excitation light is found in our experiments. Its mechanism is studied. The results indicate that free radicals are the main reason for the photosensitive breakage. Furthermore, the mechanical properties of the microtubules are probed with a dual-optical tweezers system. It is found that the fluorescence-labeled microtubules are much easier to extend compared with those without fluorescence. Such microtubules can be extended by 30%, and the force for breaking them up is only several piconewtons. In addition, we find that the breakup of the protofilaments is not simultaneous but step-by-step, which further confirms that the interaction between protofilaments is fairly weak. PMID:16387782

  16. Multiscale Polar Theory of Microtubule and Motor-Protein Assemblies

    PubMed Central

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, M. D.; Shelley, Michael J.

    2015-01-01

    Microtubules and motor proteins are building blocks of self-organized subcellular biological structures such as the mitotic spindle and the centrosomal microtubule array. These same ingredients can form new “bioactive” liquid-crystalline fluids that are intrinsically out of equilibrium and which display complex flows and defect dynamics. It is not yet well understood how microscopic activity, which involves polarity-dependent interactions between motor proteins and microtubules, yields such larger-scale dynamical structures. In our multiscale theory, Brownian dynamics simulations of polar microtubule ensembles driven by cross-linking motors allow us to study microscopic organization and stresses. Polarity sorting and cross-link relaxation emerge as two polar-specific sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. The results connect local polar structure to flow structures and defect dynamics. PMID:25679909

  17. Multiscale polar theory of microtubule and motor-protein assemblies

    DOE PAGES

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; ...

    2015-01-27

    Microtubules and motor proteins are building blocks of self-organized subcellular biological structures such as the mitotic spindle and the centrosomal microtubule array. These same ingredients can form new “bioactive” liquid-crystalline fluids that are intrinsically out of equilibrium and which display complex flows and defect dynamics. It is not yet well understood how microscopic activity, which involves polarity-dependent interactions between motor proteins and microtubules, yields such larger-scale dynamical structures. In our multiscale theory, Brownian dynamics simulations of polar microtubule ensembles driven by cross-linking motors allow us to study microscopic organization and stresses. Polarity sorting and cross-link relaxation emerge as two polar-specificmore » sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. Finally, the results connect local polar structure to flow structures and defect dynamics.« less

  18. Multiscale polar theory of microtubule and motor-protein assemblies

    SciTech Connect

    Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; Betterton, Meredith D.; Shelley, Michael J.

    2015-01-27

    Microtubules and motor proteins are building blocks of self-organized subcellular biological structures such as the mitotic spindle and the centrosomal microtubule array. These same ingredients can form new “bioactive” liquid-crystalline fluids that are intrinsically out of equilibrium and which display complex flows and defect dynamics. It is not yet well understood how microscopic activity, which involves polarity-dependent interactions between motor proteins and microtubules, yields such larger-scale dynamical structures. In our multiscale theory, Brownian dynamics simulations of polar microtubule ensembles driven by cross-linking motors allow us to study microscopic organization and stresses. Polarity sorting and cross-link relaxation emerge as two polar-specific sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. Finally, the results connect local polar structure to flow structures and defect dynamics.

  19. Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle.

    PubMed

    Nannas, Natalie J; O'Toole, Eileen T; Winey, Mark; Murray, Andrew W

    2014-12-15

    The length of the mitotic spindle varies among different cell types. A simple model for spindle length regulation requires balancing two forces: pulling, due to micro-tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles. In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore-microtubule interactions generate an inward force to balance forces that elongate the spindle. Electron microscopy shows that manipulating kinetochore number alters the number of spindle microtubules: adding extra kinetochores increases the number of spindle microtubules, suggesting kinetochore-based regulation of microtubule number.

  20. Microfilament Distribution in Maize Meiotic Mutants Correlates with Microtubule Organization.

    PubMed Central

    Staiger, CJ; Cande, WZ

    1991-01-01

    Microtubules and microfilaments often codistribute in plants; their presumed interaction can be tested with drugs although it is not always clear that these are without side effects. In this study, we exploited mutants defective in meiotic cell division to investigate in a noninvasive way the relationship between the two cytoskeletal elements. By staining unfixed, permeabilized cells with rhodamine-phalloidin, spatial and temporal changes in microfilament distribution during maize meiosis were examined. In wild-type microsporocytes, a microtubule array that radiates from the nucleus disappeared during spindle formation and returned at late telophase. This result differed from the complex cytoplasmic microfilament array that is present at all stages, including karyokinesis and cytokinesis. During division, a second class of microfilaments also was observed in the spindle and phragmoplast. To analyze this apparent association of microtubules and microfilaments, we examined several meiotic mutants known to have stage-specific disruptions in their microtubule arrays. Two mutations that altered the number or form of meiotic spindles also led to a dramatic reorganization of F-actin. In contrast, rearrangement of nonspindle, cytoplasmic microtubules did not lead to concomitant changes in F-actin distribution. These results suggested that microtubules and microfilaments interact in a cell cycle-specific and site-specific fashion during higher plant meiosis. PMID:12324607

  1. A "MICROTUBULE" IN A BACTERIUM

    PubMed Central

    van Iterson, Woutera; Hoeniger, Judith F. M.; van Zanten, Eva Nijman

    1967-01-01

    A study of the anchorage of the flagella in swarmers of Proteus mirabilis led to the incidental observation of microtubules. These microtubules were found in thin sections and in whole mount preparations of cells from which most of the content had been released by osmotic shock before staining negatively with potassium phosphotungstate (PTA). The microtubules are in negatively stained preparations about 200 A wide, i.e. somewhat thicker than the flagella (approximately 130 A). They are thus somewhat thinner than most microtubules recorded for other cells. They are referred to as microtubules because of their smooth cylindrical wall, or cortex, surrounding a hollow core which is readily filled with PTA when stained negatively. Since this is probably the first time that such a structure is described inside a bacterium, we do not know for certain whether it represents a normal cell constituent or an abnormality, for instance of the type of "polysheaths" (16). PMID:10976198

  2. Equimolar heterodimers in microtubules

    PubMed Central

    1982-01-01

    Two equimolar beta chains can be resolved from sea urchin sperm flagellar and scallop gill ciliary tubulins, and from certain brain tubulins as well, using the Triton X-100-acid-urea polyacrylamide gel system commonly used for histone analysis. The beta chains are identified as such from their mobility on urea-free SDS PAGE, from amino acid composition, and from tryptic peptide distribution. Scallop beta chains have almost identical amino acid profiles but they differ by one tryptic peptide. Optimal conditions for beta chain resolution are very species-dependent, with some closely related species showing either maximal or no beta chain separation. In addition, beef brain tubulin on Triton X-100-acid-urea electrophoresis and scallop gill ciliary tubulin upon isoelectric focusing in the presence of SDS show two approximately equimolar alpha chains. These data, indicating equimolar amounts of two potentially different tubulin heterodimers from a variety of microtubule types, support a model for microtubule structure wherein protofilaments consist of alternating heterodimers of two kinds, generating a 16-nm (2-dimer) axial repeat. PMID:7202008

  3. Changes in microtubule stability and density in myelin-deficient shiverer mouse CNS axons

    NASA Technical Reports Server (NTRS)

    Kirkpatrick, L. L.; Witt, A. S.; Payne, H. R.; Shine, H. D.; Brady, S. T.

    2001-01-01

    Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelin-deficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.

  4. TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux

    PubMed Central

    Fu, Jingyan; Bian, Minglei; Xin, Guangwei; Deng, Zhaoxuan; Luo, Jia; Guo, Xiao; Chen, Hao; Wang, Yao; Jiang, Qing

    2015-01-01

    A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux. PMID:26240182

  5. Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton.

    PubMed

    Herreros, L; Rodríguez-Fernandez, J L; Brown, M C; Alonso-Lebrero, J L; Cabañas, C; Sánchez-Madrid, F; Longo, N; Turner, C E; Sánchez-Mateos, P

    2000-08-25

    Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules. alpha-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal LIM domain as a bait. In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of alpha-tubulin with the C terminus of paxillin. Another member of the tubulin family, gamma-tubulin, bound to both the N and the C terminus of paxillin. The interaction between paxillin and both alpha- and gamma-tubulin in vivo was confirmed by co-immunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with alpha- and gamma-tubulin. The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands. These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton, and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.

  6. Isolation of microtubule-based motor proteins by ATP release from paclitaxel-stabilized microtubules.

    PubMed

    Sloboda, Roger D

    2015-02-02

    The α-β-tubulin heterodimer is asymmetric, and when asymmetric subunits assemble in a head-to-tail fashion, they produce a polymer that is itself asymmetric. Microtubules are therefore polar polymers having a head (or plus) end and a tail (or minus) end. Both ends can be distinguished kinetically because they add and lose subunits at different rates. Because of this inherent asymmetry, translocation of a particle along a microtubule from the head to the tail is a different molecular event than is translocation from the minus to the plus end. Currently, two classes of microtubule-dependent motor proteins are recognized: Those that are plus-end-directed (i.e., kinesin-like) and those that are minus-end-directed (dynein-like). The kinesin family of proteins in humans contains at least 14 classes of kinesins, a grouping based on tertiary and quaternary structure considerations, as well as on enzymatic activity. The dyneins are organized into two groups: Axonemal dyneins and cytoplasmic dyneins. This protocol provides methods for the enrichment of kinesin or cytoplasmic dynein, based on the differential interactions of each type of motor protein with microtubules in the presence of different nucleotides. For a cleaner preparation of motor proteins, the protocol includes steps for the further separation of kinesin and dynein from one another by sucrose gradient centrifugation.

  7. A coarse-grained model of microtubule self-assembly

    NASA Astrophysics Data System (ADS)

    Regmi, Chola; Cheng, Shengfeng

    Microtubules play critical roles in cell structures and functions. They also serve as a model system to stimulate the next-generation smart, dynamic materials. A deep understanding of their self-assembly process and biomechanical properties will not only help elucidate how microtubules perform biological functions, but also lead to exciting insight on how microtubule dynamics can be altered or even controlled for specific purposes such as suppressing the division of cancer cells. Combining all-atom molecular dynamics (MD) simulations and the essential dynamics coarse-graining method, we construct a coarse-grained (CG) model of the tubulin protein, which is the building block of microtubules. In the CG model a tubulin dimer is represented as an elastic network of CG sites, the locations of which are determined by examining the protein dynamics of the tubulin and identifying the essential dynamic domains. Atomistic MD modeling is employed to directly compute the tubulin bond energies in the surface lattice of a microtubule, which are used to parameterize the interactions between CG building blocks. The CG model is then used to study the self-assembly pathways, kinetics, dynamics, and nanomechanics of microtubules.

  8. Microtubule acetylation promotes kinesin-1 binding and transport.

    PubMed

    Reed, Nathan A; Cai, Dawen; Blasius, T Lynne; Jih, Gloria T; Meyhofer, Edgar; Gaertig, Jacek; Verhey, Kristen J

    2006-11-07

    Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking.

  9. Self-assembly of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Aranson, Igor; Tsimring, Lev

    2005-03-01

    We derive a model describing spatio-temporal assembly of an array of microtubules interacting via molecular motors. Starting from a stochastic model of inelastic polar rods with a generic anisotropic interaction kernel we obtain a set of equations for the local rods concentration and orientation. At large enough mean density of rods and concentration of motors, the model describes orientational instability. We demonstrate that the orientational instability leads to the formation of vortices and (for large density and/or kernel anisotropy) asters seen in recent experiments.

  10. Self-organization of microtubules and motors.

    SciTech Connect

    Aranson, I. S.; Tsimring, L. S.; Materials Science Division; Univ. of California at San Diego

    2006-01-01

    Here we introduce a model for spatio-temporal self-organization of an ensemble of microtubules interacting via molecular motors. Starting from a generic stochastic model of inelastic polar rods with an anisotropic interaction kernel we derive a set of equations for the local rods concentration and orientation. At large enough mean density of rods and concentration of motors, the model describes orientational instability. We demonstrate that the orientational instability leads to the formation of vortices and (for large density and/or kernel anisotropy) asters seen in recent experiments. The corresponding phase diagram of vortexasters transitions is in qualitative agreement with experiment.

  11. Dual detection of chromosomes and microtubules by the chromosomal passenger complex drives spindle assembly.

    PubMed

    Tseng, Boo Shan; Tan, Lei; Kapoor, Tarun M; Funabiki, Hironori

    2010-06-15

    Chromosome-dependent spindle assembly requires the chromosomal recruitment and activation of Aurora B, the kinase subunit of the chromosomal passenger complex (CPC). It remains unclear how the chromosome-activated kinase spatially transmits signals to organize the micron-scale spindle. Here we reveal that the CPC must detect two structures, chromosomes and microtubules, to support spindle assembly in Xenopus egg extracts. While Aurora B is enriched on chromosomes in metaphase, we establish that a fraction of Aurora B is targeted to the metaphase spindle and phosphorylates microtubule-bound substrates. We demonstrate that chromosomally activated Aurora B must be targeted to microtubules to drive spindle assembly. Moreover, although the CPC-microtubule interaction can activate Aurora B, which further promotes microtubule assembly, this positive feedback is not initiated without chromosomes. We propose that the dual detection of chromosomes and microtubules by the CPC is a critical step in assembling spindles around and only around chromosomes.

  12. Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

    PubMed

    Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

    2015-08-10

    Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles.

  13. A plus-end raft to control microtubule dynamics and function.

    PubMed

    Galjart, Niels; Perez, Franck

    2003-02-01

    Cells require a properly oriented and organised microtubule array to transmit positional information. Recent data have revealed a heterogeneous population of microtubule-binding proteins that accumulates mainly at distal ends of polymerising microtubules. Two mechanisms may account for this concentration: transient immobilisation, which involves association of proteins with growing ends, followed by release more proximally; and deposition at ends via a molecular motor. As with lipid rafts, protein concentration at distal ends may allow a cascade of interactions in the restricted area of a microtubule plus end. This may, in turn, control the dynamic behaviour of this cytoskeletal network and its anchoring to other structures.

  14. Influence of dense quantum plasmas on fine-structure splitting of Lyman doublets of hydrogenic systems

    SciTech Connect

    De, Madhab Ray, Debasis

    2015-05-15

    Relativistic calculations are performed to study the effects of oscillatory quantum plasma screening on the fine-structure splitting between the components of Lyman-α and β line doublets of atomic hydrogen and hydrgen-like argon ion within dense quantum plasmas, where the effective two-body (electron–nucleus) interaction is modeled by the Shukla–Eliasson oscillatory exponential cosine screened-Coulomb potential. The numerical solutions of the radial Dirac equation for the quantum plasma-embedded atomic systems reveal that the oscillatory quantum screening effect suppresses the doublet (energy) splitting substantially and the suppression becomes more prominent at large quantum wave number k{sub q}. In the absence of the oscillatory cosine screening term, much larger amount of suppression is noticed at larger values of k{sub q}, and the corresponding results represent the screening effect of an exponential screened-Coulomb two-body interaction. The Z{sup 4} scaling of the Lyman doublet splitting in low-Z hydrogen isoelectronic series of ions in free space is violated in dense quantum plasma environments. The relativistic data for the doublet splitting in the zero screening (k{sub q} = 0) case are in very good agreement with the NIST reference data, with slight discrepancies (∼0.2%) arising from the neglect of the quantum electrodynamic effects.

  15. Influence of dense quantum plasmas on fine-structure splitting of Lyman doublets of hydrogenic systems

    NASA Astrophysics Data System (ADS)

    De, Madhab; Ray, Debasis

    2015-05-01

    Relativistic calculations are performed to study the effects of oscillatory quantum plasma screening on the fine-structure splitting between the components of Lyman-α and β line doublets of atomic hydrogen and hydrgen-like argon ion within dense quantum plasmas, where the effective two-body (electron-nucleus) interaction is modeled by the Shukla-Eliasson oscillatory exponential cosine screened-Coulomb potential. The numerical solutions of the radial Dirac equation for the quantum plasma-embedded atomic systems reveal that the oscillatory quantum screening effect suppresses the doublet (energy) splitting substantially and the suppression becomes more prominent at large quantum wave number kq. In the absence of the oscillatory cosine screening term, much larger amount of suppression is noticed at larger values of kq, and the corresponding results represent the screening effect of an exponential screened-Coulomb two-body interaction. The Z4 scaling of the Lyman doublet splitting in low-Z hydrogen isoelectronic series of ions in free space is violated in dense quantum plasma environments. The relativistic data for the doublet splitting in the zero screening (kq = 0) case are in very good agreement with the NIST reference data, with slight discrepancies (˜0.2%) arising from the neglect of the quantum electrodynamic effects.

  16. Discrete and continuous symmetries in multi-Higgs-doublet models

    SciTech Connect

    Ferreira, P. M.; Silva, Joao P.

    2008-12-01

    We consider the Higgs sector of multi-Higgs-doublet models in the presence of simple symmetries relating the various fields. We construct basis-invariant observables which may in principle be used to detect these symmetries for any number of doublets. A categorization of the symmetries into classes is required, which we perform in detail for the case of two and three Higgs doublets.

  17. Doublet-singlet model and unitarity

    NASA Astrophysics Data System (ADS)

    Cynolter, G.; Kovács, J.; Lendvai, E.

    2016-12-01

    We study the renormalizable singlet-doublet fermionic extension of the Standard Model (SM). In this model, the new vector-like fermions couple to the gauge bosons and to the Higgs via new Yukawa couplings that allow for nontrivial mixing in the new sector, providing a stable, neutral dark matter candidate. Approximate analytic formulae are given for the mass spectrum around the blind spots, where the dark matter candidate coupling to h or Z vanishes. We calculate the two particle scattering amplitudes in the model, impose the perturbative unitarity constraints and establish bounds on the Yukawa couplings.

  18. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2011-08-20

    electronic properties of a single Microtubule Google Mountain view campus, workshop on quantum biology 22 October 2010 http://www.youtube.com/watch?v...Chemists, Tsukuba, Japan March 1-3, (2011) 3. Quantum aspects of microtubule: Direct experimental evidence for the existence of quantum states in...microtubule, Towards a science of consciousness May 2-8 (2011), Sweden 4. Electromagnetic energy of cells and microtubule: how microtubule research will

  19. Twisted custodial symmetry in two-Higgs-doublet models.

    PubMed

    Gérard, J-M; Herquet, M

    2007-06-22

    In the standard model for electroweak interactions, the Higgs sector is known to display a custodial symmetry protecting the mass relation m(W(+/-))(2) = m(W(3))(2) from large corrections. When considering extensions of the scalar sector, this symmetry has to be introduced by hand in order to pass current electroweak precision tests in a natural way. In this Letter, we implement a generalized custodial symmetry in the two-Higgs-doublet model. Assuming the invariance of the potential under CP transformations, we prove the existence of a new custodial scenario characterized by m(H(+/-))(2) = m(H(0))(2) instead of m(H(+/-))(2) = m(A(0))(2). Consequently, the pseudoscalar A(0) may be much lighter than the charged H(+/-), giving rise to interesting phenomenology.

  20. Impact of C24:0 on actin-microtubule interaction in human neuronal SK-N-BE cells: evaluation by FRET confocal spectral imaging microscopy after dual staining with rhodamine-phalloidin and tubulin tracker green

    PubMed Central

    Zarrouk, Amira; Nury, Thomas; Dauphin, Aurélien; Frère, Perrine; Riedinger, Jean-Marc; Bachelet, Claude-Marie; Frouin, Frédérique; Moreau, Thibault; Hammami, Mohamed; Kahn, Edmond; Lizard, Gérard

    2015-01-01

    Summary Disorganization of the cytoskeleton of neurons has major consequences on the transport of neuro-transmitters via the microtubule network. The interaction of cytoskeleton proteins (actin and tubulin) was studied in neuronal SK-N-BE cells treated with tetracosanoic acid (C24:0), which is cytotoxic and increased in Alzheimer’s disease patients. When SK-N-BE cells were treated with C24:0, mitochondrial dysfunctions and a non-apoptotic mode of cell death were observed. Fluorescence microscopy revealed shrunken cells with perinuclear condensation of actin and tubulin. After staining with rhodamine-phalloidin and with an antibody raised against α-/β-tubulin, modifications of F-actin and α-/β-tubulin levels were detected by flow cytometry. Lower levels of α-tubulin were found by Western blotting. In C24:0-treated cells, spectral analysis and fluorescence recovery after photo-bleaching (FRAP) measured by confocal microscopy proved the existence of fluorescence resonance energy transfer (FRET) when actin and tubulin were stained with tubulin tracker and rhodamine-phalloidin demonstrating actin and tubulin co-localization/interaction. In control cells, no FRET was observed. Our data demonstrate quantitative changes in actin and tubulin, and modified interactions between actin and tubulin in SK-N-BE cells treated with C24:0. They also show that FRET confocal imaging microscopy is an interesting method for specifying the impact of cytotoxic compounds on cytoskeleton proteins. PMID:26214025

  1. Microtubule organization during human parthenogenesis.

    PubMed

    Terada, Yukihiro; Hasegawa, Hisataka; Ugajin, Tomohisa; Murakami, Takashi; Yaegashi, Nobuo; Okamura, Kunihiro

    2009-04-01

    In human fertilization, the sperm centrosome plays a crucial role as a microtubule organizing center (MTOC). We studied microtubule organization during human parthenogenesis, which occurs when a human egg undergoes cleavage without a sperm centrosome. Multiple cytoplasmic asters were organized in the human oocyte after parthenogenetic activation, indicating that multiple MTOC are present in the human oocyte cytoplasm and function like a human sperm centrosome during parthenogenesis.

  2. Multi-modal microtubule binding by the Ndc80 kinetochore complex

    PubMed Central

    Alushin, Gregory M.; Musinipally, Vivek; Matson, Daniel; Tooley, John; Stukenberg, P. Todd; Nogales, Eva

    2012-01-01

    Summary The Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular “head” formed by tandem calponin-homology domains and an 80 amino-acid unstructured “tail” that contains sites of phospho-regulation by the Aurora B kinase. Using biochemical, cell biological, and electron microscopy analyses, we have dissected the tail’s roles in microtubule binding and mediating cooperative interactions between Ndc80 complexes. Two segments of the tail that contain Aurora B sites become ordered at interfaces; one with tubulin and the second with an adjacent Ndc80 head on the microtubule surface, forming interactions which are disrupted by phosphorylation. We propose a model in which Ndc80’s interaction with either growing or shrinking microtubule ends can be tuned by the phosphorylation state of its tail. PMID:23085714

  3. Intact Microtubules Preserve Transient Receptor Potential Vanilloid 1 (TRPV1) Functionality through Receptor Binding*

    PubMed Central

    Storti, Barbara; Bizzarri, Ranieri; Cardarelli, Francesco; Beltram, Fabio

    2012-01-01

    The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a protein currently under scrutiny as a pharmacological target for pain management therapies. Recently, the role of TRPV1-microtubule interaction in transducing nociception stimuli to cells by cytoskeletal rearrangement was proposed. In this work, we investigate TRPV1-microtubule interaction in living cells under the resting or activated state of TRPV1, as well as in presence of structurally intact or depolymerized cytoskeletal microtubules. We combined a toolbox of high resolution/high sensitivity fluorescence imaging techniques (such as FRET, correlation spectroscopy, and fluorescence anisotropy) to monitor TRPV1 aggregation status, membrane mobility, and interaction with microtubules. We found that TRPV1 is a dimeric membrane protein characterized by two populations with different diffusion properties in basal condition. After stimulation with resiniferatoxin, TRPV1 dimers tetramerize. The tetramers and the slower population of TRPV1 dimers bind dynamically to intact microtubules but not to tubulin dimers. Upon microtubule disassembly, the interaction with TRPV1 is lost thereby inducing receptor self-aggregation with partial loss of functionality. Intact microtubules play an essential role in maintaining TRPV1 functionality toward activation stimuli. This previously undisclosed property mirrors the recently reported role of TRPV1 in modulating microtubule assembly/disassembly and suggests the participation of these two players in a feedback cycle linking nociception and cytoskeletal remodeling. PMID:22262838

  4. Characterization of rat brain crude extract microtubule assembly: correlation of cold stability with the phosphorylation state of a microtubule-associated 64K protein.

    PubMed

    Margolis, R L; Rauch, C T

    1981-07-21

    We have conducted preliminary investigations into the control of microtubule assembly in rat brain crude extract supernatants. The rationale for these experiments is that microtubules interact with many proteins and are undoubtedly subject to physiological control mechanisms that are lost during tubulin purification. A more complete understanding of the cellular regulation of microtubules must include the physiology of these proteins. Assembly can be monitored in rat brain crude extract high-speed supernatants by measuring the increase in solution turbidity. We find that assembly is maximal in both rate and extent in the absence of added nucleotide. Increasing concentrations of either adenosine 5'-triphosphate (ATP) or guanosine 5'-triphosphate (GTP) inhibit both initiation and elongation of microtubules. GTP appears necessary for assembly and is apparently replenished from an intrinsic energy source during the time course of the assembly reaction. Inhibition of GTP production prevents microtubule assembly, and addition of exogenous GTP will reverse the blockage. Enzymatic removal of GTP at steady state causes a rapid depolymerization to the cold-stable microtubule level. Both GTP production and microtubule assembly display periodic oscillatory maxima. Cold-stable microtubules, which are always present in rat brain crude extract preparations, are rapidly made labile by addition of ATP. Analysis of proteins in cold-stable and cold-labile microtubule fractions shows changes in protein phosphorylation but not in the microtubule-associated protein composition. The tentative conclusion is that the state of phosphorylation of a 64K protein, designated the "switch protein", determines the cold stability or lability, and therefore the dimer association and dissociation rates, of crude extract microtubules.

  5. Elve Doublets: The Ionospheric Fingerprints of Compact Intracloud Discharges

    NASA Astrophysics Data System (ADS)

    da Silva, C. L.; Marshall, R. A.; Pasko, V. P.

    2015-12-01

    Compact intracloud discharges (CIDs) persist to date as one of the most mysterious lightning manifestations. CIDs are known to be the strongest natural sources of radio-frequency radiation on Earth. At VHF frequencies, approximately above 30 MHz, their emitted power is ten times stronger than that of other lightning processes. The well-known strength of CIDs in VHF contrasts with the lack of substantial optical measurements. CID's VLF/LF electric field change waveforms resemble one full cycle of a distorted sine function, with the first half-cycle being (a few times) larger-amplitude and shorter-duration than the second. For this reason CIDs have been dubbed narrow bipolar events (NBEs). NBE waveshapes are strikingly similar to the largest initial breakdown pulses (IBPs) that occur during the earlier stages of a conventional lightning flash, called classic IBPs. The similarity between classic IBP and NBE far-field waveforms, combined with the fact that positive-polarity NBEs frequently appear as the first event in an otherwise regular positive intracloud discharge, may be indicative that the source of these two E-field pulse types share the same physical mechanism inside thunderclouds [da Silva and Pasko, JGR, 120, 4989-5009, 2015]. In this presentation, we introduce a novel way to investigate CIDs. We show evidence that CIDs can produce an unique ionospheric signature, named "elve doublets". These signatures are characterized by a pair of elves separated in time by 80-160 microseconds. Our analysis combines fast photometric elve data, equivalent-transmission-line models to describe the dynamics of CID source currents, and FDTD modeling of electromagnetic wave propagation in the Earth-ionosphere waveguide accounting for its nonlinear interaction with the lower ionosphere [Marshall et al., GRL, 42, 2015, doi:10.1002/2015GL064862]. We show that typical (negative-polarity) CID altitudes, between 14-22 km, explain the time delay observed in elve doublets, where the

  6. Microtubules in Plants

    PubMed Central

    Hashimoto, Takashi

    2015-01-01

    Microtubules (MTs) are highly conserved polar polymers that are key elements of the eukaryotic cytoskeleton and are essential for various cell functions. αβ-tubulin, a heterodimer containing one structural GTP and one hydrolysable and exchangeable GTP, is the building block of MTs and is formed by the sequential action of several molecular chaperones. GTP hydrolysis in the MT lattice is mechanistically coupled with MT growth, thus giving MTs a metastable and dynamic nature. MTs adopt several distinct higher-order organizations that function in cell division and cell morphogenesis. Small molecular weight compounds that bind tubulin are used as herbicides and as research tools to investigate MT functions in plant cells. The de novo formation of MTs in cells requires conserved γ-tubulin-containing complexes and targeting/activating regulatory proteins that contribute to the geometry of MT arrays. Various MT regulators and tubulin modifications control the dynamics and organization of MTs throughout the cell cycle and in response to developmental and environmental cues. Signaling pathways that converge on the regulation of versatile MT functions are being characterized. PMID:26019693

  7. Self-repair promotes microtubule rescue

    PubMed Central

    Gaillard, Jérémie; John, Karin; Blanchoin, Laurent; Théry, Manuel

    2016-01-01

    Summary The dynamic instability of microtubules is characterised by slow growth phases stochastically interrupted by rapid depolymerisations called catastrophes. Rescue events can arrest the depolymerisation and restore microtubule elongation. However the origin of these rescue events remain unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints. PMID:27617929

  8. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton.

    PubMed

    Krendel, Mira; Zenke, Frank T; Bokoch, Gary M

    2002-04-01

    Regulation of the actin cytoskeleton by microtubules is mediated by the Rho family GTPases. However, the molecular mechanisms that link microtubule dynamics to Rho GTPases have not, as yet, been identified. Here we show that the Rho guanine nucleotide exchange factor (GEF)-H1 is regulated by an interaction with microtubules. GEF-H1 mutants that are deficient in microtubule binding have higher activity levels than microtubule-bound forms. These mutants also induce Rho-dependent changes in cell morphology and actin organization. Furthermore, drug-induced microtubule depolymerization induces changes in cell morphology and gene expression that are similar to the changes induced by the expression of active forms of GEF-H1. Furthermore, these effects are inhibited by dominant-negative versions of GEF-H1. Thus, GEF-H1 links changes in microtubule integrity to Rho-dependent regulation of the actin cytoskeleton.

  9. Zampanolide, a potent new microtubule stabilizing agent, covalently reacts with the taxane luminal site in both tubulin α,β-heterodimers and microtubules

    PubMed Central

    Field, Jessica J.; Pera, Benet; Calvo, Enrique; Canales, Angeles; Zurwerra, Didier; Trigili, Chiara; Rodríguez-Salarichs, Javier; Matesanz, Ruth; Kanakkanthara, Arun; Wakefield, St. John; Singh, A. Jonathan; Jiménez-Barbero, Jesús; Northcote, Peter; Miller, John H.; López, Juan Antonio; Hamel, Ernest; Barasoain, Isabel; Altmann, Karl-Heinz; Díaz, José Fernando

    2012-01-01

    Summary Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to β-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,β-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates β-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism. PMID:22726683

  10. Scalar doublet models confront τ and b anomalies

    NASA Astrophysics Data System (ADS)

    Cline, James M.

    2016-04-01

    There are indications of a possible breakdown of the standard model, suggesting that τ lepton interactions violate flavor universality, particularly through B meson decays. BABAR, Belle, and LHCb report high ratios of B →D(*)τ ν . There are long-standing excesses in B →τ ν and W →τ ν decays and a deficit in inclusive τ to strange decays. We investigate whether two Higgs doublet models with the most general allowed couplings to quarks, and a large coupling to τ leptons, can explain these anomalies while respecting other flavor constraints and technical naturalness. Fits to B →D(*)τ ν data require couplings of the new Higgs doublet to down-type quarks, opening the door to many highly constrained flavor-changing neutral current processes. We confront these challenges by introducing a novel ansatz that relates the new up- and down-type Yukawa couplings, and demonstrate viable values of the couplings that are free from fine-tuning. LEP and LHC searches for new Higgs bosons decaying via H0→τ+τ- and H±→τ±ν allow a window of masses mH=[100 - 125 ] GeV and m±˜100 GeV that is consistent with the predictions of our model. Contamination of the W+→τ+ν signal by H+→τ+ν decays at LEP could explain the apparent W →τ ν excess. We predict that the branching ratio for Bs→τ+τ- is not far below its current limit of several percent. An alternative model with decays of B →D(*)τ νs to a sterile neutrino is also argued to be viable.

  11. Push or Pull? -- Cryo-Electron Microscopy of Microtubule's Dynamic Instability and Its Roles in the Kinetochore

    NASA Astrophysics Data System (ADS)

    Wang, Hong-Wei

    2009-03-01

    Microtubule is a biopolymer made up of alpha-beta-tubulin heterodimers. The tubulin dimers assemble head-to-tail as protofilaments and about 13 protofilaments interact laterally to form a hollow cylindrical structure which is the microtubule. As the major cytoskeleton in all eukaryotic cells, microtubules have the intrinsic property to switch stochastically between growth and shrinkage phases, a phenomenon termed as their dynamic instability. Microtubule's dynamic instability is closely related to the types of nucleotide (GTP or GDP) that binds to the beta-tubulin. We have biochemically trapped two types of assembly states of tubulin with GTP or GDP bound representing the polymerizing and depolymerizing ends of microtubules respectively. Using cryo-electron microscopy, we have elucidated the structures of these intermediate assemblies, showing that tubulin protofilaments demonstrate various curvatures and form different types of lateral interactions depending on the nucleotide states of tubulin and the temperature. Our work indicates that during the microtubule's dynamic cycle, tubulin undergoes various assembly states. These states, different from the straight microtubule, lend the highly dynamic and complicated behavior of microtubules. Our study of microtubule's interaction with certain kinetochore complexes suggests that the intermediate assemblies are responsible for specific mechanical forces that are required during the mitosis or meiosis. Our discoveries strongly suggest that a microtubule is a molecular machine rather than a simple cellular scaffold.

  12. Simulation studies of self-organization of microtubules and molecular motors.

    SciTech Connect

    Jian, Z.; Karpeev, D.; Aranson, I. S.; Bates, P. W.; Michigan State Univ.

    2008-05-01

    We perform Monte Carlo type simulation studies of self-organization of microtubules interacting with molecular motors. We model microtubules as stiff polar rods of equal length exhibiting anisotropic diffusion in the plane. The molecular motors are implicitly introduced by specifying certain probabilistic collision rules resulting in realignment of the rods. This approximation of the complicated microtubule-motor interaction by a simple instant collision allows us to bypass the 'computational bottlenecks' associated with the details of the diffusion and the dynamics of motors and the reorientation of microtubules. Consequently, we are able to perform simulations of large ensembles of microtubules and motors on a very large time scale. This simple model reproduces all important phenomenology observed in in vitro experiments: Formation of vortices for low motor density and raylike asters and bundles for higher motor density.

  13. Simulation studies of self-organization of microtubules and molecular motors

    NASA Astrophysics Data System (ADS)

    Jia, Zhiyuan; Karpeev, Dmitry; Aranson, Igor S.; Bates, Peter W.

    2008-05-01

    We perform Monte Carlo type simulation studies of self-organization of microtubules interacting with molecular motors. We model microtubules as stiff polar rods of equal length exhibiting anisotropic diffusion in the plane. The molecular motors are implicitly introduced by specifying certain probabilistic collision rules resulting in realignment of the rods. This approximation of the complicated microtubule-motor interaction by a simple instant collision allows us to bypass the “computational bottlenecks” associated with the details of the diffusion and the dynamics of motors and the reorientation of microtubules. Consequently, we are able to perform simulations of large ensembles of microtubules and motors on a very large time scale. This simple model reproduces all important phenomenology observed in in vitro experiments: Formation of vortices for low motor density and raylike asters and bundles for higher motor density.

  14. Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle

    PubMed Central

    Nannas, Natalie J.; O’Toole, Eileen T.; Winey, Mark; Murray, Andrew W.

    2014-01-01

    The length of the mitotic spindle varies among different cell types. A simple model for spindle length regulation requires balancing two forces: pulling, due to micro­tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles. In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore–microtubule interactions generate an inward force to balance forces that elongate the spindle. Electron microscopy shows that manipulating kinetochore number alters the number of spindle microtubules: adding extra kinetochores increases the number of spindle microtubules, suggesting kinetochore-based regulation of microtubule number. PMID:25318669

  15. Possible multiple chiral doublet bands in 107Ag

    NASA Astrophysics Data System (ADS)

    Qi, B.; Jia, H.; Zhang, N. B.; Liu, C.; Wang, S. Y.

    2013-08-01

    Two pairs of nearly degenerate doublet bands in 107Ag are studied via the relativistic mean-field (RMF) theory and the multiparticle plus rotor model (PRM), which suggests these bands as two distinct sets of chiral doublet bands. For the suggested πg9/2-1⊗νh11/22 and πg9/2-1⊗νh11/2d5/2 configurations, the favorable triaxial deformation γ for nuclear chirality can be obtained from the configuration-fixed constrained triaxial RMF calculations. Adopting the PRM, the data available are reproduced very well for the two pairs of doublet bands. Chiral geometry is further conformed by analyzing the angular momentum components. We suggest that two pairs of doublet bands in 107Ag would be another example of multiple chiral doublet bands.

  16. Partially natural two Higgs doublet models

    SciTech Connect

    Draper, Patrick; Haber, Howard E.; Ruderman, Joshua T.

    2016-06-21

    It is possible that the electroweak scale is low due to the fine-tuning of microscopic parameters, which can result from selection effects. The experimental discovery of new light fundamental scalars other than the Standard Model Higgs boson would seem to disfavor this possibility, since generically such states imply parametrically worse fine-tuning with no compelling connection to selection effects. We discuss counterexamples where the Higgs boson is light because of fine-tuning, and a second scalar doublet is light because a discrete symmetry relates its mass to the mass of the Standard Model Higgs boson. Our examples require new vectorlike fermions at the electroweak scale, and the models possess a rich electroweak vacuum structure. Furthermore, the mechanism that we discuss does not protect a small CP-odd Higgs mass in split or high-scale supersymmetry-breaking scenarios of the MSSM due to an incompatibility between the discrete symmetries and holomorphy.

  17. Electromagnetic transitions in multiple chiral doublet bands

    NASA Astrophysics Data System (ADS)

    Jia, Hui; Qi, Bin; Wang, Shou-Yu; Wang, Shuo; Liu, Chen

    2016-12-01

    Multiple chiral doublet bands (MχD) in the 80, 130 and 190 mass regions are studied by the model of γ = 90° triaxial rotor coupled with identical symmetric proton-neutron configurations. By selecting a suitable basis, the calculated wave functions are explicitly exhibited to be symmetric under the operator Â, which is defined as rotation by 90° about the 3-axis with the exchange of valance proton and neutron. We found that both M1 and E2 transitions are allowed between levels with different values of A, while they are forbidden between levels with same values of A. Such a selection rule holds true for MχD in different mass regions. Supported by National Natural Science Foundation of China (11675094, 11622540, 11545011, 11405096, 11461141001, U1432119), Shandong Natural Science Foundation (ZR2014AQ012), and Young Scholars Program of Shandong University, Weihai (2015WHWLJH01)

  18. Microtubule defects & Neurodegeneration.

    PubMed

    Baird, Fiona J; Bennett, Craig L

    2013-12-06

    One of the major challenges facing the long term survival of neurons is their requirement to maintain efficient axonal transport over long distances. In humans as large, long-lived vertebrates, the machinery maintaining neuronal transport must remain efficient despite the slow accumulation of cell damage during aging. Mutations in genes encoding proteins which function in the transport system feature prominently in neurologic disorders. Genes known to cause such disorders and showing traditional Mendelian inheritance have been more readily identified. It has been more difficult, however, to isolate factors underlying the complex genetics contributing to the more common idiopathic forms of neurodegenerative disease. At the heart of neuronal transport is the rail network or scaffolding provided by neuron specific microtubules (MTs). The importance of MT dynamics and stability is underscored by the critical role tau protein plays in MT-associated stabilization versus the dysfunction seen in Alzheimer's disease, frontotemporal dementia and other tauopathies. Another example of the requirement for tight regulation of MT dynamics is the need to maintain balanced levels of post-translational modification of key MT building-blocks such as α-tubulin. Tubulins require extensive polyglutamylation at their carboxyl-terminus as part of a novel post-translational modification mechanism to signal MT growth versus destabilization. Dramatically, knock-out of a gene encoding a deglutamylation family member causes an extremely rapid cell death of Purkinje cells in the ataxic mouse model, pcd. This review will examine a range of neurodegenerative conditions where current molecular understanding points to defects in the stability of MTs and axonal transport to emphasize the central role of MTs in neuron survival.

  19. Multifunctional Microtubule-Associated Proteins in Plants

    PubMed Central

    Krtková, Jana; Benáková, Martina; Schwarzerová, Kateřina

    2016-01-01

    Microtubules (MTs) are involved in key processes in plant cells, including cell division, growth and development. MT-interacting proteins modulate MT dynamics and organization, mediating functional and structural interaction of MTs with other cell structures. In addition to conventional microtubule-associated proteins (MAPs) in plants, there are many other MT-binding proteins whose primary function is not related to the regulation of MTs. This review focuses on enzymes, chaperones, or proteins primarily involved in other processes that also bind to MTs. The MT-binding activity of these multifunctional MAPs is often performed only under specific environmental or physiological conditions, or they bind to MTs only as components of a larger MT-binding protein complex. The involvement of multifunctional MAPs in these interactions may underlie physiological and morphogenetic events, e.g., under specific environmental or developmental conditions. Uncovering MT-binding activity of these proteins, although challenging, may contribute to understanding of the novel functions of the MT cytoskeleton in plant biological processes. PMID:27148302

  20. Tubulin tyrosine nitration regulates microtubule organization in plant cells

    PubMed Central

    Blume, Yaroslav B.; Krasylenko, Yuliya A.; Demchuk, Oleh M.; Yemets, Alla I.

    2013-01-01

    During last years, selective tyrosine nitration of plant proteins gains importance as well-recognized pathway of direct nitric oxide (NO) signal transduction. Plant microtubules are one of the intracellular signaling targets for NO, however, the molecular mechanisms of NO signal transduction with the involvement of cytoskeletal proteins remain to be elucidated. Since biochemical evidence of plant α-tubulin tyrosine nitration has been obtained recently, potential role of this posttranslational modification in regulation of microtubules organization in plant cell is estimated in current paper. It was shown that 3-nitrotyrosine (3-NO2-Tyr) induced partially reversible Arabidopsis primary root growth inhibition, alterations of root hairs morphology and organization of microtubules in root cells. It was also revealed that 3-NO2-Tyr intensively decorates such highly dynamic microtubular arrays as preprophase bands, mitotic spindles and phragmoplasts of Nicotiana tabacum Bright Yellow-2 (BY-2) cells under physiological conditions. Moreover, 3D models of the mitotic kinesin-8 complexes with the tail of detyrosinated, tyrosinated and tyrosine nitrated α-tubulin (on C-terminal Tyr 450 residue) from Arabidopsis were reconstructed in silico to investigate the potential influence of tubulin nitrotyrosination on the molecular dynamics of α-tubulin and kinesin-8 interaction. Generally, presented data suggest that plant α-tubulin tyrosine nitration can be considered as its common posttranslational modification, the direct mechanism of NO signal transduction with the participation of microtubules under physiological conditions and one of the hallmarks of the increased microtubule dynamics. PMID:24421781

  1. Spatial organization of the Ran pathway by microtubules in mitosis

    PubMed Central

    Oh, Doogie; Yu, Che-Hang; Needleman, Daniel J.

    2016-01-01

    Concentration gradients of soluble proteins are believed to be responsible for control of morphogenesis of subcellular systems, but the mechanisms that generate the spatial organization of these subcellular gradients remain poorly understood. Here, we use a newly developed multipoint fluorescence fluctuation spectroscopy technique to study the ras-related nuclear protein (Ran) pathway, which forms soluble gradients around chromosomes in mitosis and is thought to spatially regulate microtubule behaviors during spindle assembly. We found that the distribution of components of the Ran pathway that influence microtubule behaviors is determined by their interactions with microtubules, resulting in microtubule nucleators being localized by the microtubules whose formation they stimulate. Modeling and perturbation experiments show that this feedback makes the length of the spindle insensitive to the length scale of the Ran gradient, allows the spindle to assemble outside the peak of the Ran gradient, and explains the scaling of the spindle with cell size. Such feedback between soluble signaling pathways and the mechanics of the cytoskeleton may be a general feature of subcellular organization. PMID:27439876

  2. Self-organized pattern formation in motor-microtubule mixtures

    NASA Astrophysics Data System (ADS)

    Sankararaman, Sumithra; Menon, Gautam I.; Sunil Kumar, P. B.

    2004-09-01

    We model the stable self-organized patterns obtained in the nonequilibrium steady states of mixtures of molecular motors and microtubules. In experiments [Nédélec , Nature (London) 389, 305 (1997); Surrey , Science 292, 1167 (2001)] performed in a quasi-two-dimensional geometry, microtubules are oriented by complexes of motor proteins. This interaction yields a variety of patterns, including arrangements of asters, vortices, and disordered configurations. We model this system via a two-dimensional vector field describing the local coarse-grained microtubule orientation and two scalar density fields associated to molecular motors. These scalar fields describe motors which either attach to and move along microtubules or diffuse freely within the solvent. Transitions between single aster, spiral, and vortex states are obtained as a consequence of confinement, as parameters in our model are varied. For systems in which the effects of confinement can be neglected, we present a map of nonequilibrium steady states, which includes arrangements of asters and vortices separately as well as aster-vortex mixtures and fully disordered states. We calculate the steady state distribution of bound and free motors in aster and vortex configurations of microtubules and compare these to our simulation results, providing qualitative arguments for the stability of different patterns in various regimes of parameter space. We study the role of crowding or “saturation” effects on the density profiles of motors in asters, discussing the role of such effects in stabilizing single asters. We also comment on the implications of our results for experiments.

  3. Tryprostatin A, a specific and novel inhibitor of microtubule assembly.

    PubMed

    Usui, T; Kondoh, M; Cui, C B; Mayumi, T; Osada, H

    1998-08-01

    We have investigated the cell cycle inhibition mechanism and primary target of tryprostatin A (TPS-A) purified from Aspergillus fumigatus. TPS-A inhibited cell cycle progression of asynchronously cultured 3Y1 cells in the M phase in a dose- and time-dependent manner. In contrast, TPS-B (the demethoxy analogue of TPS-A) showed cell-cycle non-specific inhibition on cell growth even though it inhibited cell growth at lower concentrations than TPS-A. TPS-A treatment induced the reversible disruption of the cytoplasmic microtubules of 3Y1 cells as observed by indirect immunofluorescence microscopy in the range of concentrations that specifically inhibited M-phase progression. TPS-A inhibited the assembly in vitro of microtubules purified from bovine brains (40% inhibition at 250 microM); however, there was little or no effect on the self-assembly of purified tubulin when polymerization was induced by glutamate even at 250 microM TPS-A. TPS-A did not inhibit assembly promoted by taxol or by digestion of the C-terminal domain of tubulin. However, TPS-A blocked the tubulin assembly induced by inducers interacting with the C-terminal domain, microtubule-associated protein 2 (MAP2), tau and poly-(l-lysine). These results indicate that TPS-A is a novel inhibitor of MAP-dependent microtubule assembly and, through the disruption of the microtubule spindle, specifically inhibits cell cycle progression at the M phase.

  4. Kinetochore–microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint

    PubMed Central

    Etemad, Banafsheh; Kuijt, Timo E. F.; Kops, Geert J. P. L.

    2015-01-01

    The spindle assembly checkpoint (SAC) is a genome surveillance mechanism that protects against aneuploidization. Despite profound progress on understanding mechanisms of its activation, it remains unknown what aspect of chromosome–spindle interactions is monitored by the SAC: kinetochore–microtubule attachment or the force generated by dynamic microtubules that signals stable biorientation of chromosomes? To answer this, we uncoupled these two processes by expressing a non-phosphorylatable version of the main microtubule-binding protein at kinetochores (HEC1-9A), causing stabilization of incorrect kinetochore–microtubule attachments despite persistent activity of the error-correction machinery. The SAC is fully functional in HEC1-9A-expressing cells, yet cells in which chromosomes cannot biorient but are stably attached to microtubules satisfy the SAC and exit mitosis. SAC satisfaction requires neither intra-kinetochore stretching nor dynamic microtubules. Our findings support the hypothesis that in human cells the end-on interactions of microtubules with kinetochores are sufficient to satisfy the SAC without the need for microtubule-based pulling forces. PMID:26621779

  5. Optimization of isopolar microtubule arrays.

    PubMed

    Agayan, Rodney R; Tucker, Robert; Nitta, Takahiro; Ruhnow, Felix; Walter, Wilhelm J; Diez, Stefan; Hess, Henry

    2013-02-19

    Isopolar arrays of aligned cytoskeletal filaments are components in a number of designs of hybrid nanodevices incorporating biomolecular motors. For example, a combination of filament arrays and motor arrays can form an actuator or a molecular engine resembling an artificial muscle. Here, isopolar arrays of microtubules are fabricated by flow alignment, and their quality is characterized by their degree of alignment. We find, in agreement with our analytical models, that the degree of alignment is ultimately limited by thermal forces, while the kinetics of the alignment process are influenced by the flow strength, the microtubule stiffness, the gliding velocity, and the tip length. Strong flows remove microtubules from the surface and reduce the filament density, suggesting that there is an optimal flow strength for the fabrication of ordered arrays.

  6. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2014-03-05

    single Microtubule Google Mountain view campus, workshop on quantum biology 22 October 2010 3. Paul Davies Beyond Center at Arizona State University...Phoenix) Phoenix, workshop on quantum biology and cancer research, Experimental studies on single microtubule, 25-27 October 2010, Tempe, Arizona...State University, USA 4. Quantum aspects of microtubule: Direct experimental evidence for the existence of quantum states in microtubule, Towards a

  7. How dynein moves along microtubules

    PubMed Central

    Bhabha, Gira; Johnson, Graham T.; Schroeder, Courtney M.; Vale, Ronald D.

    2015-01-01

    Cytoplasmic dynein, a member of the AAA family of ATPases, drives the processive movement of numerous intracellular cargos towards the minus end of microtubules. Here, we summarize the structural and motile properties of dynein and highlight features that distinguish this motor from kinesin-1 and myosin V, two well-studied transport motors. Integrating information from recent crystal and cryo-EM structures as well as high-resolution single molecule studies, we also discuss models for how dynein biases its movement in one direction along a microtubule track, and present a movie that illustrates these principles. PMID:26678005

  8. Microtubule Severing Stymied by Free Tubulin

    NASA Astrophysics Data System (ADS)

    Ross, Jennifer; Bailey, Megan

    2015-03-01

    Proper organization of the microtubule cytoskeletal network is required to perform many necessary cellular functions including mitosis, cell development, and cell motility. Network organization is achieved through filament remodeling by microtubule-associated proteins (MAPs) that control microtubule dynamics. MAPs that stabilize are relatively well understood, while less is known about destabilizing MAPs, such as severing enzymes. Katanin, the first-discovered microtubule-severing enzyme, is a AAA + enzyme that oligomerizes into hexamers and uses ATP hydrolysis to sever microtubules. Using quantitative fluorescence imaging on reconstituted microtubule severing assays in vitro we investigate how katanin can regulate microtubule dynamics. Interestingly, we find microtubule dynamics inhibits katanin severing activity; dynamic microtubules are not severed. Using systematic experiments introducing free tubulin into the assays we find that free tubulin can compete for microtubule filaments for the katanin proteins. Our work indicates that katanin could function best on stabile microtubules or stabile regions of microtubules in cells in regions where free tubulin is sequesters, low, or depleted.

  9. Doublet craters and the tidal disruption of binary asteroids

    NASA Technical Reports Server (NTRS)

    Melosh, H. J.; Stansberry, J. A.

    1991-01-01

    An evaluation is conducted of the possibility that the tidal disruption of a population of contact binary asteroids can account for terrestrial-impact 'doublet' craters. Detailed orbital integrations indicate that while such asteroids are often disrupted by tidal forces outside the Roche limit, the magnitude of the resulting separations is too small to account for the observed doublet craters. It is hypothesized that an initial population of km-scale earth-crossing objects encompassing 10-20 percent binaries must be responsible for doublet impacts, as may be verified by future observations of earth-approaching asteroids.

  10. Dear microtubule, I see you

    PubMed Central

    Nogales, Eva

    2016-01-01

    This essay summarizes my personal journey toward the atomic visualization of microtubules and a mechanistic understanding of how these amazing polymers work. During this journey, I have been witness and partaker in the blooming of a technique I love—cryo-electron microscopy. PMID:27799495

  11. Termination of Protofilament Elongation by Eribulin Induces Lattice Defects that Promote Microtubule Catastrophes.

    PubMed

    Doodhi, Harinath; Prota, Andrea E; Rodríguez-García, Ruddi; Xiao, Hui; Custar, Daniel W; Bargsten, Katja; Katrukha, Eugene A; Hilbert, Manuel; Hua, Shasha; Jiang, Kai; Grigoriev, Ilya; Yang, Chia-Ping H; Cox, David; Horwitz, Susan Band; Kapitein, Lukas C; Akhmanova, Anna; Steinmetz, Michel O

    2016-07-11

    Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation of individual protofilaments can potentially lead to an altered microtubule-end structure that promotes sudden depolymerization, termed catastrophe [1-4]. However, how the dynamics of individual protofilaments relates to overall growth persistence has remained unclear. Here, we used the microtubule targeting anti-cancer drug Eribulin [5-7] to explore the consequences of stalled protofilament elongation on microtubule growth. Using X-ray crystallography, we first revealed that Eribulin binds to a site on β-tubulin that is required for protofilament plus-end elongation. Based on the structural information, we engineered a fluorescent Eribulin molecule. We demonstrate that single Eribulin molecules specifically interact with microtubule plus ends and are sufficient to either trigger a catastrophe or induce slow and erratic microtubule growth in the presence of EB3. Interestingly, we found that Eribulin increases the frequency of EB3 comet "splitting," transient events where a slow and erratically progressing comet is followed by a faster comet. This observation possibly reflects the "healing" of a microtubule lattice. Because EB3 comet splitting was also observed in control microtubules in the absence of any drugs, we propose that Eribulin amplifies a natural pathway toward catastrophe by promoting the arrest of protofilament elongation.

  12. Sizable NSI from the SU(2)L scalar doublet-singlet mixing and the implications in DUNE

    NASA Astrophysics Data System (ADS)

    Forero, David V.; Huang, Wei-Chih

    2017-03-01

    We propose a novel and simple mechanism where sizable effects of non-standard interactions (NSI) in neutrino propagation are induced from the mixing between an electrophilic second Higgs doublet and a charged singlet. The mixing arises from a dimensionful coupling of the scalar doublet and singlet to the standard model Higgs boson. In light of the small mass, the light mass eigenstate from the doublet-singlet mixing can generate much larger NSI than those induced by the heavy eigenstate. We show that a sizable NSI ɛ eτ (˜0.3) can be attained without being excluded by a variety of experimental constraints. Furthermore, we demonstrate that NSI can mimic effects of the Dirac CP phase in the neutrino mixing matrix but they can potentially be disentangled by future long-baseline neutrino experiments, such as the Deep Underground Neutrino Experiment (DUNE).

  13. A Stochastic Multiscale Model That Explains the Segregation of Axonal Microtubules and Neurofilaments in Neurological Diseases

    PubMed Central

    Xue, Chuan; Shtylla, Blerta; Brown, Anthony

    2015-01-01

    The organization of the axonal cytoskeleton is a key determinant of the normal function of an axon, which is a long thin projection of a neuron. Under normal conditions two axonal cytoskeletal polymers, microtubules and neurofilaments, align longitudinally in axons and are interspersed in axonal cross-sections. However, in many neurotoxic and neurodegenerative disorders, microtubules and neurofilaments segregate apart from each other, with microtubules and membranous organelles clustered centrally and neurofilaments displaced to the periphery. This striking segregation precedes the abnormal and excessive neurofilament accumulation in these diseases, which in turn leads to focal axonal swellings. While neurofilament accumulation suggests an impairment of neurofilament transport along axons, the underlying mechanism of their segregation from microtubules remains poorly understood for over 30 years. To address this question, we developed a stochastic multiscale model for the cross-sectional distribution of microtubules and neurofilaments in axons. The model describes microtubules, neurofilaments and organelles as interacting particles in a 2D cross-section, and is built upon molecular processes that occur on a time scale of seconds or shorter. It incorporates the longitudinal transport of neurofilaments and organelles through this domain by allowing stochastic arrival and departure of these cargoes, and integrates the dynamic interactions of these cargoes with microtubules mediated by molecular motors. Simulations of the model demonstrate that organelles can pull nearby microtubules together, and in the absence of neurofilament transport, this mechanism gradually segregates microtubules from neurofilaments on a time scale of hours, similar to that observed in toxic neuropathies. This suggests that the microtubule-neurofilament segregation can be a consequence of the selective impairment of neurofilament transport. The model generates the experimentally testable

  14. Partially natural two Higgs doublet models

    DOE PAGES

    Draper, Patrick; Haber, Howard E.; Ruderman, Joshua T.

    2016-06-21

    It is possible that the electroweak scale is low due to the fine-tuning of microscopic parameters, which can result from selection effects. The experimental discovery of new light fundamental scalars other than the Standard Model Higgs boson would seem to disfavor this possibility, since generically such states imply parametrically worse fine-tuning with no compelling connection to selection effects. We discuss counterexamples where the Higgs boson is light because of fine-tuning, and a second scalar doublet is light because a discrete symmetry relates its mass to the mass of the Standard Model Higgs boson. Our examples require new vectorlike fermions atmore » the electroweak scale, and the models possess a rich electroweak vacuum structure. Furthermore, the mechanism that we discuss does not protect a small CP-odd Higgs mass in split or high-scale supersymmetry-breaking scenarios of the MSSM due to an incompatibility between the discrete symmetries and holomorphy.« less

  15. Microtubule catastrophe from protofilament dynamics.

    PubMed

    Jemseena, V; Gopalakrishnan, Manoj

    2013-09-01

    The disappearance of the guanosine triphosphate- (GTP) tubulin cap is widely believed to be the forerunner event for the growth-shrinkage transition ("catastrophe") in microtubule filaments in eukaryotic cells. We study a discrete version of a stochastic model of the GTP cap dynamics, originally proposed by Flyvbjerg, Holy, and Leibler [Phys. Rev. Lett. 73, 2372 (1994)]. Our model includes both spontaneous and vectorial hydrolysis, as well as dissociation of a nonhydrolyzed dimer from the filament after incorporation. In the first part of the paper, we apply this model to a single protofilament of a microtubule. A catastrophe transition is defined for each protofilament, similarly to the earlier one-dimensional models, the frequency of occurrence of which is then calculated under various conditions but without explicit assumption of steady-state conditions. Using a perturbative approach, we show that the leading asymptotic behavior of the protofilament catastrophe in the limit of large growth velocities is remarkably similar across different models. In the second part of the paper, we extend our analysis to the entire filament by making a conjecture that a minimum number of such transitions are required to occur for the onset of microtubule catastrophe. The frequency of microtubule catastrophe is then determined using numerical simulations and compared with analytical and semianalytical estimates made under steady-state and quasi-steady-state assumptions, respectively, for the protofilament dynamics. A few relevant experimental results are analyzed in detail and compared with predictions from the model. Our results indicate that loss of GTP cap in two to three protofilaments is necessary to trigger catastrophe in a microtubule.

  16. Effective-range function for doublet nd scattering from an analysis of modern data

    SciTech Connect

    Orlov, Yu. V. Nikitina, L. I.

    2006-04-15

    The parameters of the generalized effective-range function K(k{sup 2}) having a pole are found by using the results that were obtained by calculating the S-wave phase shift {delta}(E) for doublet nd scattering and the triton binding energy on the basis of Faddeev equations and within the N/D method and which were presented in the literature. The convergence of the expansion of K(k{sup 2}) in powers of momentum is studied. The binding energy of the virtual triton and the residues of the partial-wave scattering amplitudes at the poles corresponding to the bound and virtual states are calculated. Correlations between the binding energies of the bound and virtual states of the triton, on one hand, and the doublet scattering length for nd interaction, on the other hand, are considered. The function K(k{sup 2}) is also calculated within a two-body model featuring various potentials.

  17. Low Scale Thermal Leptogenesis in Neutrinophilic Higgs Doublet Models

    NASA Astrophysics Data System (ADS)

    Haba, N.; Seto, O.

    2011-06-01

    It is well-known that leptogenesis in low energy scale is difficult in the conventional Type-I seesaw mechanism with hierarchical right-handed neutrino masses. We show that in a class of two Higgs doublet model, where one Higgs doublet generates masses of quarks and charged leptons whereas the other Higgs doublet with a tiny vacuum expectation value generates neutrino Dirac masses, large Yukawa couplings lead to a large enough CP asymmetry of the right-handed neutrino decay. Thermal leptogenesis suitably works at the low energy scale as keeping no enhancement of lepton number violating wash-out effects. We will also point out that thermal leptogenesis works well without confronting the gravitino problem in a supersymmetric neutrinophilic Higgs doublet model with gravity mediated supersymmetry breaking. Neutralino dark matter and baryon asymmetry generation by thermal leptogenesis are easily compatible in our setup.

  18. Pathway of Force Production by the Kinesin-Microtubule ATPase

    NASA Astrophysics Data System (ADS)

    Johnson, Kenneth

    2007-03-01

    Kinesin is the smallest of the molecular motors, consisting of a dimer of motor domains that interact with microtubules and ATP to generate motion towards the plus ends of microtubules for fast axonal transport of membranous organelles. It operates via an alternating site ATPase pathway in which the binding of ATP to one motor domain stimulates the release of ADP from the neighboring domain as the motor walks ``hand over hand'' along the microtubule surface. This alternating site pathway is accomplished in part due to strain that distinguishes the leading from the lagging motor domains when both are bound to the microtubule. This strain leads to a weak nucleotide binding state in the leading motor and a strong nucleotide binding state in the lagging motor. The ATPase activity is linked to alternating weak and strong nucleotide binding states that are coupled to association and dissociation at the microtubule surface to produce a force for forward motion. Strain in the leading motor domain appears to be due to the disruption of the ``neck linker'' in the leading motor. Release of the trailing motor domain from the microtubule surface is the rate-limiting step and, by relaxing the tension, allows the leading domain to bind ATP and continue the cycle and forward motion. Although many of the rate constants for steps in this pathway are known, details regarding the structural and thermodynamic basis for the coupling of ATP hydrolysis to force production remain to be established. I will review our current understanding and describe some of our early attempts to resolve intermediates during movement using single molecule fluorescence methods. In collaboration with Tim Scholz and Bernhard Brenner, Hannover Medical School.

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

    PubMed

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

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

  20. Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly.

    PubMed

    Ayoub, Ahmed T; Klobukowski, Mariusz; Tuszynski, Jack A

    2015-06-01

    Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder's axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability.

  1. Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly

    PubMed Central

    Ayoub, Ahmed T.; Klobukowski, Mariusz; Tuszynski, Jack A.

    2015-01-01

    Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder's axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability. PMID:26030285

  2. Evidence for Octupole Correlations in Multiple Chiral Doublet Bands

    NASA Astrophysics Data System (ADS)

    Liu, C.; Wang, S. Y.; Bark, R. A.; Zhang, S. Q.; Meng, J.; Qi, B.; Jones, P.; Wyngaardt, S. M.; Zhao, J.; Xu, C.; Zhou, S.-G.; Wang, S.; Sun, D. P.; Liu, L.; Li, Z. Q.; Zhang, N. B.; Jia, H.; Li, X. Q.; Hua, H.; Chen, Q. B.; Xiao, Z. G.; Li, H. J.; Zhu, L. H.; Bucher, T. D.; Dinoko, T.; Easton, J.; Juhász, K.; Kamblawe, A.; Khaleel, E.; Khumalo, N.; Lawrie, E. A.; Lawrie, J. J.; Majola, S. N. T.; Mullins, S. M.; Murray, S.; Ndayishimye, J.; Negi, D.; Noncolela, S. P.; Ntshangase, S. S.; Nyakó, B. M.; Orce, J. N.; Papka, P.; Sharpey-Schafer, J. F.; Shirinda, O.; Sithole, P.; Stankiewicz, M. A.; Wiedeking, M.

    2016-03-01

    Two pairs of positive-and negative-parity doublet bands together with eight strong electric dipole transitions linking their yrast positive- and negative-parity bands have been identified in 78Br. They are interpreted as multiple chiral doublet bands with octupole correlations, which is supported by the microscopic multidimensionally-constrained covariant density functional theory and triaxial particle rotor model calculations. This observation reports the first example of chiral geometry in octupole soft nuclei.

  3. Arabidopsis MICROTUBULE-ASSOCIATED PROTEIN18 functions in directional cell growth by destabilizing cortical microtubules.

    PubMed

    Wang, Xia; Zhu, Lei; Liu, Baoquan; Wang, Che; Jin, Lifeng; Zhao, Qian; Yuan, Ming

    2007-03-01

    Microtubule-associated proteins (MAPs) play important roles in the regulation of microtubule function in cells. We describe Arabidopsis thaliana MAP18, which binds to microtubules and inhibits tubulin polymerization in vitro and colocalizes along cortical microtubules as patches of dot-like structures. MAP18 is expressed mostly in the expanding cells. Cells overexpressing MAP18 in Arabidopsis exhibit various growth phenotypes with loss of polarity. Cortical microtubule arrays were significantly altered in cells either overexpressing MAP18 or where it had been downregulated by RNA interference (RNAi). The cortical microtubules were more sensitive to treatment with microtubule-disrupting drugs when MAP18 was overexpressed, but more resistant when MAP18 was eliminated in cells expressing MAP18 RNAi. Our study demonstrated that MAP18 may play a role in regulating directional cell growth and cortical microtubule organization by destabilizing microtubules.

  4. Microtubule-microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes.

    PubMed

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill; Gelfand, Vladimir I

    2016-08-23

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

  5. Doublets and other allied well patterns

    SciTech Connect

    Brigham, W.E.

    1997-06-01

    Whenever a liquid is injected into an infinite reservoir containing liquid with the same flow properties, the equations of flow are well known. The pressures in such a system vary over time and distance (radius) in ways that depend on the formation and liquid flow properties. Such equations are well known--they form the basis for the voluminous well-testing literature in petroleum engineering and ground water hydrology. Suppose there are two wells--one an injector and one a producer--with identical rates. The behavior of this system can be calculated using superposition; which merely means that the results can be added independently of each other. When this is done, the remarkable result is that after a period of time there is a region that approaches steady state flow. Thereafter, the pressures and flow velocities in this region stay constant. The size of this region increases with time. This ``steady state`` characteristic can be used to solve a number of interesting and useful problems, both in heat transfer and in fluid flow. The heat transfer problems can be addressed because the equations are identical in form. A number of such problems are solved herein for doublet systems. In addition, concepts are presented to help solve other cases that flow logically from the problems solved herein. It is not necessary that only two wells be involved. It turns out that any time the total injection and production are equal, the system approaches steady state. This idea is also addressed in these notes. A number of useful multiwell cases are addressed to present the flavor of such solutions.

  6. Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin.

    PubMed

    Gupta, Kamlesh K; Li, Chunlei; Duan, Aranda; Alberico, Emily O; Kim, Oleg V; Alber, Mark S; Goodson, Holly V

    2013-12-17

    Regulation of microtubule dynamic instability is crucial for cellular processes, ranging from mitosis to membrane transport. Stathmin (also known as oncoprotein 18/Op18) is a prominent microtubule destabilizer that acts preferentially on microtubule minus ends. Stathmin has been studied intensively because of its association with multiple types of cancer, but its mechanism of action remains controversial. Two models have been proposed. One model is that stathmin promotes microtubule catastrophe indirectly, and does so by sequestering tubulin; the other holds that stathmin alters microtubule dynamics by directly destabilizing growing microtubules. Stathmin's sequestration activity is well established, but the mechanism of any direct action is mysterious because stathmin binds to microtubules very weakly. To address these issues, we have studied interactions between stathmin and varied tubulin polymers. We show that stathmin binds tightly to Dolastatin-10 tubulin rings, which mimic curved tubulin protofilaments, and that stathmin depolymerizes stabilized protofilament-rich polymers. These observations lead us to propose that stathmin promotes catastrophe by binding to and acting upon protofilaments exposed at the tips of growing microtubules. Moreover, we suggest that stathmin's minus-end preference results from interactions between stathmin's N terminus and the surface of α-tubulin that is exposed only at the minus end. Using computational modeling of microtubule dynamics, we show that these mechanisms could account for stathmin's observed activities in vitro, but that both the direct and sequestering activities are likely to be relevant in a cellular context. Taken together, our results suggest that stathmin can promote catastrophe by direct action on protofilament structure and interactions.

  7. Probing a self-assembled fd virus membrane with a microtubule

    NASA Astrophysics Data System (ADS)

    Xie, Sheng; Pelcovits, Robert A.; Hagan, Michael F.

    2016-06-01

    The self-assembly of highly anisotropic colloidal particles leads to a rich variety of morphologies whose properties are just beginning to be understood. This article uses computer simulations to probe a particle-scale perturbation of a commonly studied colloidal assembly, a monolayer membrane composed of rodlike fd viruses in the presence of a polymer depletant. Motivated by experiments currently in progress, we simulate the interaction between a microtubule and a monolayer membrane as the microtubule "pokes" and penetrates the membrane face-on. Both the viruses and the microtubule are modeled as hard spherocylinders of the same diameter, while the depletant is modeled using ghost spheres. We find that the force exerted on the microtubule by the membrane is zero either when the microtubule is completely outside the membrane or when it has fully penetrated the membrane. The microtubule is initially repelled by the membrane as it begins to penetrate but experiences an attractive force as it penetrates further. We assess the roles played by translational and rotational fluctuations of the viruses and the osmotic pressure of the polymer depletant. We find that rotational fluctuations play a more important role than the translational ones. The dependence on the osmotic pressure of the depletant of the width and height of the repulsive barrier and the depth of the attractive potential well is consistent with the assumed depletion-induced attractive interaction between the microtubule and viruses. We discuss the relevance of these studies to the experimental investigations.

  8. Nerve Growth Factor Promotes Reorganization of the Axonal Microtubule Array at Sites of Axon Collateral Branching

    PubMed Central

    Ketschek, Andrea; Jones, Steven; Spillane, Mirela; Korobova, Farida; Svitkina, Tatyana; Gallo, Gianluca

    2015-01-01

    The localized debundling of the axonal microtubule array and the entry of microtubules into axonal filopodia are two defining features of collateral branching. We report that nerve growth factor (NGF), a branch inducing signal, increases the frequency of microtubule debundling along the axon shaft of chicken embryonic sensory neurons. Sites of debundling correlate strongly with the localized targeting of microtubules into filopodia. Platinum replica electron microscopy suggests physical interactions between debundled microtubules and axonal actin filaments. However, as evidenced by depolymerization of actin filaments and inhibition of myosin II, actomyosin force generation does not promote debundling. In contrast, loss of actin filaments or inhibition of myosin II activity promotes debundling, indicating that axonal actomyosin forces suppress debundling. MAP1B is a microtubule associated protein that represses axon branching. Following treatment with NGF, microtubules penetrating filopodia during the early stages of branching exhibited lower levels of associated MAP1B. NGF increased and decreased the levels of MAP1B phosphorylated at a GSK-3β site (pMAP1B) along the axon shaft and within axonal filopodia, respectively. The levels of MAP1B and pMAP1B were not altered at sites of debundling, relative to the rest of the axon. Unlike the previously determined effects of NGF on the axonal actin cytoskeleton, the effects of NGF on microtubule debundling were not affected by inhibition of protein synthesis. Collectively, these data indicate that NGF promotes localized axonal microtubule debundling, that actomyosin forces antagonize microtubule debundling and that NGF regulates pMAP1B in axonal filopodia during the early stages of collateral branch formation. PMID:25846486

  9. Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy.

    PubMed

    Yan, Si; Guo, Changmiao; Hou, Guangjin; Zhang, Huilan; Lu, Xingyu; Williams, John Charles; Polenova, Tatyana

    2015-11-24

    Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

  10. Microtubule-associated proteins from Antarctic fishes.

    PubMed

    Detrich, H W; Neighbors, B W; Sloboda, R D; Williams, R C

    1990-01-01

    Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.

  11. Neutral Higgs boson pair production in photon-photon annihilation in the two Higgs doublet model

    SciTech Connect

    Arhrib, Abdesslam; Benbrik, Rachid; Chen, C.-H.; Santos, Rui

    2009-07-01

    We study double Higgs production in photon-photon collisions as a probe of the new dynamics of Higgs interactions in the framework of two Higgs doublet models. We analyze neutral Higgs bosons production and decay in the fusion processes, {gamma}{gamma}{yields}S{sub i}S{sub j}, S{sub i}=h{sup 0}, H{sup 0}, A{sup 0}, and show that both h{sup 0}h{sup 0} and A{sup 0}A{sup 0} production can be enhanced by threshold effects in the region E{sub {gamma}}{sub {gamma}}{approx_equal}2m{sub H{+-}}. Resonant effects due to the heavy Higgs H{sup 0} can also play a role in the cross section enhancement when it is allowed to decay to two light CP-even h{sup 0} or to two light CP-odd A{sup 0} scalars. We have scanned the allowed parameter space of the two Higgs doublet model and found a vast region of the parameter space where the cross section is 2 orders of magnitude above the standard model cross section. We further show that the standard model experimental analysis can be used to discover or to constrain the two Higgs doublet model parameter space.

  12. Renormalization Group Analysis of Yukawa Parameters with One and Two Higgs Doublets, and Flavor Gauge Theory

    NASA Astrophysics Data System (ADS)

    Cvetič, G.; Kim, C. S.

    We assume that the standard model (SM) breaks down around some energy Λ and is replaced there by a new (Higgsless) flavor gauge theory (FGT) with fewer input parameters in the interactions corresponding to the Yukawa sector of SM. This would imply more symmetry for the values of the Yukawa (running) parameters of SM at E Λ, possibly by a (approximate) flavor democracy (for the quark mass sector). We investigate this possibility by studying the renormalization group equations (RGE's) for the quark Yukawa couplings of SM with one and two Higgs doublets, by running them from the known physical values at low energies (E 1 GeV) to Λ (> 1 TeV) and comparing the resulting quark masses mq (E Λ) for various mt and υU/υD. Unlike previous investigations of these RGE's, we do not implement the requirement mt(Λpole) = ∞. We found that SM with two Higgs doublets (type 2) is most likely to experience a gradual transition to FGT. Our results also shed more light on the adequacy and deficiencies of the usual RGE approaches within TMSM and related models. We also found that, independent of the assumption of a transition mechanism to FGT, mt phy< ˜ 200 GeV for Λpole≪ ΛPlanck in most cases of SM with two Higgs doublets.

  13. Basis invariant conditions for supersymmetry in the two-Higgs-doublet model

    SciTech Connect

    Ferreira, P. M.; Haber, Howard E.; Silva, Joao P.

    2010-07-01

    The minimal supersymmetric standard model involves a rather restrictive Higgs potential with two Higgs fields. Recently, the full set of classes of symmetries allowed in the most general two-Higgs-doublet model was identified; these classes do not include the supersymmetric limit as a particular class. Thus, a physically meaningful definition of the supersymmetric limit must involve the interaction of the Higgs sector with other sectors of the theory. Here we show how one can construct basis invariant probes of supersymmetry involving both the Higgs sector and the gaugino-Higgsino-Higgs interactions.

  14. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

    SciTech Connect

    Nieznanski, Krzysztof . E-mail: k.nieznanski@nencki.gov.pl; Podlubnaya, Zoya A.; Nieznanska, Hanna

    2006-10-13

    A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for First time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of {approx}50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers.

  15. Microtubule detyrosination guides chromosomes during mitosis

    PubMed Central

    Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K.; Magiera, Maria M.; Zaytsev, Anatoly V.; Pereira, Ana L.; Janke, Carsten; Grishchuk, Ekaterina L.; Maiato, Helder

    2015-01-01

    Before chromosomes segregate into daughter cells they align at the mitotic spindle equator, a process known as chromosome congression. CENP-E/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically towards the equator. Here we found that congression of pole-proximal chromosomes depended on specific post-translational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. PMID:25908662

  16. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2012-02-15

    microtubule: Direct experimental evidence for the existence of quantum states in microtubule, Towards a science of consciousness May 2-8 (2011), Sweden 4... quantum jump, sinusoidal switching of microtubule through various σS levels suggests that the spring-like expansion/contraction is the final defense...re-appears. To unravel the origin of quantum jump, we transmit thermal phonons along the Si-surface so that they propagate transversely across

  17. A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle

    PubMed Central

    1995-01-01

    Immunofluorescence microscopy revealed the presence of protein phosphatase 2A (PP2A) on microtubules in neuronal and nonneuronal cells. Interphase and mitotic spindle microtubules, as well as centrosomes, were all labeled with antibodies against individual PP2A subunits, showing that the AB alpha C holoenzyme is associated with microtubules. Biochemical analysis showed that PP2A could be reversibly bound to microtubules in vitro and that approximately 75% of the PP2A in cytosolic extracts could interact with microtubules. The activity of microtubule-associated PP2A was differentially regulated during the cell cycle. Enzymatic activity was high during S phase and intermediate during G1, while the activity in G2 and M was 20-fold lower than during S phase. The amount of microtubule-bound PP2A remained constant throughout the cell cycle, implying that cell cycle regulation of its enzymatic activity involves factors other than microtubules. These results raise the possibility that PP2A regulates cell cycle-dependent microtubule functions, such as karyokinesis and membrane transport. PMID:7896877

  18. Ectopic A-lattice seams destabilize microtubules.

    PubMed

    Katsuki, Miho; Drummond, Douglas R; Cross, Robert A

    2014-01-01

    Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

  19. Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

    PubMed Central

    Nunes Bastos, Ricardo; Gandhi, Sapan R.; Baron, Ryan D.; Gruneberg, Ulrike; Nigg, Erich A.

    2013-01-01

    Anaphase central spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the central spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the central spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase central spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to central spindle size control in anaphase. PMID:23940115

  20. Direct visualization of the microtubule lattice seam both in vitro and in vivo

    PubMed Central

    1994-01-01

    Microtubules are constructed from alpha- and beta-tubulin heterodimers that are arranged into protofilaments. Most commonly there are 13 or 14 protofilaments. A series of structural investigations using both electron microscopy and x-ray diffraction have indicated that there are two potential lattices (A and B) in which the tubulin subunits can be arranged. Electron microscopy has shown that kinesin heads, which bind only to beta-tubulin, follow a helical path with a 12-nm pitch in which subunits repeat every 8-nm axially, implying a primarily B-type lattice. However, these helical symmetry parameters are not consistent with a closed lattice and imply that there must be a discontinuity or "seam" along the microtubule. We have used quick-freeze deep-etch electron microscopy to obtain the first direct evidence for the presence of this seam in microtubules formed either in vivo or in vitro. In addition to a conventional single seam, we have also rarely found microtubules in which there is more than one seam. Overall our data indicates that microtubules have a predominantly B lattice, but that A lattice bonds between tubulin subunits are found at the seam. The cytoplasmic microtubules in mouse nerve cells also have predominantly B lattice structure and A lattice bonds at the seam. These observations have important implications for the interaction of microtubules with MAPs and with motor proteins, and for example, suggest that kinesin motors may follow a single protofilament track. PMID:7806574

  1. The Microtubule Binding Properties of CENP-E’s C-terminus and CENP-F

    PubMed Central

    Musinipally, Vivek; Howes, Stuart; Alushin, Gregory M.; Nogales, Eva

    2013-01-01

    CENP-E (Centromere Protein E) and CENP-F, also known as mitosin, are large, multi-functional proteins associated with the outer kinetochore. CENP-E features a well-characterized kinesin motor domain at its N-terminus and a second microtubule-binding domain at its C-terminus of unknown function. CENP-F is important for the formation of proper kinetochore-microtubule attachment and, like CENP-E, contains two microtubule-binding domains at its termini. While the importance of these proteins is known, the details of their interactions with microtubules have not yet been investigated. We have biochemically and structurally characterized the microtubule-binding properties of the amino- and carboxyl-terminal domains of CENP-F as well as the carboxyl-terminal (non-kinesin) domain of CENP-E. CENP-E’s C-terminus and CENP-F’s N-terminus bind microtubules with similar affinity to the well-characterized Ndc80 complex, while CENP-F’s C-terminus shows much lower affinity. Electron microscopy analysis reveals that all of these domains engage the microtubule surface in a disordered manner, suggesting that these factors have no favored binding geometry and may allow for initial side-on attachments early in mitosis. PMID:23892111

  2. Depletion of JMJD5 sensitizes tumor cells to microtubule-destabilizing agents by altering microtubule stability.

    PubMed

    Wu, Junyu; He, Zhimin; Wang, Da-Liang; Sun, Fang-Lin

    2016-11-01

    Microtubules play essential roles in mitosis, cell migration, and intracellular trafficking. Drugs that target microtubules have demonstrated great clinical success in cancer treatment due to their capacity to impair microtubule dynamics in both mitotic and interphase stages. In a previous report, we demonstrated that JMJD5 associated with mitotic spindle and was required for proper mitosis. However, it remains elusive whether JMJD5 could regulate the stability of cytoskeletal microtubules and whether it affects the efficacy of microtubule-targeting agents. In this study, we find that JMJD5 localizes not only to the nucleus, a fraction of it also localizes to the cytoplasm. JMJD5 depletion decreases the acetylation and detyrosination of α-tubulin, both of which are markers of microtubule stability. In addition, microtubules in JMJD5-depleted cells are more sensitive to nocodazole-induced depolymerization, whereas JMJD5 overexpression increases α-tubulin detyrosination and enhances the resistance of microtubules to nocodazole. Mechanistic studies revealed that JMJD5 regulates MAP1B protein levels and that MAP1B overexpression rescued the microtubule destabilization induced by JMJD5 depletion. Furthermore, JMJD5 depletion significantly promoted apoptosis in cancer cells treated with the microtubule-targeting anti-cancer drugs vinblastine or colchicine. Together, these findings suggest that JMJD5 is required to regulate the stability of cytoskeletal microtubules and that JMJD5 depletion increases the susceptibility of cancer cells to microtubule-destabilizing agents.

  3. Nonlinear dynamics of dipoles in microtubules: Pseudospin model.

    PubMed

    Nesterov, Alexander I; Ramírez, Mónica F; Berman, Gennady P; Mavromatos, Nick E

    2016-06-01

    We perform a theoretical study of the dynamics of the electric field excitations in a microtubule by taking into consideration the realistic cylindrical geometry, dipole-dipole interactions of the tubulin-based protein heterodimers, the radial electric field produced by the solvent, and a possible degeneracy of energy states of individual heterodimers. The consideration is done in the frame of the classical pseudospin model. We derive the system of nonlinear dynamical partial differential equations of motion for interacting dipoles and the continuum version of these equations. We obtain the solutions of these equations in the form of snoidal waves, solitons, kinks, and localized spikes. Our results will help to achieve a better understanding of the functional properties of microtubules including the motor protein dynamics and the information transfer processes. Our considerations are based on classical dynamics. Some speculations on the role of possible quantum effects are also made.

  4. Nonlinear dynamics of dipoles in microtubules: Pseudospin model

    NASA Astrophysics Data System (ADS)

    Nesterov, Alexander I.; Ramírez, Mónica F.; Berman, Gennady P.; Mavromatos, Nick E.

    2016-06-01

    We perform a theoretical study of the dynamics of the electric field excitations in a microtubule by taking into consideration the realistic cylindrical geometry, dipole-dipole interactions of the tubulin-based protein heterodimers, the radial electric field produced by the solvent, and a possible degeneracy of energy states of individual heterodimers. The consideration is done in the frame of the classical pseudospin model. We derive the system of nonlinear dynamical partial differential equations of motion for interacting dipoles and the continuum version of these equations. We obtain the solutions of these equations in the form of snoidal waves, solitons, kinks, and localized spikes. Our results will help to achieve a better understanding of the functional properties of microtubules including the motor protein dynamics and the information transfer processes. Our considerations are based on classical dynamics. Some speculations on the role of possible quantum effects are also made.

  5. The role of TOG domains in microtubule plus end dynamics.

    PubMed

    Slep, Kevin C

    2009-10-01

    The XMAP215 (Xenopus microtubule-associated protein 215) and CLASP [CLIP-170 (cytoskeletal linker protein 170) associated protein] microtubule plus end tracking families play central roles in the regulation of interphase microtubule dynamics and the proper formation of mitotic spindle architecture and flux. XMAP215 members comprise N-terminally-arrayed hexa-HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor) repeats known as TOG (tumour overexpressed gene) domains. Higher eukaryotic XMAP215 members are monomeric and have five TOG domains. Yeast counterparts are dimeric and have two TOG domains. Structure determination of the TOG domain reveals that the six HEAT repeats are aligned to form an oblong scaffold. The TOG domain face composed of intra-HEAT loops forms a contiguous, conserved tubulin-binding surface. Nested within the conserved intra-HEAT loop 1 is an invariant, signature, surface-exposed tryptophan residue that is a prime determinant in the TOG domain-tubulin interaction. The arrayed organization of TOG domains is critical for the processive mechanism of XMAP215, indicative that multiple tubulin/microtubule-binding sites are required for plus end tracking activity. The CLASP family has been annotated as containing a single N-terminal TOG domain. Using XMAP215 TOG domain structure determinants as a metric to analyse CLASP sequence, it is anticipated that CLASP contains two additional cryptic TOGL (TOG-like) domains. The presence of additional TOGL domains implicates CLASP as an ancient XMAP215 relative that uses a similar, multi-TOG-based mechanism to processively track microtubule ends.

  6. Two-Higgs-doublet models with Minimal Flavour Violation

    SciTech Connect

    Carlucci, Maria Valentina

    2010-12-22

    The tree-level flavour-changing neutral currents in the two-Higgs-doublet models can be suppressed by protecting the breaking of either flavour or flavour-blind symmetries, but only the first choice, implemented by the application of the Minimal Flavour Violation hypothesis, is stable under quantum corrections. Moreover, a two-Higgs-doublet model with Minimal Flavour Violation enriched with flavour-blind phases can explain the anomalies recently found in the {Delta}F = 2 transitions, namely the large CP-violating phase in B{sub s} mixing and the tension between {epsilon}{sub K} and S{sub {psi}KS}.

  7. Candidates for chiral doublet bands in 136Nd

    NASA Astrophysics Data System (ADS)

    Mergel, E.; Petrache, C. M.; Lo Bianco, G.; Hübel, H.; Domscheit, J.; Roßbach, D.; Schönwaßer, G.; Nenoff, N.; Neußer, A.; Görgen, A.; Becker, F.; Bouchez, E.; Houry, M.; Hürstel, A.; Le Coz, Y.; Lucas, R.; Theisen, Ch.; Korten, W.; Bracco, A.; Blasi, N.; Camera, F.; Leoni, S.; Hannachi, F.; Lopez-Martens, A.; Rejmund, M.; Gassmann, D.; Reiter, P.; Thirolf, P. G.; Astier, A.; Buforn, N.; Meyer, M.; Redon, N.; Stezowski, O.

    The even-even nucleus 136Nd was studied via in-beam γ-ray spectroscopy using the 16O + 125Te reaction at 100 MeV and the EUROBALL array. One new dipole band was observed. Together with a previously identified dipole band, whose position in the level scheme is revised, the new band forms a doublet structure similar to the recently observed chiral bands in the odd-odd neighboring nuclei. This would be the first case of a chiral doublet in an even-even nucleus.

  8. Two Higgs doublet models with an S3 symmetry

    NASA Astrophysics Data System (ADS)

    Cogollo, D.; Silva, João P.

    2016-05-01

    We study all implementations of the S3 symmetry in the two Higgs doublet models with quarks, consistent with nonzero quark masses and a Cabibbo-Kobayashi-Maskawa (CKM) matrix, which is not block diagonal. We study the impact of the various soft-breaking terms and vacuum expectation values and find an interesting relation between the mixing angles α and β . We also show that, in this minimal setting, only two types of assignments are possible: Either all field sectors are in singlets or all field sectors have a doublet.

  9. MCF7 microtubules: Cancer microtubules with relatively slow and stable dynamic in vitro.

    PubMed

    Feizabadi, Mitra Shojania; Rosario, Brandon

    2017-03-04

    There is known to be significant diversity of β-tubulin isoforms in cells. However, whether the functions of microtubules that are polymerized from different distributions of beta isotypes become distinct from one another are still being explored. Of particular interest, recent studies have identified the role that different beta tubulin isotypes carry in regulating the functions of some of the molecular motors along MCF7, or breast cancer, microtubules. That being said, how the specific distribution of beta tubulin isotypes impacts the MCF7 microtubules' dynamic is not well understood. The current study was initiated to directly quantify the in vitro dynamic and polymerization parameters of single MCF7 microtubules and then compare them with those obtained from neuronal microtubules polymerized from porcine brain tubulin. Surprisingly, unlike porcine brain microtubules, this type of cancer microtubule showed a relatively stable and slow dynamic. The comparison between the subsequently fast and unstable dynamic of porcine brain microtubules with the significantly slow and relatively stable dynamic of MCF7 microtubules suggests that beta tubulin isotypes may not only influence the microtubule based functionalities of some molecular motors, but also may change the microtubule's intrinsic dynamic.

  10. Microtubule motors: moving forward on many fronts.

    PubMed

    Allan, Viki

    2009-07-08

    Microtubule motors drive the movement of many different cargoes in eukaryotic cells. A combination of in vitro and in vivo approaches has led to a better understanding of their mechanism of action and function and are also revealing that the microtubule track itself may have an important role to play in directing cargo movement within the cell.

  11. Microtubule Control of Metabolism in Prostate Cancer

    DTIC Science & Technology

    2013-06-01

    Prostate Cancer PRINCIPAL INVESTIGATOR: Lynne Cassimeris CONTRACTING ORGANIZATION...Microtubule Control of Metabolism in Prostate Cancer 5b. GRANT NUMBER W81XWH-12-1-0071 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT...SUPPLEMENTARY NOTES 14. ABSTRACT The current standard chemotherapy treatment for metastatic castrate-resistant prostate cancer is the microtubule

  12. Mechanical Properties of Doubly Stabilized Microtubule Filaments

    PubMed Central

    Hawkins, Taviare L.; Sept, David; Mogessie, Binyam; Straube, Anne; Ross, Jennifer L.

    2013-01-01

    Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5′ [(α, β)-methyleno] triphosphate, guanosine-5′-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules. PMID:23561528

  13. Mechanical properties of doubly stabilized microtubule filaments.

    PubMed

    Hawkins, Taviare L; Sept, David; Mogessie, Binyam; Straube, Anne; Ross, Jennifer L

    2013-04-02

    Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5' [(α, β)-methyleno] triphosphate, guanosine-5'-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules.

  14. Hypothesis: NDL proteins function in stress responses by regulating microtubule organization

    PubMed Central

    Khatri, Nisha; Mudgil, Yashwanti

    2015-01-01

    N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants. PMID:26583023

  15. On complex, curved trajectories in microtubule gliding

    NASA Astrophysics Data System (ADS)

    Gosselin, Pierre; Mohrbach, Hervé; Kulić, Igor M.; Ziebert, Falko

    2016-04-01

    We study the dynamics of microtubules in gliding assays. These biofilaments are typically considered as purely semiflexible, hence their trajectories under the action of motors covering the substrate have been regarded so far as straight, modulo fluctuations. However, this is not always the case experimentally, where microtubules are known to move on large scale circles or spirals, or even display quite regular wavy trajectories and more complex dynamics. Incorporating recent experimental evidence for a (small) preferred curvature as well as the microtubules' well established lattice twist into a dynamic model for microtubule gliding, we could reproduce both types of trajectories. Interestingly, as a function of the microtubules' length we found length intervals of stable rings alternating with regions where wavy and more complex dynamics prevails. Finally, both types of dynamics (rings and waves) can be rationalized by considering simple limits of the full model.

  16. Tubulin bistability and polymorphic dynamics of microtubules.

    PubMed

    Mohrbach, Hervé; Johner, Albert; Kulić, Igor M

    2010-12-31

    Based on the hypothesis that the GDP-tubulin dimer is a conformationally bistable molecule-rapidly fluctuating between a discrete curved and a straight state-we develop a model for polymorphic dynamics of the microtubule lattice. We show that GDP-tubulin bistability consistently explains unusual dynamic fluctuations, the apparent length-stiffness relation of grafted taxol-stabilized microtubules, and the curved-helical appearance of microtubules in general. When clamped by one end the microtubules undergo an unusual zero energy motion-in its effect reminiscent of a limited rotational hinge. We conclude that microtubules exist in highly cooperative energy-degenerate helical states and discuss possible implications in vivo.

  17. FLUCTUATING MOTOR FORCES BEND GROWING MICROTUBULES

    PubMed Central

    Shekhar, Nandini; Neelam, Srujana; Wu, Jun; Ladd, Anthony JC; Dickinson, Richard B.; Lele, Tanmay P.

    2013-01-01

    Despite their rigidity, microtubules in living cells bend significantly during polymerization resulting in greater curvature than can be explained by thermal forces alone. However, the source of the non-thermal forces that bend growing microtubules remains obscure. We analyzed the motion of microtubule tips in NIH-3T3 fibroblasts expressing EGFP-EB1, a fluorescent +TIP protein that specifically binds to the growing ends of microtubules. We found that dynein inhibition significantly reduced the deviation of the growing tip from its initial trajectory. Inhibiting myosin modestly reduced tip fluctuations, while simultaneous myosin and dynein inhibition caused no further decrease in fluctuations compared to dynein inhibition alone. Our results can be interpreted with a model in which dynein linkages play a key role in generating and transmitting fluctuating forces that bend growing microtubules. PMID:24039637

  18. Visualizing individual microtubules by bright field microscopy

    NASA Astrophysics Data System (ADS)

    Gutiérrez-Medina, Braulio; Block, Steven M.

    2010-11-01

    Microtubules are slender (˜25 nm diameter), filamentous polymers involved in cellular structure and organization. Individual microtubules have been visualized via fluorescence imaging of dye-labeled tubulin subunits and by video-enhanced, differential interference-contrast microscopy of unlabeled polymers using sensitive CCD cameras. We demonstrate the imaging of unstained microtubules using a microscope with conventional bright field optics in conjunction with a webcam-type camera and a light-emitting diode illuminator. The light scattered by microtubules is image-processed to remove the background, reduce noise, and enhance contrast. The setup is based on a commercial microscope with a minimal set of inexpensive components, suitable for implementation in a student laboratory. We show how this approach can be used in a demonstration motility assay, tracking the gliding motions of microtubules driven by the motor protein kinesin.

  19. Targeting Microtubules for Wound Repair

    PubMed Central

    Charafeddine, Rabab A.; Nosanchuk, Joshua D.; Sharp, David J.

    2016-01-01

    Significance: Fast and seamless healing is essential for both deep and chronic wounds to restore the skin and protect the body from harmful pathogens. Thus, finding new targets that can both expedite and enhance the repair process without altering the upstream signaling milieu and causing serious side effects can improve the way we treat wounds. Since cell migration is key during the different stages of wound healing, it presents an ideal process and intracellular structural machineries to target. Recent Advances and Critical Issues: The microtubule (MT) cytoskeleton is rising as an important structural and functional regulator of wound healing. MTs have been reported to play different roles in the migration of the various cell types involved in wound healing. Specific microtubule regulatory proteins (MRPs) can be targeted to alter a section or subtype of the MT cytoskeleton and boost or hinder cell motility. However, inhibiting intracellular components can be challenging in vivo, especially using unstable molecules, such as small interfering RNA. Nanoparticles can be used to protect these unstable molecules and topically deliver them to the wound. Utilizing this approach, we recently showed that fidgetin-like 2, an uncharacterized MRP, can be targeted to enhance cell migration and wound healing. Future Directions: To harness the full potential of the current MRP therapeutic targets, studies should test them with different delivery platforms, dosages, and skin models. Screening for new MT effectors that boost cell migration in vivo would also help find new targets for skin repair. PMID:27785378

  20. Active contraction of microtubule networks

    PubMed Central

    Foster, Peter J; Fürthauer, Sebastian; Shelley, Michael J; Needleman, Daniel J

    2015-01-01

    Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction. DOI: http://dx.doi.org/10.7554/eLife.10837.001 PMID:26701905

  1. Cellular effects of curcumin on Plasmodium falciparum include disruption of microtubules.

    PubMed

    Chakrabarti, Rimi; Rawat, Parkash S; Cooke, Brian M; Coppel, Ross L; Patankar, Swati

    2013-01-01

    Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC50 of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.

  2. Dark matter with topological defects in the Inert Doublet Model

    SciTech Connect

    Hindmarsh, Mark; Kirk, Russell; No, Jose Miguel; West, Stephen M.

    2015-05-26

    We examine the production of dark matter by decaying topological defects in the high mass region m{sub DM}≫m{sub W} of the Inert Doublet Model, extended with an extra U(1) gauge symmetry. The density of dark matter states (the neutral Higgs states of the inert doublet) is determined by the interplay of the freeze-out mechanism and the additional production of dark matter states from the decays of topological defects, in this case cosmic strings. These decays increase the predicted relic abundance compared to the standard freeze-out only case, and as a consequence the viable parameter space of the Inert Doublet Model can be widened substantially. In particular, for a given dark matter annihilation rate lower dark matter masses become viable. We investigate the allowed mass range taking into account constraints on the energy injection rate from the diffuse γ-ray background and Big Bang Nucleosynthesis, together with constraints on the dark matter properties coming from direct and indirect detection limits. For the Inert Doublet Model high-mass region, an inert Higgs mass as low as ∼200 GeV is permitted. There is also an upper limit on string mass per unit length, and hence the symmetry breaking scale, from the relic abundance in this scenario. Depending on assumptions made about the string decays, the limits are in the range 10{sup 12} GeV to 10{sup 13} GeV.

  3. Dark matter with topological defects in the Inert Doublet Model

    SciTech Connect

    Hindmarsh, Mark; No, Jose Miguel; Kirk, Russell; West, Stephen M. E-mail: russell.kirk.2008@live.rhul.ac.uk E-mail: stephen.west@rhul.ac.uk

    2015-05-01

    We examine the production of dark matter by decaying topological defects in the high mass region m{sub DM} >> m{sub W} of the Inert Doublet Model, extended with an extra U(1) gauge symmetry. The density of dark matter states (the neutral Higgs states of the inert doublet) is determined by the interplay of the freeze-out mechanism and the additional production of dark matter states from the decays of topological defects, in this case cosmic strings. These decays increase the predicted relic abundance compared to the standard freeze-out only case, and as a consequence the viable parameter space of the Inert Doublet Model can be widened substantially. In particular, for a given dark matter annihilation rate lower dark matter masses become viable. We investigate the allowed mass range taking into account constraints on the energy injection rate from the diffuse γ-ray background and Big Bang Nucleosynthesis, together with constraints on the dark matter properties coming from direct and indirect detection limits. For the Inert Doublet Model high-mass region, an inert Higgs mass as low as ∼ 200 GeV is permitted. There is also an upper limit on string mass per unit length, and hence the symmetry breaking scale, from the relic abundance in this scenario. Depending on assumptions made about the string decays, the limits are in the range 10{sup 12} GeV to 10{sup 13} GeV.

  4. Effects of fluid propagation on occurrence of doublet earthquakes

    NASA Astrophysics Data System (ADS)

    Mastrolembo V., Brunella; Rinaldi, Antonio Pio; Urpi, Luca; Rivalta, Eleonora; Passarelli, Luigi

    2016-04-01

    Most earthquake sequences consist of a main large event preceded and followed by a series of smaller magnitude quakes commonly referred as to fore- and after-shocks. However, seismic catalogs report many examples of earthquake sequences featuring two or more main events of comparable magnitude Such events are often referred as doublet earthquakes and are particularly observed in environments characterized by a large number of faults. Doublet earthquakes occur all over the world representing a significant issue in terms of seismic hazard assessment after large events. Some examples of doublets are: the 2012 Emilia-Romagna sequence (Italy), during which a magnitude 5.9 event occurred on May 20th, followed by a magnitude 5.8 event on May 29th; the 1992 Landers earthquake in California, which has been associated to the Big Bear earthquake, that hit about three hours later after the mainshock; the 2006 November 15th M8.3 event along the Kuril arc followed by a M8.1 event on 13 January 2007 is one of the largest great doublet earthquake on record. The spatial distribution of aftershocks usually well correlates with the coseismic (static) Coulomb stress change, while the observed time delay of aftershocks, as well as their diffusive-like behavior, have been explained as due to additional physical processes such as post-seismic relaxation, afterslip, poro-elastic effect, as well as induced fluid propagation. In this work we first perform an analysis of the available worldwide seismic catalogs in order to identify a number of doublet earthquakes based on a spatial and temporal distance correlation. Then we perform a parametric study to identify the main characteristics of every couple of events and extrapolate the common relations between time delay, hypocentral distances, geological, as well as hydrogeological parameters and fluids content. Numerical simulations are then carried out to study the time delay occurring between two events as related to hydrogeological and

  5. Cross-linking of microtubules by microtubule-associated proteins (MAPs) from the brine shrimp, Artemia.

    PubMed

    Campbell, E J; MacKinlay, S A; MacRae, T H

    1989-05-01

    Microtubules induced with taxol to assemble in cell-free extracts of the brine shrimp, Artemia, are cross-linked by microtubule-associated proteins (MAPs). When the MAPs, extracted from taxol-stabilized microtubules with 1 M-NaCl are co-assembled with purified Artemia or mammalian neural tubulin, reconstitution of cross-linking between microtubules occurs. The most prominent non-tubulin protein associated with reconstituted cross-linked microtubules has a molecular weight of 49,000 but we cannot yet exclude the possibility that other proteins may be responsible for the cross-linking. Cross-linkers are separated by varying distances while cross-linked microtubules, prepared under different conditions, are 6.9-7.7 nm apart. Cross-linking of microtubules by MAPs occurs whether MAPs are added to assembling tubulin or to microtubules, and it is not disrupted by ATP. The MAPs are heat-sensitive and do not stabilize microtubules to cold. Immunological characterization of Artemia MAPs on Western blots indicates that Artemia lack MAP 1, MAP 2 and tau. Our results clearly demonstrate that Artemia contain novel MAPs with the ability to cross-link microtubules from phylogenetically disparate organisms in an ATP-independent manner.

  6. The Human Centriolar Protein CEP135 Contains a Two-Stranded Coiled-Coil Domain Critical for Microtubule Binding.

    PubMed

    Kraatz, Sebastian; Guichard, Paul; Obbineni, Jagan M; Olieric, Natacha; Hatzopoulos, Georgios N; Hilbert, Manuel; Sen, Indrani; Missimer, John; Gönczy, Pierre; Steinmetz, Michel O

    2016-07-20

    Centrioles are microtubule-based structures that play important roles notably in cell division and cilium biogenesis. CEP135/Bld10p family members are evolutionarily conserved microtubule-binding proteins important for centriole formation. Here, we analyzed in detail the microtubule-binding activity of human CEP135 (HsCEP135). X-ray crystallography and small-angle X-ray scattering in combination with molecular modeling revealed that the 158 N-terminal residues of HsCEP135 (HsCEP135-N) form a parallel two-stranded coiled-coil structure. Biochemical, cryo-electron, and fluorescence microscopy analyses revealed that in vitro HsCEP135-N interacts with tubulin, protofilaments, and microtubules and induces the formation of microtubule bundles. We further identified a 13 amino acid segment spanning residues 96-108, which represents a major microtubule-binding site in HsCEP135-N. Within this segment, we identified a cluster of three lysine residues that contribute to the microtubule bundling activity of HsCEP135-N. Our results provide the first structural information on CEP135/Bld10p proteins and offer insights into their microtubule-binding mechanism.

  7. Reovirus Cell Entry Requires Functional Microtubules

    PubMed Central

    Mainou, Bernardo A.; Zamora, Paula F.; Ashbrook, Alison W.; Dorset, Daniel C.; Kim, Kwang S.; Dermody, Terence S.

    2013-01-01

    ABSTRACT Mammalian reovirus binds to cell-surface glycans and junctional adhesion molecule A and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within the endocytic compartment, reovirus undergoes stepwise disassembly, allowing release of the transcriptionally active viral core into the cytoplasm. To identify cellular mediators of reovirus infectivity, we screened a library of small-molecule inhibitors for the capacity to block virus-induced cytotoxicity. In this screen, reovirus-induced cell killing was dampened by several compounds known to impair microtubule dynamics. Microtubule inhibitors were assessed for blockade of various stages of the reovirus life cycle. While these drugs did not alter reovirus cell attachment or internalization, microtubule inhibitors diminished viral disassembly kinetics with a concomitant decrease in infectivity. Reovirus virions colocalize with microtubules and microtubule motor dynein 1 during cell entry, and depolymerization of microtubules results in intracellular aggregation of viral particles. These data indicate that functional microtubules are required for proper sorting of reovirus virions following internalization and point to a new drug target for pathogens that use the endocytic pathway to invade host cells. PMID:23820395

  8. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

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

    2016-01-01

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

  9. The novel microtubule-destabilizing drug BAL27862 binds to the colchicine site of tubulin with distinct effects on microtubule organization.

    PubMed

    Prota, Andrea E; Danel, Franck; Bachmann, Felix; Bargsten, Katja; Buey, Rubén M; Pohlmann, Jens; Reinelt, Stefan; Lane, Heidi; Steinmetz, Michel O

    2014-04-17

    Microtubule-targeting agents are widely used for the treatment of cancer and as tool compounds to study the microtubule cytoskeleton. BAL27862 is a novel microtubule-destabilizing drug that is currently undergoing phase I clinical evaluation as the prodrug BAL101553. The drug is a potent inhibitor of tumor cell growth and shows a promising activity profile in a panel of human cancer models resistant to clinically relevant microtubule-targeting agents. Here, we evaluated the molecular mechanism of the tubulin-BAL27862 interaction using a combination of cell biology, biochemistry and structural biology methods. Tubulin-binding assays revealed that BAL27862 potently inhibited tubulin assembly at 37 °C with an IC50 of 1.4 μM and bound to unassembled tubulin with a stoichiometry of 1 mol/mol tubulin and a dissociation constant of 244±30 nM. BAL27862 bound to tubulin independently of vinblastine, without the formation of tubulin oligomers. The kinetics of BAL27862 binding to tubulin were distinct from those of colchicine, with evidence of competition between BAL27862 and colchicine for binding. Determination of the tubulin-BAL27862 structure by X-ray crystallography demonstrated that BAL27862 binds to the same site as colchicine at the intradimer interface. Comparison of crystal structures of tubulin-BAL27862 and tubulin-colchicine complexes shows that the binding mode of BAL27862 to tubulin is similar to that of colchicine. However, comparative analyses of the effects of BAL27862 and colchicine on the microtubule mitotic spindle and in tubulin protease-protection experiments suggest different outcomes of tubulin binding. Taken together, our data define BAL27862 as a potent, colchicine site-binding, microtubule-destabilizing agent with distinct effects on microtubule organization.

  10. Microtubule nucleation remote from centrosomes may explain how asters span large cells.

    PubMed

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

    2014-12-16

    A major challenge in cell biology is to understand how nanometer-sized molecules can organize micrometer-sized cells in space and time. One solution in many animal cells is a radial array of microtubules called an aster, which is nucleated by a central organizing center and spans the entire cytoplasm. Frog (here Xenopus laevis) embryos are more than 1 mm in diameter and divide with a defined geometry every 30 min. Like smaller cells, they are organized by asters, which grow, interact, and move to precisely position the cleavage planes. It has been unclear whether asters grow to fill the enormous egg by the same mechanism used in smaller somatic cells, or whether special mechanisms are required. We addressed this question by imaging growing asters in a cell-free system derived from eggs, where asters grew to hundreds of microns in diameter. By tracking marks on the lattice, we found that microtubules could slide outward, but this was not essential for rapid aster growth. Polymer treadmilling did not occur. By measuring the number and positions of microtubule ends over time, we found that most microtubules were nucleated away from the centrosome and that interphase egg cytoplasm supported spontaneous nucleation after a time lag. We propose that aster growth is initiated by centrosomes but that asters grow by propagating a wave of microtubule nucleation stimulated by the presence of preexisting microtubules.

  11. Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

    PubMed Central

    Pilhofer, Martin; Ladinsky, Mark S.; McDowall, Alasdair W.; Petroni, Giulio; Jensen, Grant J.

    2011-01-01

    Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as “bacterial microtubules” (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening. PMID:22162949

  12. ATPase Cycle of the Nonmotile Kinesin NOD Allows Microtubule End Tracking and Drives Chromosome Movement

    SciTech Connect

    Cochran, J.; Sindelar, C; Mulko, N; Collins, K; Kong, S; Hawley, R; Kull, F

    2009-01-01

    Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile kinesin-10. We have determined the X-ray crystal structure of the NOD catalytic domain in the ADP- and AMPPNP-bound states. These structures reveal an alternate conformation of the microtubule binding region as well as a nucleotide-sensitive relay of hydrogen bonds at the active site. Additionally, a cryo-electron microscopy reconstruction of the nucleotide-free microtubule-NOD complex shows an atypical binding orientation. Thermodynamic studies show that NOD binds tightly to microtubules in the nucleotide-free state, yet other nucleotide states, including AMPPNP, are weakened. Our pre-steady-state kinetic analysis demonstrates that NOD interaction with microtubules occurs slowly with weak activation of ADP product release. Upon rapid substrate binding, NOD detaches from the microtubule prior to the rate-limiting step of ATP hydrolysis, which is also atypical for a kinesin. We propose a model for NOD's microtubule plus-end tracking that drives chromosome movement.

  13. Antibody against tuberlin: the specific visualization of cytoplasmic microtubules in tissue culture cells.

    PubMed Central

    Weber, K; Pollack, R; Bibring, T

    1975-01-01

    Cytoplasmic microtubules in tissue culture cells can be directly visualized by immunofluorescence microscopy. Antibody against tubulin from the outer doublets of sea urchin sperm flagella decorates a network of fine cytoplasmic fibers in a variety of cell lines of human, monkey, rat, mouse, and chicken origin. These fibers are separate and of uniform thickness and are seen throughout the cytoplasm. The fibers disappear either in a medium containing colchicine or after subjection of the cells to low temperature. The same treatments do not destroy the microfilamentous structures that are visualized by means of antibody against actin. When tryspin-treated enucleated cells are replated and then stained with antibody against tubulin, the fibers can be seen to traverse the entire enucleated cytoplasm. Images PMID:804694

  14. A mechanism for reorientation of cortical microtubule arrays driven by microtubule severing.

    PubMed

    Lindeboom, Jelmer J; Nakamura, Masayoshi; Hibbel, Anneke; Shundyak, Kostya; Gutierrez, Ryan; Ketelaar, Tijs; Emons, Anne Mie C; Mulder, Bela M; Kirik, Viktor; Ehrhardt, David W

    2013-12-06

    Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array.

  15. The microtubule catastrophe promoter Sentin delays stable kinetochore–microtubule attachment in oocytes

    PubMed Central

    Głuszek, A. Agata; Cullen, C. Fiona; Li, Wenjing; Battaglia, Rachel A.; Radford, Sarah J.; Costa, Mariana F.; McKim, Kim S.; Goshima, Gohta

    2015-01-01

    The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore–microtubule attachment in oocytes. PMID:26668329

  16. Insights into Antiparallel Microtubule Crosslinking by PRC1, a Conserved Nonmotor Microtubule Binding Protein

    SciTech Connect

    Subramanian, Radhika; Wilson-Kubalek, Elizabeth M.; Arthur, Christopher P.; Bick, Matthew J.; Campbell, Elizabeth A.; Darst, Seth A.; Milligan, Ronald A.; Kapoor, Tarun M.

    2010-09-03

    Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks are compliant and do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish crosslinks that selectively mark antiparallel overlap in dynamic cytoskeletal networks.

  17. Hierarchical Bionanotubes Formed By the Self Assembly of Microtubules With Cationic Membranes Or Polypeptides

    SciTech Connect

    Raviv, U.; Needleman, D.J.; Ewert, K.K.; Safinya, C.R.

    2009-06-05

    At present there is a surge in interest in biophysical research aimed at elucidating collective interactions between cellular proteins and associated biomolecules leading to supramolecular structures, with the ultimate goal of relating structure to function. The nerve cell cytoskeleton provides a rich example of highly ordered bundles and networks of interacting neurofilaments, microtubules and filamentous actin, where the nature of the interactions, structures and structure-function correlations remain poorly understood. We present synchrotron X-ray diffraction and electron microscopy data, in reconstituted protein systems from the bovine central nervous system, which reveal unexpected structures not predicted by current electrostatic theories. By mixing preassembled microtubules with charged membranes or polypeptides we found hierarchical bionanotubes made of microtubules coated by lipid bilayers or polypeptides, which in turn are coated with a third layer of tubulin oligomers forming rings or spirals.

  18. Assembly of Caenorhabditis elegans acentrosomal spindles occurs without evident microtubule-organizing centers and requires microtubule sorting by KLP-18/kinesin-12 and MESP-1

    PubMed Central

    Wolff, Ian D.; Tran, Michael V.; Mullen, Timothy J.; Villeneuve, Anne M.; Wignall, Sarah M.

    2016-01-01

    Although centrosomes contribute to spindle formation in most cell types, oocytes of many species are acentrosomal and must organize spindles in their absence. Here we investigate this process in Caenorhabditis elegans, detailing how acentrosomal spindles form and revealing mechanisms required to establish bipolarity. Using high-resolution imaging, we find that in meiosis I, microtubules initially form a “cage-like” structure inside the disassembling nuclear envelope. This structure reorganizes so that minus ends are sorted to the periphery of the array, forming multiple nascent poles that then coalesce until bipolarity is achieved. In meiosis II, microtubules nucleate in the vicinity of chromosomes but then undergo similar sorting and pole formation events. We further show that KLP-18/kinesin-12 and MESP-1, previously shown to be required for spindle bipolarity, likely contribute to bipolarity by sorting microtubules. After their depletion, minus ends are not sorted outward at the early stages of spindle assembly and instead converge. These proteins colocalize on microtubules, are interdependent for localization, and can interact, suggesting that they work together. We propose that KLP-18/kinesin-12 and MESP-1 form a complex that functions to sort microtubules of mixed polarity into a configuration in which minus ends are away from the chromosomes, enabling formation of nascent poles. PMID:27559133

  19. Rigidity of microtubules is increased by stabilizing agents

    PubMed Central

    1995-01-01

    Microtubules are rigid polymers that contribute to the static mechanical properties of cells. Because microtubules are dynamic structures whose polymerization is regulated during changes in cell shape, we have asked whether the mechanical properties of microtubules might also be modulated. We measured the flexural rigidity, or bending stiffness, of individual microtubules under a number of different conditions that affect the stability of microtubules against depolymerization. The flexural rigidity of microtubules polymerized with the slowly hydrolyzable nucleotide analogue guanylyl-(alpha, beta)- methylene-diphosphonate was 62 +/- 9 x 10(-24) Nm2 (weighted mean +/- SEM); that of microtubules stabilized with tau protein was 34 +/- 3 x 10(-24) Nm2; and that of microtubules stabilized with the antimitotic drug taxol was 32 +/- 2 x 10(-24) Nm2. For comparison, microtubules that were capped to prevent depolymerization, but were not otherwise stabilized, had a flexural rigidity of 26 +/- 2 x 10(-24) Nm2. Decreasing the temperature from 37 degrees C to approximately 25 degrees C, a condition that makes microtubules less stable, decreased the stiffness of taxol-stabilized microtubules by one-third. We thus find that the more stable a microtubule, the higher its flexural rigidity. This raises the possibility that microtubule rigidity may be regulated in vivo. In addition, the high rigidity of an unstabilized, GDP-containing microtubule suggests that a large amount of energy could be stored as mechanical strain energy in the protein lattice for subsequent force generation during microtubule depolymerization. PMID:7642706

  20. Yukawa alignment in the two-Higgs-doublet model

    SciTech Connect

    Pich, Antonio; Tuzon, Paula

    2009-11-01

    In multi-Higgs-doublet models the alignment in flavor space of the relevant Yukawa matrices guarantees the absence of tree-level flavor-changing couplings of the neutral scalar fields. We analyze the consequences of this condition within the two-Higgs-doublet model and show that it leads to a generic Yukawa structure which contains as particular cases all known specific implementations of the model based on Z{sub 2} symmetries. All possible freedom in the Yukawa sector gets parametrized in terms of three complex couplings {sigma}{sub f}. In spite of having flavor conservation in the neutral scalar couplings, the phases of these three parameters represent potential new sources of CP violation.

  1. Study the most favorable shapes of electrostatic quadrupole doublet lenses

    NASA Astrophysics Data System (ADS)

    Hussein, O. A.; Sise, O.

    2017-02-01

    The optical properties of an electrostatic quadrupole doublet lens with two different electrode shapes were studied with the aid of computer simulation. The optimal electrode voltages of the electrostatic quadrupole lenses which give the stigmatic image in both planes simultaneously were found for both concave cylindrical electrode shape and plan electrode shape of the operation mode: parallel to point focusing. The effect of electrode shape on the image properties was investigated, and the aberration figures were studied. The results showed that under the same operation condition and the geometrical dimensions, the changing of the electrode shape of the electrostatic quadrupole doublet lenses lead to important differences in the optical properties of the lenses and the characteristics of the systems.

  2. A search for close-mass lepton doublet

    SciTech Connect

    Riles, J.K.

    1989-04-01

    Described is a search for a heavy charged lepton with an associated neutrino of nearly the same mass, together known as a close-mass lepton doublet. The search is conducted in e/sup +/e/sup/minus// annihilation data taken with the Mark II detector at a center-of-mass energy of 29 GeV. In order to suppress contamination from conventional two-photon reactions, the search applies a novel, radiative-tagging technique. Requiring the presence of an isolated, energetic photon allows exploration for lepton doublets with a mass splitting smaller than that previously accessible to experiment. No evidence for such a new lepton has been found, enabling limits to be placed on allowed mass combinations. Mass differences as low as 250-300 MeV are excluded for charged lepton masses up to 10 GeV. 78 refs., 64 figs., 8 tabs.

  3. Microtubules, polarity and vertebrate neural tube morphogenesis

    PubMed Central

    Cearns, Michael D.; Escuin, Sarah; Alexandre, Paula; Greene, Nicholas D. E.; Copp, Andrew J.

    2016-01-01

    Microtubules are key cellular components, long known to participate in morphogenetic events that shape the developing embryo. However, the links between the cellular functions of microtubules, their effects on cell shape and polarity and their role in large-scale morphogenesis remain poorly understood. Here, we examine these relationships with respect to two strategies for generating the vertebrate neural tube: bending and closure of the mammalian neural plate, and cavitation of the teleost neural rod. The latter process has been compared to ‘secondary’ neurulation that generates the caudal spinal cord in mammals. Microtubules align along the apico-basal axis of the mammalian neuroepithelium early in neural tube closure, participating functionally in interkinetic nuclear migration which indirectly impacts on cell shape. Whether microtubules play other functional roles in mammalian neurulation remains unclear. In the zebrafish, microtubules are important for defining the neural rod midline prior to its cavitation, both by localizing apical proteins at the tissue midline and by orienting cell division through a mirror-symmetric microtubule apparatus that helps to further define the medial localization of apical polarity proteins. Par proteins have been implicated in centrosome positioning in neuroepithelia as well as in the control of polarized morphogenetic movements in the neural rod. Understanding of microtubule functions during early nervous system development has so far been limited, partly by techniques that fail to distinguish ‘cause’ from ‘effect’. Future developments will likely rely on novel ways to selectively impair microtubule function in order to investigate the roles they play. PMID:27025884

  4. The Salmonella effector SseJ disrupts microtubule dynamics when ectopically expressed in normal rat kidney cells

    PubMed Central

    Raines, Sally A.; Hodgkinson, Michael R.; Dowle, Adam A.

    2017-01-01

    Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ’s acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family. PMID:28235057

  5. Asymmetric inelastic inert doublet dark matter from triplet scalar leptogenesis

    NASA Astrophysics Data System (ADS)

    Arina, Chiara; Sahu, Narendra

    2012-01-01

    The nature of dark matter (DM) particles and the mechanism that provides their measured relic abundance are currently unknown. In this paper we investigate inert scalar and vector like fermion doublet DM candidates with a charge asymmetry in the dark sector, which is generated by the same mechanism that provides the baryon asymmetry, namely baryogenesis-via-leptogenesis induced by decays of scalar triplets. At the same time the model gives rise to neutrino masses in the ballpark of oscillation experiments via type II seesaw. We discuss possible sources of depletion of asymmetry in the DM and visible sectors and solve the relevant Boltzmann equations for quasi-equilibrium decay of triplet scalars. A Monte-Carlo-Markov-Chain analysis is performed for the whole parameter space. The survival of the asymmetry in the dark sector leads to inelastic scattering off nuclei. We then apply Bayesian statistic to infer the model parameters favoured by the current experimental data, in particular the DAMA annual modulation and XENON100 exclusion limit. The latter strongly disfavours asymmetric scalar doublet DM of mass O(TeV) as required by DM-DM¯ oscillations, while an asymmetric vector like fermion doublet DM with mass around 100 GeV is a good candidate for DAMA annual modulation yet satisfying the constraints from XENON100 data.

  6. Phenomenology of a Flavorful Two Higgs Doublet Model

    NASA Astrophysics Data System (ADS)

    Tuckler, Douglas; Altmannshofer, Wolfgang; Eby, Joshua; Gori, Stefania; Lotito, Matteo; Martone, Mario

    2017-01-01

    The discovery of the Higgs boson in 2012 gave us the final piece of the SM, and since then there has been a large effort to understand its properties. Current experimental sensitivities allow us to probe the couplings of the Higgs to the 3rd generation of quarks and leptons, and measurements tell us that the Higgs is responsible for generating their mass. However, a lot less is known about the origin of mass of the 1st and 2nd generations: measurements of their couplings to the Higgs are out of experimental reach. With limited experimental sensitivities, one might be led to ask: is the origin of mass of the 1st and 2nd generation fermions due to the SM Higgs at all? In this talk, the idea that the mass of the 1st and 2nd generation fermions is not due to the SM Higgs, but a second source of electroweak symmetry breaking, is investigated. This can be realized simply by a two Higgs doublet model (2HDM), where one doublet couples mainly to the 3rd generations fermions while the second doublet couples mainly to the 1st and 2nd generation. We will see how a non-standard Yukawa texture leads to phenomenology that is markedly different from well studied 2HDMs, enhancing the collider signatures involving 2nd generation quarks and leptons.

  7. Vacuum topology of the two Higgs doublet model

    NASA Astrophysics Data System (ADS)

    Battye, Richard A.; Brawn, Gary D.; Pilaftsis, Apostolos

    2011-08-01

    We perform a systematic study of generic accidental Higgs-family and CP symmetries that could occur in the two-Higgs-doublet-model potential, based on a Majorana scalar-field formalism which realizes a subgroup of {text{GL}}left( {8,mathbb{C}} right) . We derive the general conditions of convexity and stability of the scalar potential and present analytical solutions for two non-zero neutral vacuum expectation values of the Higgs doublets for a typical set of six symmetries, in terms of the gauge-invariant parameters of the theory. By means of a homotopy-group analysis, we identify the topological defects associated with the spontaneous symmetry breaking of each symmetry, as well as the massless Goldstone bosons emerging from the breaking of the continuous symmetries. We find the existence of domain walls from the breaking of Z2, CP1 and CP2 discrete symmetries, vortices in models with broken U(1)PQ and CP3 symmetries and a global monopole in the SO(3)HF-broken model. The spatial profile of the topological defect solutions is studied in detail, as functions of the potential parameters of the two-Higgs doublet model. The application of our Majorana scalar-field formalism in studying more general scalar potentials that are not constrained by the U(1)Y hypercharge symmetry is discussed. In particular, the same formalism may be used to properly identify seven additional symmetries that may take place in a U(1)Y-invariant scalar potential.

  8. Critical Assessment of TD-DFT for Excited States of Open-Shell Systems: I. Doublet-Doublet Transitions.

    PubMed

    Li, Zhendong; Liu, Wenjian

    2016-01-12

    A benchmark set of 11 small radicals is set up to assess the performance of time-dependent density functional theory (TD-DFT) for the excited states of open-shell systems. Both the unrestricted (U-TD-DFT) and spin-adapted (X-TD-DFT) formulations of TD-DFT are considered. For comparison, the well-established EOM-CCSD (equation-of-motion coupled-cluster with singles and doubles) is also used. In total, 111 low-lying singly excited doublet states are accessed by all the three approaches. Taking the MRCISD+Q (multireference configuration interaction with singles and doubles plus the Davidson correction) results as the benchmark, it is found that both U-TD-DFT and EOM-CCSD perform well for those states dominated by singlet-coupled single excitations (SCSE) from closed-shell to open-shell, open-shell to vacant-shell, or closed-shell to vacant-shell orbitals. However, for those states dominated by triplet-coupled single excitations (TCSE) from closed-shell to vacant-shell orbitals, both U-TD-DFT and EOM-CCSD fail miserably due to severe spin contaminations. In contrast, X-TD-DFT provides balanced descriptions of both SCSE and TCSE. As far as the functional dependence is concerned, it is found that, when the Hartree-Fock ground state does not suffer from the instability problem, both global hybrid (GH) and range-separated hybrid (RSH) functionals perform grossly better than pure density functionals, especially for Rydberg and charge-transfer excitations. However, if the Hartree-Fock ground state is instable or nearly instable, GH and RSH tend to underestimate severely the excitation energies. The SAOP (statistically averaging of model orbital potentials) performs more uniformly than any other density functionals, although it generally overestimates the excitation energies of valence excitations. Not surprisingly, both EOM-CCSD and adiabatic TD-DFT are incapable of describing excited states with substantial double excitation characters.

  9. Collider and dark matter searches in the inert doublet model from Peccei-Quinn symmetry

    NASA Astrophysics Data System (ADS)

    Alves, Alexandre; Camargo, Daniel A.; Dias, Alex G.; Longas, Robinson; Nishi, Celso C.; Queiroz, Farinaldo S.

    2016-10-01

    Weakly Interacting Massive Particles (WIMPs) and axions are arguably the most compelling dark matter candidates in the literature. Could they coexist as dark matter particles? More importantly, can they be incorporated in a well motivated framework in agreement with experimental data? In this work, we show that this two component dark matter can be realized in the Inert Doublet Model in an elegant and natural manner by virtue of the spontaneous breaking of a Peccei-Quinn U(1) P Q symmetry into a residual {Z}_2 symmetry. The WIMP stability is guaranteed by the {Z}_2 symmetry and a new dark matter component, the axion, arises. There are two interesting outcomes: (i) vector-like quarks needed to implement the Peccei-Quinn symmetry in the model may act as a portal between the dark sector and the SM fields with a supersymmetry-type phenomenology at colliders; (ii) two-component Inert Doublet Model re-opens the phenomenologically interesting 100-500 GeV mass region. We show that the model can successfully realize a two component dark matter framework and at the same time avoid low and high energy physics constraints such as monojet and dijet plus missing energy, as well as indirect and direct dark matter detection bounds.

  10. Isospin-violating dark-matter-nucleon scattering via two-Higgs-doublet-model portals

    SciTech Connect

    Drozd, Aleksandra; Grzadkowski, Bohdan; Gunion, John F.; Jiang, Yun

    2016-10-24

    We show that in a multi-Higgs model in which one Higgs fits the LHC 125 GeV state, one or more of the other Higgs bosons can mediate DM-nucleon interactions with maximal DM isospin violation being possible for appropriate Higgs-quark couplings, independent of the nature of DM. We then consider the explicit example of a Type II two-Higgs-doublet model, identifying the h or H as the 125 GeV state while the H or h, respectively, mediates DM-nucleon interactions. Finally, we show that if a stable scalar, S, is added then it can be a viable light DM candidate with correct relic density while obeying all direct and indirect detection limits.

  11. Single file diffusion in microtubules

    NASA Astrophysics Data System (ADS)

    Rutenberg, Andrew; Farrell, Spencer; Brown, Aidan

    2015-03-01

    We investigate the single file diffusion (SFD) of large particles entering a confined tubular geometry, such as luminal diffusion of proteins inside microtubules or flagella. While single-file effects have no effect on particle density, we report significant single-file effects for individually-tracked tracer particle motion. Both exact and approximate ordering statistics of particles entering semi-infinite tubes agree well with our stochastic simulations. Considering initially empty semi-infinite tubes, with particles entering at one end starting from an initial time t = 0 , tracked particles display super-diffusive effective exponents just after they enter the system and trends towards diffusive exponents at later times. Equivalently, if diffusive exponents are assumed the effective diffusivity is reduced at early times and enhanced at later times through a logarithmic factor logN , where N is the number of particles in the tube. When we number each particle from the first (n = 1) to the most recent (n = N), we find good scaling collapse of the effective diffusivity for all n. Techniques that track individual particles, or local groups of particles, such as photo-activation or photobleaching, will exhibit single-file effects.

  12. Microtubules self-repair in response to mechanical stress

    NASA Astrophysics Data System (ADS)

    Schaedel, Laura; John, Karin; Gaillard, Jérémie; Nachury, Maxence V.; Blanchoin, Laurent; Théry, Manuel

    2015-11-01

    Microtubules--which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport--can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses.

  13. Organization of neuronal microtubules in the nematode Caenorhabditis elegans

    PubMed Central

    1979-01-01

    We have studied the organization of microtubules in neurons of the nematode Caenorhabditis elegans. Six neurons, which we call the microtubule cells, contain bundles of darkly staining microtubules which can be followed easily in serial-section electron micrographs. Reconstruction of individual microtubules in these cells indicate that most, if not all, microtubules are short compared with the length of the cell process. Average microtubule length varies characteristically with cell type. The arrangement of microtubules gives an overall polarity to each bundle: the distal ends of the microtubles are on the outside of the bundle, whereas the proximal ends are preferentially inside. The distal and proximal ends each have a characteristic appearance indicating that these microtubules may have a polarity of their own. Short microtubules in processes of other neurons in C. elegans have also been observed. PMID:479300

  14. [Microtubules in the nerve cells: morphological and functional aspects].

    PubMed

    Vorob'ev, V S; Portuganov, V V

    1980-10-01

    The modern literature concerning ultrastructure and cytochemistry of microtubules in the nervous tissue is reviewed. Common features of cytological and biochemical organization of microtubules in different parts of the nervous system of the vertebrates and invertebrates are analysed: the similarity of ultrastructure of microtubules and their molecular organization (tubulin and its alpha- and beta-monomeres), the ability of microtubules to assemble and disassemble, to bind specifically with poisons--colchicine and vinblastine, participation of microtubules in the neuroplastic transport. The authors' data on space arrangement of microtubules within cytoplasm of the neuronal processes (dendrites and unmyelinated axons in the central and peripheral nevous system) are presented. Some literature and personal results concerning ultrastructure of neurofilaments and microtubules in the myelinated nerve fibres are also considered. The functional significance of microtubules in the nervous system is discussed with special reference to facts and hypotheses on a possible role of microtubules in the propagation of nerve impulse.

  15. Microtubule organization is determined by the shape of epithelial cells

    PubMed Central

    Gomez, Juan Manuel; Chumakova, Lyubov; Bulgakova, Natalia A.; Brown, Nicholas H.

    2016-01-01

    Interphase microtubule organization is critical for cell function and tissue architecture. In general, physical mechanisms are sufficient to drive microtubule organization in single cells, whereas cells within tissues are thought to utilize signalling mechanisms. By improving the imaging and quantitation of microtubule alignment within developing Drosophila embryos, here we demonstrate that microtubule alignment underneath the apical surface of epithelial cells follows cell shape. During development, epidermal cell elongation and microtubule alignment occur simultaneously, but by perturbing cell shape, we discover that microtubule organization responds to cell shape, rather than the converse. A simple set of microtubule behaviour rules is sufficient for a computer model to mimic the observed responses to changes in cell surface geometry. Moreover, we show that microtubules colliding with cell boundaries zip-up or depolymerize in an angle-dependent manner, as predicted by the model. Finally, we show microtubule alignment responds to cell shape in diverse epithelia. PMID:27779189

  16. Microtubules self-repair in response to mechanical stress

    PubMed Central

    Schaedel, Laura; John, Karin; Gaillard, Jérémie; Nachury, Maxence V.; Blanchoin, Laurent; Théry, Manuel

    2015-01-01

    Microtubules - which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport - can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of larger damages, which further decrease microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses. PMID:26343914

  17. Intercourse between cell wall and cytoplasm exemplified by arabinogalactan proteins and cortical microtubules.

    PubMed

    Driouich, Azeddine; Baskin, Tobias I

    2008-12-01

    How does a plant cell sense and respond to the status of its cell wall? Intercourse between cell wall and cytoplasm has long been supposed to involve arabinogalactan proteins, in part because many of them are anchored to the plasma membrane. Disrupting arabinogalactan proteins has recently been shown to disrupt the array of cortical microtubules present just inside the plasma membrane, implying that microtubules and arabinogalactan proteins interact. In this article, we assess possibilities for how this interaction might be mediated. First, we consider microdomains in the plasma membrane (lipid rafts), which have been alleged to link internal and external regions of the plasma membrane; however, the characteristics and even the existence of these domains remains controversial. Next, we point out that disrupting the synthesis of cellulose also can disrupt microtubules and consider whether arabinogalactan proteins are part of a network linking microtubules and nascent microfibrils. Finally, we outline several signaling cascades that could transmit information from arabinogalactan proteins to microtubules through channels of cellular communication. These diverse possibilities highlight the work that remains to be done before we can understand how plant cells communicate across their membranes.

  18. History-dependent catastrophes regulate axonal microtubule behavior.

    PubMed

    Stepanova, Tatiana; Smal, Ihor; van Haren, Jeffrey; Akinci, Umut; Liu, Zhe; Miedema, Marja; Limpens, Ronald; van Ham, Marco; van der Reijden, Michael; Poot, Raymond; Grosveld, Frank; Mommaas, Mieke; Meijering, Erik; Galjart, Niels

    2010-06-08

    In Chinese hamster ovary cells, microtubules originate at the microtubule organizing center (MTOC) and grow persistently toward the cell edge, where they undergo catastrophe. In axons, microtubule dynamics must be regulated differently because microtubules grow parallel to the plasma membrane and there is no MTOC. GFP-tagged microtubule plus end tracking proteins (+TIPs) mark the ends of growing neuronal microtubules. Their fluorescent "comet-like" pattern reflects turnover of +TIP binding sites. Using GFP-tagged +TIPs and fluorescence-based segmentation and tracking tools, we show that axonal microtubules grow with a constant average velocity and that they undergo catastrophes at random positions, yet in a programmed fashion. Using protein depletion approaches, we find that the +TIPs CLIP-115 and CLIP-170 affect average microtubule growth rate and growth distance in neurons but not the duration of a microtubule growth event. In N1E-115 neuroblastoma cells, we find that EB1, the core +TIP, regulates microtubule growth rate, growth distance, and duration, consistent with in vitro data. Combined, our data suggest that CLIPs influence the axonal microtubule/tubulin ratio, whereas EB1 stimulates microtubule growth and structural transitions at microtubule ends, thereby regulating microtubule catastrophes and the turnover of +TIP binding sites.

  19. Phenomenology of the inert (2+1) and (4+2) Higgs doublet models

    NASA Astrophysics Data System (ADS)

    Keus, Venus; King, Stephen F.; Moretti, Stefano

    2014-10-01

    We make a phenomenological study of a model with two inert doublets plus one Higgs doublet [I(2+1)HDM] which is symmetric under a Z2 group, preserved after electroweak symmetry breaking by the vacuum alignment (0,0,v). This model may be regarded as an extension to the model with one inert doublet plus one Higgs doublet [I(1+1)HDM], by the addition of an extra inert scalar doublet. The neutral fields from the two inert doublets provide a viable dark matter (DM) candidate which is stabilized by the conserved Z2 symmetry. We study the new Higgs decay channels offered by the scalar fields from the extra doublets and their effect on the standard model Higgs couplings, including a new decay channel into (off-shell) photon(s) plus missing energy, which distinguishes the I(2+1)HDM from the I(1+1)HDM. Motivated by supersymmetry, which requires an even number of doublets, we then extend this model into a model with four inert doublets plus two Higgs doublets [I(4+2)HDM] and study the phenomenology of the model with the vacuum alignment (0,0,0,0,v ,v). This scenario offers a wealth of Higgs signals, the most distinctive ones being cascade decays of heavy Higgs states into inert ones. Finally, we also remark that the smoking-gun signature of all the considered models is represented by invisible Higgs decays into the lightest inert Higgs bosons responsible for DM.

  20. Non-centrosomal nucleation mediated by augmin organizes microtubules in post-mitotic neurons and controls axonal microtubule polarity

    PubMed Central

    Sánchez-Huertas, Carlos; Freixo, Francisco; Viais, Ricardo; Lacasa, Cristina; Soriano, Eduardo; Lüders, Jens

    2016-01-01

    Neurons display a highly polarized microtubule network that mediates trafficking throughout the extensive cytoplasm and is crucial for neuronal differentiation and function. In newborn migrating neurons, the microtubule network is organized by the centrosome. During neuron maturation, however, the centrosome gradually loses this activity, and how microtubules are organized in more mature neurons remains poorly understood. Here, we demonstrate that microtubule organization in post-mitotic neurons strongly depends on non-centrosomal nucleation mediated by augmin and by the nucleator γTuRC. Disruption of either complex not only reduces microtubule density but also microtubule bundling. These microtubule defects impair neurite formation, interfere with axon specification and growth, and disrupt axonal trafficking. In axons augmin does not merely mediate nucleation of microtubules but ensures their uniform plus end-out orientation. Thus, the augmin-γTuRC module, initially identified in mitotic cells, may be commonly used to generate and maintain microtubule configurations with specific polarity. PMID:27405868

  1. Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

    PubMed

    Margolis, R L; Rauch, C T; Pirollet, F; Job, D

    1990-12-01

    STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.

  2. Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

    PubMed Central

    Volkov, Vladimir A.; Zaytsev, Anatoly V.; Grishchuk, Ekaterina L.

    2014-01-01

    Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion. PMID:24686554

  3. Distinct pose of discodermolide in taxol binding pocket drives a complementary mode of microtubule stabilization.

    PubMed

    Khrapunovich-Baine, Marina; Menon, Vilas; Verdier-Pinard, Pascal; Smith, Amos B; Angeletti, Ruth Hogue; Fiser, Andras; Horwitz, Susan Band; Xiao, Hui

    2009-12-15

    The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.

  4. Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

    PubMed Central

    Pampaloni, Francesco; Lattanzi, Gianluca; Jonáš, Alexandr; Surrey, Thomas; Frey, Erwin; Florin, Ernst-Ludwig

    2006-01-01

    Microtubules are hollow cylindrical structures that constitute one of the three major classes of cytoskeletal filaments. On the mesoscopic length scale of a cell, their material properties are characterized by a single stiffness parameter, the persistence length ℓp. Its value, in general, depends on the microscopic interactions between the constituent tubulin dimers and the architecture of the microtubule. Here, we use single-particle tracking methods combined with a fluctuation analysis to systematically study the dependence of ℓp on the total filament length L. Microtubules are grafted to a substrate with one end free to fluctuate in three dimensions. A fluorescent bead is attached proximally to the free tip and is used to record the thermal fluctuations of the microtubule's end. The position distribution functions obtained with this assay allow the precise measurement of ℓp for microtubules of different contour length L. Upon varying L between 2.6 and 47.5 μm, we find a systematic increase of ℓp from 110 to 5,035 μm. At the same time we verify that, for a given filament length, the persistence length is constant over the filament within the experimental accuracy. We interpret this length dependence as a consequence of a nonnegligible shear deflection determined by subnanometer relative displacement of adjacent protofilaments. Our results may shine new light on the function of microtubules as sophisticated nanometer-sized molecular machines and give a unified explanation of seemingly uncorrelated spreading of microtubules' stiffness previously reported in literature. PMID:16801537

  5. Theory of self-assembly of microtubules and motors.

    SciTech Connect

    Aranson, I. S.; Tsimring, L. S.; Materials Science Division; Univ. California at San Diego

    2006-01-01

    We derive a model describing spatiotemporal organization of an array of microtubules interacting via molecular motors. Starting from a stochastic model of inelastic polar rods with a generic anisotropic interaction kernel, we obtain a set of equations for the local rods concentration and orientation. At large enough mean density of rods and concentration of motors, the model describes an orientational instability. We demonstrate that the orientational instability leads to the formation of vortices and (for large density and/or kernel anisotropy) asters seen in recent experiments. We derive the specific form of the interaction kernel from the detailed analysis of microscopic interaction of two filaments mediated by a moving molecular motor and extend our results to include variable motor density and motor attachment to the substrate.

  6. Theory of self-assembly of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Aranson, Igor S.; Tsimring, Lev S.

    2006-09-01

    We derive a model describing spatiotemporal organization of an array of microtubules interacting via molecular motors. Starting from a stochastic model of inelastic polar rods with a generic anisotropic interaction kernel, we obtain a set of equations for the local rods concentration and orientation. At large enough mean density of rods and concentration of motors, the model describes an orientational instability. We demonstrate that the orientational instability leads to the formation of vortices and (for large density and/or kernel anisotropy) asters seen in recent experiments. We derive the specific form of the interaction kernel from the detailed analysis of microscopic interaction of two filaments mediated by a moving molecular motor and extend our results to include variable motor density and motor attachment to the substrate.

  7. Physical basis of large microtubule aster growth

    PubMed Central

    Ishihara, Keisuke; Korolev, Kirill S; Mitchison, Timothy J

    2016-01-01

    Microtubule asters - radial arrays of microtubules organized by centrosomes - play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks. DOI: http://dx.doi.org/10.7554/eLife.19145.001 PMID:27892852

  8. Gauged Two Higgs Doublet Model confronts the LHC 750 GeV diphoton anomaly

    NASA Astrophysics Data System (ADS)

    Huang, Wei-Chih; Tsai, Yue-Lin Sming; Yuan, Tzu-Chiang

    2016-08-01

    In light of the recent 750 GeV diphoton anomaly observed at the LHC, we study the possibility of accommodating the deviation from the standard model prediction based on the recently proposed Gauged Two Higgs Doublet Model. The model embeds two Higgs doublets into a doublet of a non-abelian gauge group SU(2)H, while the standard model SU(2)L right-handed fermion singlets are paired up with new heavy fermions to form SU(2)H doublets, and SU(2)L left-handed fermion doublets are singlets under SU(2)H. An SU(2)H scalar doublet, which provides masses to the new heavy fermions as well as the SU(2)H gauge bosons, can be produced via gluon fusion and subsequently decays into two photons with the new fermions circulating the triangle loops to account for the deviation from the standard model prediction.

  9. Perturbative unitarity bounds in composite two-Higgs doublet models

    NASA Astrophysics Data System (ADS)

    De Curtis, Stefania; Moretti, Stefano; Yagyu, Kei; Yildirim, Emine

    2016-09-01

    We study bounds from perturbative unitarity in a composite two-Higgs doublet model (C2HDM) based on the spontaneous breakdown of a global symmetry S O (6 )→S O (4 )×S O (2 ) at the compositeness scale f . The eight pseudo-Nambu-Goldstone bosons (pNGBs) emerging from such a dynamics are identified as two isospin doublet Higgs fields. We calculate the S -wave amplitude for all possible two-to-two-body elastic (pseudo)scalar boson scatterings at energy scales √{s } reachable at the Large Hadron Collider (LHC) and beyond it, including the longitudinal components of weak gauge boson states as the corresponding pNGB states. In our calculation, the Higgs potential is assumed to have the same form as that in the elementary two-Higgs doublet model (E2HDM) with a discrete Z2 symmetry, which is expected to be generated at the one-loop level via the Coleman-Weinberg mechanism. We find that the S -wave amplitude matrix can be block-diagonalized with maximally 2 ×2 submatrices in a way similar to the E2HDM case as long as we only keep the contributions from O (ξ s ) and O (ξ0s0) in the amplitudes, where ξ =vSM2/f2 and vSM≃246 GeV , which is an appropriate approximation for our analysis. By requiring the C2HDM to satisfy perturbative unitarity at energies reachable by the LHC, we derive bounds on its parameters such as ξ and the masses of extra Higgs bosons present in the scenario alongside the Standard Model-like Higgs state discovered in 2012.

  10. The oncoprotein HBXIP promotes migration of breast cancer cells via GCN5-mediated microtubule acetylation.

    PubMed

    Li, Leilei; Liu, Bowen; Zhang, Xiaodong; Ye, Lihong

    2015-03-13

    We have documented that the oncoprotein hepatitis B X-interacting protein (HBXIP) is able to promote migration of breast cancer cells. A subset of acetylated microtubules that accumulates in the cell leading edge is necessary for cell polarization and directional migration. In this study, we explored the hypothesis that HBXIP contributes to migration of breast cancer cells by supporting microtubule acetylation in breast cancer cells. We found that HBXIP could induce acetylated microtubules accumulating into the leading protrusion in wound-induced directional migration in breast cancer cells by immunofluorescence staining analysis. Interestingly, HBXIP was able to increase the acetylation of α-tubulin in the cells by immunofluorescence staining and Western blot analysis. Furthermore, we observed that acetyltransferase GCN5 was involved in the event that HBXIP induced increase of acetylated microtubules and their expansion in protrusions in breast cancer cells by Western blot analysis and immunofluorescence staining. Moreover, GCN5 was required for the HBXIP-enhanced migration of breast cancer cells by wound healing assay. Thus, we conclude that HBXIP promotes the migration of breast cancer cells through modulating microtubule acetylation mediated by GCN5. Therapeutically, HBXIP may serve as a novel target in breast cancer.

  11. The spectraplakin Short stop is an essential microtubule regulator involved in epithelial closure in Drosophila

    PubMed Central

    Takács, Zsanett; Vilmos, Péter; Lénárt, Péter; Röper, Katja; Erdélyi, Miklós

    2017-01-01

    ABSTRACT Dorsal closure of the Drosophila embryonic epithelium provides an excellent model system for the in vivo analysis of molecular mechanisms regulating cytoskeletal rearrangements. In this study, we investigated the function of the Drosophila spectraplakin Short stop (Shot), a conserved cytoskeletal structural protein, during closure of the dorsal embryonic epithelium. We show that Shot is essential for the efficient final zippering of the opposing epithelial margins. By using isoform-specific mutant alleles and genetic rescue experiments with truncated Shot variants, we demonstrate that Shot functions as an actin–microtubule cross-linker in mediating zippering. At the leading edge of epithelial cells, Shot regulates protrusion dynamics by promoting filopodia formation. Fluorescence recovery after photobleaching (FRAP) analysis and in vivo imaging of microtubule growth revealed that Shot stabilizes dynamic microtubules. The actin- and microtubule-binding activities of Shot are simultaneously required in the same molecule, indicating that Shot is engaged as a physical crosslinker in this process. We propose that Shot-mediated interactions between microtubules and actin filaments facilitate filopodia formation, which promotes zippering by initiating contact between opposing epithelial cells. PMID:28062848

  12. Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK

    PubMed Central

    Cross, Marie K.; Powers, Maureen A.

    2011-01-01

    During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere–associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly. PMID:21209315

  13. Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis.

    PubMed

    Nam, Sang-Chul

    2016-11-01

    Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Here, Tau, a microtubule associated protein, was analyzed to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins' localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. It is found that Tau has a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation.

  14. Wafer-level fabrication of arrays of glass lens doublets

    NASA Astrophysics Data System (ADS)

    Passilly, Nicolas; Perrin, Stéphane; Albero, Jorge; Krauter, Johann; Gaiffe, Olivier; Gauthier-Manuel, Ludovic; Froehly, Luc; Lullin, Justine; Bargiel, Sylwester; Osten, Wolfgang; Gorecki, Christophe

    2016-04-01

    Systems for imaging require to employ high quality optical components in order to dispose of optical aberrations and thus reach sufficient resolution. However, well-known methods to get rid of optical aberrations, such as aspherical profiles or diffractive corrections are not easy to apply to micro-optics. In particular, some of these methods rely on polymers which cannot be associated when such lenses are to be used in integrated devices requiring high temperature process for their further assembly and separation. Among the different approaches, the most common is the lens splitting that consists in dividing the focusing power between two or more optical components. In here, we propose to take advantage of a wafer-level technique, devoted to the generation of glass lenses, which involves thermal reflow in silicon cavities to generate lens doublets. After the convex lens sides are generated, grinding and polishing of both stack sides allow, on the first hand, to form the planar lens backside and, on the other hand, to open the silicon cavity. Nevertheless, silicon frames are then kept and thinned down to form well-controlled and auto-aligned spacers between the lenses. Subsequent accurate vertical assembly of the glass lens arrays is performed by anodic bonding. The latter ensures a high level of alignment both laterally and axially since no additional material is required. Thanks to polishing, the generated lens doublets are then as thin as several hundreds of microns and compatible with micro-opto-electro-systems (MOEMS) technologies since they are only made of glass and silicon. The generated optical module is then robust and provide improved optical performances. Indeed, theoretically, two stacked lenses with similar features and spherical profiles can be almost diffraction limited whereas a single lens characterized by the same numerical aperture than the doublet presents five times higher wavefront error. To demonstrate such assumption, we fabricated glass

  15. Vacuum structure of the Higgs complex singlet-doublet model

    NASA Astrophysics Data System (ADS)

    Ferreira, P. M.

    2016-11-01

    The complex singlet-doublet model is a popular theory to account for dark matter and electroweak baryogenesis, wherein the Standard Model particle content is supplemented by a complex scalar gauge singlet, with certain discrete symmetries imposed. The scalar potential which results thereof can have seven different types of minima at tree level, which may coexist for specific choices of parameters. There is therefore the possibility that a given minimum is not global but rather a local one, and may tunnel to a deeper extremum, thus causing vacuum instability. This rich vacuum structure is explained and discussed in detail.

  16. Microtubules move the nucleus to quiescence.

    PubMed

    Laporte, Damien; Sagot, Isabelle

    2014-01-01

    The nucleus is a cellular compartment that hosts several macro-molecular machines displaying a highly complex spatial organization. This tight architectural orchestration determines not only DNA replication and repair but also regulates gene expression. In budding yeast microtubules play a key role in structuring the nucleus since they condition the Rabl arrangement in G1 and chromosome partitioning during mitosis through their attachment to centromeres via the kinetochore proteins. Recently, we have shown that upon quiescence entry, intranuclear microtubules emanating from the spindle pole body elongate to form a highly stable bundle that spans the entire nucleus. Here, we examine some molecular mechanisms that may underlie the formation of this structure. As the intranuclear microtubule bundle causes a profound re-organization of the yeast nucleus and is required for cell survival during quiescence, we discuss the possibility that the assembly of such a structure participates in quiescence establishment.

  17. Orthotropic elastic shell model for buckling of microtubules.

    PubMed

    Wang, C Y; Ru, C Q; Mioduchowski, A

    2006-11-01

    In view of the fact that microtubules exhibit strong anisotropic elastic properties, an orthotropic elastic shell model for microtubules is developed to study buckling behavior of microtubules. The predicted critical pressure is found to agree well with recent unexplained experimental data on pressure-induced buckling of microtubules [Needleman, Phys. Rev. Lett. 93, 198104 (2004); Biophys. J. 89, 3410 (2005)] which are lower than that predicted by the isotropic shell model by four orders of magnitude. General buckling behavior of microtubules under axial compression or radial pressure is studied. The results show that the isotropic shell model greatly overestimates the bucking loads of microtubules, except columnlike axially compressed buckling of long microtubules (of length-to-diameter ratio larger than, say, 150). In particular, the present results also offer a plausible explanation for the length dependency of flexibility of microtubules reported in the literature.

  18. Understanding force-generating microtubule systems through in vitro reconstitution

    PubMed Central

    Kok, Maurits; Dogterom, Marileen

    2016-01-01

    ABSTRACT Microtubules switch between growing and shrinking states, a feature known as dynamic instability. The biochemical parameters underlying dynamic instability are modulated by a wide variety of microtubule-associated proteins that enable the strict control of microtubule dynamics in cells. The forces generated by controlled growth and shrinkage of microtubules drive a large range of processes, including organelle positioning, mitotic spindle assembly, and chromosome segregation. In the past decade, our understanding of microtubule dynamics and microtubule force generation has progressed significantly. Here, we review the microtubule-intrinsic process of dynamic instability, the effect of external factors on this process, and how the resulting forces act on various biological systems. Recently, reconstitution-based approaches have strongly benefited from extensive biochemical and biophysical characterization of individual components that are involved in regulating or transmitting microtubule-driven forces. We will focus on the current state of reconstituting increasingly complex biological systems and provide new directions for future developments. PMID:27715396

  19. Dynamic microtubules and the texture of plant cell walls.

    PubMed

    Lloyd, Clive

    2011-01-01

    The relationship between microtubules and cell-wall texture has had a fitful history in which progress in one area has not been matched by progress in the other. For example, the idea that wall texture arises entirely from self-assembly, independently of microtubules, originated with electron microscopic analyses of fixed cells that gave no clue to the ability of microtubules to reorganize. Since then, live-cell studies have established the surprising dynamicity of plant microtubules involving collisions, changes in angle, parallelization, and rotation of microtubule tracks. Combined with proof that cellulose synthases do track along shifting microtubules, this offers more realistic models for the dynamic influence of microtubules on wall texture than could have been imagined in the electron microscopic era-the era from which most ideas on wall texture originate. This review revisits the classical literature on wall organization from the vantage point of current knowledge of microtubule dynamics.

  20. Biomolecular motor modulates mechanical property of microtubule.

    PubMed

    Kabir, Arif Md Rashedul; Inoue, Daisuke; Hamano, Yoshimi; Mayama, Hiroyuki; Sada, Kazuki; Kakugo, Akira

    2014-05-12

    The microtubule (MT) is the stiffest cytoskeletal filamentous protein that takes part in a wide range of cellular activities where its mechanical property plays a crucially significant role. How a single biological entity plays multiple roles in cell has been a mystery for long time. Over the recent years, it has been known that modulation of the mechanical property of MT by different cellular agents is the key to performing manifold in vivo activities by MT. Studying the mechanical property of MT thus has been a prerequisite in understanding how MT plays such diversified in vivo roles. However, the anisotropic structure of MT has been an impediment in obtaining a precise description of the mechanical property of MT along its longitudinal and lateral directions that requires employment of distinct experimental approach and has not been demonstrated yet. In this work, we have developed an experimental system that enabled us to investigate the effect of tensile stress on MT. By using our newly developed system, (1) we have determined the Young's modulus of MT considering its deformation under applied tensile stress and (2) a new role of MT associated motor protein kinesin in modulating the mechanical property of MT was revealed for the first time. Decrease in Young's modulus of MT with the increase in interaction with kinesin suggests that kinesin has a softening effect on MT and thereby can modulate the rigidity of MT. This work will be an aid in understanding the modulation of mechanical property of MTs by MT associated proteins and might also help obtain a clear insight of the endurance and mechanical instability of MTs under applied stress.

  1. Microtubule guidance tested through controlled cell geometry

    PubMed Central

    Huda, Sabil; Soh, Siowling; Pilans, Didzis; Byrska-Bishop, Marta; Kim, Jiwon; Wilk, Gary; Borisy, Gary G.; Kandere-Grzybowska, Kristiana; Grzybowski, Bartosz A.

    2012-01-01

    Summary In moving cells dynamic microtubules (MTs) target and disassemble substrate adhesion sites (focal adhesions; FAs) in a process that enables the cell to detach from the substrate and propel itself forward. The short-range interactions between FAs and MT plus ends have been observed in several experimental systems, but the spatial overlap of these structures within the cell has precluded analysis of the putative long-range mechanisms by which MTs growing through the cell body reach FAs in the periphery of the cell. In the work described here cell geometry was controlled to remove the spatial overlap of cellular structures thus allowing for unambiguous observation of MT guidance. Specifically, micropatterning of living cells was combined with high-resolution in-cell imaging and gene product depletion by means of RNA interference to study the long-range MT guidance in quantitative detail. Cells were confined on adhesive triangular microislands that determined cell shape and ensured that FAs localized exclusively at the vertices of the triangular cells. It is shown that initial MT nucleation at the centrosome is random in direction, while the alignment of MT trajectories with the targets (i.e. FAs at vertices) increases with an increasing distance from the centrosome, indicating that MT growth is a non-random, guided process. The guided MT growth is dependent on the presence of FAs at the vertices. The depletion of either myosin IIA or myosin IIB results in depletion of F-actin bundles and spatially unguided MT growth. Taken together our findings provide quantitative evidence of a role for long-range MT guidance in MT targeting of FAs. PMID:22992457

  2. Amyloid-beta peptide binds to microtubule-associated protein 1B (MAP1B).

    PubMed

    Gevorkian, Goar; Gonzalez-Noriega, Alfonso; Acero, Gonzalo; Ordoñez, Jorge; Michalak, Colette; Munguia, Maria Elena; Govezensky, Tzipe; Cribbs, David H; Manoutcharian, Karen

    2008-05-01

    Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer's disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer's disease.

  3. Arrangement of high molecular weight associated proteins on purified mammalian brain microtubules

    PubMed Central

    1977-01-01

    The arrangement of the high molecular weight proteins associated with the walls of reconstituted mammalian brain microtubules has been investigated by electron microscopy of negatively stained preparations. The images are found to be consistent with an arrangement whereby the high molecular weight molecules are spaced 12 tubulin dimers apart, i.e., 960 A, along each protofilament of the microtubule, in agreement with the relative stoichiometry of tubulin and high molecular weight protein. Molecules on neighbouring protofilaments seem to be staggered so that they give rise to a helical superlattice, which can be superimposed on the underlying tubulin lattice. In micrographs of disintegrating tubules there is some indication of lateral interactions between neighbouring high molecular weight molecules. When the microtubules are depolymerized into a mixture of short spirals and rings, the high molecular weight proteins appear to remain attached to their respective protofilaments. PMID:65355

  4. Titanium dioxide nanoparticles alter cellular morphology via disturbing the microtubule dynamics

    NASA Astrophysics Data System (ADS)

    Mao, Zhilei; Xu, Bo; Ji, Xiaoli; Zhou, Kun; Zhang, Xuemei; Chen, Minjian; Han, Xiumei; Tang, Qiusha; Wang, Xinru; Xia, Yankai

    2015-04-01

    Titanium dioxide (TiO2) nanoparticles (NPs) have been widely used in our daily lives, for example, in the areas of sunscreens, cosmetics, toothpastes, food products, and nanomedical reagents. Recently, increasing concern has been raised about their neurotoxicity, but the mechanisms underlying such toxic effects are still unknown. In this work, we employed a human neuroblastoma cell line (SH-SY5Y) to study the effects of TiO2 NPs on neurological systems. Our results showed that TiO2 NPs did not affect cell viability but induced noticeable morphological changes until 100 μg ml-1. Immunofluorescence detection showed disorder, disruption, retraction, and decreased intensity of the microtubules after TiO2 NPs treatment. Both α and β tubule expressions did not change in the TiO2 NP-treated group, but the percentage of soluble tubules was increased. A microtubule dynamic study in living cells indicated that TiO2 NPs caused a lower growth rate and a higher shortening rate of microtubules as well as shortened lifetimes of de novo microtubules. TiO2 NPs did not cause changes in the expression and phosphorylation state of tau proteins, but a tau-TiO2 NP interaction was observed. TiO2 NPs could interact with tubule heterodimers, microtubules and tau proteins, which led to the instability of microtubules, thus contributing to the neurotoxicity of TiO2 NPs.Titanium dioxide (TiO2) nanoparticles (NPs) have been widely used in our daily lives, for example, in the areas of sunscreens, cosmetics, toothpastes, food products, and nanomedical reagents. Recently, increasing concern has been raised about their neurotoxicity, but the mechanisms underlying such toxic effects are still unknown. In this work, we employed a human neuroblastoma cell line (SH-SY5Y) to study the effects of TiO2 NPs on neurological systems. Our results showed that TiO2 NPs did not affect cell viability but induced noticeable morphological changes until 100 μg ml-1. Immunofluorescence detection showed disorder

  5. AMYLOID-β PEPTIDE BINDS TO MICROTUBULE-ASSOCIATED PROTEIN 1B (MAP1B)

    PubMed Central

    Gevorkian, Goar; Gonzalez-Noriega, Alfonso; Acero, Gonzalo; Ordoñez, Jorge; Michalak, Colette; Munguia, Maria Elena; Govezensky, Tzipe; Cribbs, David H.; Manoutcharian, Karen

    2008-01-01

    Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer’s disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer’s disease. PMID:18079022

  6. Mutations in TRAF3IP1/IFT54 reveal a new role for IFT proteins in microtubule stabilization.

    PubMed

    Bizet, Albane A; Becker-Heck, Anita; Ryan, Rebecca; Weber, Kristina; Filhol, Emilie; Krug, Pauline; Halbritter, Jan; Delous, Marion; Lasbennes, Marie-Christine; Linghu, Bolan; Oakeley, Edward J; Zarhrate, Mohammed; Nitschké, Patrick; Garfa-Traore, Meriem; Serluca, Fabrizio; Yang, Fan; Bouwmeester, Tewis; Pinson, Lucile; Cassuto, Elisabeth; Dubot, Philippe; Elshakhs, Neveen A Soliman; Sahel, José A; Salomon, Rémi; Drummond, Iain A; Gubler, Marie-Claire; Antignac, Corinne; Chibout, Salahdine; Szustakowski, Joseph D; Hildebrandt, Friedhelm; Lorentzen, Esben; Sailer, Andreas W; Benmerah, Alexandre; Saint-Mezard, Pierre; Saunier, Sophie

    2015-10-21

    Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies.

  7. Microtubule-binding protein doublecortin-like kinase 1 (DCLK1) guides kinesin-3-mediated cargo transport to dendrites.

    PubMed

    Lipka, Joanna; Kapitein, Lukas C; Jaworski, Jacek; Hoogenraad, Casper C

    2016-02-01

    In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule-binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin-3 (KIF1) and kinesin-4 (KIF21) subfamily that can also target dendrites. We found that microtubule-binding protein doublecortin-like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1-dependent dense-core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule-binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.

  8. The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme.

    PubMed

    Alford, Lea M; Stoddard, Daniel; Li, Jennifer H; Hunter, Emily L; Tritschler, Douglas; Bower, Raqual; Nicastro, Daniela; Porter, Mary E; Sale, Winfield S

    2016-06-01

    We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of ∼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc.

  9. Fluorescent microtubules break up under illumination

    PubMed Central

    1988-01-01

    We have synthesized three new fluorescent analogues of tubulin, using fluorescein or rhodamine groups attached to N-hydroxy-succinimidyl esters, and have partially characterized the properties of these analogues. We have also further characterized the tubulin derivatized with dichlorotriazinyl-aminofluorescein that has previously been used in this and other laboratories. Our results show that all four analogues assemble into microtubules which break up when exposed to light of the wavelengths that excite fluorescence. This sensitivity places severe constraints on the use of these analogues in studies of microtubule dynamics. PMID:3417772

  10. Colloidal Stabilization of Neurofilaments and Microtubules

    DTIC Science & Technology

    2002-06-01

    Alzheimer’s disease . To address this problem we used a set of biophysical methods, including atomic force microscopy, to investigate interfilament potentials. There are several main conclusions from the work under this award. First, microtubule associated proteins behave as though they are largely unstructured and can give rise to a long range repulsive force that is predominantly entropic in origin. This is an important finding that provides a biophysical mechanism that explains how microtubule spacing is maintained. Second, treating the unstructured proteins domains

  11. Molecular Modeling Approaches to Study the Binding Mode on Tubulin of Microtubule Destabilizing and Stabilizing Agents

    NASA Astrophysics Data System (ADS)

    Botta, Maurizio; Forli, Stefano; Magnani, Matteo; Manetti, Fabrizio

    Tubulin targeting agents constitute an important class of anticancer drugs. By acting either as microtubule stabilizers or destabilizers, they disrupt microtubule dynamics, thus inducing mitotic arrest and, ultimately, cell death by apoptosis. Three different binding sites, whose exact location on tubulin has been experimentally detected, have been identified so far for antimitotic compound targeting microtubules, namely the taxoid, the colchicine and the vinka alkaloid binding site. A number of ligand- and structure-based molecular modeling studies in this field has been reported over the years, aimed at elucidating the binding modes of both stabilizing and destabilizing agent, as well as the molecular features responsible for their efficacious interaction with tubulin. Such studies are described in this review, focusing on information provided by different modeling approaches on the structural determinants of antitubulin agents and the interactions with the binding pockets on tubulin emerged as fundamental for antitumor activity.To describe molecular modeling approaches applied to date to molecules known to bind microtubules, this paper has been divided into two main parts: microtubule destabilizing (Part 1) and stabilizing (Part 2) agents. The first part includes structure-based and ligand-based approaches to study molecules targeting colchicine (1.1) and vinca alkaloid (1.2) binding sites, respectively. In the second part, the studies performed on microtubule-stabilizing antimitotic agents (MSAA) are described. Starting from the first representative compound of this class, paclitaxel, molecular modeling studies (quantitative structure-activity relationships - QSAR - and structure-based approaches), performed on natural compounds acting with the same mechanism of action and temptative common pharmacophoric hypotheses for all of these compounds, are reported.

  12. GDP-tubulin incorporation into growing microtubules modulates polymer stability.

    PubMed

    Valiron, Odile; Arnal, Isabelle; Caudron, Nicolas; Job, Didier

    2010-06-04

    Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule "structural plasticity."

  13. Microtubules and the endoplasmic reticulum are highly interdependent structures

    PubMed Central

    1986-01-01

    The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system. PMID:3533956

  14. Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau

    PubMed Central

    1994-01-01

    Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter- repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site- directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult- specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons. PMID:8120098

  15. Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau.

    PubMed

    Goode, B L; Feinstein, S C

    1994-03-01

    Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter-repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site-directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult-specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons.

  16. Probing the inert doublet dark matter model with Cherenkov telescopes

    SciTech Connect

    Garcia-Cely, Camilo; Gustafsson, Michael; Ibarra, Alejandro E-mail: michael.gustafsson@theorie.physik.uni-goettingen.de

    2016-02-01

    We present a detailed study of the annihilation signals of the inert dark matter doublet model in its high mass regime. Concretely, we study the prospects to observe gamma-ray signals of the model in current and projected Cherenkov telescopes taking into account the Sommerfeld effect and including the contribution to the spectrum from gamma-ray lines as well as from internal bremsstrahlung. We show that present observations of the galactic center by the H.E.S.S. instrument are able to exclude regions of the parameter space that give the correct dark matter relic abundance. In particular, models with the charged and the neutral components of the inert doublet nearly degenerate in mass have strong gamma-ray signals. Furthermore, for dark matter particle masses above 1 TeV, we find that the non-observation of the continuum of photons generated by the hadronization of the annihilation products typically give stronger constraints on the model parameters than the sharp spectral features associated to annihilation into monochromatic photons and the internal bremsstrahlung process. Lastly, we also analyze the interplay between indirect and direct detection searches for this model, concluding that the prospects for the former are more promising. In particular, we find that the upcoming Cherenkov Telescope Array will be able to probe a significant part of the high mass regime of the model.

  17. The CTA aims at the Inert Doublet Model

    SciTech Connect

    Queiroz, Farinaldo S.; Yaguna, Carlos E. E-mail: carlos.yaguna@mpi-hd.mpg.de

    2016-02-01

    We show that the Cherenkov Telescope Array (CTA) can realistically challenge the Inert Doublet Model, one of the simplest and best known models of dark matter. Specifically, the CTA may exclude its heavy regime up to dark matter masses of 800 GeV and probe a large fraction of the remaining viable parameter space at even higher masses. Two features of the Inert Doublet Model make it particularly suitable for CTA searches. First, the dark matter mass (in the heavy regime) must be larger than 500 GeV. Second, the dark matter annihilation cross section, σ v, is always larger than the thermal one, reaching values as high as 10{sup −25} cm{sup 3}s{sup −1}. This higher value of σv is the result of the unavoidable coannihilation effects that determine the relic density via thermal freeze-out in the early Universe. We find that with 100 hours of Galactic Center exposure, CTA's expected limit widely surpasses, even after the inclusion of systematic errors, current and projected bounds from Fermi-LAT and HESS on this model.

  18. Next-to-minimal two Higgs Doublet Model

    DOE PAGES

    Chen, Chien -Yi; Freid, Michael; Sher, Marc

    2014-04-07

    The simplest extension of the Two Higgs Doublet Model is the addition of a real scalar singlet, S. The effects of mixing between the singlet and the doublets can be manifested in two ways. It can modify the couplings of the 126 GeV Higgs boson, h, and it can lead to direct detection of the heavy Higgs at the LHC. In this paper, we show that in the type-I Model, for heavy Higgs masses in the 200-600 GeV range, the latter effect will be detected earlier than the former for most of parameter space. Should no such Higgs be discoveredmore » in this mass range, then the upper limit on the mixing will be sufficiently strong such that there will be no significant effects on the couplings of the h for most of parameter space. Thus, the reverse is true in the type-II model, the limits from measurements of the couplings of the h will dominate over the limits from non-observation of the heavy Higgs.« less

  19. Next-to-minimal two Higgs Doublet Model

    SciTech Connect

    Chen, Chien -Yi; Freid, Michael; Sher, Marc

    2014-04-07

    The simplest extension of the Two Higgs Doublet Model is the addition of a real scalar singlet, S. The effects of mixing between the singlet and the doublets can be manifested in two ways. It can modify the couplings of the 126 GeV Higgs boson, h, and it can lead to direct detection of the heavy Higgs at the LHC. In this paper, we show that in the type-I Model, for heavy Higgs masses in the 200-600 GeV range, the latter effect will be detected earlier than the former for most of parameter space. Should no such Higgs be discovered in this mass range, then the upper limit on the mixing will be sufficiently strong such that there will be no significant effects on the couplings of the h for most of parameter space. Thus, the reverse is true in the type-II model, the limits from measurements of the couplings of the h will dominate over the limits from non-observation of the heavy Higgs.

  20. The effect of human microtubule-associated-protein tau on the assembly structure of microtubules and its ionic strength dependence

    NASA Astrophysics Data System (ADS)

    Choi, M. C.; Raviv, U.; Miller, H. P.; Gaylord, M. R.; Kiris, E.; Ventimiglia, D.; Needleman, D. J.; Chung, P. J.; Deek, J.; Lapointe, N.; Kim, M. W.; Wilson, L.; Feinstein, S. C.; Safinya, C. R.

    2010-03-01

    Microtubules (MTs), 25 nm protein nanotubes, are among the major filamentous elements of the eukaryotic cytoskeleton involved in intracellular trafficking, cell division and the establishment and maintenance of cell shape. Microtubule-associated-protein tau regulates tubulin assembly, MT dynamics and stability. Aberrant tau action has long been correlated with numerous neurodegenerative diseases, including Alzheimer's, and fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) Using synchrotron small angle x-ray scattering (SAXS) and binding assay, we examine the effects of tau on the assembly structure of taxol-stabilized MTs. We find that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius of MTs with increasing the tau/tubulin molar ratio. Additionally, tau-MT interactions are mediated to a large extent via electrostatic interactions: the binding affinity of tau to MTs is ionic strength dependent. Supported by DOE-BES DE-FG02-06ER46314, NSF DMR-0803103, NIH NS35010, NIH NS13560. (Ref) M.C. Choi, S.C. Feinstein, and C.R. Safinya et al. Biophys. J. 97; 519 (2009).

  1. Vesicle deformation by microtubules: A phase diagram

    NASA Astrophysics Data System (ADS)

    Emsellem, Virginie; Cardoso, Olivier; Tabeling, Patrick

    1998-10-01

    The experimental investigation of vesicles deformed by the growth of encapsulated microtubules shows that the axisymmetric morphologies can be classified into ovals, lemons, φ, cherries, dumbbells, and pearls. A geometrical phase diagram is established. Numerical minimization of the elastic energy of the membrane reproduces satisfactorily well the observed morphologies and the corresponding phase diagram.

  2. Microtubule Initiation from the Nuclear Surface Controls Cortical Microtubule Growth Polarity and Orientation in Arabidopsis thaliana

    PubMed Central

    Ambrose, Chris; Wasteneys, Geoffrey O.

    2014-01-01

    The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth. PMID:25008974

  3. Sliding of STOP proteins on microtubules: a model system for diffusion-dependent microtubule motility.

    PubMed

    Margolis, R L; Job, D; Pabion, M; Rauch, C T

    1986-01-01

    STOP proteins, of 145 kD, act substoichiometrically to block end-wise disassembly of microtubules. STOPs bind to microtubules either during microtubule assembly or when added at steady state, and when binding to the polymers is apparently irreversible. They are not measurably lost from polymers under competition conditions, and there is no measurable exchange between polymers. Nonetheless, STOP proteins exhibit an extraordinary behavior: they "slide" laterally on the surface of the microtubule. Displacement is assayed by forming hybrid microtubules in which cold stable or cold labile region subunits are labeled. Displacement of STOPs on the polymer with time will cause labeled subunits of cold-stable regions to become increasingly cold labile in a manner reciprocal to cold stabilization of previously cold-labile subunits. Because equilibrium exchange of STOP proteins onto and off the polymers can be ruled out, the displacement of STOPs relative to subunits can only be explained by lateral diffusion or "sliding." Axonal transport and mitotic mechanisms were discussed as implications of such a lateral translocation mechanism for microtubule-dependent motility.

  4. Doublet Production in the Development of Medieval and Modern Spanish: New Approaches to Phonolexical Duplication

    ERIC Educational Resources Information Center

    Haney, Darren W.

    2011-01-01

    This dissertation offers new approaches to an old and well-known problem in the study of the development of Romance varieties: duplicate lexis or doublets. Traditional analyses of duplication are narrow in scope both in what qualifies as a doublet (the popular/learned opposition has dominated, to the exclusion of other pairs) and in channels of…

  5. Organelles are transported on sliding microtubules in Reticulomyxa.

    PubMed

    Orokos, D D; Cole, R W; Travis, J L

    2000-12-01

    Organelles and plasma membrane domains appear to be transported along Reticulomyxa's microtubule cytoskeleton. Previously we demonstrated that organelle and cell surface transport share the same enzymatic properties and suggested that both are powered by the same cytoplasmic dynein. Motility analysis in Reticulomyxa is complicated by the fact that the microtubules also are motile and appear to "slide" bidirectionally throughout the network. We have utilized laser ablation to address this frame-of-reference problem as to how each transport component (microtubule sliding vs. organelle translocations) contributes to reactivated bidirectional translocation of organelles along the microtubule cytoskeleton. Laser ablation was used to cut microtubule bundles from lysed networks into 4-15-microm segments. After examining these reactivated cut fragments, it appears that the majority of organelles did not move relative to microtubule fragments, but remained attached to microtubules and moved as the microtubules slid. Microtubule sliding stops after 1-2 min and cannot be reactivated even when perfused with fresh ATP. Furthermore, once sliding stops, organelle transport also stops. Our findings indicate that the majority of Reticulomyxa pseudopodial organelles do not move along the surface of the microtubules, rather it is the sliding of the microtubules to which they are attached that moves them.

  6. Microtubule Dynamics Control Tail Retraction in Migrating Vascular Endothelial Cells†

    PubMed Central

    Ganguly, Anutosh; Yang, Hailing; Zhang, Hong; Cabral, Fernando; Patel, Kamala D.

    2014-01-01

    Drugs that target microtubules are potent inhibitors of angiogenesis but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; but, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish rather than elongated cells, rapid but non-directional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells while more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay. PMID:24107446

  7. Loop formation of microtubules during gliding at high density

    NASA Astrophysics Data System (ADS)

    Liu, Lynn; Tüzel, Erkan; Ross, Jennifer L.

    2011-09-01

    The microtubule cytoskeleton, including the associated proteins, forms a complex network essential to multiple cellular processes. Microtubule-associated motor proteins, such as kinesin-1, travel on microtubules to transport membrane bound vesicles across the crowded cell. Other motors, such as cytoplasmic dynein and kinesin-5, are used to organize the cytoskeleton during mitosis. In order to understand the self-organization processes of motors on microtubules, we performed filament-gliding assays with kinesin-1 motors bound to the cover glass with a high density of microtubules on the surface. To observe microtubule organization, 3% of the microtubules were fluorescently labeled to serve as tracers. We find that microtubules in these assays are not confined to two dimensions and can cross one other. This causes microtubules to align locally with a relatively short correlation length. At high density, this local alignment is enough to create 'intersections' of perpendicularly oriented groups of microtubules. These intersections create vortices that cause microtubules to form loops. We characterize the radius of curvature and time duration of the loops. These different behaviors give insight into how crowded conditions, such as those in the cell, might affect motor behavior and cytoskeleton organization.

  8. Self-organisation and forces in the microtubule cytoskeleton.

    PubMed

    Nédélec, François; Surrey, Thomas; Karsenti, Eric

    2003-02-01

    Modern microscopy techniques allow us to observe specifically tagged proteins in live cells. We can now see directly that many cellular structures, for example mitotic spindles, are in fact dynamic assemblies. Their apparent stability results from out-of-equilibrium stochastic interactions at the molecular level. Recent studies have shown that the spindles can form even after centrosomes are destroyed, and that they can even form around DNA-coated beads devoid of kinetochores. Moreover, conditions have been produced in which microtubule asters interact even in the absence of chromatin. Together, these observations suggest that the spindle can be experimentally deconstructed, and that its defining characteristics can be studied in a simplified context, in the absence of the full division machinery.

  9. Directional loading of the kinesin motor molecule as it buckles a microtubule.

    PubMed Central

    Gittes, F; Meyhöfer, E; Baek, S; Howard, J

    1996-01-01

    Single kinesin motor molecules were observed to buckle the microtubules along which they moved in a modified in vitro gliding assay. In this assay a central portion of the microtubule was clamped to the glass substrate via biotin-streptavidin bonds, while the plus end of the microtubule was free to interact with motors adsorbed at low density to the substrate. A statistical analysis of the length of microtubules buckled by single motors showed a decreasing probability of buckling for loads greater than 4-6 pN parallel to the filament. This is consistent with kinesin stalling forces found in other experiments. A detailed analysis of some buckling events allowed us to estimate both the magnitude and direction of the loading force as it developed a perpendicular component tending to pull the motor away from the microtubule. We also estimated the motor speed as a function of this changing vector force. The kinesin motors consistently reached unexpectedly high speeds as the force became nonparallel to the direction of motor movement. Our results suggest that a perpendicular component of load does not hinder the kinesin motor, but on the contrary causes the motor to move faster against a given parallel load. Because the perpendicular force component speeds up the motor but does no net work, perpendicular force acts as a mechanical catalyst for the reaction. A simple explanation is that there is a spatial motion of the kinesin molecule during its cycle that is rate-limiting under load; mechanical catalysis results if this motion is oriented away from the surface of the microtubule. Images FIGURE 2 PMID:8770218

  10. Dual Role for Microtubules in Regulating Cortical Contractility during Cytokinesis

    PubMed Central

    Murthy, Kausalya; Wadsworth, Patricia

    2008-01-01

    Microtubules stimulate contractile ring formation in the equatorial cortex and simultaneously suppress contractility in the polar cortex; how they accomplish these differing activities is incompletely understood. We measured the behavior of GFP-actin in mammalian cells treated with nocodazole under conditions that either completely eliminate microtubules or selectively disassemble astral microtubules. Selective disassembly of astral microtubules resulted functional contractile rings that were wider than controls and had altered dynamic activity, as measured by FRAP. Complete microtubule disassembly or selective loss of astral microtubules resulted in wave-like contractile behavior of actin in the non-equatorial cortex and mislocalization of myosin II and Rho. FRAP experiments showed that both contractility and actin polymerization contributed to the wave-like behavior of actin. Wave-like, contractile behavior in anaphase cells was Rho-dependent. We conclude that dynamic astral microtubules function to suppress Rho activation in the nonequatorial cortex, limiting the contractile activity of the polar cortex. PMID:18559890

  11. Mathematical modeling of microtubule dynamics: insights into physiology and disease.

    PubMed

    Buxton, Gavin A; Siedlak, Sandra L; Perry, George; Smith, Mark A

    2010-12-01

    Computer models of microtubule dynamics have provided the basis for many of the theories on the cellular mechanics of the microtubules, their polymerization kinetics, and the diffusion of tubulin and tau. In the three-dimensional model presented here, we include the effects of tau concentration and the hydrolysis of GTP-tubulin to GDP-tubulin and observe the emergence of microtubule dynamic instability. This integrated approach simulates the essential physics of microtubule dynamics in a cellular environment. The model captures the structure of the microtubules as they undergo steady state dynamic instabilities in this simplified geometry, and also yields the average number, length, and cap size of the microtubules. The model achieves realistic geometries and simulates cellular structures found in degenerating neurons in disease states such as Alzheimer disease. Further, this model can be used to simulate microtubule changes following the addition of antimitotic drugs which have recently attracted attention as chemotherapeutic agents.

  12. Modelling the role of catastrophe, crossover and katanin-mediated severing in the self-organisation of plant cortical microtubules.

    PubMed

    Mace, Alex; Wang, Wenjia

    2015-12-01

    Plant cortical microtubules can form ordered arrays through interactions among themselves. When an incident microtubule collides with a barrier microtubule it may entrain if below a certain angle. Else it undergoes collision induced catastrophe (CIC) or crosses over the barrier microtubule. It has been proposed that katanin is necessary to create order by severing these crossover sites. The authors present a three-state computational model using Arabidopsis thaliana data to show how spontaneous catastrophe, the probability of CIC versus crossover, and katanin-mediated severing at the crossover sites affect microtubule ordering. The results of the systematic simulations show that (1), the microtubule order is more sensitive to the catastrophe rate than the rescue rate; (2), at 21°C, peak order is observed at 0.3 CIC and order decreases as CIC increases; and (3) at 0.2 CIC, katanin severing acting uniformly at all crossover sites is able to create order within a biologically reasonable time frame, but at lower CICs this becomes unrealistically fast. This would imply that at lower CIC levels preferential crossover site targeting and severing activity regulators would be required for katanin to bring about order.

  13. Monoclonal antibodies to kinesin heavy and light chains stain vesicle- like structures, but not microtubules, in cultured cells

    PubMed Central

    1989-01-01

    Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane- bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface. PMID:2522455

  14. The kinesin-2 family member KIF3C regulates microtubule dynamics and is required for axon growth and regeneration.

    PubMed

    Gumy, Laura F; Chew, Daniel J; Tortosa, Elena; Katrukha, Eugene A; Kapitein, Lukas C; Tolkovsky, Aviva M; Hoogenraad, Casper C; Fawcett, James W

    2013-07-10

    Axon regeneration after injury requires the extensive reconstruction, reorganization, and stabilization of the microtubule cytoskeleton in the growth cones. Here, we identify KIF3C as a key regulator of axonal growth and regeneration by controlling microtubule dynamics and organization in the growth cone. KIF3C is developmentally regulated. Rat embryonic sensory axons and growth cones contain undetectable levels of KIF3C protein that is locally translated immediately after injury. In adult neurons, KIF3C is axonally transported from the cell body and is enriched at the growth cone where it preferentially binds to tyrosinated microtubules. Functionally, the interaction of KIF3C with EB3 is necessary for its localization at the microtubule plus-ends in the growth cone. Depletion of KIF3C in adult neurons leads to an increase in stable, overgrown and looped microtubules because of a strong decrease in the microtubule frequency of catastrophes, suggesting that KIF3C functions as a microtubule-destabilizing factor. Adult axons lacking KIF3C, by RNA interference or KIF3C gene knock-out, display an impaired axonal outgrowth in vitro and a delayed regeneration after injury both in vitro and in vivo. Murine KIF3C knock-out embryonic axons grow normally but do not regenerate after injury because they are unable to locally translate KIF3C. These data show that KIF3C is an injury-specific kinesin that contributes to axon growth and regeneration by regulating and organizing the microtubule cytoskeleton in the growth cone.

  15. Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells.

    PubMed

    Ory, Eleanor; Bhandary, Lekhana; Boggs, Amanda; Chakrabarti, Kristi; Parker, Joshua; Losert, Wolfgang; Martin, Stuart S

    2017-01-16

    The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems - growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

  16. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells

    PubMed Central

    1990-01-01

    During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule- nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the

  17. Effects of organotin compounds on mitosis, spindle structure, toxicity and in vitro microtubule assembly.

    PubMed

    Jensen, K G; Onfelt, A; Wallin, M; Lidums, V; Andersen, O

    1991-09-01

    Di- and tri-methyl, -butyl and phenyl tin, all as chlorides were tested for toxicity and spindle disturbances in V79 Chinese hamster cells and for effects on in vitro assembly of bovine brain tubulin. The V79 cells were treated for 30 min and in general, loss of a stainable spindle could be demonstrated at slightly higher concentrations than c-mitosis. Both these effects were observed at low, non-toxic concentrations. The c-mitotic activity of the compounds was found to increase with increasing lipophilicity and it was best described by a regression on both lipophilicity (partition coefficient octanol/water) and loss of spindle stain. All compounds showed a concentration dependent inhibition of microtubule assembly and all but diphenyltin induced disassembly of preassembled microtubules. An effect on the rate of polymerization was suggested for tributyl- and triphenyltin. The results further indicate that the inhibition of microtubule assembly is through direct interaction with tubulin but does not involve the sulfhydryls of the protein. Thus, the organotins seem to act through two different cooperative mechanisms, inhibition of microtubule assembly and interaction with hydrophobic sites. The latter mechanism might involve Cl-/OH- exchange across cellular membranes. Previous studies have demonstrated chromosomal supercontraction and aneuploidy in human lymphocytes exposed to low concentrations of organotin in vitro and it is suggested that exposure to these compounds may increase the risk of aneuploidy in humans.

  18. Characterizing and engineering microtubule properties for use in hybrid nanodevices

    NASA Astrophysics Data System (ADS)

    Jeune-Smith, Yolaine

    The emergence of nanotechnology in materials science research has had a major impact in biotechnology. Nature provides novel materials and structures that can be redesigned and reassembled for engineering purposes. One system in particular is the intracellular transport system consisting of the kinesin motor protein and microtubule. For synthetic devices, either the bead geometry (kinesin motors walking along a microtubule coated surface) or the gliding geometry (microtubules gliding over a kinesin-coated surface) is used. Molecular shuttles, utilizing the gliding geometry, have the potential for use in hybrid nanodevices such as biosensors. The kinesin-powered molecular shuttle has been extensively studied. Advances have been made in controlling activation of the kinesin motors, guiding movement of kinesin motors and cargo loading onto the molecular shuttles. In this dissertation the interest in molecular shuttle development is extended with a research focus on the microtubule filament. The microtubule is a central element in the molecular shuttle. The sensing capabilities and limitations of molecular shuttles are tied to the microtubules. It would be desired to have nanodevices with molecular shuttles of predictable size, speed and lifetime. Three materials properties of the microtubules are examined. First, the microtubule length distribution is measured and compared to the length distribution of synthetic polymers. Post polymerization processing techniques, shearing and annealing, are utilized to try to reduce the polydispersity index of the microtubule length distribution. Second, the effect of kinesin activity on the lifetime of the microtubules is observed and quantified. Degradation of microtubules is monitored as a function of kinesin activity and time. Lastly, the effect of cargo loading on microtubule gliding speed is measured to gain insight on the mechanism of cargo attachment. These property behaviors will play a role in the final development of

  19. Active doublet method for measuring small changes in physical properties

    DOEpatents

    Roberts, Peter M.; Fehler, Michael C.; Johnson, Paul A.; Phillips, W. Scott

    1994-01-01

    Small changes in material properties of a work piece are detected by measuring small changes in elastic wave velocity and attenuation within a work piece. Active, repeatable source generate coda wave responses from a work piece, where the coda wave responses are temporally displaced. By analyzing progressive relative phase and amplitude changes between the coda wave responses as a function of elapsed time, accurate determinations of velocity and attenuation changes are made. Thus, a small change in velocity occurring within a sample region during the time periods between excitation origin times (herein called "doublets") will produce a relative delay that changes with elapsed time over some portion of the scattered waves. This trend of changing delay is easier to detect than an isolated delay based on a single arrival and provides a direct measure of elastic wave velocity changes arising from changed material properties of the work piece.

  20. A two-Higgs-doublet model facing experimental hints

    NASA Astrophysics Data System (ADS)

    Crivellin, Andreas; Heeck, Julian; Stoffer, Peter

    2016-04-01

    Physics beyond the Standard Model has so far eluded our experimental probes. Nevertheless, a number of interesting anomalies have accumulated that can be taken as hints towards new physics: BaBar, Belle, and LHCb have found deviations of approximately 3:8σ in B → Dτν and B → D*τν; the anomalous magnetic moment of the muon differs by about 3σ from the theoretic prediction; the branching ratio for τ → μνν is about 2σ above the Standard Model expectation; and CMS and ATLAS found hints for a non-zero decay rate of h → μτ at 2.6σ. Here we consider these processes within a lepton-specific two-Higgs doublet model with additional non-standard Yukawa couplings and show how (and which of) these excesses can be accommodated.

  1. The electroweak phase transition in the Inert Doublet Model

    SciTech Connect

    Blinov, Nikita; Profumo, Stefano; Stefaniak, Tim

    2015-07-21

    We study the strength of a first-order electroweak phase transition in the Inert Doublet Model (IDM), where particle dark matter (DM) is comprised of the lightest neutral inert Higgs boson. We improve over previous studies in the description and treatment of the finite-temperature effective potential and of the electroweak phase transition. We focus on a set of benchmark models inspired by the key mechanisms in the IDM leading to a viable dark matter particle candidate, and illustrate how to enhance the strength of the electroweak phase transition by adjusting the masses of the yet undiscovered IDM Higgs states. We argue that across a variety of DM masses, obtaining a strong enough first-order phase transition is a generic possibility in the IDM. We find that due to direct dark matter searches and collider constraints, a sufficiently strong transition and a thermal relic density matching the universal DM abundance is possible only in the Higgs funnel regime.

  2. The electroweak phase transition in the Inert Doublet Model

    SciTech Connect

    Blinov, Nikita; Profumo, Stefano; Stefaniak, Tim E-mail: profumo@ucsc.edu

    2015-07-01

    We study the strength of a first-order electroweak phase transition in the Inert Doublet Model (IDM), where particle dark matter (DM) is comprised of the lightest neutral inert Higgs boson. We improve over previous studies in the description and treatment of the finite-temperature effective potential and of the electroweak phase transition. We focus on a set of benchmark models inspired by the key mechanisms in the IDM leading to a viable dark matter particle candidate, and illustrate how to enhance the strength of the electroweak phase transition by adjusting the masses of the yet undiscovered IDM Higgs states. We argue that across a variety of DM masses, obtaining a strong enough first-order phase transition is a generic possibility in the IDM. We find that due to direct dark matter searches and collider constraints, a sufficiently strong transition and a thermal relic density matching the universal DM abundance is possible only in the Higgs funnel regime.

  3. Parity-doublet representation of Majorana fermions and neutron oscillation

    NASA Astrophysics Data System (ADS)

    Fujikawa, Kazuo; Tureanu, Anca

    2016-12-01

    We present a parity-doublet theorem for the representation of the intrinsic parity of Majorana fermions, which is expected to be useful also in condensed matter physics, and it is illustrated to provide a criterion of neutron-antineutron oscillation in a Bardeen-Cooper-Schrieffer type of effective theory with Δ B =2 baryon number-violating terms. The C P violation in the present effective theory causes no direct C P -violating effects in the oscillation itself, which is demonstrated by the exact solution, although it influences the neutron electric dipole moment in the leading order of small Δ B =2 parameters. An analog of Bogoliubov transformation, which preserves P and C P , is crucial in the analysis.

  4. Inhibition of microtubule formation by uremic toxins: action mechanism and hypothesis about the active component.

    PubMed

    Braguer, D; Gallice, P; Monti, J P; Murisasco, A; Crevat, A

    1986-04-01

    We show in vitro inhibitory effect of a mixture of uremic toxins on tubulin 6S polymerization. It proves the existence of a direct interaction protein-toxin where micro-tubule associated proteins are not involved. A similar phenomenom could occur in uremic neuropathy. The action mechanism of this interaction is quite different from that of classical tubulin inhibitors: Vinca alcaloïdes and colchicine. Finally we hypothesize about the active molecule.

  5. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila

    PubMed Central

    Duncan, Jason E.; Lytle, Nikki K.; Zuniga, Alfredo; Goldstein, Lawrence S. B.

    2013-01-01

    Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila. The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila, which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila, we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport. PMID:23840848

  6. Spindle pole body-anchored Kar3 drives the nucleus along microtubules from another nucleus in preparation for nuclear fusion during yeast karyogamy.

    PubMed

    Gibeaux, Romain; Politi, Antonio Z; Nédélec, François; Antony, Claude; Knop, Michael

    2013-02-01

    Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule-microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion.

  7. Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts Schizosaccharomyces versatilis and Schizosaccharomyces pombe.

    PubMed

    Svoboda, A; Bähler, J; Kohli, J

    1995-11-01

    Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the

  8. Magnetic properties of a Kramers doublet. An univocal bridge between experimental results and theoretical predictions.

    PubMed

    Alonso, P J; Martínez, J I

    2015-06-01

    The magnetic response of a Kramers doublet is analyzed in a general case taking into account only the formal properties derived from time reversal operation. It leads to a definition of a matrix G (gyromagnetic matrix) whose expression depends on the chosen reference frame and on the Kramers conjugate basis used to describe the physical system. It is shown that there exists a reference frame and a suitable Kramers conjugate basis that gives a diagonal form for the G-matrix with all non-null elements having the same sign. A detailed procedure for obtaining this canonical expression of G is presented when the electronic structure of the KD is known regardless the level of the used theory. This procedure provides a univocal way to compare the theoretical predictions with the experimental results obtained from a complete set of magnetic experiments. In this way the problems arising from ambiguities in the g-tensor definition are overcome. This procedure is extended to find a spin-Hamiltonian suitable for describing the magnetic behavior of a pair of weakly coupled Kramers systems in the multispin scheme when the interaction between the two moieties as well as the individual Zeeman interaction are small enough as compared with ligand field splitting. Explicit relations between the physical interaction and the parameters of such a spin-Hamiltonian are also obtained.

  9. Role of the microtubule cytoskeleton in gravisensing Chara rhizoids.

    PubMed

    Braun, M; Sievers, A

    1994-04-01

    The arrangement of the microtubule cytoskeleton in tip-growing and gravisensing Chara rhizoids has been documented by immunofluorescence microscopy. Predominantly axially oriented undulating bundles of cortical microtubules were found in the basal zone of the rhizoids and colocalized with the microfilament bundles underlying the cytoplasmic streaming. Microtubules penetrate the subapical zone, forming a three-dimensional network that envelops the nucleus and organelles. Microtubules are present up to 5 to 10 microns basal from the apical cytoplasmic region containing the statoliths. No microtubules were found in the apical zone of the rhizoid which is the site of tip growth and gravitropism. Depolymerization of microtubules by application of oryzalin does not affect cytoplasmic streaming and gravitropic growth until the relatively stationary and polarly organized apical and subapical cytoplasm is converted into streaming cytoplasm. When the statoliths and the apical cytoplasm are included in the cytoplasmic streaming, tip growth and gravitropism are stopped. Oryzalin-induced disruption of the microtubule cytoskeleton also results in a rearrangement of the dense network of apical and subapical microfilaments into thicker bundles, whereas disruption of the microfilament cytoskeleton by cytochalasin D had no effect on the organization of the microtubule cytoskeleton. It is, therefore, concluded that the arrangement of microtubules is essential for the polar cytoplasmic zonation and the functionally polar organization of the actin cytoskeleton which is responsible for the motile processes in rhizoids. Microtubules are not involved in the primary events of gravitropism in Chara rhizoids.

  10. Neurodegeneration and microtubule dynamics: death by a thousand cuts

    PubMed Central

    Dubey, Jyoti; Ratnakaran, Neena; Koushika, Sandhya P.

    2015-01-01

    Microtubules form important cytoskeletal structures that play a role in establishing and maintaining neuronal polarity, regulating neuronal morphology, transporting cargo, and scaffolding signaling molecules to form signaling hubs. Within a neuronal cell, microtubules are found to have variable lengths and can be both stable and dynamic. Microtubule associated proteins, post-translational modifications of tubulin subunits, microtubule severing enzymes, and signaling molecules are all known to influence both stable and dynamic pools of microtubules. Microtubule dynamics, the process of interconversion between stable and dynamic pools, and the proportions of these two pools have the potential to influence a wide variety of cellular processes. Reduced microtubule stability has been observed in several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), and tauopathies like Progressive Supranuclear Palsy. Hyperstable microtubules, as seen in Hereditary Spastic Paraplegia (HSP), also lead to neurodegeneration. Therefore, the ratio of stable and dynamic microtubules is likely to be important for neuronal function and perturbation in microtubule dynamics might contribute to disease progression. PMID:26441521

  11. Size scaling of microtubule asters in confinement

    NASA Astrophysics Data System (ADS)

    Pelletier, James; Field, Christine; Krutkramelis, Kaspars; Fakhri, Nikta; Oakey, John; Gatlin, Jay; Mitchison, Timothy

    Microtubule asters are radial arrays of microtubules (MTs) nucleated around organizing centers (MTOCs). Across a wide range of cell types and sizes, aster positioning influences cellular organization. To investigate aster size and positioning, we reconstituted dynamic asters in Xenopus cytoplasmic extract, confined in fluorous oil microfluidic emulsions. In large droplets, we observed centering of MTOCs. In small droplets, we observed a breakdown in natural positioning, with MTOCs at the droplet edge and buckled or bundled MTs along the interface. In different systems, asters are positioned by different forces, such as pushing due to MT polymerization, or pulling due to bulk or cortical dynein. To estimate different contributions to aster positioning, we biochemically perturbed dynactin function, or MT or actin polymerization. We used carbon nanotubes to measure molecular motions and forces in asters. These experimental results inform quantitative biophysical models of aster size and positioning in confinement. JFP was supported by a Fannie and John Hertz Graduate Fellowship.

  12. Dynamic Concentration of Motors in Microtubule Arrays

    NASA Astrophysics Data System (ADS)

    Nédélec, François; Surrey, Thomas; Maggs, A. C.

    2001-04-01

    We present experimental and theoretical studies of the dynamics of molecular motors in microtubule arrays and asters. By solving a convection-diffusion equation we find that the density profile of motors in a two-dimensional aster is characterized by continuously varying exponents. Simulations are used to verify the assumptions of the continuum model. We observe the concentration profiles of kinesin moving in quasi-two-dimensional artificial asters by fluorescent microscopy and compare with our theoretical results.

  13. Self-organization of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Ndlec, F. J.; Surrey, T.; Maggs, A. C.; Leibler, S.

    1997-09-01

    Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly `pathways'.

  14. Molecular Communication: Simulation of Microtubule Topology

    NASA Astrophysics Data System (ADS)

    Moore, Michael J.; Enomoto, Akihiro; Nakano, Tadashi; Kayasuga, Atsushi; Kojima, Hiroaki; Sakakibara, Hitoshi; Oiwa, Kazuhiro; Suda, Tatsuya

    Molecular communication is one method for communication among biological nanomachines. Nanomachines are artificial or biological nano-scale devices that perform simple computation, sensing, or actuation. Future applications using nanomachines may require various communication mechanisms. For example, broadcast is one primitive communication for transmission from one sender to many receivers. In this paper, we discuss preliminary work on designing a molecular communication system that is adapted from the molecular motor transport mechanism existing in biological cells. In the proposed molecular motor mechanism, a sender releases information molecules, and molecular motors transport the information molecules along microtubule filaments to receiver nanomachines up to hundreds of micrometers away. This paper describes some possible arrangements for microtubule filaments and simulations to evaluate sending of one information molecule to many receivers. The simulation results indicate that the proposed molecular motor system transports simulated information molecules (100nm radius spheres) more quickly than a diffusion-only communication and that placement of receivers at the plus-end of microtubules results in lower propagation delay.

  15. Tuning microtubule-based transport via filamentous MAPs: the problem of dynein

    PubMed Central

    Vershinin, Michael; Xu, Jing; Razafsky, David S.; King, Stephen J.; Gross, Steven P.

    2010-01-01

    We recently proposed that regulating the single-to-multiple motor transition was a likely strategy for regulating kinesin-based transport in vivo. Here, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein-based transport. We show that tau’s regulation of kinesin function can proceed without interfering with dynein-based transport. Surprisingly, at extremely high tau levels—where kinesin cannot bind microtubules—dynein can still contact microtubules. The difference between tau’s effects on kinesin- and dynein-based motility suggests that tau can be used to tune relative amounts of plus-end and minus-end directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to microtubules. We show that this isoform-specific effect is not due to steric interference of tau’s projection domains, but rather due to tau’s interactions with the motor at the microtubule surface. Nonetheless, we do observe a modest steric interference effect of tau away from the microtubule and discuss the potential implications of this for molecular motor structure. PMID:18373727

  16. NuSAP modulates the dynamics of kinetochore microtubules by attenuating MCAK depolymerisation activity

    PubMed Central

    Li, Chenyu; Zhang, Yajun; Yang, Qiaoyun; Ye, Fan; Sun, Stella Ying; Chen, Ee Sin; Liou, Yih-Cherng

    2016-01-01

    Nucleolar and spindle-associated protein (NuSAP) is a microtubule-associated protein that functions as a microtubule stabiliser. Depletion of NuSAP leads to severe mitotic defects, however the mechanism by which NuSAP regulates mitosis remains elusive. In this study, we identify the microtubule depolymeriser, mitotic centromere-associated kinesin (MCAK), as a novel binding partner of NuSAP. We show that NuSAP regulates the dynamics and depolymerisation activity of MCAK. Phosphorylation of MCAK by Aurora B kinase, a component of the chromosomal passenger complex, significantly enhances the interaction of NuSAP with MCAK and modulates the effects of NuSAP on the depolymerisation activity of MCAK. Our results reveal an underlying mechanism by which NuSAP controls kinetochore microtubule dynamics spatially and temporally by modulating the depolymerisation function of MCAK in an Aurora B kinase-dependent manner. Hence, this study provides new insights into the function of NuSAP in spindle formation during mitosis. PMID:26733216

  17. Arabidopsis AUGMIN Subunit8 Is a Microtubule Plus-End Binding Protein That Promotes Microtubule Reorientation in Hypocotyls[C][W

    PubMed Central

    Cao, Lingyan; Wang, Linhai; Zheng, Min; Cao, Hong; Ding, Lian; Zhang, Xiaolan; Fu, Ying

    2013-01-01

    In plant cells, cortical microtubules provide tracks for cellulose-synthesizing enzymes and regulate cell division, growth, and morphogenesis. The role of microtubules in these essential cellular processes depends on the spatial arrangement of the microtubules. Cortical microtubules are reoriented in response to changes in cell growth status and cell shape. Therefore, an understanding of the mechanism that underlies the change in microtubule orientation will provide insight into plant cell growth and morphogenesis. This study demonstrated that AUGMIN subunit8 (AUG8) in Arabidopsis thaliana is a novel microtubule plus-end binding protein that participates in the reorientation of microtubules in hypocotyls when cell elongation slows down. AUG8 bound to the plus ends of microtubules and promoted tubulin polymerization in vitro. In vivo, AUG8 was recruited to the microtubule branch site immediately before nascent microtubules branched out. It specifically associated with the plus ends of growing cortical microtubules and regulated microtubule dynamics, which facilitated microtubule reorientation when microtubules changed their growth trajectory or encountered obstacle microtubules during microtubule reorientation. This study thus reveals a novel mechanism underlying microtubule reorientation that is critical for modulating cell elongation in Arabidopsis. PMID:23735294

  18. Microtubule-based gold nanowires and nanowire arrays.

    PubMed

    Zhou, Jing C; Gao, Yao; Martinez-Molares, Alfredo A; Jing, Xiaoye; Yan, Dong; Lau, Joseph; Hamasaki, Toshikazu; Ozkan, Cengiz S; Ozkan, Mihrimah; Hu, Evelyn; Dunn, Bruce

    2008-09-01

    Biological structures are attractive as templates to form nanoscale architectures for electronics because of their dimensions and the ability to interact with inorganic materials. In this study, we report the fabrication and electrical properties of microtubule (MT)-templated Au nanowires, and methods for assembling Au nanowire arrays based on these templates. The adsorption of MTs on silicon substrates is an effective means for preserving the conformation of the MT and provides a convenient platform for electrical measurements. To improve the metallization of MTs, a photochemical route for gold reduction is adapted, which leads to continuous coverage. The conductivity values measured on micrometer-long nanowires are similar to those reported for other biotemplated gold nanowires. A protocol for fabricating arrays of MT-templated gold nanowires is demonstrated.

  19. Shape-motion relationships of centering microtubule asters.

    PubMed

    Tanimoto, Hirokazu; Kimura, Akatsuki; Minc, Nicolas

    2016-03-28

    Although mechanisms that contribute to microtubule (MT) aster positioning have been extensively studied, still little is known on how asters move inside cells to faithfully target a cellular location. Here, we study sperm aster centration in sea urchin eggs, as a stereotypical large-scale aster movement with extreme constraints on centering speed and precision. By tracking three-dimensional aster centration dynamics in eggs with manipulated shapes, we show that aster geometry resulting from MT growth and interaction with cell boundaries dictates aster instantaneous directionality, yielding cell shape-dependent centering trajectories. Aster laser surgery and modeling suggest that dynein-dependent MT cytoplasmic pulling forces that scale to MT length function to convert aster geometry into directionality. In contrast, aster speed remains largely independent of aster size, shape, or absolute dynein activity, which suggests it may be predominantly determined by aster growth rate rather than MT force amplitude. These studies begin to define the geometrical principles that control aster movements.

  20. Conformational mechanism for the stability of microtubule-kinetochore attachments.

    PubMed

    Bertalan, Zsolt; La Porta, Caterina A M; Maiato, Helder; Zapperi, Stefano

    2014-07-15

    Regulating the stability of microtubule (MT)-kinetochore attachments is fundamental to avoiding mitotic errors and ensuring proper chromosome segregation during cell division. Although biochemical factors involved in this process have been identified, their mechanics still need to be better understood. Here we introduce and simulate a mechanical model of MT-kinetochore interactions in which the stability of the attachment is ruled by the geometrical conformations of curling MT-protofilaments entangled in kinetochore fibrils. The model allows us to reproduce, with good accuracy, in vitro experimental measurements of the detachment times of yeast kinetochores from MTs under external pulling forces. Numerical simulations suggest that geometrical features of MT-protofilaments may play an important role in the switch between stable and unstable attachments.

  1. The 2008 May 29 earthquake doublet in SW Iceland

    NASA Astrophysics Data System (ADS)

    Decriem, J.; Árnadóttir, T.; Hooper, A.; Geirsson, H.; Sigmundsson, F.; Keiding, M.; Ófeigsson, B. G.; Hreinsdóttir, S.; Einarsson, P.; LaFemina, P.; Bennett, R. A.

    2010-05-01

    On 2008 May 29 an earthquake doublet shook the southwestern part of Iceland. The first main shock originated beneath Mt Ingólfsfjall, located near the western margin of the South Iceland Seismic Zone (SISZ) approximately 40 km east of the capital Reykjavík. Immediate aftershock activity was recorded by the SIL seismic network, operated by the Icelandic Meteorological Office (IMO), with both N-S and E-W structures illuminated over a broad area. A continuous GPS (CGPS) network, also operated by the IMO, recorded coseismic offsets with up to 200 mm of horizontal motion at the closest stations. We estimate the coseismic surface deformation observed by campaign and continuous GPS and satellite radar data (InSAR). We invert the geodetic data to find the optimal geometry, location and slip on the main faults, accounting for variation in the elastic parameters of the crust with depth. Our models indicate that most of the slip occurred on two N-S structures spaced ~5 km apart. From a joint inversion of GPS and InSAR data for variable slip models we find that most of the slip for the first (Ingólfsfjall) event was concentrated at 2-4 km depth with a maximum of 1.9 m, whereas the slip on the second (Kross) fault was located deeper, at 3-6 km depth with up to 1.4 m of motion. The models give similar geodetic moments for the two main events, equivalent to a moment magnitude of Mw5.8 and Mw5.9 for the first and second event, respectively. Our estimated composite moment therefore equals a Mw6.1 for the doublet, smaller than the Mw6.3 estimated from teleseismic data (e.g. NEIC and Harvard). The geodetic data support rupture on two main faults and analysis of high-rate (1Hz) CGPS data suggests that slip on the second fault initiated within 3 s of the first main shock. Static Coulomb failure stress calculations indicate that the first event caused a stress increase in the area of the main asperity (i.e. at the location of the largest slip patch) on the second fault. However, we

  2. Dimer model for Tau proteins bound in microtubule bundles

    NASA Astrophysics Data System (ADS)

    Hall, Natalie; Kluber, Alexander; Hayre, N. Robert; Singh, Rajiv; Cox, Daniel

    2013-03-01

    The microtubule associated protein tau is important in nucleating and maintaining microtubule spacing and structure in neuronal axons. Modification of tau is implicated as a later stage process in Alzheimer's disease, but little is known about the structure of tau in microtubule bundles. We present preliminary work on a proposed model for tau dimers in microtubule bundles (dimers are the minimal units since there is one microtubule binding domain per tau). First, a model of tau monomer was created and its characteristics explored using implicit solvent molecular dynamics simulation. Multiple simulations yield a partially collapsed form with separate positively/negatively charged clumps, but which are a factor of two smaller than required by observed microtubule spacing. We argue that this will elongate in dimer form to lower electrostatic energy at a cost of entropic ``spring'' energy. We will present preliminary results on steered molecular dynamics runs on tau dimers to estimate the actual force constant. Supported by US NSF Grant DMR 1207624.

  3. Cellular Samurai: katanin and the severing of microtubules.

    PubMed

    Quarmby, L

    2000-08-01

    Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

  4. Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells

    PubMed Central

    1990-01-01

    When viewed by light microscopy the mitotic spindle in newt pneumocytes assembles in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which can be much larger than the forming spindle. This unique optical property has allowed us to examine the behavior of individual microtubules, at the periphery of asters in highly flattened living prometaphase cells, by video-enhanced differential interference-contrast light microscopy and digital image processing. As in interphase newt pneumocytes (Cassimeris, L., N. K. Pryer, and E. D. Salmon. 1988. J. Cell Biol. 107:2223-2231), centrosomal (i.e., astral) microtubules in prometaphase cells appear to exhibit dynamic instability, elongating at a mean rate of 14.3 +/- 5.1 microns/min (N = 19) and shortening at approximately 16 microns/min. Under favorable conditions the initial interaction between a kinetochore and the forming spindle can be directly observed. During this process the unattached chromosome is repeatedly probed by microtubules projecting from one of the polar regions. When one of these microtubules contacts the primary constriction the chromosome rapidly undergoes poleward translocation. Our observations on living mitotic cells directly demonstrate, for the first time, that chromosome attachment results from an interaction between astral microtubules and the kinetochore. PMID:2391359

  5. Design, manufacturing, performance and application of wide angle aspherical achromatic doublet

    NASA Astrophysics Data System (ADS)

    Melich, Radek; Procháska, František; Tomka, David; Rail, Zdeněk.; Bartoňíček, Jiří; Pleštil, Jan; Šrajer, Bohdan

    2016-11-01

    The paper describes an achromatic Steinhal type doublet that employs an aspherical surface to allow wide angle imaging. A design criteria, optimization techniques and tolerancing of the doublet are described. Further a manufacturing process of the system and achieved optical performance measurement is discussed. Benefits of the wide angle imaging doublet are recently planned to be used in automotive industry application, namely for optimizing of head-light performance and their final evaluation. The final device is planned to be part of the production line.

  6. 2HDMC — two-Higgs-doublet model calculator

    NASA Astrophysics Data System (ADS)

    Eriksson, David; Rathsman, Johan; Stål, Oscar

    2010-04-01

    We describe version 1.0.6 of the public C++ code 2HDMC, which can be used to perform calculations in a general, CP-conserving, two-Higgs-doublet model (2HDM). The program features simple conversion between different parametrizations of the 2HDM potential, a flexible Yukawa sector specification with choices of different Z-symmetries or more general couplings, a decay library including all two-body — and some three-body — decay modes for the Higgs bosons, and the possibility to calculate observables of interest for constraining the 2HDM parameter space, as well as theoretical constraints from positivity and unitarity. The latest version of the 2HDMC code and full documentation is available from: http://www.isv.uu.se/thep/MC/2HDMC. New version program summaryProgram title: 2HDMC Catalogue identifier: AEFI_v1_1 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEFI_v1_1.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU GPL No. of lines in distributed program, including test data, etc.: 12 110 No. of bytes in distributed program, including test data, etc.: 92 731 Distribution format: tar.gz Programming language: C++ Computer: Any computer running Linux Operating system: Linux RAM: 5 Mb Catalogue identifier of previous version: AEFI_v1_0 Journal reference of previous version: Comput. Phys. Comm. 180 (2010) 189 Classification: 11.1 External routines: GNU Scientific Library ( http://www.gnu.org/software/gsl/) Does the new version supersede the previous version?: Yes Nature of problem: Determining properties of the potential, calculation of mass spectrum, couplings, decay widths, oblique parameters, muon g-2, and collider constraints in a general two-Higgs-doublet model. Solution method: From arbitrary potential and Yukawa sector, tree-level relations are used to determine Higgs masses and couplings. Decay widths are calculated at leading order, including FCNC decays when applicable. Decays to off

  7. Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro

    SciTech Connect

    Walker, R.A.; Inoue, S.; Salmon, E.D.

    1989-03-01

    The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a GTP cap. A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of stabilizing cap, possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

  8. Cortical microtubule arrays are initiated from a nonrandom prepattern driven by atypical microtubule initiation.

    PubMed

    Lindeboom, Jelmer J; Lioutas, Antonios; Deinum, Eva E; Tindemans, Simon H; Ehrhardt, David W; Emons, Anne Mie C; Vos, Jan W; Mulder, Bela M

    2013-03-01

    The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.

  9. A divergent canonical WNT-signaling pathway regulates microtubule dynamics

    PubMed Central

    Ciani, Lorenza; Krylova, Olga; Smalley, Matthew J.; Dale, Trevor C.; Salinas, Patricia C.

    2004-01-01

    Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β). In the canonical WNT pathway, the negative regulator Axin forms a complex with β-catenin and GSK-3β, resulting in β-catenin degradation. Inhibition of GSK-3β by DVL increases β-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3β through a transcription- and β-catenin–independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3β–mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3β, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability. PMID:14734535

  10. Vinblastine suppresses dynamics of individual microtubules in living interphase cells.

    PubMed Central

    Dhamodharan, R; Jordan, M A; Thrower, D; Wilson, L; Wadsworth, P

    1995-01-01

    We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells. Images PMID:8534917

  11. Polar organizers and girdling bands of microtubules are associated with gamma-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta).

    PubMed

    Brown, R C; Lemmon, B E

    2006-05-01

    Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of gamma-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants.

  12. INSIGHTS INTO ANTI-PARALLEL MICROTUBULE CROSSLINKING BY PRC1, A CONSERVED NON-MOTOR MICROTUBULE BINDING PROTEIN

    PubMed Central

    Subramanian, Radhika; Wilson-Kubalek, Elizabeth M.; Arthur, Christopher P.; Bick, Matthew J.; Campbell, Elizabeth A.; Darst, Seth A.; Milligan, Ronald A.; Kapoor, Tarun M.

    2010-01-01

    SUMMARY Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish compliant crosslinks that selectively `mark' antiparallel overlap in dynamic cytoskeletal networks. PMID:20691902

  13. Muon g - 2 in the aligned two Higgs doublet model

    SciTech Connect

    Han, Tao; Kang, Sin Kyu; Sayre, Joshua

    2016-02-16

    In this paper, we study the Two-Higgs-Doublet Model with the aligned Yukawa sector (A2HDM) in light of the observed excess measured in the muon anomalous magnetic moment. We take into account the existing theoretical and experimental constraints with up-to-date values and demonstrate that a phenomenologically interesting region of parameter space exists. With a detailed parameter scan, we show a much larger region of viable parameter space in this model beyond the limiting case Type X 2HDM as obtained before. It features the existence of light scalar states with masses 3 GeV ≲ mH ≲ 50 GeV, or 10 GeV ≲ mA ≲ 130 GeV, with enhanced couplings to tau leptons. The charged Higgs boson is typically heavier, with 200 GeV ≲ mH+ ≲ 630 GeV. The surviving parameter space is forced into the CP-conserving limit by EDM constraints. Some Standard Model observables may be significantly modified, including a possible new decay mode of the SMlike Higgs boson to four taus. Lastly, we comment on future measurements and direct searches for those effects at the LHC as tests of the model.

  14. Higgs Doublet Decay as the Origin of the Baryon Asymmetry

    NASA Astrophysics Data System (ADS)

    Hambye, Thomas; Teresi, Daniele

    2016-08-01

    We consider a question that curiously had not been properly considered thus far: in the standard seesaw model, what is the minimum value the mass of a right-handed (RH) neutrino must have for allowing successful leptogenesis via C P -violating decays? Answering this question requires us to take into account a number of thermal effects. We show that, for low RH neutrino masses, and thanks to these effects, leptogenesis turns out to proceed efficiently from the decay of the standard model scalar doublet components into a RH neutrino and a lepton. Such decays produce the asymmetry at low temperatures, slightly before sphaleron decoupling. If the RH neutrino has thermalized prior to producing the asymmetry, this mechanism turns out to lead to the bound mN>2 GeV . If, instead, the RH neutrinos have not thermalized, leptogenesis from these decays is enhanced further and can be easily successful, even at lower scales. This Higgs-decay leptogenesis new mechanism works without requiring an interplay of flavor effects and/or cancellations of large Yukawa couplings in the neutrino mass matrix. Last but not least, such a scenario turns out to be testable, from direct production of the RH neutrino(s).

  15. Exploring collider signatures of the inert Higgs doublet model

    NASA Astrophysics Data System (ADS)

    Datta, Amitava; Ganguly, Nabanita; Khan, Najimuddin; Rakshit, Subhendu

    2017-01-01

    We revisit the multilepton (m l )+ ET+X signatures of the inert doublet model (IDM) of dark matter in future LHC experiments for m =3 , 4 and simulate, for the first time, the m =5 case. Here X stands for any number of jets. We illustrate these signals with benchmark points consistent with the usual constraints like unitarity, perturbativity, the precision electroweak data, the observed dark matter relic density of the Universe and, most importantly, the stringent LHC constraints from the post-Higgs (h ) discovery era like the measured Mh and the upper bound on the invisible width of h decay, which were not included in earlier analyses of multilepton signatures. We find that if the IDM is embedded in a grand desert scenario so that the unitarity constraint holds up to a very high scale, the whole of the highly restricted parameter space allowed by the above constraints can be probed at the LHC via the 3 l signal for an integrated luminosity ˜3000 fb-1 . On the other hand, if any new physics shows up at a scale ˜10 TeV , only a part of the enlarged allowed parameter space can be probed. The 4 l and 5 l signals can help to discriminate among different IDM scenarios as and when sufficient integrated luminosity accumulates.

  16. The doublet majoron model and solar neutrino oscillations

    NASA Astrophysics Data System (ADS)

    Bertolini, S.; Santamaria, A.

    1988-12-01

    We present a minimal extension of the standard electroweak theory which, as a consequence of the spontaneous breaking of lepton number and the radiative origin of the neutrino mass, offers a natural framework for the solution of the solar neutrino problem through matter-enhanced neutrino oscillations (Mikheyev-Smirnov-Wolfenstein mechanism). Indeed, we show that the presently available astrophysical bounds on the lepton-breaking vacuum expectation value naturally lead to neutrino masses in the required regime. The fact that the Majoron belongs to an SU(2)L doublet and not a triplet has relevant phenomenological implications. In particular, the scalar contribution to the Z0 width is four times smaller than in the triplet model and equivalent to 1/2 a neutrino-antineutrino mode. Relevant effects related to the presence of two physical singly charged scalars, both at the quantum and tree level, are studied. As a result we find that the model is tightly constrained by present data. In particular, for a wide range of parameters, the decay μ --> eγ is within two orders of magnitude from the present experimental limit. Also at Department de Física Teòrica, Universitat de València and IFIC, Universitat de València-CSIC, Spain.

  17. Muon g - 2 in the aligned two Higgs doublet model

    DOE PAGES

    Han, Tao; Kang, Sin Kyu; Sayre, Joshua

    2016-02-16

    In this paper, we study the Two-Higgs-Doublet Model with the aligned Yukawa sector (A2HDM) in light of the observed excess measured in the muon anomalous magnetic moment. We take into account the existing theoretical and experimental constraints with up-to-date values and demonstrate that a phenomenologically interesting region of parameter space exists. With a detailed parameter scan, we show a much larger region of viable parameter space in this model beyond the limiting case Type X 2HDM as obtained before. It features the existence of light scalar states with masses 3 GeV ≲ mH ≲ 50 GeV, or 10 GeV ≲more » mA ≲ 130 GeV, with enhanced couplings to tau leptons. The charged Higgs boson is typically heavier, with 200 GeV ≲ mH+ ≲ 630 GeV. The surviving parameter space is forced into the CP-conserving limit by EDM constraints. Some Standard Model observables may be significantly modified, including a possible new decay mode of the SMlike Higgs boson to four taus. Lastly, we comment on future measurements and direct searches for those effects at the LHC as tests of the model.« less

  18. Lausannevirus, a giant amoebal virus encoding histone doublets.

    PubMed

    Thomas, Vincent; Bertelli, Claire; Collyn, François; Casson, Nicola; Telenti, Amalio; Goesmann, Alexander; Croxatto, Antony; Greub, Gilbert

    2011-06-01

    Large viruses infecting algae or amoebae belong to the NucleoCytoplasmic Large DNA Viruses (NCLDV) and present genotypic and phenotypic characteristics that have raised major interest among microbiologists. Here, we describe a new large virus discovered in Acanthamoeba castellanii co-culture of an environmental sample. The virus, referred to as Lausannevirus, has a very limited host range, infecting Acanthamoeba spp. but being unable to infect other amoebae and mammalian cell lines tested. Within A. castellanii, this icosahedral virus of about 200 nm exhibits a development cycle similar to Mimivirus, with an eclipse phase 2 h post infection and a logarithmic growth leading to amoebal lysis in less than 24 h. The 346 kb Lausannevirus genome presents similarities with the recently described Marseillevirus, sharing 89% of genes, and thus belongs to the same family as confirmed by core gene phylogeny. Interestingly, Lausannevirus and Marseillevirus genomes both encode three proteins with predicted histone folds, including two histone doublets, that present similarities to eukaryotic and archaeal histones. The discovery of Lausannevirus and the analysis of its genome provide some insight in the evolution of these large amoebae-infecting viruses.

  19. Dark matter physics in neutrino specific two Higgs doublet model

    NASA Astrophysics Data System (ADS)

    Baek, Seungwon; Nomura, Takaaki

    2017-03-01

    Although the seesaw mechanism is a natural explanation for the small neutrino masses, there are cases when the Majorana mass terms for the right-handed neutrinos are not allowed due to symmetry. In that case, if neutrino-specific Higgs doublet is introduced, neutrinos become Dirac particles and their small masses can be explained by its small VEV. We show that the same symmetry, which we assume a global U(1) X , can also be used to explain the stability of dark matter. In our model, a new singlet scalar breaks the global symmetry spontaneously down to a discrete Z 2 symmetry. The dark matter particle, lightest Z 2-odd fermion, is stabilized. We discuss the phenomenology of dark matter: relic density, direct detection, and indirect detection. We find that the relic density can be explained by a novel Goldstone boson channel or by resonance channel. In the most region of parameter space considered, the direct detections is suppressed well below the current experimental bound. Our model can be further tested in indirect detection experiments such as FermiLAT gamma ray searches or neutrinoless double beta decay experiments.

  20. Diverse rupture processes in the 2015 Peru deep earthquake doublet

    PubMed Central

    Ye, Lingling; Lay, Thorne; Kanamori, Hiroo; Zhan, Zhongwen; Duputel, Zacharie

    2016-01-01

    Earthquakes in deeply subducted oceanic lithosphere can involve either brittle or dissipative ruptures. On 24 November 2015, two deep (606 and 622 km) magnitude 7.5 and 7.6 earthquakes occurred 316 s and 55 km apart. The first event (E1) was a brittle rupture with a sequence of comparable-size subevents extending unilaterally ~50 km southward with a rupture speed of ~4.5 km/s. This earthquake triggered several aftershocks to the north along with the other major event (E2), which had 40% larger seismic moment and the same duration (~20 s), but much smaller rupture area and lower rupture speed than E1, indicating a more dissipative rupture. A minor energy release ~12 s after E1 near the E2 hypocenter, possibly initiated by the S wave from E1, and a clear aftershock ~165 s after E1 also near the E2 hypocenter, suggest that E2 was likely dynamically triggered. Differences in deep earthquake rupture behavior are commonly attributed to variations in thermal state between subduction zones. However, the marked difference in rupture behavior of the nearby Peru doublet events suggests that local variations of stress state and material properties significantly contribute to diverse behavior of deep earthquakes. PMID:27386585

  1. Diverse rupture processes in the 2015 Peru deep earthquake doublet.

    PubMed

    Ye, Lingling; Lay, Thorne; Kanamori, Hiroo; Zhan, Zhongwen; Duputel, Zacharie

    2016-06-01

    Earthquakes in deeply subducted oceanic lithosphere can involve either brittle or dissipative ruptures. On 24 November 2015, two deep (606 and 622 km) magnitude 7.5 and 7.6 earthquakes occurred 316 s and 55 km apart. The first event (E1) was a brittle rupture with a sequence of comparable-size subevents extending unilaterally ~50 km southward with a rupture speed of ~4.5 km/s. This earthquake triggered several aftershocks to the north along with the other major event (E2), which had 40% larger seismic moment and the same duration (~20 s), but much smaller rupture area and lower rupture speed than E1, indicating a more dissipative rupture. A minor energy release ~12 s after E1 near the E2 hypocenter, possibly initiated by the S wave from E1, and a clear aftershock ~165 s after E1 also near the E2 hypocenter, suggest that E2 was likely dynamically triggered. Differences in deep earthquake rupture behavior are commonly attributed to variations in thermal state between subduction zones. However, the marked difference in rupture behavior of the nearby Peru doublet events suggests that local variations of stress state and material properties significantly contribute to diverse behavior of deep earthquakes.

  2. HILI destabilizes microtubules by suppressing phosphorylation and Gigaxonin-mediated degradation of TBCB

    PubMed Central

    Tan, Hao; Liao, Hua; Zhao, Lianfang; Lu, Yilu; Jiang, Siyuan; Tao, Dachang; Liu, Yunqiang; Ma, Yongxin

    2017-01-01

    Human PIWIL2, aka HILI, is a member of PIWI protein family and overexpresses in various tumors. However, the underlying mechanisms of HILI in tumorigenesis remain largely unknown. TBCB has a critical role in regulating microtubule dynamics and is overexpressed in many cancers. Here we report that HILI inhibits Gigaxonin-mediated TBCB ubiquitination and degradation by interacting with TBCB, promoting the binding between HSP90 and TBCB, and suppressing the interaction between Gigaxonin and TBCB. Meanwhile, HILI can also reduce phosphorylation level of TBCB induced by PAK1. Our results showed that HILI suppresses microtubule polymerization and promotes cell proliferation, migration and invasion via TBCB for the first time, revealing a novel mechanism for HILI in tumorigenesis. PMID:28393858

  3. The Dynamics of Microtubule/Motor-Protein Assemblies in Biology and Physics

    NASA Astrophysics Data System (ADS)

    Shelley, Michael J.

    2016-01-01

    Many important processes in the cell are mediated by stiff microtubule polymers and the active motor proteins moving on them. This includes the transport of subcellular structures (nuclei, chromosomes, organelles) and the self-assembly and positioning of the mitotic spindle. Little is understood of these processes, but they present fascinating problems in fluid-structure interactions. Microtubules and motor proteins are also the building blocks of new biosynthetic active suspensions driven by motor-protein activity. These reduced systems can be probed—and modeled—more easily than can the fully biological ones and demonstrate their own aspects of self-assembly and complex dynamics. I review recent work modeling such systems as fluid-structure interaction problems and as multiscale complex fluids.

  4. Twelve protofilament taxol-induced microtubules assembled from purified tublin. A synchrotron X-ray scattering study in comparison with glycerol- and map-induced microtubules

    NASA Astrophysics Data System (ADS)

    Andreu, J. M.; Garcia de Ancos, J.; Medrano, F. J.; Gil, R.; Diaz, J. F.; Nogales, E.; Towns-Andrews, E.; Pantos, E.; Bordas, J.

    1991-05-01

    The X-ray solution scattering profiles of taxol microtubules made of purified tubulin and control microtubules, assembled either from purified tubulin in glycerol buffer (a non-specific enhancer of the polymerization of tubulin) or from microtubule protein (a preparation containing tubulin plus microtubule associated proteins), were obtained to 3.3 nm resolution. These profiles show features of the microtubule wall structure which had not been observed in solution before. Comparison of the different profiles indicated that the structure of the microtubule wall is very similar in the three types of microtubules to the resolution of the measurements, however the mean diameter of the taxol microtubules is smaller than that of the control microtubules, by approximately one protofilament less. Actually, only 12 protofilament computer models of microtubules could fit the position of the maxima in the experimental scattering profile of the taxol microtubules. Having only 12 protofilaments implies a discontinuity on the microtubule wall, irrespective of whether the lateral contacts follow the A or B microtubule lattice, and also requires adjustment of the normal lattice to one protofilament axis with respect to the cylinder axis. The fact that the majority of these taxol microtubules assembled from purified tubulin have 12 protofilaments has been visualized by electron micrographs of tannic acid stained microtubule thin sections, and is fully consistent with the microtubule wall projections (fringe patterns) observed in negatively stained and cryo-electron microscopy specimens, which correspond to a 12 protofilament-three start lattice type.

  5. T violation in Kμ3 decay in a general two-Higgs-doublet model

    NASA Astrophysics Data System (ADS)

    Godina Nava, J. J.

    1996-02-01

    We calculate the transverse muon polarization in the K+μ3 process arising from the Yukawa couplings of charged Higgs bosons in a general two-Higgs-doublet model where spontaneous violation of CP invariance is present.

  6. STAT3 Inhibition by Microtubule-Targeted Drugs: Dual Molecular Effects of Chemotherapeutic Agents

    PubMed Central

    Walker, Sarah R.; Chaudhury, Mousumi; Frank, David A.

    2011-01-01

    To improve the effectiveness of anti-cancer therapies, it is necessary to identify molecular targets that are essential to a tumor cell but dispensable in a normal cell. Increasing evidence indicates that the transcription factor STAT3, which regulates the expression of genes controlling proliferation, survival, and self-renewal, constitutes such a target. Recently it has been found that STAT3 can associate with the cytoskeleton. Since many of the tumors in which STAT3 is activated, such as breast cancer and ovarian cancer, are responsive to drugs that target microtubules, we examined the effect of these compounds on STAT3. We found that microtubule stabilizers, such as paclitaxel, or microtubule inhibitors, such as vinorelbine, decrease the activating tyrosine phosphorylation of STAT3 in tumor cells and inhibit the expression of STAT3 target genes. Paclitaxel decreases the association between STAT3 and microtubules, and appears to decrease STAT3 phosphorylation through induction of a negative feedback regulator. The cytotoxic activity of paclitaxel in breast cancer cell lines correlates with its ability to decrease STAT3 phosphorylation. However, consistent with the necessity for expression of a negative regulator, treatment of resistant MDA-MB-231 cells with the DNA demethylating agent 5-azacytidine restores the ability of paclitaxel to block STAT3-dependent gene expression. Finally, the combination of paclitaxel and agents that directly target STAT3 has beneficial effects in killing STAT3-dependent cell lines. Thus, microtubule-targeted agents may exert some of their effects by inhibiting STAT3, and understanding this interaction may be important for optimizing rational targeted cancer therapies. PMID:21949561

  7. Microtubule-regulating proteins and cAMP-dependent signaling in neuroblastoma differentiation.

    PubMed

    Muñoz-Llancao, Pablo; de Gregorio, Cristian; Las Heras, Macarena; Meinohl, Christopher; Noorman, Kevin; Boddeke, Erik; Cheng, Xiaodong; Lezoualc'h, Frank; Schmidt, Martina; Gonzalez-Billault, Christian

    2017-03-01

    Neurons are highly differentiated cells responsible for the conduction and transmission of information in the nervous system. The proper function of a neuron relies on the compartmentalization of their intracellular domains. Differentiated neuroblastoma cells have been extensively used to study and understand the physiology and cell biology of neuronal cells. Here, we show that differentiation of N1E-115 neuroblastoma cells is more pronounced upon exposure of a chemical analog of cyclic AMP (cAMP), db-cAMP. We next analysed the expression of key microtubule-regulating proteins in differentiated cells and the expression and activation of key cAMP players such as EPAC, PKA and AKAP79/150. Most of the microtubule-promoting factors were up regulated during differentiation of N1E-115 cells, while microtubule-destabilizing proteins were down regulated. We observed an increase in tubulin post-translational modifications related to microtubule stability. As expected, db-cAMP increased PKA- and EPAC-dependent signalling. Consistently, pharmacological modulation of EPAC activity instructed cell differentiation, number of neurites, and neurite length in N1E-115 cells. Moreover, disruption of the PKA-AKAP interaction reduced these morphometric parameters. Interestingly, PKA and EPAC act synergistically to induce neuronal differentiation in N1E-115. Altogether these results show that the changes observed in the differentiation of N1E-115 cells proceed by regulating several microtubule-stabilizing factors, and the acquisition of a neuronal phenotype is a process involving concerted although independent functions of EPAC and PKA.

  8. The microtubule plus-end tracking protein ARMADILLO-REPEAT KINESIN1 promotes microtubule catastrophe in Arabidopsis.

    PubMed

    Eng, Ryan Christopher; Wasteneys, Geoffrey O

    2014-08-01

    Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1's function is redundant in cells other than those forming root hairs.

  9. Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly.

    PubMed

    Hung, Liang-Yi; Chen, Hua-Ling; Chang, Ching-Wen; Li, Bor-Ran; Tang, Tang K

    2004-06-01

    We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.

  10. Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules

    PubMed Central

    1987-01-01

    The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti- centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole- resistant microtubules in MDCK cells contained detyrosinated alpha- tubulin (glu tubulin). On the other hand

  11. Effects of fluoro-doxorubicin (ME2303) on microtubules: influence of different classes of microtubule-associated proteins.

    PubMed

    Fromes, Y; Gounon, P; Tapiero, H; Fellous, A

    1996-08-01

    Anthracyclines are among the most useful agents for the treatment of neoplastic disease, but their clinical use is limited by progressive cardiomyopathy. A few studies have suggested the role of microtubules for the understanding of this toxicity. By using kinetic and structural studies, we demonstrate the disorganizing action of fluoro-doxorubicin, a novel anthracycline, on the microtubule system. Microtubules have a rich and complex composition in relation to their numerous functions in cells. In the present study, we investigate the role of two major microtubule-associated protein (MAP) families, Tau and MAP2. Both MAP families are responsible for the properties of different classes of microtubules. We show the differential effect of fluoro-doxorubicin on these two classes of microtubules. Furthermore, we show that fluoro-doxorubicin is able to affect the capacity of purified tubulin to form normal microtubules. This study confirms that anthracyclines may interfer with the microtubule organization. We suggest that some classes of microtubules, with regard to their MAP composition, may be affected more specifically in cardiac myocytes.

  12. The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization

    PubMed Central

    Miller, Rita K.; Matheos, Dina; Rose, Mark D.

    1999-01-01

    In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)– Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Δ, pea2Δ, bud6Δ, and bni1Δ, required for normal polarization and actin cytoskeleton functions and, of these, bni1Δ affected Kar9p localization most severely. Like kar9Δ, bni1Δ mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Δ, the bni1Δ mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Δ bni1Δ double mutants suggested that Kar9p retained some function in bni1Δ mitotic cells. Unlike the polarization mutants, kar9Δ shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Δ. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks. PMID:10085294

  13. Stability of the normal vacuum in multi-Higgs-doublet models

    SciTech Connect

    Barroso, A.; Ferreira, P. M.; Santos, R.; Silva, Joao P.

    2006-10-15

    We show that the vacuum structure of a generic multi-Higgs-doublet model shares several important features with the vacuum structure of the two and three Higgs-doublet model. In particular, one can still define the usual charge breaking, spontaneous CP breaking, and normal (charge and CP preserving) stationary points. We analyze the possibility of charge or spontaneous CP breaking by studying the relative depth of the potential in each of the possible stationary points.

  14. MARK4 is a novel microtubule-associated proteins/microtubule affinity-regulating kinase that binds to the cellular microtubule network and to centrosomes.

    PubMed

    Trinczek, Bernhard; Brajenovic, Miro; Ebneth, Andreas; Drewes, Gerard

    2004-02-13

    The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease.

  15. Characterization of the role of calcium in regulating the microtubule-destabilizing activity of MDP25.

    PubMed

    Qin, Tao; Li, Jiejie; Yuan, Ming; Mao, Tonglin

    2012-07-01

    Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins. Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.

  16. Microtubules in protozoan cells. III. Ultrastructural changes during disintegration and reformation of heliozoan microtubules.

    PubMed

    Toyohara, A; Shigenaka, Y; Mohri, H

    1978-08-01

    In the heliozoan, Echinosphaerium nucleofilum strain MA, cold temperature (2 degrees C) induced axopodial retraction to about 36% of the initial length after 3 h. By electron microscopy, it was found that such axopodial shortening is accompanied by degradation of axonemal microtubules (25 nm in diameter), followed by the appearance of macrotubles (37 nm in diameter) and filamentous structures (14 nm in diameter) of a tubular and twisted appearance. All of these structures (microtubules, macrotubules and filamentous structures) were found to be depolymerized completely by applying 10 mM colchicine for 1-2 h, and to be replaced by regions with low electron density. Axopodial re-extension was induced rapidly by returning the cold-treated organisms to room temperature (20 degrees C). At a very early stage of axopodial re-extension, the filamentous structures were often observed to be continuous with the macrotubules. At a late stage of axopodial re-extension, the reforming axoneme was composed only of normal microtubules, while the macrotubules and filamentous structures had disappeared. On the basis of these results, the processes of disintegration and reformation of microtubules are discussed.

  17. Video microscopy analysis of the polymerization dynamics of individual microtubules

    NASA Astrophysics Data System (ADS)

    Salmon, E. D.

    1991-05-01

    We have developed methods using video-enhanced differential interference contrast light microscopy (VE-DIC) to measure the association and dissociation rate constants and transition frequencies of microtubule dynamic instability for microtubules assembled from pure tubulin, plus brain microtubule assoicated proteins (MAPs), and for microtubule assembly in living cells and cytosol extracts. Following nucleation, a microtubule end is seen to elongate at constant velocity until it abruptly begins rapid shortening, a transition termed catastrophe. The microtubule either disappears, or converts back to the elongation phase, a transition termed rescue. Catastrophes and rescues occur stochastically and infrequently in comparison to the durations of the elongation and shortening phases. In purified tubulin preparations from both mammalian brain and sea urchin embryos, the elongation and shortening phases exhibit distinctly different association and dissociation rate constants; in particular, the rate of dissocation during rapid shortening can be 100 times or more greater than during elongation particularly at high Mg2+. Brain MAPs (MAP2 and Tau) promote faster elongation, but suppress dynamic instability mainly by decreasing the frequency of catastrophe and increasing the frequency of rescue. In contrast, there are unknown factors in living dividing cells and in extracts from dividing cells which enhance dynamic instability by producing high frequencies of catastrophe (.01-.05 sec-1) at fast elongation velocities (10 μm min-1). Using a microscope perfusion chamber, we have shown for microtubules assembled from pure tubulin that dilution induces rapid shortening within several seconds independent of the elongation velocity or microtubule length. Thus, the stabilizing cap at elongating microtubule ends is small and sensitive to transient changes in the rate of tubulin association, even at high elongation velocities. This means that substantial changes in microtubule

  18. Response of a doublet to a nearby dc electrode of uniform potential

    NASA Astrophysics Data System (ADS)

    Wirth, Christopher L.; Nuthalapati, Sri Harsha

    2016-10-01

    The electric-field-assisted directed assembly of spherical colloidal particles near an electrode has been studied for nearly three decades. Recently, focus has shifted to the electric-field-assisted assembly and propulsion of nonspherical (i.e., anisotropic) particles. This paper describes calculations and results for a doublet of asymmetric ζ potential and size responding to a dc electric field. The doublet experienced a net vertical force that depended on both the asymmetry in ζ potential and lobe size. In addition, the doublet experienced a net lateral force perpendicular to the applied electric field. The lateral force depended on the difference in ζ potential of the two lobes, the lobe size asymmetry, and also the angle of inclination of the doublet. The net force was used to predict an apparent lateral velocity, which was found to be perpendicular to the applied electric field. In addition, the particle experienced rotation from a net torque that depended on the lobe size asymmetry and also the angle of inclination of the doublet. The magnitude of the predicted velocity was of the same order of magnitude as has been observed for particles responding to ac electric fields in experiments. These results demonstrate that lobe ζ potential, lobe size, and orientation of a colloidal doublet can be tuned to achieve propulsion.

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

    PubMed Central

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

    2010-01-01

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

  20. Assembly of bipolar microtubule structures by passive cross-linkers and molecular motors.

    PubMed

    Johann, D; Goswami, D; Kruse, K

    2016-06-01

    During cell division, sister chromatids are segregated by the mitotic spindle, a bipolar assembly of interdigitating antiparallel polar filaments called microtubules. The spindle contains the midzone, a stable region of overlapping antiparallel microtubules, that is essential for maintaining bipolarity. Although a lot is known about the molecular players involved, the mechanism underlying midzone formation and maintenance is still poorly understood. We study the interaction of polar filaments that are cross-linked by molecular motors moving directionally and by passive cross-linkers diffusing along microtubules. Using a particle-based stochastic model, we find that the interplay of motors and passive cross-linkers can generate a stable finite overlap between a pair of antiparallel polar filaments. We develop a mean-field theory to study this mechanism in detail and investigate the influence of steric interactions between motors and passive cross-linkers on the overlap dynamics. In the presence of interspecies steric interactions, passive cross-linkers mimic the behavior of molecular motors and stable finite overlaps are generated even for non-cross-linking motors. Finally, we develop a mean-field theory for a bundle of aligned polar filaments and show that they can self-organize into a spindlelike pattern. Our work suggests possible ways as to how cells can generate spindle midzones and control their extensions.

  1. Assembly of bipolar microtubule structures by passive cross-linkers and molecular motors

    NASA Astrophysics Data System (ADS)

    Johann, D.; Goswami, D.; Kruse, K.

    2016-06-01

    During cell division, sister chromatids are segregated by the mitotic spindle, a bipolar assembly of interdigitating antiparallel polar filaments called microtubules. The spindle contains the midzone, a stable region of overlapping antiparallel microtubules, that is essential for maintaining bipolarity. Although a lot is known about the molecular players involved, the mechanism underlying midzone formation and maintenance is still poorly understood. We study the interaction of polar filaments that are cross-linked by molecular motors moving directionally and by passive cross-linkers diffusing along microtubules. Using a particle-based stochastic model, we find that the interplay of motors and passive cross-linkers can generate a stable finite overlap between a pair of antiparallel polar filaments. We develop a mean-field theory to study this mechanism in detail and investigate the influence of steric interactions between motors and passive cross-linkers on the overlap dynamics. In the presence of interspecies steric interactions, passive cross-linkers mimic the behavior of molecular motors and stable finite overlaps are generated even for non-cross-linking motors. Finally, we develop a mean-field theory for a bundle of aligned polar filaments and show that they can self-organize into a spindlelike pattern. Our work suggests possible ways as to how cells can generate spindle midzones and control their extensions.

  2. Building the Microtubule Cytoskeleton Piece by Piece*

    PubMed Central

    Alfaro-Aco, Ray; Petry, Sabine

    2015-01-01

    The microtubule (MT) cytoskeleton gives cells their shape, organizes the cellular interior, and segregates chromosomes. These functions rely on the precise arrangement of MTs, which is achieved by the coordinated action of MT-associated proteins (MAPs). We highlight the first and most important examples of how different MAP activities are combined in vitro to create an ensemble function that exceeds the simple addition of their individual activities, and how the Xenopus laevis egg extract system has been utilized as a powerful intermediate between cellular and purified systems to uncover the design principles of self-organized MT networks in the cell. PMID:25957410

  3. Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern.

    PubMed

    Rank, Katherine C; Chen, Chun Ju; Cope, Julia; Porche, Ken; Hoenger, Andreas; Gilbert, Susan P; Rayment, Ivan

    2012-06-25

    Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.

  4. The effects of the phospholipase D-antagonist 1-butanol on seedling development and microtubule organisation in Arabidopsis.

    PubMed

    Gardiner, John; Collings, David A; Harper, John D I; Marc, Jan

    2003-07-01

    The organisation of plant microtubules into distinct arrays during the cell cycle requires interactions with partner proteins. Having recently identified a 90-kDa phospholipase D (PLD) that associates with microtubules and the plasma membrane [Gardiner et al. (2001) Plant Cell 13: 2143], we exposed seeds and young seedlings of Arabidopsis to 1-butanol, a specific inhibitor of PLD-dependent production of the signalling molecule phosphatidic acid (PA). When added to agar growth media, 0.2% 1-butanol strongly inhibited the emergence of the radicle and cotyledons, while 0.4% 1-butanol effectively blocked germination. When normal seedlings were transferred onto media containing 0.2% and 0.4% 1-butanol, the inhibitor retarded root growth by about 40% and 90%, respectively, by reducing cell elongation. Inhibited plants showed significant swelling in the root elongation zone, bulbous or branched root hairs, and modified cotyledon morphology. Confocal immunofluorescence microscopy of root tips revealed that 1-butanol disrupted the organisation of interphase cortical microtubules. Butanol isomers that do not inhibit PLD-dependent PA production, 2- and 3-butanol, had no effect on seed germination, seedling growth, or microtubule organisation. We propose that production of PA by PLD may be required for normal microtubule organisation and hence normal growth in Arabidopsis.

  5. Activation of microtubule dynamics increases neuronal growth via the nerve growth factor (NGF)- and Gαs-mediated signaling pathways.

    PubMed

    Sarma, Tulika; Koutsouris, Athanasia; Yu, Jiang Zhu; Krbanjevic, Aleksandar; Hope, Thomas J; Rasenick, Mark M

    2015-04-17

    Signals that activate the G protein Gαs and promote neuronal differentiation evoke Gαs internalization in rat pheochromocytoma (PC12) cells. These agents also significantly increase Gαs association with microtubules, resulting in an increase in microtubule dynamics because of the activation of tubulin GTPase by Gαs. To determine the function of Gαs/microtubule association in neuronal development, we used real-time trafficking of a GFP-Gαs fusion protein. GFP-Gαs concentrates at the distal end of the neurites in differentiated living PC12 cells as well as in cultured hippocampal neurons. Gαs translocates to specialized membrane compartments at tips of growing neurites. A dominant-negative Gα chimera that interferes with Gαs binding to tubulin and activation of tubulin GTPase attenuates neurite elongation and neurite number both in PC12 cells and primary hippocampal neurons. This effect is greatest on differentiation induced by activated Gαs. Together, these data suggest that activated Gαs translocates from the plasma membrane and, through interaction with tubulin/microtubules in the cytosol, is important for neurite formation, development, and outgrowth. Characterization of neuronal G protein dynamics and their contribution to microtubule dynamics is important for understanding the molecular mechanisms by which G protein-coupled receptor signaling orchestrates neuronal growth and differentiation.

  6. Saccharomyces cerevisiae Cells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

    PubMed Central

    Brachat, Arndt; Kilmartin, John V.; Wach, Achim; Philippsen, Peter

    1998-01-01

    Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating. PMID:9571234

  7. A biophysical model of how α-tubulin carboxy-terminal tails tune kinesin-1 processivity along microtubule.

    PubMed

    Sataric, Miljko V; Sekulic, Dalibor L; Zdravkovic, Slobodan; Ralevic, Nebojsa M

    2017-03-12

    It appears that so-called post-translational modifications of tubulin heterodimers are mostly focussed at positions of amino acid sequences of carboxy-terminal tails. These changes have very profound effects on microtubule functions especially in connection with cellular traffic in terms of motor proteins. In this study, we elaborated the biophysical model aimed to explain the strategy governing these subtle interplays between structural and functional properties of microtubules. We relied onto Langevin equations including fluctuation-dissipation processes. In that context we found out that small interaction between a charged motor neck domain and oppositely charged carboxy-terminal tail of the α-tubulin plays the decisive role in tuning kinesin-1 motor processivity along microtubules.

  8. Dynamic model of the force driving kinesin to move along microtubule-Simulation with a model system

    NASA Astrophysics Data System (ADS)

    Chou, Y. C.; Hsiao, Yi-Feng; To, Kiwing

    2015-09-01

    A dynamic model for the motility of kinesin, including stochastic-force generation and step formation is proposed. The force driving the motion of kinesin motor is generated by the impulse from the collision between the randomly moving long-chain stalk and the ratchet-shaped outer surface of microtubule. Most of the dynamical and statistical features of the motility of kinesin are reproduced in a simulation system, with (a) ratchet structures similar to the outer surface of microtubule, (b) a bead chain connected to two heads, similarly to the stalk of the real kinesin motor, and (c) the interaction between the heads of the simulated kinesin and microtubule. We also propose an experiment to discriminate between the conventional hand-over-hand model and the dynamic model.

  9. Passive athermalization of doublets in 8-13 micron waveband

    NASA Astrophysics Data System (ADS)

    Schuster, Norbert

    2014-10-01

    Passive athermalization of lenses has become a key-technology for automotive and other outdoor applications using modern uncooled 25, 17 and 12 micron pixel pitch bolometer arrays. Typical pixel counts for thermal imaging are 384x288 (qVGA), 640x480 (VGA), and 1024x768 (XGA). Two lens arrangements (called Doublets) represent a cost effective way to satisfy resolution requirements of these detectors with F-numbers 1.4 or faster. Thermal drift of index of refraction and the geometrical changes (in lenses and housing) versus temperature defocus the initial image plane from the detector plane. The passive athermalization restricts this drop of spatial resolution in a wide temperature range (typically -40°C…+80°C) to an acceptable value without any additional external refocus. In particular, lenses with long focal lengths and high apertures claim athermalization. A careful choice of lens and housing materials and a sophistical dimensioning lead to three different principles of passivation: The Passive Mechanical Athermalization (PMA) shifts the complete lens cell, the Passive Optical and Mechanical Athermalization (POMA) shifts only one lens inside the housing, the Passive Optical Athermalization (POA) works without any mechanism. All three principles will be demonstrated for a typical narrow-field lens (HFOV about 12°) with high aperture (aperture based F-number 1.3) for the actual uncooled reference detector (17micron VGA). Six design examples using different combinations of lens materials show the impact on spatial lens resolution, on overall length, and on weight. First order relations are discussed. They give some hints for optimization solutions. Pros and cons of different passive athermalization principles are evaluated in regards of housing design, availability of materials and costing. Examples with a convergent GASIR®1-lens in front distinguish by best resolution, short overall length, and lowest weight.

  10. Competing Kondo Effects in Non-Kramers Doublet Systems

    NASA Astrophysics Data System (ADS)

    Kusunose, Hiroaki

    2016-06-01

    In non-Kramers Kondo systems with quadrupolar degrees of freedom, an ordinary magnetic Kondo effect can compete with the quadrupolar Kondo effect. We discuss such competition keeping PrT2Zn20 (T = Ir, Rh) and PrT2Al20 (T = V, Ti) in mind, where the Γ3 non-Kramers crystalline-electric-field (CEF) doublet ground state is realized in a Pr3+ ion with a (4f)2 configuration under cubic symmetry. The quadrupolar Kondo effect can be described by the two-channel Kondo model, which leads to the local non-Fermi-liquid (NFL) ground state, while the magnetic Kondo effect favors the ordinary local Fermi-liquid (FL) ground state. On the basis of the minimal extended two-channel Kondo model including the magnetic Kondo coupling as well, we investigate the competition and resulting thermodynamics, and orbital/magnetic and single-particle excitation spectra by Wilson's numerical renormalization group (NRG) method. There is a first-order transition between the NFL and FL ground states. In addition to these two states, the alternative FL state accompanied by a free magnetic spin appears in the intermediate temperature range, which eventually reaches the true NFL ground state, as a consequence of the stronger competition between the magnetic and quadrupolar Kondo effects. In this peculiar state, the magnetic susceptibility shows a Curie-like behavior, while the orbital fluctuation exhibits the FL behavior. Moreover, the single-particle spectra yield a more singular behavior. Implications to the Pr 1-2-20 systems are briefly discussed.

  11. Leading at the Front: How EB Proteins Regulate Microtubule Dynamics

    NASA Astrophysics Data System (ADS)

    Hawkins, Taviare

    2012-02-01

    Microtubules are the most rigid of the cytoskeletal filaments, they provide the cell's scaffolding, form the byways on which motor proteins transport intracellular cargo and reorganize to form the mitotic spindle when the cell needs to divide. These biopolymers are composed of alpha and beta tubulin monomers that create hollow cylindrical nanotubes with an outer diameter of 25 nm and an inner diameter of 17 nm. At steady state concentrations, microtubules undergo a process known as dynamic instability. During dynamic instability the length of individual microtubules is changing as the filament alternates between periods of growth to shrinkage (catastrophe) and shrinkage to growth (rescue). This process can be enhanced or diminished with the addition of microtubule associated proteins (MAPs). MAPs are microtubule binding proteins that stabilize, destabilize, or nucleate microtubules. We will discuss the effects of the stabilizing end-binding proteins (EB1, EB2 and EB3), on microtubule dynamics observed in vitro. The EBs are a unique family of MAPs known to tip track and enhance microtubule growth by stabilizing the ends. This is a different mechanism than those employed by structural MAPs such as tau or MAP4.

  12. Microtubules: dynamically unstable stochastic phase-switching polymers

    NASA Astrophysics Data System (ADS)

    Zakharov, P. N.; Arzhanik, V. K.; Ulyanov, E. V.; Gudimchuk, N. B.; Ataullakhanov, F. I.

    2016-08-01

    One of the simplest molecular motors, a biological microtubule, is reviewed as an example of a highly nonequilibrium molecular machine capable of stochastic transitions between slow growth and rapid disassembly phases. Basic properties of microtubules are described, and various approaches to simulating their dynamics, from statistical chemical kinetics models to molecular dynamics models using the Metropolis Monte Carlo and Brownian dynamics methods, are outlined.

  13. Microtubule distribution in gravitropic protonemata of the moss Ceratodon

    NASA Technical Reports Server (NTRS)

    Schwuchow, J.; Sack, F. D.; Hartmann, E.

    1990-01-01

    Tip cells of dark-grown protonemata of the moss Ceratodon purpureus are negatively gravitropic (grow upward). They possess a unique longitudinal zonation: (1) a tip group of amylochloroplasts in the apical dome, (2) a plastid-free zone, (3) a zone of significant plastid sedimentation, and (4) a zone of mostly non-sedimenting plastids. Immunofluorescence of vertical cells showed microtubules distributed throughout the cytoplasm in a mostly axial orientation extending through all zones. Optical sectioning revealed a close spatial association between microtubules and plastids. A majority (two thirds) of protonemata gravistimulated for > 20 min had a higher density of microtubules near the lower flank compared to the upper flank in the plastid-free zone. This apparent enrichment of microtubules occurred just proximal to sedimented plastids and near the part of the tip that presumably elongates more to produce curvature. Fewer than 5% of gravistimulated protonemata had an enrichment in microtubules near the upper flank, whereas 14% of vertical protonemata were enriched near one of the side walls. Oryzalin and amiprophos-methyl (APM) disrupted microtubules, gravitropism, and normal tip growth and zonation, but did not prevent plastid sedimentation. We hypothesize that a microtubule redistribution plays a role in gravitropism in this protonema. This appears to be the first report of an effect of gravity on microtubule distribution in plants.

  14. Microtubule-severing enzymes at the cutting edge.

    PubMed

    Sharp, David J; Ross, Jennifer L

    2012-06-01

    ATP-dependent severing of microtubules was first reported in Xenopus laevis egg extracts in 1991. Two years later this observation led to the purification of the first known microtubule-severing enzyme, katanin. Katanin homologs have now been identified throughout the animal kingdom and in plants. Moreover, members of two closely related enzyme subfamilies, spastin and fidgetin, have been found to sever microtubules and might act alongside katanins in some contexts (Roll-Mecak and McNally, 2010; Yu et al., 2008; Zhang et al., 2007). Over the past few years, it has become clear that microtubule-severing enzymes contribute to a wide range of cellular activities including mitosis and meiosis, morphogenesis, cilia biogenesis and disassembly, and migration. Thus, this group of enzymes is revealing itself to be among the most important of the microtubule regulators. This Commentary focuses on our growing understanding of how microtubule-severing enzymes contribute to the organization and dynamics of diverse microtubule arrays, as well as the structural and biophysical characteristics that afford them the unique capacity to catalyze the removal of tubulin from the interior microtubule lattice. Our goal is to provide a broader perspective, focusing on a limited number of particularly informative, representative and/or timely findings.

  15. A RhoGEF and Rho family GTPase-activating protein complex links the contractile ring to cortical microtubules at the onset of cytokinesis.

    PubMed

    Somers, W Gregory; Saint, Robert

    2003-01-01

    The mechanism that positions the cytokinetic contractile ring is unknown, but derives from the spindle midzone. We show that an interaction between the Rho GTP exchange factor, Pebble, and the Rho family GTPase-activating protein, RacGAP50C, connects the contractile ring to cortical microtubules at the site of furrowing in D. melanogaster cells. Pebble regulates actomyosin organization, while RacGAP50C and its binding partner, the Pavarotti kinesin-like protein, regulate microtubule bundling. All three factors are required for cytokinesis. As furrowing begins, these proteins colocalize to a cortical equatorial ring. We propose that RacGAP50C-Pavarotti complexes travel on cortical microtubules to the cell equator, where they associate with the Pebble RhoGEF to position contractile ring formation and coordinate F-actin and microtubule remodeling during cytokinesis.

  16. Microtubule patterning in the presence of moving motor proteins.

    PubMed

    White, D; de Vries, G; Martin, J; Dawes, A

    2015-10-07

    Cytoskeletal polymers such as microtubules (MTs) interact with motor proteins to form higher-order structures. In vitro experiments have shown that MT patterns such as asters, bundles, and vortices can form under the influence of a single type of dynamic motor protein. MTs also can form anti-parallel bundles, similar to bundles that form the mitotic spindle during cell division, under the influence of two types of moving motors with opposite directionality. Despite the importance of MT structures, their mechanism of formation is not yet understood. We develop an integro-partial differential equation model to describe the dynamic interactions between MTs and moving motor proteins. Our model takes into account motor protein speed, processivity, density, and directionality, as well as MT treadmilling and reorganization due to interactions with motors. Simulation results show that plus-end directed motor proteins can form vortex patterns at low motor density, while minus-end directed motor proteins form aster patterns at similar densities. Also, motor proteins with opposite directionality are able to organize MTs into anti-parallel bundles. Our model is able to provide a quantitative and qualitative description of MT patterning, providing insights into possible mechanisms of spindle formation.

  17. Calmodulin immunolocalization to cortical microtubules is calcium independent

    SciTech Connect

    Fisher, D.D.; Cyr, R.J.

    1992-12-31

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 {mu}M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 {mu}M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  18. Calmodulin immunolocalization to cortical microtubules is calcium independent

    SciTech Connect

    Fisher, D.D.; Cyr, R.J.

    1992-01-01

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 [mu]M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  19. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

    PubMed

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W; Gelfand, Vladimir I

    2014-07-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

  20. Assembly and Positioning of Microtubule Asters in Microfabricated Chambers

    NASA Astrophysics Data System (ADS)

    Holy, Timothy E.; Dogterom, Marileen; Yurke, Bernard; Leibler, Stanislas

    1997-06-01

    Intracellular organization depends on a variety of molecular assembly processes; while some of these have been studied in simplified cell-free systems, others depend on the confined geometry of cells and cannot be reconstructed using bulk techniques. To study the latter processes in vitro, we fabricated microscopic chambers that simulate the closed environment of cells. We used these chambers to study the positioning of microtubule asters. Microtubule assembly alone, without the action of molecular motors, is sufficient to position asters. Asters with short microtubules move toward the position expected from symmetry; however, once the microtubules become long enough to buckle, symmetry is broken. Calculations and experiments show that the bending-energy landscape has multiple minima. Microtubule dynamic instability modifies the landscape over time and allows asters to explore otherwise inaccessible configurations.

  1. Molecular mechanism of action of microtubule-stabilizing anticancer agents.

    PubMed

    Prota, Andrea E; Bargsten, Katja; Zurwerra, Didier; Field, Jessica J; Díaz, José Fernando; Altmann, Karl-Heinz; Steinmetz, Michel O

    2013-02-01

    Microtubule-stabilizing agents (MSAs) are efficacious chemotherapeutic drugs widely used for the treatment of cancer. Despite the importance of MSAs for medical applications and basic research, their molecular mechanisms of action on tubulin and microtubules remain elusive. We determined high-resolution crystal structures of αβ-tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane pocket of β-tubulin and used their respective side chains to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. Further, our results offer fundamental structural insights into the control mechanisms of microtubule dynamics.

  2. SEROLOGICAL SIMILARITY OF FLAGELLAR AND MITOTIC MICROTUBULES

    PubMed Central

    Fulton, Chandler; Kane, R. E.; Stephens, R. E.

    1971-01-01

    An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins. PMID:4106543

  3. Transient Pinning and Pulling: A Mechanism for Bending Microtubules

    PubMed Central

    Kent, Ian A.; Rane, Parag S.; Dickinson, Richard B.; Ladd, Anthony J. C.; Lele, Tanmay P.

    2016-01-01

    Microtubules have a persistence length of the order of millimeters in vitro, but inside cells they bend over length scales of microns. It has been proposed that polymerization forces bend microtubules in the vicinity of the cell boundary or other obstacles, yet bends develop even when microtubules are polymerizing freely, unaffected by obstacles and cell boundaries. How these bends are formed remains unclear. By tracking the motions of microtubules marked by photobleaching, we found that in LLC-PK1 epithelial cells local bends develop primarily by plus-end directed transport of portions of the microtubule contour towards stationary locations (termed pinning points) along the length of the microtubule. The pinning points were transient in nature, and their eventual release allowed the bends to relax. The directionality of the transport as well as the overall incidence of local bends decreased when dynein was inhibited, while myosin inhibition had no observable effect. This suggests that dynein generates a tangential force that bends microtubules against stationary pinning points. Simulations of microtubule motion and polymerization accounting for filament mechanics and dynein forces predict the development of bends of size and shape similar to those observed in cells. Furthermore, simulations show that dynein-generated bends at a pinning point near the plus end can cause a persistent rotation of the tip consistent with the observation that bend formation near the tip can change the direction of microtubule growth. Collectively, these results suggest a simple physical mechanism for the bending of growing microtubules by dynein forces accumulating at pinning points. PMID:26974838

  4. Rnd1 regulates axon extension by enhancing the microtubule destabilizing activity of SCG10.

    PubMed

    Li, Ying-Hua; Ghavampur, Sharang; Bondallaz, Percy; Will, Lena; Grenningloh, Gabriele; Püschel, Andreas W

    2009-01-02

    The GTPase Rnd1 affects actin dynamics antagonistically to Rho and has been implicated in the regulation of neurite outgrowth, dendrite development, and axon guidance. Here we show that Rnd1 interacts with the microtubule regulator SCG10. This interaction requires a central domain of SCG10 comprising about 40 amino acids located within the N-terminal-half of a putative alpha-helical domain and is independent of phosphorylation at the four identified phosphorylation sites that regulate SCG10 activity. Rnd1 enhances the microtubule destabilizing activity of SCG10 and both proteins colocalize in neurons. Knockdown of Rnd1 or SCG10 by RNAi suppressed axon extension, indicating a critical role for both proteins during neuronal differentiation. Overexpression of Rnd1 in neurons induces the formation of multiple axons. The effect of Rnd1 on axon extension depends on SCG10. These results indicate that SCG10 acts as an effector downstream of Rnd1 to regulate axon extensions by modulating microtubule organization.

  5. Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules

    PubMed Central

    Wang, Xinnan; Shaw, W. Robert; Tsang, Hilda T. H.; Reid, Evan; O'Kane, Cahir J.

    2008-01-01

    Summary To understand the functions of SPG6, mutated in the neurodegenerative disease hereditary spastic paraplegia, and of ichthyin, mutated in autosomal recessive congenital ichthyosis, we have studied their Drosophila ortholog, spichthyin (Spict). Spict is found on early endosomes. Loss of Spict leads to upregulation of BMP signaling and expansion of the neuromuscular junction. BMP signaling is also necessary for a normal microtubule cytoskeleton and axonal transport; analysis of loss and gain-of-function phenotypes suggests that Spict antagonizes this function of BMP signaling. Spict interacts with BMP receptors and promotes their internalization from the plasma membrane, suggesting that it inhibits BMP signaling by regulating BMP receptor traffic. This is the first demonstration of a role for an SPG protein or ichthyin family member in a specific signaling pathway, and suggests disease mechanisms for hereditary spastic paraplegia that involve dependence of the microtubule cytoskeleton on BMP signaling. PMID:17220882

  6. Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules.

    PubMed

    Wang, Xinnan; Shaw, W Robert; Tsang, Hilda T H; Reid, Evan; O'Kane, Cahir J

    2007-02-01

    To understand the functions of NIPA1, mutated in the neurodegenerative disease hereditary spastic paraplegia, and of ichthyin, mutated in autosomal recessive congenital ichthyosis, we have studied their Drosophila melanogaster ortholog, spichthyin (Spict). Spict is found on early endosomes. Loss of Spict leads to upregulation of bone morphogenetic protein (BMP) signaling and expansion of the neuromuscular junction. BMP signaling is also necessary for a normal microtubule cytoskeleton and axonal transport; analysis of loss- and gain-of-function phenotypes indicate that Spict may antagonize this function of BMP signaling. Spict interacts with BMP receptors and promotes their internalization from the plasma membrane, implying that it inhibits BMP signaling by regulating BMP receptor traffic. This is the first demonstration of a role for a hereditary spastic paraplegia protein or ichthyin family member in a specific signaling pathway, and implies disease mechanisms for hereditary spastic paraplegia that involve dependence of the microtubule cytoskeleton on BMP signaling.

  7. [Dynamics of cytoskeleton microtubules in higher plant meiosis. II. Perinuclear band formation].

    PubMed

    Shamina, N V; Dorogova, N V; Seriukova, E G

    2003-01-01

    Analyses of correspondent meiotic abnormalities is a good tool for studying cytoskeletal rearrangements during plant cell division. The paper reports on the wheat x wheatgrass F1 hybrids, showing various abnormalities during organization of the prophase perinuclear band of microtubules (PNB) in male meiosis. Based on these data, it may be concluded that the perinuclear system of microtubules (MT) in higher plant meiosis is formed from fibrils of the radial system as a result of their translocation in the cell cytoplasm space. According to our data, at this stage the radial MT arrays pass through the following consequence of events: separating from the nuclear envelope, 2) approaching, 3) tangential orientation to the nuclear surface, 4) bending, 5) co-orientation, lateral interaction. As a result, a flat ring of well organized concentric bent MT bundles encircling the nucleus meridionally is organized.

  8. Stimulation of Vesicular Stomatitis Virus in vitro RNA Synthesis by Microtubule-Associated Proteins

    NASA Astrophysics Data System (ADS)

    Hill, Virginia M.; Harmon, Shirley A.; Summers, Donald F.

    1986-08-01

    Microtubule-associated proteins purified from bovine brains stimulated the in vitro transcription and replication reactions of vesicular stomatitis virus. The products of these reactions were intact messenger or genome-sized RNA species. A preparation from HeLa cells containing tubulin and microtubule-associated proteins also stimulated vesicular stomatitis virus transcription in vitro. This observation is in accord with previous studies, which suggested that a host cell factor was involved with the function of the vesicular stomatitis virus RNA polymerase, and others that indicated that several animal viruses displayed an association with host cell cytoskeletal elements during their replication cycles. We show evidence in this report of a host cell protein that seems to have a functional role in interacting with the virion polymerase.

  9. A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain*

    PubMed Central

    McNaughton, Lynn; Tikhonenko, Irina; Banavali, Nilesh K.; LeMaster, David M.; Koonce, Michael P.

    2010-01-01

    Dynein interacts with microtubules through a dedicated binding domain that is dynamically controlled to achieve high or low affinity, depending on the state of nucleotide bound in a distant catalytic pocket. The active sites for microtubule binding and ATP hydrolysis communicate via conformational changes transduced through a ∼10-nm length antiparallel coiled-coil stalk, which connects the binding domain to the roughly 300-kDa motor core. Recently, an x-ray structure of the murine cytoplasmic dynein microtubule binding domain (MTBD) in a weak affinity conformation was published, containing a covalently constrained β+ registry for the coiled-coil stalk segment (Carter, A. P., Garbarino, J. E., Wilson-Kubalek, E. M., Shipley, W. E., Cho, C., Milligan, R. A., Vale, R. D., and Gibbons, I. R. (2008) Science 322, 1691–1695). We here present an NMR analysis of the isolated MTBD from Dictyostelium discoideum that demonstrates the coiled-coil β+ registry corresponds to the low energy conformation for this functional region of dynein. Addition of sequence encoding roughly half of the coiled-coil stalk proximal to the binding tip results in a decreased affinity of the MTBD for microtubules. In contrast, addition of the complete coiled-coil sequence drives the MTBD to the conformationally unstable, high affinity binding state. These results suggest a thermodynamic coupling between conformational free energy differences in the α and β+ registries of the coiled-coil stalk that acts as a switch between high and low affinity conformations of the MTBD. A balancing of opposing conformations in the stalk and MTBD enables potentially modest long-range interactions arising from ATP binding in the motor core to induce a relaxation of the MTBD into the stable low affinity state. PMID:20351100

  10. Association of actin filaments with axonal microtubule tracts.

    PubMed

    Bearer, E L; Reese, T S

    1999-02-01

    Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 microm with some at least as long as 3.5 microm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.

  11. Poleward microtubule flux mitotic spindles assembled in vitro

    PubMed Central

    1991-01-01

    In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase. PMID:1999464

  12. An improved quantitative analysis method for plant cortical microtubules.

    PubMed

    Lu, Yi; Huang, Chenyang; Wang, Jia; Shang, Peng

    2014-01-01

    The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD) algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1) image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM) algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies.

  13. Oscillatory fluid flow influences primary cilia and microtubule mechanics.

    PubMed

    Espinha, Lina C; Hoey, David A; Fernandes, Paulo R; Rodrigues, Hélder C; Jacobs, Christopher R

    2014-07-01

    Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues, such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity.

  14. Jupiter, a new Drosophila protein associated with microtubules.

    PubMed

    Karpova, Nina; Bobinnec, Yves; Fouix, Sylvaine; Huitorel, Philippe; Debec, Alain

    2006-05-01

    In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.

  15. A thermodynamic model of microtubule assembly and disassembly.

    PubMed

    Piette, Bernard M A G; Liu, Junli; Peeters, Kasper; Smertenko, Andrei; Hawkins, Timothy; Deeks, Michael; Quinlan, Roy; Zakrzewski, Wojciech J; Hussey, Patrick J

    2009-08-11

    Microtubules are self-assembling polymers whose dynamics are essential for the normal function of cellular processes including chromosome separation and cytokinesis. Therefore understanding what factors effect microtubule growth is fundamental to our understanding of the control of microtubule based processes. An important factor that determines the status of a microtubule, whether it is growing or shrinking, is the length of the GTP tubulin microtubule cap. Here, we derive a Monte Carlo model of the assembly and disassembly of microtubules. We use thermodynamic laws to reduce the number of parameters of our model and, in particular, we take into account the contribution of water to the entropy of the system. We fit all parameters of the model from published experimental data using the GTP tubulin dimer attachment rate and the lateral and longitudinal binding energies of GTP and GDP tubulin dimers at both ends. Also we calculate and incorporate the GTP hydrolysis rate. We have applied our model and can mimic published experimental data, which formerly suggested a single layer GTP tubulin dimer microtubule cap, to show that these data demonstrate that the GTP cap can fluctuate and can be several microns long.

  16. Selective adhesion of functional microtubules to patterned silane surfaces.

    PubMed Central

    Turner, D C; Chang, C; Fang, K; Brandow, S L; Murphy, D B

    1995-01-01

    We show that microtubule polymers can be immobilized selectively on lithographically patterned silane surfaces while retaining their native properties. Silane films were chemisorbed on polished silicon wafers or glass coverslips and patterned using a deep UV lithographic process developed at the Naval Research Laboratory. Hydrocarbon and fluorocarbon alkyl silanes, as well as amino and thiol terminal alkyl silanes, were investigated as substrates for microtubule adhesion with retention of biological activity. Microtubules were found to adhere strongly to amine terminal silanes while retaining the ability to act as substrates for the molecular motor protein kinesin. Aminosilane patterns with linewidths varying from 1 to 50 microns were produced lithographically and used to produce patterns of selectively adhered microtubules. Microtubules were partially aligned on the patterned lines by performing the immobilization in a fluid flow field. Patterns were imaged with atomic force microscopy and differential interference contrast microscopy. Motility assays were carried out using kinesin-coated beads and observed with differential interference contrast microscopy. Kinesin bead movement on the patterned microtubules was comparable to movement on microtubule control surfaces. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:8599684