Sample records for axonemal microtubule doublets

  1. 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.

  2. 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

  3. Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas.

    PubMed

    Finst, R J; Kim, P J; Griffis, E R; Quarmby, L M

    2000-06-01

    A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca(2+)-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca(2+)-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca(2+)/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca(2+)-activated, Ca(2+)-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing. PMID:10806107

  4. Displacement-Weighted Velocity Analysis of Gliding Assays Reveals that Chlamydomonas Axonemal Dynein Preferentially Moves Conspecific Microtubules

    PubMed Central

    Alper, Joshua D.; Tovar, Miguel; Howard, Jonathon

    2013-01-01

    In vitro gliding assays, in which microtubules are observed to glide over surfaces coated with motor proteins, are important tools for studying the biophysics of motility. Gliding assays with axonemal dyneins have the unusual feature that the microtubules exhibit large variations in gliding speed despite measures taken to eliminate unsteadiness. Because axonemal dynein gliding assays are usually done using heterologous proteins, i.e., dynein and tubulin from different organisms, we asked whether the source of tubulin could underlie the unsteadiness. By comparing gliding assays with microtubules polymerized from Chlamydomonas axonemal tubulin with those from porcine brain tubulin, we found that the unsteadiness is present despite matching the source of tubulin to the source of dynein. We developed a novel, to our knowledge, displacement-weighted velocity analysis to quantify both the velocity and the unsteadiness of gliding assays systematically and without introducing bias toward low motility. We found that the quantified unsteadiness is independent of tubulin source. In addition, we found that the short Chlamydomonas microtubules translocate significantly faster than their porcine counterparts. By modeling the effect of length on velocity, we propose that the observed effect may be due to a higher rate of binding of Chlamydomonas axonemal dynein to Chlamydomonas microtubules than to porcine microtubules. PMID:23663842

  5. Determination of Microtubule Polarity in Vitroby the Use of Video-Enhanced Differential-Interference Contrast Light Microscopy and ChlamydomonasFlagellar Axonemal Pieces

    Microsoft Academic Search

    Chris T. Gamblin

    1995-01-01

    Microtubules nucleated by sea urchin sperm-tail axonemes have polar ends that differ both functionally and structurally but cannot be distinguished from one another when viewed by light microscopy. Ambiguity and circularity surround any classification of microtubule polarity by conventional methods.Chlamydomonasflagellar axonemal pieces have distinct morphological differences at their plus- and minus-ends, and microtubules nucleated from these pieces can be distinguished

  6. 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. PMID:24574454

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

    PubMed Central

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

    2014-01-01

    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. PMID:24574454

  8. Integrated Control of Axonemal Dynein AAA+ Motors

    PubMed Central

    King, Stephen M.

    2012-01-01

    Axonemal dyneins are AAA+ enzymes that convert ATP hydrolysis to mechanical work. This leads to the sliding of doublet microtubules with respect to each other and ultimately the generation of ciliary/flagellar beating. However, in order for useful work to be generated, the action of individual dynein motors must be precisely controlled. In addition, cells modulate the motility of these organelles through a variety of second messenger systems and these signals too must be integrated by the dynein motors to yield an appropriate output. This review describes the current status of efforts to understand dynein control mechanisms and their connectivity focusing mainly on studies of the outer dynein arm from axonemes of the unicellular biflagellate green alga Chlamydomonas. PMID:22406539

  9. Functional diversity of axonemal dyneins as assessed by in vitro and in vivo motility assays of Chlamydomonas mutants.

    PubMed

    Kamiya, Ritsu; Yagi, Toshiki

    2014-10-01

    This review outlines the current knowledge of the functional diversity of axonemal dyneins, as revealed by studies with the model organism Chlamydomonas. Axonemal dyneins, which comprise outer and inner dynein arms, power cilia and flagella beating by producing sliding movements between adjacent outer-doublet microtubules. Outer- and inner-arm dyneins have traditionally been considered similar in structure and function. However, recent evidence suggests that they differ rather strikingly in subunit composition, axonemal arrangement, and molecular motor properties. We posit that these arms make up two largely independent motile systems; whereas outer-arm dynein can generate axonemal beating by itself under certain conditions, inner-arm dynein can generate beating only in cooperation with the central pair/radial spokes. This conclusion is supported by genome analyses of various organisms. Outer-arm dynein appears to be particularly important for nodal cilia of mammalian embryos that function for determination of left-right body asymmetry. PMID:25284382

  10. Rebinding of Tetrahymena 13 S and 21 S dynein ATPases to extracted doublet microtubules. The inner row and outer row dynein arms.

    PubMed

    Warner, F D; Perreault, J G; McIlvain, J H

    1985-08-01

    Ciliary axonemes from Tetrahymena contain a second salt-extractable ATPase distinguishable from outer arm 21 S dynein by sedimentation velocity (congruent to 13 S), electrophoretic mobility and substrate specificity. As characterized by turbidimetric assay, gel electrophoresis in the presence of sodium dodecyl sulphate, ATPase activity and electron microscopy, the 13 S dynein ATPase rebinds to extracted doublet microtubules. Compared to structural-side (ATP-insensitive) 21 S dynein binding, which is moderately specific for the 24 nm outer row arm position, rebinding of 13 S dynein is highly specific but for the inner row arm position. However, 13 S dynein rebinds to the A subfibre with a spacing that coincides with the triplet spacing of the radial spokes (24-32-40 nm periods; 96 nm repeat). All of the major protein components present in the 13 S or 21 S fractions rebind to extracted doublets under conditions that both restore and activate dynein ATPase activity. Unlike active-side (ATP-sensitive) rebound 21 S dynein, rebound 13 S dynein is completely insensitive to dissociation by ATP-vanadate and does not independently decorate the B subfibre. The saturation profile for rebinding of 13 S dynein exhibits a lack of cooperativity between binding events (h = 1.0) similar to structural-side rebinding of 21 S dynein. At low 21 S/doublet stoichiometry there is no measureable competition between the 13 S and 21 S dyneins for binding sites on the A subfibre lattice, although at saturating concentrations of 21 S dynein, rebinding of 13 S dynein is blocked completely. PMID:2935546

  11. Morphological changes of wrasse sperm axoneme after their motility initiation observed with use of atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Shimizu, Hideaki; Majima, Toshikazu; Takai, Hiroyuki; Inaba, Kazuo; Tomie, Toshihisa

    1995-03-01

    The sperm of bambooleaf wrasse, a marine teleost, are immotile when they are diluted in a solution isotonic to the seminal plasma, but they begin to swim when they are suspended in sea water. What changes arise in morphology of the sperm cell after the motility initiation? The semen collected from the abdomen of a matured wrasse was mixed with either thinned sea water or sea water. A drop of the same specimen was placed on a cleaned silicon wafer, respectively. After fixed chemically, they were rinsed with distilled water and dried naturally in room temperature. These samples were examined carefully with use of an atomic force microscopy. Although the axonemes of intact sperms were found to be crushed as if the axonemes were cut open along doublet microtubules. The motility initiated sperm was strong enough to resist the force caused by surface tension of water in the drying process and could maintain the structure of the axoneme. These experimental facts suggest that the binding characteristics in the structure of the axoneme after the initiation of the motility were clearly changed stronger that before.

  12. Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme

    PubMed Central

    Owa, Mikito; Furuta, Akane; Usukura, Jiro; Arisaka, Fumio; King, Stephen M.; Witman, George B.; Kamiya, Ritsu; Wakabayashi, Ken-ichi

    2014-01-01

    Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA–DC has an ellipsoidal shape ?24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity. PMID:24979786

  13. Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme.

    PubMed

    Owa, Mikito; Furuta, Akane; Usukura, Jiro; Arisaka, Fumio; King, Stephen M; Witman, George B; Kamiya, Ritsu; Wakabayashi, Ken-ichi

    2014-07-01

    Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA-DC has an ellipsoidal shape ?24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity. PMID:24979786

  14. Ciliary reversal without rotation of axonemal structures in ctenophore comb plates

    PubMed Central

    1981-01-01

    We have used a newly discovered reversal response of ctenophore comb plates to investigate the structural mechanisms controlling the direction of ciliary bending. High K+ concentrations cause cydippid larvae of the ctenophore Pleurobrachia to swim backward. High-speed cine films of backward-swimming animals show a 180 degree reversal in beat direction of the comb plates. Ion substitution and blocking experiments with artificial seawaters demonstrate that ciliary reversal is a Ca++-dependent response. Comb plate cilia possess unique morphological markers for numbering specific outer-doublet microtubules and identifying the sidedness of the central pair. Comb plates of forward- and backward-swimming ctenophores were frozen in different stages of the beat cycle by an "instantaneous fixation" method. Analysis of transverse and longitudinal sections of instantaneously fixed cilia showed that the assembly of outer doublets does not twist during ciliary reversal. This directly confirms the existence of radial switching mechanism regulating the sequence of active sliding on opposite sides of the axoneme. We also found that the axis of the central pair always remains perpendicular to the plane of bending; more importantly, the ultrastructural marker showed that the central pair does not rotate during a 180 degree reversal in beat direction. Thus, the orientation of the central pair does not control the direction of ciliary bending (i.e., the pattern of active sliding around the axoneme). We discuss the validity of this finding for three-dimensional as well as two-dimensional ciliary beat cycles and conclude that models of central-pair function based on correlative data alone must now be re- examined in light of these new findings on causal relations. PMID:6114102

  15. Structural Analysis of Mutations in the Drosophila ?2-Tubulin Isoform Reveals Regions in the ?-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 ?Tub85D, the gene that encodes the testis-specific ?2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the ?2-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 ?-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 ?-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 ?3, a developmentally regulated Drosophila ?-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type ?2-tubulin. We show here by complementation analysis that ?3 and the B2t(6) product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the ?2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the ?2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the ?2 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 comparisons of vertebrate ?-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type ?-tubulins. We propose that the integrity of this structure in the Drosophila testis ?2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics. PMID:7705629

  16. Glutamylated and glycylated tubulin isoforms in the aberrant sperm axoneme of the gall-midge fly, Asphondylia ruebsaameni.

    PubMed

    Mencarelli, Caterina; Caroti, Daniela; Bré, Marie-Hélène; Levilliers, Nicolette; Mercati, David; Robbins, Leonard G; Dallai, Romano

    2004-07-01

    The axonemal organization expressed in the sperm flagella of the cecidomyiid dipteran Asphondylia ruebsaameni is unconventional, being characterized by the presence of an exceedingly high number of microtubular doublets and by the absence of both the inner dynein arms and the central pair/radial spoke complex. Consequently, its motility, both in vivo and in vitro, is also peculiar. Using monoclonal antibodies directed against posttranslational modifications, we have analyzed the presence and distribution of glutamylated and glycylated tubulin isoforms in this aberrant axonemal structure, and compared them with those of a reference insect species (Apis mellifera), endowed with a conventional axoneme. Our results have shown that the unorthodox structure and motility of the Asphondylia axoneme are concomitant with: (1). a very low glutamylation extent in the alpha-tubulin subunit, (2). a high level of glutamylation in the beta-subunit, (3). an extremely low total extent of glycylation, with regard to both monoglycylated and polyglycylated sites, either in alpha- or in beta-tubulin, (4). the presence of a strong labeling of glutamylated tubulin isoforms at the proximal end of the axoneme, and (5). a uniform distribution of glutamylated as well as glycylated isoforms along the rest of the axoneme. Thus, our data indicate that tubulin molecular heterogeneity is much lower in the Asphondylia axoneme than in the conventional 9+2 axoneme with regard to both isoform content and isoform distribution along the axoneme. PMID:15146535

  17. An Integrative Model of Internal Axoneme Mechanics and External Fluid Dynamics in Ciliary Beating

    Microsoft Academic Search

    ROBERT H. DILLON; LISA J. FAUCI

    2000-01-01

    We present a fluid-mechanical model of an individual cilium which incorporates discrete representations of the dynein arms, the passive elastic structure of the axoneme including the microtubules and nexin links. This model, based upon the immersed boundary method (Peskin, 1977), couples the internal force generation of the molecular motors through the passive elastic structure with the external fluid mechanics governed

  18. Morphogenesis of the giant sperm axoneme in Asphondylia ruebsaameni Kertesz (Diptera, Cecidomyiidae).

    PubMed

    Mencarelli, C; Lupetti, P; Rosetto, M; Dallai, R

    2000-04-01

    The formation of the sperm giant axoneme of the gall-midge fly Asphondylia ruebsaameni is described here. The axoneme consists of a great number of microtubular doublets (up to 2,500) arranged in a double spiral wrapping around an axial cluster of mitochondria. Each microtubular doublet is provided with an outer arm only. In the early spermatid the occurrence of a large system of curved multi-layered filamentous material associated with membranous cisternae has been observed in the perinuclear region. Such a system extends throughout the cytoplasm to contact the plasma membrane. The filamentous material appears to act as a nucleating centre for the assembly of the microtubular doublets, which initially have a submembranous location and later are distributed in the interior of the cell. After their assembly, microtubular doublets are associated pairwise and are arranged in a single microtubular row with a zig-zag configuration. This configuration changes during spermiogenesis as a consequence both of a rotation of the microtubular doublet pairs and a compaction of the axonemal complex due to the elimination of the excess cytoplasm. As a result of this process, a double parallel spiral of microtubular doublets is formed. PMID:10855705

  19. Cloning, Localization, and Axonemal Function of Tetrahymena Centrin

    PubMed Central

    Guerra, Charles; Wada, Yuuko; Leick, Vagn; Bell, Aaron; Satir, Peter

    2003-01-01

    Centrin, an EF hand Ca2+ binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca2+ control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca2+ or the Ca2+-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca2+, increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca2+ responses, including ciliary reversal or chemotaxis. PMID:12529441

  20. EFFECT OF SOLUTION COMPOSITION AND PROTEOLYSIS ON THE CONFORMATION OF AXONEMAL COMPONENTS

    PubMed Central

    Zobel, C. Richard

    1973-01-01

    The effect of solution composition and enzymic proteolysis on axonemes prepared from the sperm of sea urchins, Tripneustes gratilla, has been investigated. Aliquots of axonemes, prepared by treatment of sperm with Triton X-100 and differential centrifugation, were transferred to solutions of different composition with and without intervening tryptic proteolysis, and the particle conformations observed by dark-field and electron microscopy. In most solutions particles in partially digested preparations underwent conformational transformations to coiled or helix-like forms. Proteolysis was accompanied by an increase in the ATPase activity of the digest: by centrifuging down the insoluble digestion products it was shown that digestion resulted in the appearance of ATPase activity in the soluble phase with a concomitant decrease in ATPase activity in the pellet fraction. Gel electrophoresis showed this corresponded to the appearance of dynein in the supernatant and a decrease in dynein associated with the insoluble fraction. Supernatant dynein had a greater specific ATPase activity than dynein extracted from axonemes. Observations on specimens prepared for electron microscopy by thin sectioning allowed a rough correlation to be made between the dark-field observations, chemical analyses, and morphological alterations attendant with the proteolysis and solution conditions. It is concluded that in the intact axoneme the doublet tubules are under considerable tension and that proteolytic destruction of physical restraining elements allows spontaneous conformational alterations of the digestion products. In addition, proteolysis increases the specific ATPase activity of dynein and removes a portion of it from the axonemal structure. PMID:4271496

  1. Torque generation by axonemal outer-arm Dynein.

    PubMed

    Yamaguchi, Shin; Saito, Kei; Sutoh, Miki; Nishizaka, Takayuki; Toyoshima, Yoko Y; Yajima, Junichiro

    2015-02-17

    Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (???) as well as proteolytically generated two-headed (??) and one-headed (?) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in microtubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed ??? and one-headed ? exhibited significantly longer pitch. In contrast, the pitch driven by two-headed ?? did not display this sensitivity. In the assays on lawns containing mixtures of ? and ?? at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of ? in the mixture increased. Even small proportions of ?-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (???) but is an intrinsic property of the subparticles of axonemal dyneins and also suggest that each subparticle may have distinct mechanical properties. PMID:25692592

  2. Tubulin polyglutamylation regulates axonemal motility by modulating activities of inner-arm dyneins.

    PubMed

    Kubo, Tomohiro; Yanagisawa, Haru-aki; Yagi, Toshiki; Hirono, Masafumi; Kamiya, Ritsu

    2010-03-01

    Tubulin polyglutamylation is a modification that adds multiple glutamates to the gamma-carboxyl group of a glutamate residue in the C-terminal tails of alpha- and beta-tubulin [1, 2]. This modification has been implicated in the regulation of axonal transport and ciliary motility. However, its molecular function in cilia remains unknown. Here, using a novel Chlamydomonas reinhardtii mutant (tpg1) that lacks a homolog of human TTLL9, a glutamic acid ligase enzyme [3], we found that the lack of a long polyglutamate side chain in alpha-tubulin moderately weakens flagellar motility without noticeably impairing the axonemal structure. Furthermore, the double mutant of tpg1 with oda2, a mutation that leads to loss of outer-arm dynein, completely lacks motility. More surprisingly, when treated with protease and ATP, the axoneme of this paralyzed double mutant displayed faster microtubule sliding than the motile oda2 axoneme. These and other results suggest that polyglutamylation directly regulates microtubule-dynein interaction mainly by modulating the function of inner-arm dyneins. PMID:20188560

  3. Release of intact microtubule-capping structures from Tetrahymena cilia

    E-print Network

    Suprenant, Kathy A.; Dentler, William L., Jr

    1988-12-01

    microtubule caps, are selectively and independently released from the axoneme by CaCl2 and MgCl2 but not by MgSO4, ZnCl2, NaCl, KCl, or KI. The release of the caps and filaments is specific for Ca+2, Mg+2, and Cl- and is not simply a function of ionic strength...

  4. XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover

    PubMed Central

    1994-01-01

    We have used video-enhanced DIC microscopy to examine the effects of XMAP, a Mr 215,000 microtubule-associated protein from Xenopus eggs (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), on the dynamic instability of microtubules nucleated from axoneme fragments in vitro. Our results indicate that XMAP substantially alters the parameters of microtubule assembly at plus ends. Specifically, addition of 0.2 microM XMAP resulted in (a) 7-10-fold increase in elongation velocity, (b) approximately threefold increase in shortening velocity, and (c) near elimination of rescue (the switch from rapid shortening to elongation). Thus, addition of XMAP resulted in the assembly of longer, but more dynamic, microtubules from the plus ends of axonemes which upon catastrophe disassembled back to the axoneme nucleation site. In agreement with previous observations (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), the effects of XMAP on the minus end were much less dramatic, with only a 1.5-3-fold increase in elongation velocity. These results indicate that XMAP, unlike brain MAPs, promotes both polymer assembly and turnover, and suggests that the interaction of XMAP with tubulin and the function of XMAP in vivo may differ from previously characterized MAPs. PMID:7962080

  5. Polarity and asymmetry in the arrangement of dynein and related structures in the Chlamydomonas axoneme

    PubMed Central

    Bui, Khanh Huy; Yagi, Toshiki; Yamamoto, Ryosuke; Kamiya, Ritsu

    2012-01-01

    Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer–inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella. PMID:22945936

  6. Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis

    E-print Network

    Dentler, William L., Jr

    1980-02-01

    - nemal microtubules)? Certainly, the principal method used to isolate the protein, detergent-ex- traction of the whole cilium, should solubilize both membrane and matrix proteins . The axonemal microtubules, however, were not significantly dis- rupted... with tubulin obtained by solubilization of ciliary microtubules . The tryptic peptide maps are not, however, identical. Similar results were reported in membrane fractions prepared by detergent ex- traction of scallop gill cilia (42) . Other differences also...

  7. A Metastable Intermediate State of Microtubule Dynamic Instability That Differs Significantly between Plus and Minus Ends

    Microsoft Academic Search

    P. T. Tran; R. A. Walker; E. D. Salmon

    1997-01-01

    The current two-state GTP cap model of mi- crotubule dynamic instability proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation while loss of this GTP-tubulin cap converts the microtubule end to shortening. How- ever, when this model was directly tested by using a UV microbeam to sever axoneme-nucleated micro- tubules and thereby remove the microtubule's

  8. The Small GTPase Rsg1 is important for the cytoplasmic localization and axonemal dynamics of intraflagellar transport proteins

    PubMed Central

    2013-01-01

    Background Cilia are small, microtubule-based protrusions important for development and homeostasis. We recently demonstrated that the planar cell polarity effector protein Fuz is a critical regulator of axonemal intraflagellar transport dynamics and localization. Here, we report our findings on the role of the small GTPase Rsg1, a known binding partner of Fuz, and its role in the dynamics and cytoplasmic localization of intraflagellar transport proteins. Results We find that Rsg1 loss of function leads to impaired axonemal IFT dynamics in multiciliated cells. We further show that Rsg1 is required for appropriate cytoplasmic localization of the retrograde IFT-A protein IFT43. Finally, we show that Rsg1 governs the apical localization of basal bodies, the anchoring structures of cilia. Conclusions Our data suggest that Rsg1 is a regulator of multiple aspects of ciliogenesis, including apical trafficking of basal bodies and the localization and dynamics intraflagellar transport proteins. PMID:24192041

  9. A novel role of centrin in flagellar motility: stabilizing an inner-arm dynein motor in the flagellar axoneme

    PubMed Central

    Li, Ziyin

    2015-01-01

    Centrin is an evolutionarily conserved EF-hand calcium-binding protein found in the centriole of animals and the basal body of flagellated organisms. It was originally discovered in the flagellated unicellular green alga Chlamydomonas reinhardtii, where it associates with flagellum-associated structures and regulates basal body duplication and flagellar motility. Centrin constitutes a light chain of three inner-arm dynein complexes in the flagellar axoneme in Chlamydomonas, and presumably regulates the activity of the inner-arm dynein for flagellar motility. In the ciliated organism Tetrahymena, centrin also associates with the inner-arm dynein and appears to regulate the microtubule sliding velocity of the inner-arm dynein. Using Trypanosoma brucei as the model organism, we discovered that centrin maintains the stability of an inner-arm dynein in the flagellar axoneme [Wei et al., (2014) Nat. Commun 5: 4060]. T. brucei expresses five centrins, three of which, TbCentrin1, 2, and 4, associate with the flagellar basal body, but no centrin was found to regulate cell motility. We found that TbCentrin3 associates tightly with the flagellum and that RNAi of TbCentrin3 compromised cell motility. Biochemical approaches further showed that TbCentrin3 interacts with TbIAD5-1, an inner-arm dynein in the flagellar axoneme. Knockdown of TbIAD5-1 also caused defective cell motility. Strikingly, depletion of TbCentrin3 or depletion of TbIAD5-1 resulted in disassembly of the complex from the axoneme and subsequent degradation of the complex in the cytosol. Our findings identified a novel role of TbCentrin3 in cell motility by stabilizing TbIAD5-1 in the axoneme, which likely is well conserved in other flagellated and ciliated organisms, such as Chlamydomonas and Tetrahymena where centrin is also known to associate with inner-arm dyneins.

  10. Pih1d3 is required for cytoplasmic preassembly of axonemal dynein in mouse sperm

    PubMed Central

    Dong, Fenglan; Shinohara, Kyosuke; Botilde, Yanick; Nabeshima, Ryo; Asai, Yasuko; Fukumoto, Akemi; Hasegawa, Toshiaki; Matsuo, Moe; Takeda, Hiroyuki; Shiratori, Hidetaka; Nakamura, Tetsuya

    2014-01-01

    Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain–containing protein, develop normally but manifest male sterility. Pih1d3?/? sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins. PMID:24421334

  11. A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas flagellum

    PubMed Central

    1994-01-01

    The kinesin superfamily of mechanochemical proteins has been implicated in a wide variety of cellular processes. We have begun studies of kinesins in the unicellular biflagellate alga, Chlamydomonas reinhardtii. A full-length cDNA, KLP1, has been cloned and sequenced, and found to encode a new member of the kinesin superfamily. An antibody was raised against the nonconserved tail region of the Klp1 protein, and it was used to probe for Klp1 in extracts of isolated flagella and in situ. Immunofluorescence of whole cells indicated that Klp1 was present in both the flagella and cell bodies. In wild-type flagella, Klp1 was found tightly to the axoneme; immunogold labeling of wild-type axonemal whole mounts showed that Klp1 was restricted to one of the two central pair microtubules at the core of the axoneme. Klp1 was absent from the flagella of mutants lacking the central pair microtubules, but was present in mutant flagella from pf16 cells, which contain an unstable C1 microtubule, indicating that Klp1 was bound to the C2 central pair microtubule. Localization of Klp1 to the C2 microtubule was confirmed by immunogold labeling of negatively stained and thin-sectioned axonemes. These findings suggest that Klp1 may play a role in rotation or twisting of the central pair microtubules. PMID:8207060

  12. Concentration dependence of variability in growth rates of microtubules.

    PubMed Central

    Pedigo, Susan; Williams, Robley C

    2002-01-01

    Growth and shortening of microtubules in the course of their polymerization and depolymerization have previously been observed to occur at variable rates. To gain insight into the meaning of this prominent variability, we studied the way in which its magnitude depends on the growth rate of experimentally observed and computer-simulated microtubules. The dynamic properties of plus-ended microtubules nucleated by pieces of Chlamydomonas flagellar axonemes were observed in real time by video-enhanced differential interference contrast light microscopy at differing tubulin concentrations. By means of a Monte Carlo algorithm, populations of microtubules were simulated that had similar growth and dynamic properties to the experimentally observed microtubules. By comparison of the experimentally observed and computer-simulated populations of microtubules, we found that 1) individual microtubules displayed an intrinsic variability that did not change as the rate of growth for a population increased, and 2) the variability was approximately fivefold greater than predicted by a simple model of subunit addition and loss. The model used to simulate microtubule growth has no provision for incorporation of lattice defects of any type, nor sophisticated geometry of the growing end. Thus, these as well as uncontrolled experimental variables were eliminated as causes for the prominent variability. PMID:12324403

  13. 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

  14. An Electron Microscope Study of the Axonemal Ultrastructure in Human Spermatozoa From Male Smokers and Nonsmokers

    Microsoft Academic Search

    Panayiotis M. Zavos; Juan R. Correa; Christos S. Karagounis; Andrea Ahparaki; Christa Phoroglou; Clair L. Hicks; Panayota N. Zarmakoupis-Zavos

    1998-01-01

    Objective: To investigate possible abnormalities or deterioration of the sperm axonemal ultrastructure in men who have smoked a large quantity of cigarettes (>20 per day) for a prolonged period.Design: Semen specimens were collected by patients via masturbation; qualitative characteristics of the sperm were assessed and ultrastructural analysis of the sperm axoneme was performed using standard operating procedures for electron transmission

  15. Splice-Site Mutations in the Axonemal Outer Dynein Arm Docking Complex Gene CCDC114 Cause Primary Ciliary Dyskinesia

    PubMed Central

    Onoufriadis, Alexandros; Paff, Tamara; Antony, Dinu; Shoemark, Amelia; Micha, Dimitra; Kuyt, Bertus; Schmidts, Miriam; Petridi, Stavroula; Dankert-Roelse, Jeanette E.; Haarman, Eric G.; Daniels, Johannes M.A.; Emes, Richard D.; Wilson, Robert; Hogg, Claire; Scambler, Peter J.; Chung, Eddie M.K.; Pals, Gerard; Mitchison, Hannah M.

    2013-01-01

    Defects in motile cilia and sperm flagella cause primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right laterality disturbances, usually as a result of loss of the outer dynein arms (ODAs) that power cilia/flagella beating. Here, we identify loss-of-function mutations in CCDC114 causing PCD with laterality malformations involving complex heart defects. CCDC114 is homologous to DCC2, an ODA microtubule-docking complex component of the biflagellate alga Chlamydomonas. We show that CCDC114 localizes along the entire length of human cilia and that its deficiency causes a complete absence of ciliary ODAs, resulting in immotile cilia. Thus, CCDC114 is an essential ciliary protein required for microtubular attachment of ODAs in the axoneme. Fertility is apparently not greatly affected by CCDC114 deficiency, and qPCR shows that this may explained by low transcript expression in testis compared to ciliated respiratory epithelium. One CCDC114 mutation, c.742G>A, dating back to at least the 1400s, presents an important diagnostic and therapeutic target in the isolated Dutch Volendam population. PMID:23261303

  16. Essential and synergistic roles of RP1 and RP1L1 in rod photoreceptor axoneme and retinitis pigmentosa

    PubMed Central

    Yamashita, Tetsuji; Liu, Jiewu; Gao, Jiangang; LeNoue, Sean; Wang, Changguan; Kaminoh, Jack; Bowne, Sara J.; Sullivan, Lori S.; Daiger, Stephen P.; Zhang, Kang; Fitzgerald, Malinda E.C.; Kefalov, Vladimir J.; Zuo, Jian

    2009-01-01

    Retinitis pigmentosa 1 (RP1) is a common inherited retinopathy with variable onset and severity. The RP1 gene encodes a photoreceptor-specific, microtubule-associated ciliary protein containing the doublecortin (DCX) domain. Here we show that another photoreceptor-specific Rp1-like protein (Rp1L1) in mice is also localized to the axoneme of outer segments (OS) and connecting cilia in rod photoreceptors, overlapping with Rp1. Rp1L1?/? mice display scattered OS disorganization, reduced electroretinogram amplitudes, and progressive photoreceptor degeneration, less severe and slower than in Rp1?/? mice. In single rods of Rp1L1?/?, photosensitivity is reduced, similar to that of Rp1?/?. While individual heterozygotes are normal, double heterozygotes of Rp1 and Rp1L1 exhibit abnormal OS morphology and reduced single rod photosensitivity and dark currents. The electroretinogram amplitudes of double heterozygotes are more reduced than those of individual heterozygotes combined. In support, Rp1L1 interacts with Rp1 in transfected cells and in retina pull-down experiments. Interestingly, phototransduction kinetics are normal in single rods and whole retinas of individual or double Rp1 and Rp1L1 mutant mice. Together, Rp1 and Rp1L1 play essential and synergistic roles in affecting photosensitivity and OS morphogenesis of rod photoreceptors. Our findings suggest that mutations in RP1L1 could underlie retinopathy or modify RP1 disease expression in humans. PMID:19657028

  17. Heterotrimeric Kinesin-II Is Required for the Assembly of Motile 9+2 Ciliary Axonemes on Sea Urchin Embryos

    PubMed Central

    Morris, Robert L.; Scholey, Jonathan M.

    1997-01-01

    Heterotrimeric kinesin-II is a plus end– directed microtubule (MT) motor protein consisting of distinct heterodimerized motor subunits associated with an accessory subunit. To probe the intracellular transport functions of kinesin-II, we microinjected fertilized sea urchin eggs with an anti–kinesin-II monoclonal antibody, and we observed a dramatic inhibition of ciliogenesis at the blastula stage characterized by the assembly of short, paralyzed, 9+0 ciliary axonemes that lack central pair MTs. Control embryos show no such defect and form swimming blastulae with normal, motile, 9+2 cilia that contain kinesin-II as detected by Western blotting. Injection of anti–kinesin-II into one blastomere of a two-cell embryo leads to the development of chimeric blastulae covered on one side with short, paralyzed cilia, and on the other with normal, beating cilia. We observed a unimodal length distribution of short cilia on anti–kinesin-II–injected embryos corresponding to the first mode of the trimodal distribution of ciliary lengths observed for control embryos. This short mode may represent a default ciliary assembly intermediate. We hypothesize that kinesin-II functions during ciliogenesis to deliver ciliary components that are required for elongation of the assembly intermediate and for formation of stable central pair MTs. Thus, kinesin-II plays a critical role in embryonic development by supporting the maturation of nascent cilia to generate long motile organelles capable of producing the propulsive forces required for swimming and feeding. PMID:9281580

  18. Three-dimensional structures of the flagellar dynein–microtubule complex by cryoelectron microscopy

    PubMed Central

    Oda, Toshiyuki; Hirokawa, Nobutaka; Kikkawa, Masahide

    2007-01-01

    The outer dynein arms (ODAs) of the flagellar axoneme generate forces needed for flagellar beating. Elucidation of the mechanisms underlying the chemomechanical energy conversion by the dynein arms and their orchestrated movement in cilia/flagella is of great importance, but the nucleotide-dependent three-dimensional (3D) movement of dynein has not yet been observed. In this study, we establish a new method for reconstructing the 3D structure of the in vitro reconstituted ODA–microtubule complex and visualize nucleotide-dependent conformational changes using cryoelectron microscopy and image analysis. As the complex went from the rigor state to the relaxed state, the head domain of the ? heavy chain shifted by 3.7 nm toward the B tubule and inclined 44° inwards. These observations suggest that there is a mechanism that converts head movement into the axonemal sliding motion. PMID:17438074

  19. Microtubules in root hairs.

    PubMed

    Traas, J A; Braat, P; Emons, A M; Meekes, H; Derksen, J

    1985-06-01

    The microtubules of root hairs of Raphanus sativus, Lepidium sativum, Equisetum hyemale, Limnobium stoloniferum, Ceratopteris thalictroides, Allium sativum and Urtica dioica were investigated using immunofluorescence and electron microscopy. Arrays of cortical microtubules were observed in all hairs. The microtubules in the hairs show net axial orientations, but in Allium and Urtica helical microtubule patterns are also present. Numerical parameters of microtubules in Raphanus, Equisetum and Limnobium were determined from dry-cleave preparations. The results are discussed with respect to cell wall deposition and cell morphogenesis. PMID:4066793

  20. Massive Doublet Leptons

    E-print Network

    Scott Thomas; James D. Wells

    1998-04-21

    Massive vector-like electroweak doublets are generic in many extensions of the standard model. Even though one member of the doublet is necessarily electrically charged these particles are not easily detected in collider experiments. The neutral and charged states within the doublet are split by electroweak symmetry breaking. In the absence of mixing with other states, the radiatively generated splitting is in the range 200 - 350 MeV for m > mZ/2. The charged state decays to the neutral one with an O(cm) decay length, predominantly by emission of a soft charged pion. The best possibility to detect these massive charged particles is to trigger on hard initial state radiation and search for two associated soft charged pions with displaced vertices. The mass reach for this process at LEPII is limited by luminosity rather than kinematics.

  1. Cross-reactivity of the BRAF VE1 antibody with epitopes in axonemal dyneins leads to staining of cilia.

    PubMed

    Jones, Robert T; Abedalthagafi, Malak S; Brahmandam, Mohan; Greenfield, Edward A; Hoang, Mai P; Louis, David N; Hornick, Jason L; Santagata, Sandro

    2014-11-21

    Antibodies that recognize neo-epitopes in tumor cells are valuable tools in the evaluation of tissue biopsy or resection specimens. The VE1 antibody that recognizes the V600E-mutant BRAF protein is one such example. We have recently shown that the vast majority of papillary craniopharyngiomas-tumors that arise in the sellar or suprasellar regions of the brain-harbor BRAF V600E mutations. The VE1 antibody can be effective in discriminating papillary craniopharyngioma from adamantinomatous craniopharyngioma, which harbors mutations in CTNNB1 and not BRAF. While further characterizing the use of the VE1 antibody in the differential diagnosis of suprasellar lesions, we found that the VE1 antibody stains the epithelial cells lining Rathke's cleft cysts with very strong staining of the cilia of these cells. We used targeted sequencing to show that Rathke's cleft cysts do not harbor the BRAF V600E mutation. Moreover, we found that the VE1 antibody reacts strongly with cilia in various structures-the bronchial airways, the fallopian tubes, the nasopharynx, and the epididymis-as well as with the flagella of sperm. In addition, VE1 reacts strongly with the cilia of the ependymal lining of the brain and with the cilia-containing microlumens of ependymoma tumors. There is significant sequence homology between the synthetic peptide (amino acid 596-606 of BRAF V600E: GLATEKSRWSG) that was used to generate the VE1 antibody and regions of multiple axonemal dynein heavy chain proteins (eg, DNAH2, DNAH7, and DNAH12). These proteins are major components of the axonemes of cilia and flagella where they drive the sliding of microtubules. In ELISA assays, we show that the VE1 antibody recognizes epitopes from these proteins. A familiarity with the cross-reactivity of the VE1 antibody with epitopes of proteins in cilia is of value when evaluating tissues stained with this important clinical antibody.Modern Pathology advance online publication, 21 November 2014; doi:10.1038/modpathol.2014.150. PMID:25412847

  2. The Heliogeothermal Doublet

    Microsoft Academic Search

    P. Iris; J. Adnot; R. Bonfils; D. Marchio

    1985-01-01

    Les aquifères restent en général à la même température toute l'année et représentent une très bonne source froide pour les pompes à chaleur. Malheureusement la plupart du temps, les aquifères constituent une source limitée du double point de vue hydraulique et thermique. Pour maintenir leur niveau hydraulique on doit les exploiter avec des “doublets” (un puits pour le pompage et

  3. Magnetic reconnection in doublets

    NASA Astrophysics Data System (ADS)

    Jensen, Torkil H.

    Doublet [Ohkawa, 1968] is a magnetic configuration with the prospect of being able to confine plasmas for thermonuclear fusion. Because of this prospect Doublet research was carried out at GA for a number of years. The Doublet configuration is axisymmetric and it employs a strong toroidal magnetic field. The poloidal magnetic field forms one hyperbolic magnetic axis located in the midplane and two elliptic axes above and below the midplane. Thus a separatrix with a cross sectional shape of a figure "8" is formed. Experimentally, Doublets were found subject to two sudden, mostly axisymettric deformations. During one of these the plasma split into two separate plasmas, each with one elliptic axis much like two separate tokamak plasmas. During the other the three magnetic axes tended first to merge at the midplane and secondly the plasma became unstable toward motion either up or down. These deformations involve magnetic reconnections. In the first, field lines which initially link all three magnetic axes reconnect to link only one of the elliptic magnetic axes while the reverse is true for the second type of deformation.

  4. Mutants in paramecium tetraurelia defective in their axonemal response to calcium.

    PubMed

    Hinrichsen, R D; Saimi, Y; Hennessey, T; Kung, C

    1984-01-01

    Six mutants of Paramecium tetraurelia, which display altered axonemal responses to Ca++, are described. The mutants, designated atalantas, are impaired in their ability to swim backward when stimulated by ions or heat; instead they spin very rapidly in one place. Three mutants, ataA1-3, are completely unable to swim backward. The three lines, however, can be distinguished from one another by their forward swimming velocities. The remaining three mutants are leaky. ataB swims backward briefly when stimulated, then stops and spins in place. ataC and ataD are extremely leaky and only display the spinning phenotype at elevated temperatures. An electrophysiological analysis reveals that all six mutants have normal membrane properties, including the Ca++ inward current under voltage clamp. When the membrane is disrupted so as to allow the axoneme free access to Ca++, wild-type cells swim backward, but the mutants do not. These data indicate the site(s) of lesion in the mutants is in the axoneme or in some step linking Ca++ influx and the axoneme, not within the ciliary membrane. These mutants may be useful in investigating the role of Ca++ in the regulation of axonemal motion. PMID:6478499

  5. Human FAM154A (SAXO1) is a microtubule-stabilizing protein specific to cilia and related structures.

    PubMed

    Dacheux, Denis; Roger, Benoit; Bosc, Christophe; Landrein, Nicolas; Roche, Emmanuel; Chansel, Lucie; Trian, Thomas; Andrieux, Annie; Papaxanthos-Roche, Aline; Marthan, Roger; Robinson, Derrick R; Bonhivers, Mélanie

    2015-04-01

    Cilia and flagella are microtubule-based organelles present at the surface of most cells, ranging from protozoa to vertebrates, in which these structures are implicated in processes from morphogenesis to cell motility. In vertebrate neurons, microtubule-associated MAP6 proteins stabilize cold-resistant microtubules through their Mn and Mc modules, and play a role in synaptic plasticity. Although centrioles, cilia and flagella have cold-stable microtubules, MAP6 proteins have not been identified in these organelles, suggesting that additional proteins support this role in these structures. Here, we characterize human FAM154A (hereafter referred to as hSAXO1) as the first human member of a widely conserved family of MAP6-related proteins specific to centrioles and cilium microtubules. Our data demonstrate that hSAXO1 binds specifically to centriole and cilium microtubules. We identify, in vivo and in vitro, hSAXO1 Mn modules as responsible for microtubule binding and stabilization as well as being necessary for ciliary localization. Finally, overexpression and knockdown studies show that hSAXO1 modulates axoneme length. Taken together, our findings suggest a fine regulation of hSAXO1 localization and important roles in cilium biogenesis and function. PMID:25673876

  6. An Essential Role for Katanin p80 and Microtubule Severing in Male Gamete Production

    PubMed Central

    O'Donnell, Liza; Rhodes, Danielle; Smith, Stephanie J.; Merriner, D. Jo; Clark, Brett J.; Borg, Claire; Whittle, Belinda; O'Connor, Anne E.; Smith, Lee B.; McNally, Francis J.; de Kretser, David M.; Goodnow, Chris C.; Ormandy, Chris J.; Jamsai, Duangporn; O'Bryan, Moira K.

    2012-01-01

    Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line), we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development. PMID:22654669

  7. Absence of central microtubules and transposition in the ciliary apparatus of three siblings.

    PubMed

    Bautista-Harris, G; Julià-Serdà, G; Rodríguez de Castro, F; Santana-Benitez, I; Cabrera-Navarro, P

    2000-01-01

    We studied the cases of three siblings with a history of chronic bronchitis and infertility. The electron-microscopic investigation of the tracheal and bronchial biopsies showed a ciliary defect consisting in the absence of the central microtubules and the transposition of a peripheral doublet. This is a rare and infrequently reported abnormality included in the primary ciliary diskinesia syndrome. PMID:10940803

  8. MICROTUBULE SURFACE LATTICE AND SUBUNIT STRUCTURE AND OBSERVATIONS ON REASSEMBLY

    PubMed Central

    Erickson, Harold P.

    1974-01-01

    Neuronal microtubules have been reassembled from brain tissue homogenates and purified. In reassembly from purified preparations, one of the first structures formed was a flat sheet, consisting of up to 13 longitudinal filaments, which was identified as an incomplete microtubule wall. Electron micrographs of these flat sheets and intact microtubules were analyzed by optical diffraction, and the surface lattice on which the subunits are arranged was determined to be a 13 filament, 3-start helix. A similar, and probably identical, lattice was found for outer-doublet microtubules. Finally, a 2-D image of the structure and arrangement of the microtubule subunits was obtained by processing selected images with a computer filtering and averaging system. The 40 x 50 Å morphological subunit, which has previously been seen only as a globular particle and identified as the 55,000-dalton tubulin monomer, is seen in this higher resolution reconstructed image to be elongated, and split symmetrically by a longitudinal cleft into two lobes. PMID:4855592

  9. Modeling oscillatory microtubule polymerization

    NASA Astrophysics Data System (ADS)

    Hammele, Martin; Zimmermann, Walter

    2003-02-01

    Polymerization of microtubules is ubiquitous in biological cells and under certain conditions it becomes oscillatory in time. Here, simple reaction models are analyzed that capture such oscillations as well as the length distribution of microtubules. We assume reaction conditions that are stationary over many oscillation periods, and it is a Hopf bifurcation that leads to a persistent oscillatory microtubule polymerization in these models. Analytical expressions are derived for the threshold of the bifurcation and the oscillation frequency in terms of reaction rates, and typical trends of their parameter dependence are presented. Both, a catastrophe rate that depends on the density of guanosine triphosphate liganded tubulin dimers and a delay reaction, such as the depolymerization of shrinking microtubules or the decay of oligomers, support oscillations. For a tubulin dimer concentration below the threshold, oscillatory microtubule polymerization occurs transiently on the route to a stationary state, as shown by numerical solutions of the model equations. Close to threshold, a so-called amplitude equation is derived and it is shown that the bifurcation to microtubule oscillations is supercritical.

  10. Dark matter with two inert doublets plus one Higgs doublet

    NASA Astrophysics Data System (ADS)

    Keus, Venus; King, Stephen F.; Moretti, Stefano; Sokolowska, Dorota

    2014-11-01

    Following the discovery of a Higgs boson, there has been renewed interest in the general 2-Higgs-Doublet Model (2HDM). A model with One Inert Doublet plus One Higgs Doublet (I(1+1)HDM), where one of the scalar doublets is "inert" (since it has no vacuum expectation value and does not couple to fermions) has an advantage over the 2HDM since it provides a good Dark Matter (DM) candidate, namely the lightest inert scalar. Motivated by the existence of three fermion families, here we consider a model with two scalar doublets plus one Higgs doublet (I(2+1)HDM), where the two scalar doublets are inert. The I(2+1)HDM has a richer phenomenology than either the I(1+1)HDM or the 2HDM. We discuss the new regions of DM relic density in the I(2+1)HDM with simplified couplings and address the possibility of constraining the model using recent results from the Large Hadron Collider (LHC) and DM direct detection experiments.

  11. Plasmonic achromatic doublet lens.

    PubMed

    Lee, Kyookeun; Lee, Seung-Yeol; Jung, Jaehoon; Lee, Byoungho

    2015-03-01

    An achromatic doublet lens (ADL) for surface plasmon polaritons (SPPs) is designed. Similar to the conventional ADL, the proposed plasmonic ADL is composed of two lens layers with different dispersion relations. Considering these layers as effective media, their refractive indices with respect to the free-space wavelength are calculated. Geometric parameters of the lens are initially set according to the geometrical optic theory, and then optimized by reduced dimensional calculations. The performance of proposed device is verified by using full-wave simulations and compared with a double-convex plasmonic lens to verify its achromatic characteristics. It is shown that the standard deviation of the focal length shift is reduced from 668 nm to 168 nm, after introducing the ADL. PMID:25836809

  12. Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganisation and absent inner dynein arms

    PubMed Central

    Antony, Dinu; Becker-Heck, Anita; Zariwala, Maimoona A; Schmidts, Miriam; Onoufriadis, Alexandros; Forouhan, Mitra; Wilson, Robert; Taylor-Cox, Theresa; Dewar, Ann; Jackson, Claire; Goggin, Patricia; Loges, Niki T; Olbrich, Heike; Jaspers, Martine; Jorissen, Mark; Leigh, Margaret W; Wolf, Whitney E; Daniels, M. Leigh Anne; Noone, Peader G; Ferkol, Thomas W; Sagel, Scott D; Rosenfeld, Margaret; Rutman, Andrew; Dixit, Abhijit; O’Callaghan, Christopher; Lucas, Jane S; Hogg, Claire; Scambler, Peter J; Emes, Richard D; Chung, Eddie MK; Shoemark, Amelia; Knowles, Michael R; Omran, Heymut; Mitchison, Hannah M

    2013-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed ‘radial spoke defect’. We sequenced CCDC39 and CCDC40 in 54 ‘radial spoke defect’ families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice and frameshift predicting early protein truncation, which suggests this defect is caused by ‘null’ alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganisation and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as ‘IDA and nexin-dynein regulatory complex (N-DRC) defect’, rather than ‘radial spoke defect’. PMID:23255504

  13. Time-Dependent Measure of a Nano-Scale Force-Pulse Driven by the Axonemal Dynein Motors in Individual Live Sperm Cells

    SciTech Connect

    Allen, M J; Rudd, R E; McElfresh, M W; Balhorn, R

    2009-04-23

    Nano-scale mechanical forces generated by motor proteins are crucial to normal cellular and organismal functioning. The ability to measure and exploit such forces would be important to developing motile biomimetic nanodevices powered by biological motors for Nanomedicine. Axonemal dynein motors positioned inside the sperm flagellum drive microtubule sliding giving rise to rhythmic beating of the flagellum. This force-generating action makes it possible for the sperm cell to move through viscous media. Here we report new nano-scale information on how the propulsive force is generated by the sperm flagellum and how this force varies over time. Single cell recordings reveal discrete {approx}50 ms pulses oscillating with amplitude 9.8 {+-} 2.6 nN independent of pulse frequency (3.5-19.5 Hz). The average work carried out by each cell is 4.6 x 10{sup -16} J per pulse, equivalent to the hydrolysis of {approx}5,500 ATP molecules. The mechanochemical coupling at each active dynein head is {approx}2.2 pN/ATP, and {approx}3.9 pN per dynein arm, in agreement with previously published values obtained using different methods.

  14. 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

  15. 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.

  16. Microtubule teardrop patterns

    PubMed Central

    Okeyoshi, Kosuke; Kawamura, Ryuzo; Yoshida, Ryo; Osada, Yoshihito

    2015-01-01

    Several strategies for controlling microtubule patterns are developed because of the rigidity determined from the molecular structure and the geometrical structure. In contrast to the patterns in co-operation with motor proteins or associated proteins, microtubules have a huge potential for patterns via their intrinsic flexural rigidity. We discover that a microtubule teardrop pattern emerges via self-assembly under hydrodynamic flow from the parallel bundles without motor proteins. In the growth process, the bundles ultimately bend according to the critical bending curvature. Such protein pattern formation utilizing the intrinsic flexural rigidity will provide broad understandings of self-assembly of rigid rods, not only in biomolecules, but also in supramolecules. PMID:25823414

  17. Special Seminar Microtubule Update

    E-print Network

    Bever, Thomas G.

    mitochondrial field energy to coherent electromagnetic oscillations due to nonlinear structural dynamics://sitescontent.google.com/google-workshop-on-quantum-biology/ Ten years ago we proved that particular electromagnetic oscillations occurring in cells at 8 megahertz. The microtubule coherent electromagnetic field may play a specific role in cellular functions, communication

  18. Microtubule depolymerization and tau phosphorylation.

    PubMed

    Hernández, Félix; García-García, Esther; Avila, Jesús

    2013-01-01

    Inge Grundke-Iqbal and Khalid Iqbal found a connection between microtubule associated tau and Alzheimer's disease. They described that abnormally phosphorylated tau is a component of the paired helical filaments found in the disease. Afterwards they described that tau hyperphosphorylation prevents microtubule assembly. Now trying to complement the relationship between microtubules and tau phosphorylation, we have commented on the effect of microtubule disassembly on tau phosphorylation. In this study, we investigated the role of microtubule depolymerization induced by nocodazole on tau phosphorylation in human neuroblastoma SH-SY5Y cells. Our results indicate that nocodazole provokes tau phosphorylation mediated by GSK3, as determined by using AT-8 or Tau-1 antibodies. Interestingly, total GSK3? and GSK3? phosphorylation on Ser-9 are not altered during nocodazole treatment. In addition, microtubule stabilization with taxol had similar effects, likely because taxol and tau compete for the same binding sites on microtubules, and in the presence of taxol, tau could be detached from microtubules. Thus, unbound tau from microtubles can be phosphorylated by GSK3, even if the activity of GSK3 is not altered, probably because tau unbound to microtubules could be a better substrate for the kinase than microtubule-associated tau. These findings suggest that microtubule depolymerization can be a primary event in neurodegenerative disorders like Alzheimer's disease and that tau phosphorylation takes place afterwards. PMID:23948896

  19. The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena.

    PubMed

    Edamatsu, Masaki

    2014-05-16

    Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement. PMID:24747078

  20. Are Cytoplasmic Microtubules Heteropolymers?

    PubMed Central

    Bryan, Joseph; Wilson, Leslie

    1971-01-01

    Colchicine-binding protein, considered to be microtubule protein, was purified from chick embryo brain by column chromatography in one step on DEAE-Sephadex. The active colchicine-binding unit is a dimer, MW 115,000 ± 5000, which is composed of two nonidentical monomeric units. The two subunits are separable by urea-acrylamide gel electrophoresis after they have been reduced and acetylated. Sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that the subunits both have molecular weights of 55,000 ± 2000. The amino-acid compositions of the two subunits showed statistically significant differences in six amino-acid residues. These results indicate that colchicine-sensitive cytoplasmic microtubules are heteropolymers. Images PMID:5288762

  1. The microtubule transistor

    E-print Network

    H. C. Rosu

    2007-03-26

    I point out the similarity between the microtubule experiment reported by Priel et al [Biophys. J. 90, 4639 (2006)] and the ZnO nanowire experiment of Wang et al [Nanolett. 6, 2768 (2006)]. It is quite possible that MTs are similar to a piezoelectric field effect transistor for which the role of the control gate electrode is played by the piezo-induced electric field across the width of the MT walls and their elastic bending features

  2. The ciliary inner dynein arm, I1 dynein, is assembled in the cytoplasm and transported by IFT before axonemal docking.

    PubMed

    Viswanadha, Rasagnya; Hunter, Emily L; Yamamoto, Ryosuke; Wirschell, Maureen; Alford, Lea M; Dutcher, Susan K; Sale, Winfield S

    2014-10-01

    To determine mechanisms of assembly of ciliary dyneins, we focused on the Chlamydomonas inner dynein arm, I1 dynein, also known as dynein f. I1 dynein assembles in the cytoplasm as a 20S complex similar to the 20S I1 dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 dynein preassembly in the cytoplasm. Unlike I1 dynein derived from the axoneme, the cytoplasmic 20S I1 complex will not rebind I1-deficient axonemes in vitro. To test the hypothesis that I1 dynein is transported to the distal tip of the cilia for assembly in the axoneme, we performed cytoplasmic complementation in dikaryons formed between wild-type and I1 dynein mutant cells. Rescue of I1 dynein assembly in mutant cilia occurred first at the distal tip and then proceeded toward the proximal axoneme. Notably, in contrast to other combinations, I1 dynein assembly was significantly delayed in dikaryons formed between ida7 and ida3. Furthermore, rescue of I1 dynein assembly required new protein synthesis in the ida7 × ida3 dikaryons. On the basis of the additional observations, we postulate that IDA3 is required for 20S I1 dynein transport. Cytoplasmic complementation in dikaryons using the conditional kinesin-2 mutant, fla10-1 revealed that transport of I1 dynein is dependent on kinesin-2 activity. Thus, I1 dynein complex assembly depends upon IFT for transport to the ciliary distal tip prior to docking in the axoneme. PMID:25252184

  3. Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans

    SciTech Connect

    Lye, R.J.; Porter, M.E.; Scholey, J.M.; McIntosh, J.R.

    1987-10-23

    C. elegans contains a microtubule binding protein that resembles both dynein and kinesin. This protein has a MgATPase activity and copurifies on both sucrose gradients and DEAE Sephadex columns with a polypeptide of Mr approximately 400 kd. The ATPase activity is 50% inhibited by 10 microM vanadate, 1 mM N-ethyl maleimide, or 5 mM AMP-PNP; it is enhanced 50% by 0.2% Triton. The 400 kd polypeptide is cleaved at a single site by ultraviolet light in the presence of ATP and vanadate. In these ways, the protein resembles dynein. The protein also promotes ATP-dependent translocation of microtubules or axonemes, plus ends trailing. This property is kinesin-like; however, the motility is blocked by 5 microM vanadate, 1 mM N-ethyl maleimide, 0.5 mM ATP-gamma-S, or by ATP-vanadate-UV cleavage of the 400 kd polypeptide, characteristics that differ from kinesin. We propose that this protein is a novel microtubule translocator.

  4. LETTER: Dynamics of microtubule instabilities

    NASA Astrophysics Data System (ADS)

    Antal, T.; Krapivsky, P. L.; Redner, S.

    2007-05-01

    We investigate an idealized model of microtubule dynamics that involves: (i) attachment of guanosine triphosphate (GTP) at rate ?, (ii) conversion of GTP to guanosine diphosphate (GDP) at rate 1, and (iii) detachment of GDP at rate ?. As a function of these rates, a microtubule can grow steadily or its length can fluctuate wildly. For ? = 0, we find the exact tubule and GTP cap length distributions, and power-law length distributions of GTP and GDP islands. For ? = ?, we argue that the time between catastrophes, where the microtubule shrinks to zero length, scales as e?. We also discuss the nature of the phase boundary between a growing and shrinking microtubule.

  5. 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.

  6. Discrete PIH proteins function in the cytoplasmic preassembly of different subsets of axonemal dyneins

    PubMed Central

    Yamamoto, Ryosuke; Hirono, Masafumi

    2010-01-01

    Axonemal dyneins are preassembled in the cytoplasm before being transported into cilia and flagella. Recently, PF13/KTU, a conserved protein containing a PIH (protein interacting with HSP90) domain, was identified as a protein responsible for dynein preassembly in humans and Chlamydomonas reinhardtii. This protein is involved in the preassembly of outer arm dynein and some inner arm dyneins, possibly as a cofactor of molecular chaperones. However, it is not known which factors function in the preassembly of other inner arm dyneins. Here, we analyzed a novel C. reinhardtii mutant, ida10, and found that another conserved PIH family protein, MOT48, is responsible for the formation of another subset of inner arm dyneins. A variety of organisms with motile cilia and flagella typically have three to four PIH proteins, including potential homologues of MOT48 and PF13/KTU, whereas organisms without them have no, or only one, such protein. These findings raise the possibility that multiple PIH proteins are commonly involved in the preassembly of different subsets of axonemal dyneins. PMID:20603327

  7. Why is the Higgs doublet light?

    Microsoft Academic Search

    G. Dvali

    1994-01-01

    We consider a possible mechanism for the explanation of the doublet-triplet mass hierarchy in the grand unified theories. The Higgs doublet is automatically light since it is related by a certain ``custodial'' SU(2)C symmetry to another doublet which after GUT symmetry breaking becomes an unphysical Higgs. Custodial symmetry may or may not be a part of GUT symmetry. An explicit

  8. Why is the Higgs doublet light?

    Microsoft Academic Search

    G. Dvali

    1993-01-01

    We consider a possible mechanism for the explanation of the doublet-triplet mass hierarchy in the grand unified theories. The Higgs doublet is automatically light since it is related by a certain 'custodial' SU(2)(sub C) symmetry to another doublet which after GUT symmetry breaking becomes an unphysical Higgs. Custodial symmetry may or may not be a part of GUT symmetry. An

  9. Microtubules, MAPs and Xylem Formation

    Microsoft Academic Search

    Edouard Pesquet; Clive Lloyd

    2011-01-01

    Xylem is essential for transporting water and minerals transport as well as for mechanical resistance against gravity. These key characteristics of xylem are enabled by the development of specific secondary cell walls which exhibit different patterns of thickening. Microtubules are associated with the sites at which the secondary thickenings develop and pharmacological and genetic modulation demonstrate that these cortical microtubules

  10. CMF22 Is a Broadly Conserved Axonemal Protein and Is Required for Propulsive Motility in Trypanosoma brucei

    PubMed Central

    Nguyen, HoangKim T.; Sandhu, Jaspreet; Langousis, Gerasimos

    2013-01-01

    The eukaryotic flagellum (or cilium) is a broadly conserved organelle that provides motility for many pathogenic protozoa and is critical for normal development and physiology in humans. Therefore, defining core components of motile axonemes enhances understanding of eukaryotic biology and provides insight into mechanisms of inherited and infectious diseases in humans. In this study, we show that component of motile flagella 22 (CMF22) is tightly associated with the flagellar axoneme and is likely to have been present in the last eukaryotic common ancestor. The CMF22 amino acid sequence contains predicted IQ and ATPase associated with a variety of cellular activities (AAA) motifs that are conserved among CMF22 orthologues in diverse organisms, hinting at the importance of these domains in CMF22 function. Knockdown by RNA interference (RNAi) and rescue with an RNAi-immune mRNA demonstrated that CMF22 is required for propulsive cell motility in Trypanosoma brucei. Loss of propulsive motility in CMF22-knockdown cells was due to altered flagellar beating patterns, rather than flagellar paralysis, indicating that CMF22 is essential for motility regulation and likely functions as a fundamental regulatory component of motile axonemes. CMF22 association with the axoneme is weakened in mutants that disrupt the nexin-dynein regulatory complex, suggesting potential interaction with this complex. Our results provide insight into the core machinery required for motility of eukaryotic flagella. PMID:23851336

  11. Formins and Microtubules

    PubMed Central

    Bartolini, F.; Gundersen, G. G.

    2010-01-01

    Formins have recently been recognized as prominent regulators of the microtubule (MT) cytoskeleton where they modulate the dynamics of selected MTs in interphase and mitosis. The association of formins with the MT cytoskeleton and their action on MT dynamics are relatively unexplored areas, yet growing evidence supports a direct role in their regulation of MT stability independent of their activity on actin. Formins regulate MT stability alone or in combination with accessory MT binding proteins that have previously been implicated in the stabilization of MTs downstream of polarity cues. As actin and MT arrays are typically remodeled downstream of signaling pathways that orchestrate cell shape and division, formins are emerging as excellent candidates for coordinating the responses of the cytoskeletal in diverse regulated and homeostatic processes. PMID:19631698

  12. PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella

    PubMed Central

    1996-01-01

    Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility. PMID:8636214

  13. 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

  14. The nphp-2 and arl-13 Genetic Modules Interact to Regulate Ciliogenesis and Ciliary Microtubule Patterning in C. elegans

    PubMed Central

    Warburton-Pitt, Simon R. F.; Silva, Malan; Nguyen, Ken C. Q.; Hall, David H.; Barr, Maureen M.

    2014-01-01

    Cilia are microtubule-based cellular organelles that mediate signal transduction. Cilia are organized into several structurally and functionally distinct compartments: the basal body, the transition zone (TZ), and the cilia shaft. In vertebrates, the cystoprotein Inversin localizes to a portion of the cilia shaft adjacent to the TZ, a region termed the “Inversin compartment” (InvC). The mechanisms that establish and maintain the InvC are unknown. In the roundworm C. elegans, the cilia shafts of amphid channel and phasmid sensory cilia are subdivided into two regions defined by different microtubule ultrastructure: a proximal doublet-based region adjacent to the TZ, and a distal singlet-based region. It has been suggested that C. elegans cilia also possess an InvC, similarly to mammalian primary cilia. Here we explored the biogenesis, structure, and composition of the C. elegans ciliary doublet region and InvC. We show that the InvC is conserved and distinct from the doublet region. nphp-2 (the C. elegans Inversin homolog) and the doublet region genes arl-13, klp-11, and unc-119 are redundantly required for ciliogenesis. InvC and doublet region genes can be sorted into two modules—nphp-2+klp-11 and arl-13+unc-119—which are both antagonized by the hdac-6 deacetylase. The genes of this network modulate the sizes of the NPHP-2 InvC and ARL-13 doublet region. Glutamylation, a tubulin post-translational modification, is not required for ciliary targeting of InvC and doublet region components; rather, glutamylation is modulated by nphp-2, arl-13, and unc-119. The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes. NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC. We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization. PMID:25501555

  15. Computer-assisted image analysis of human cilia and Chlamydomonas flagella reveals both similarities and differences in axoneme structure

    PubMed Central

    O’Toole, Eileen T.; Giddings, Thomas H.; Porter, Mary E.; Ostrowski, Lawrence E.

    2012-01-01

    In the past decade, investigations from several different fields have revealed the critical role of cilia in human health and disease. Because of the highly conserved nature of the basic axonemal structure, many different model systems have proven useful for the study of ciliopathies, especially the unicellular, biflagellate green alga, Chlamydomonas reinhardtii. Although the basic axonemal structure of cilia and flagella is highly conserved, these organelles often perform specialized functions unique to the cell or tissue in which they are found. These differences in function are likely reflected in differences in structural organization. In this work, we directly compare the structure of isolated axonemes from human cilia and Chlamydomonas flagella to identify similarities and differences that potentially play key roles in determining their functionality. Using transmission electron microscopy and 2D image averaging techniques, our analysis has confirmed the overall structural similarity between these two species, but also revealed clear differences in the structure of the outer dynein arms, the central pair projections, and the radial spokes. We also show how the application of 2D image averaging can clarify the underlying structural defects associated primary ciliary dyskinesia (PCD). Overall, our results document the remarkable similarity between these two structures separated evolutionarily by over a billion years, while highlighting several significant differences, and demonstrate the potential of 2D image averaging to improve the diagnosis and understanding of PCD. PMID:22573610

  16. Singlet-Doublet Dark Matter

    SciTech Connect

    Cohen, Timothy; /SLAC /Michigan U., MCTP; Kearney, John; Pierce, Aaron; /Michigan U., MCTP; 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.

  17. The CSC connects three major axonemal complexes involved in dynein regulation

    PubMed Central

    Heuser, Thomas; Dymek, Erin E.; Lin, Jianfeng; Smith, Elizabeth F.; Nicastro, Daniela

    2012-01-01

    Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ­ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility. PMID:22740634

  18. Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins

    PubMed Central

    Omran, Heymut; Kobayashi, Daisuke; Olbrich, Heike; Tsukahara, Tatsuya; Loges, Niki Tomas; Hagiwara, Haruo; Zhang, Qi; Leblond, Gerard; O’Toole, Eileen; Hara, Chikako; Mizuno, Hideaki; Kawano, Hiroyuki; Fliegauf, Manfred; Yagi, Toshiki; Koshida, Sumito; Miyawaki, Atsushi; Zentgraf, Hanswalter; Seithe, Horst; Reinhardt, Richard; Watanabe, Yoshinori; Kamiya, Ritsu; Mitchell, David R.; Takeda, Hiroyuki

    2012-01-01

    Summary Cilia/flagella are highly conserved organelles that play diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia/flagella often result in primary ciliary dyskinesia (PCD). However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a novel gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in PCD patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment. PMID:19052621

  19. DYX1C1 is required for axonemal dynein assembly and ciliary motility

    PubMed Central

    Tarkar, Aarti; Loges, Niki T.; Slagle, Christopher E.; Francis, Richard; Dougherty, Gerard W.; Tamayo, Joel V.; Shook, Brett; Cantino, Marie; Schwartz, Daniel; Jahnke, Charlotte; Olbrich, Heike; Werner, Claudius; Raidt, Johanna; Pennekamp, Petra; Abouhamed, Marouan; Hjeij, Rim; Köhler, Gabriele; Griese, Matthias; Li, You; Lemke, Kristi; Klena, Nikolas; Liu, Xiaoqin; Gabriel, George; Tobita, Kimimasa; Jaspers, Martine; Morgan, Lucy C.; Shapiro, Adam J.; Letteboer, Stef J.F.; Mans, Dorus A.; Carson, Johnny L.; Leigh, Margaret W.; Wolf, Whitney E.; Chen, Serafine; Lucas, Jane S.; Onoufriadis, Alexandros; Plagnol, Vincent; Schmidts, Miriam; Boldt, Karsten; Roepman, Ronald; Zariwala, Maimoona; Lo, Cecilia W.; Mitchison, Hannah M.; Knowles, Michael R.; Burdine, Rebecca D.; LoTurco, Joseph J.; Omran, Heymut

    2014-01-01

    SUMMARY Dyx1c1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deletion of Dyx1c1 exons 2–4 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder characterized by chronic airway disease, laterality defects, and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1c.T2A start codon mutation recovered from an ENU mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also created laterality and ciliary motility defects. In humans, recessive loss-of-function DYX1C1 mutations were identified in twelve PCD individuals. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans revealed disruptions of outer and inner dynein arms (ODA/IDA). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA/IDA assembly factor DNAAF2/KTU. Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4). PMID:23872636

  20. Disruption of cytoplasmic microtubules by ultraviolet radiation

    SciTech Connect

    Zamansky, G.B.; Perrino, B.A.; Chou, I.N. (Boston Univ. School of Medicine, MA (USA))

    1991-07-01

    Ultraviolet (UV) irradiation of cultured human skin fibroblasts causes the disassembly of their microtubules. Using indirect immunofluorescence microscopy, we have now investigated whether damage to the microtubule precursor pool may contribute to the disruption of microtubules. Exposure to polychromatic UV radiation inhibits the reassembly of microtubules during cellular recovery from cold treatment. In addition, the ability of taxol to promote microtubule polymerization and bundling is inhibited in UV-irradiated cells. However, UV irradiation of taxol-pretreated cells or in situ detergent-extracted microtubules fails to disrupt the microtubule network. These data suggest that damage to dimeric tubulin, or another soluble factor(s) required for polymerization, contributes to the disassembly of microtubules in UV-irradiated human skin fibroblasts.

  1. Microtubule defects & Neurodegeneration

    PubMed Central

    Baird, Fiona J.; Bennett, Craig L

    2014-01-01

    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 ?-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. PMID:24563812

  2. Microtubule Cytoskeleton: Navigating the Intracellular Landscape

    E-print Network

    microtubules to the actin cytoskeleton. Kar9p binds microtubules via the yeast EB1 homolog, Bim1p [4­6]. EB1/Bim1p is highly conserved and functions at microtubule plus ends [4]. Mammalian EB1 interacts

  3. A viscoelastic model for axonal microtubule rupture.

    PubMed

    Shamloo, Amir; Manuchehrfar, Farid; Rafii-Tabar, Hashem

    2015-05-01

    Axon is an important part of the neuronal cells and axonal microtubules are bundles in axons. In axons, microtubules are coated with microtubule-associated protein tau, a natively unfolded filamentous protein in the central nervous system. These proteins are responsible for cross-linking axonal microtubule bundles. Through complimentary dimerization with other tau proteins, bridges are formed between nearby microtubules creating bundles. Formation of bundles of microtubules causes their transverse reinforcement and has been shown to enhance their ability to bear compressive loads. Though microtubules are conventionally regarded as bearing compressive loads, in certain circumstances during traumatic brain injuries, they are placed in tension. In our model, microtubule bundles were formed from a large number of discrete masses. We employed Standard Linear Solid model (SLS), a viscoelastic model, to computationally simulate microtubules. In this study, we investigated the dynamic responses of two dimensional axonal microtubules under suddenly applied end forces by implementing discrete masses connected to their neighboring masses with a Standard Linear Solid unit. We also investigated the effect of the applied force rate and magnitude on the deformation of bundles. Under tension, a microtubule fiber may rupture as a result of a sudden force. Using the developed model, we could predict the critical regions of the axonal microtubule bundles in the presence of varying end forces. We finally analyzed the nature of microtubular failure under varying mechanical stresses. PMID:25835789

  4. Microtubule treadmills-possible molecular machinery

    Microsoft Academic Search

    Robert L. Margolis; Leslie Wilson

    1981-01-01

    Microtubule polymerization in vitro is the summation of different reactions occurring at each end of the polymer. In steady-state conditions in vitro, net tubulin addition on to the microtubule occurs at one end of the polymer, and net tubulin loss occurs at the opposite end. Thus, a unidirectional flux of tubulin from one end of the microtubule to the other,

  5. Absence of microtubule sliding and an analysis of spindle formation and elongation in isolated mitotic spindles from the yeast Saccharomyces cerevisiae

    PubMed Central

    1982-01-01

    Mitotic spindles were isolated from a cell division cycle mutant of the budding yeast Saccharomyces cerevisiae by the lysis of sphateroplasts on an air:buffer interface and were negatively stained with 1% gold thioglucose. Isolated spindles were incubated under conditions which promoted the sliding disintegration of parallel preparations of Tetrahymena axonemes, namely the addition of ATP to 20 microM. In no experiment was a corresponding change in microtubule organization of the spindle observed even when spindles were first pretreated with either 1-10 microgram/ml trypsin or 0.2-2% Triton X-100. During these experiments a number of spindles were isolated from cells that had passed through the imposed temperature block, and from the images obtained a detailed model of spindle formation and elongation has been constructed. Two sets of microtubules, one from each spindle pole body (SPB), completely interdigitate to form a continuous bundle, and a series of discontinuous microtubules are then nucleated by each SPB. As the spindle elongates, the number of microtubules continuous between the two SPBs decreases until, at a length of 4 micrometer, only one remains. The spindle, composed of only one microtubule, continues to elongate until it reaches the maximal nuclear dimension of 8 micrometer. The data obtained from negatively stained preparations have been verified in thin sections of wild-type cells. We suggest that, as in the later stages of mitosis only one microtubule is involved in the separation of the spindle poles, the microtubular spindle in S. cerevisiae is not a force-generating system but rather acts as a regulatory mechanism controlling the rate of separation. PMID:7050129

  6. The canonical `9+2' microtubule axoneme is the prin-cipal feature of many motile cilia and flagella and is

    E-print Network

    Schnaufer, Achim

    ), the sexual stage of the parasite life cycle requires de novo formation of flagella by male gametes to extant eukaryotes1 . Therefore the absence of cilia and flagella from yeast, many fungi, red algae, such as the biflagellate green alga Chlamydomonas reinhardtii, the interchangeability of the flagellar basal body

  7. Inert doublet model and LEP II limits

    SciTech Connect

    Lundstroem, Erik; Gustafsson, Michael; Edsjoe, Joakim [Department of Physics, Stockholm University, AlbaNova University Center, SE - 106 91 Stockholm (Sweden); INFN, Sezione di Padova, Department of Physics 'Galileo Galilei', Via Marzolo 8, I-35131, Padua (Italy) and Department of Physics, Stockholm University, AlbaNova University Center, SE - 106 91 Stockholm (Sweden); Department of Physics, Stockholm University, AlbaNova University Center, SE - 106 91 Stockholm (Sweden)

    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.

  8. Dark Two Higgs Doublet Model

    SciTech Connect

    Lee, Hye Sung [JLAB, William and Mary College; Sher, Marc [William and Mary College

    2013-06-01

    We perform a detailed study of a specific Two Higgs Doublet Model (2HDM) with a U(1) gauge symmetry, instead of a typical Z{sub 2} discrete symmetry, containing a very light gauge boson Z' (GeV scale or below). The Standard Model (SM) fermions do not carry U(1) charges, but induced couplings to the Z' (called the dark Z) are generated through mixing with the SM neutral gauge bosons. Such a light Z' could explain some astrophysical anomalies as well as the muon g-2 deviation, and has been the subject of great experimental interest. We consider the scenario in which the 125 GeV SM-like Higgs (H) is the heavier scalar state, and focus on the lighter neutral state (h) as well as charged Higgs. We analyze the constraints on the model from various experiments and predict novel channels to search for these Higgs scalars at the LHC. In particular, experiments looking for lepton-jets are among potentially important searches.

  9. Prominent Doublet Ridges on Europa

    NASA Technical Reports Server (NTRS)

    1997-01-01

    This image of Jupiter's satellite Europa was obtained from a range of 7364 miles (11851 km) by the Galileo spacecraft during its fourth orbit around Jupiter and its first close pass of Europa. The image spans 30 miles by 57 miles (48 km x 91 km) and shows features as small as 800 feet (240 meters) across, a resolution more than 150 times better than the best Voyager coverage of this area. The sun illuminates the scene from the right. The large circular feature in the upper left of the image could be the scar of a large meteorite impact. Clusters of small craters seen in the right of the image may mark sites where debris thrown from this impact fell back to the surface. Prominent doublet ridges over a mile (1.6 km) wide cross the plains in the right part of the image; younger ridges overlap older ones, allowing the sequence of formation to be determined. Gaps in ridges indicate areas where emplacement of new surface material has obliterated pre-existing terrain.

    The Jet Propulsion Laboratory, Pasadena, CA manages the mission for NASA's Office of Space Science, Washington, DC.

    This image and other images and data received from Galileo are posted on the Galileo mission home page on the World Wide Web at http://galileo.jpl.nasa.gov. Background information and educational context for the images can be found at URL http://www.jpl.nasa.gov/galileo/sepo

  10. Lymphocyte signaling converges on microtubules.

    PubMed

    Babich, Alexander; Burkhardt, Janis K

    2011-06-24

    Movement of immunoreceptor microclusters tunes lymphocyte activation, but the underlying mechanisms are incompletely understood. In this issue of Immunity, Schnyder et al. (2011) and Hashimoto-Tane et al. (2011) show that cytoplasmic dynein drives microcluster centralization along microtubules. PMID:21703536

  11. Localized modulated waves in microtubules.

    PubMed

    Zdravkovi?, Slobodan; Bugay, Aleksandr N; Aru, Guzel F; Maluckov, Aleksandra

    2014-06-01

    In the present paper, we study nonlinear dynamics of microtubules (MTs). As an analytical method, we use semi-discrete approximation and show that localized modulated solitonic waves move along MT. This is supported by numerical analysis. Both cases with and without viscosity effects are studied. PMID:24985453

  12. Molecular Biology of the Cell Vol. 5, 1051-1063, September 1994

    E-print Network

    Villefranche sur mer

    , and Human Flagellar Axonemes Claude Gagnon,*t Daniel White,* Philippe Huitorel, and Jacky Cosson* *Urology the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human doublet microtubules is responsible for ciliary and flagellar movement (Gib- bons, 1981, 1989). Dynein

  13. Microtubule catastrophe from protofilament dynamics

    NASA Astrophysics Data System (ADS)

    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.PRLTAO0031-900710.1103/PhysRevLett.73.2372 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.

  14. Application of stains-all staining to the analysis of axonemal tubulins: identification of beta-tubulin and beta-isotubulins.

    PubMed

    Nakamura, K; Masuyama, E; Wada, S; Okuno, M

    1990-01-01

    The cationic dye, Stains-all, is known to stain brain beta-tubulin blue and alpha-tubulin red (Serrano, L. et al. (1986) J. Biochem. Biophys. Methods 12, 281-287; Serrano, L. et al. (1989) Biochem. Int., 19, 235-246). The present experiments show that this stain can also be applied to detect beta-tubulin in axonemal tubulins from various sources such as cilia of protozoa, sperm flagella of echinoderm, and sperm flagella of mollusc. Furthermore, these experiments showed that it selectively stains isoforms of axonemal beta-tubulin blue following isoelectric focusing, whereas those of alpha-tubulin are stained red. These results indicate that Stains-all staining is a useful tool for electrophoretic analysis of axonemal tubulins. PMID:1704028

  15. 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. PMID:23674690

  16. A conserved flagella-associated protein in Chlamydomonas, FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9

    PubMed Central

    Kubo, Tomohiro; Yanagisawa, Haru-aki; Liu, Zhongmei; Shibuya, Rie; Hirono, Masafumi; Kamiya, Ritsu

    2014-01-01

    Tubulin undergoes various posttranslational modifications, including polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase–like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a polyglutamylation complex specialized for regulation of ciliary motility. PMID:24196831

  17. On and Around Microtubules: An Overview

    Microsoft Academic Search

    Richard H. Wade

    2009-01-01

    Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from ??-tubulin\\u000a heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the ?-tubulin subunit\\u000a facing the microtubule plus end conferring a structural polarity. The ?- and ?-tubulins are highly conserved. A third member\\u000a of the tubulin family, ?-tubulin, plays a

  18. Ectopic A-lattice seams destabilize microtubules

    PubMed Central

    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. PMID:24463734

  19. Single kinesin molecules crossbridge microtubules in vitro.

    PubMed

    Andrews, S B; Gallant, P E; Leapman, R D; Schnapp, B J; Reese, T S

    1993-07-15

    Kinesin is a cytoplasmic motor protein that moves along microtubules and can induce microtubule bundling and sliding in vitro. To determine how kinesin mediates microtubule interactions, we determined the shapes and mass distributions of squid brain kinesin, taxol-stabilized microtubules (squid and bovine), and adenosine 5'-[beta, gamma-imido]triphosphate-stabilized kinesin-microtubule complexes by high-resolution metal replication and by low-temperature, low-dose dark-field scanning transmission electron microscopy of unfixed, directly frozen preparations. Mass mapping by electron microscopy revealed kinesins loosely attached to the carbon support as asymmetrical dumbbell-shaped molecules, 40-52 nm long, with a mass of 379 +/- 15 kDa. The mass distribution and shape of these molecules suggest that these images represent kinesin in a shortened conformation. Kinesin-microtubule complexes were organized as bundles of linearly arrayed microtubules, stitched together at irregular intervals by cross-bridges typically < or = 25 nm long. The crossbridges had a mass of 360 +/- 15 kDa, consistent with one kinesin per crossbridge. These results suggest that kinesin has a second microtubule binding site in addition to the known site on the motor domain of the heavy chain; this second site may be located near the C terminus of the heavy chains or on the associated light chains. Thus, kinesin could play a role in either crosslinking or sliding microtubules. PMID:8341662

  20. Doublet III neutral beam test tank design

    SciTech Connect

    Doll, D.W.; Kamperschroer, J.H.; Bailey, E.W.

    1980-03-01

    A tank has been designed for testing the Doublet III Neutral Beam Injector which simulates the entrance and pressure conditions of the Doublet III vacuum vessel. The cylindrical shape vacuum vessel is the same size as the neutral beam injector vessel. Contained inside are a cylindrical cryopanel, a V-shaped calorimeter, and a retractable sample-holding device to be used for beam armor proof testing. The cryopanel has 4.2 m of surface for pumping the hydrogen load created by beam impingement on the calorimeter. A tank pressure of 1.3 x 10/sup -2/-1.3 x 10/sup -6/ Pa (10/sup -4/-10/sup -8/ torr) is to be maintained to simulate the Doublet III vessel pressure conditions.

  1. b quark Electric Dipole moment in the general two Higgs Doublet and three Higgs Doublet models

    E-print Network

    E. O. Iltan

    2001-05-17

    We study the Electric Dipole moment of b quark in the general two Higgs Doublet model (model III) and three Higgs Doublet model with O(2) symmetry in the Higgs sector. We analyse the dependency of this quantity to the new phase coming from the complex Yukawa couplings and masses of charged and neutral Higgs bosons. We see that the Electric Dipole moment of b quark is at the order of 10^{-20} e cm, which is an extremely large value compared to one calculated in the SM and also two Higgs Doublet model (model II) with real Yukawa couplings.

  2. The second microtubule-binding site of monomeric kid enhances the microtubule affinity.

    PubMed

    Shiroguchi, Katsuyuki; Ohsugi, Miho; Edamatsu, Masaki; Yamamoto, Tadashi; Toyoshima, Yoko Y

    2003-06-20

    Chromokinesin Kid (kinesin-like DNA-binding protein) localizes on spindles and chromosomes and has important roles in generating polar ejection force on microtubules in the metaphase. To understand these functions of Kid at the molecular level, we investigated molecular properties of Kid, its oligomeric state, interaction with microtubules, and physiological activity in vitro. Kid expressed in mammalian cells, as well as Kid expressed in Escherichia coli, was found to be monomeric. However, Kid cross-linked microtubules in an ATP-sensitive manner, suggesting that Kid has a second microtubule-binding site in addition to its motor domain. This was ascertained by binding of Kid fragments lacking the motor domain to microtubules. The interaction of the second microtubule-binding site was weak in a nucleotide-insensitive manner. KmMT of the ATPase activity of Kid was lower than that of the fragments lacking the second microtubule-binding site. Moreover, the velocity of Kid movement in vitro was not affected by the second microtubule-binding site, which is consistent with the weak binding of this site to microtubules. The second microtubule-binding site would be important to enhance the affinity to microtubules for the monomeric motor, Kid. Because the amino acid sequence of this region is highly conserved among species, it seems to have essential roles for the functions of Kid in vivo. PMID:12692123

  3. Microtubule segment stabilization by RASSF1A is required for proper microtubule dynamics and Golgi integrity.

    PubMed

    Arnette, Christopher; Efimova, Nadia; Zhu, Xiaodong; Clark, Geoffrey J; Kaverina, Irina

    2014-03-01

    The tumor suppressor and microtubule-associated protein Ras association domain family 1A (RASSF1A) has a major effect on many cellular processes, such as cell cycle progression and apoptosis. RASSF1A expression is frequently silenced in cancer and is associated with increased metastasis. Therefore we tested the hypothesis that RASSF1A regulates microtubule organization and dynamics in interphase cells, as well as its effect on Golgi integrity and cell polarity. Our results show that RASSF1A uses a unique microtubule-binding pattern to promote site-specific microtubule rescues, and loss of RASSF1A leads to decreased microtubule stability. Furthermore, RASSF1A-associated stable microtubule segments are necessary to prevent Golgi fragmentation and dispersal in cancer cells and maintain a polarized cell front. These results indicate that RASSF1A is a key regulator in the fine tuning of microtubule dynamics in interphase cells and proper Golgi organization and cell polarity. PMID:24478455

  4. Automated Two Higgs Doublet Model at NLO

    E-print Network

    Celine Degrande

    2014-12-22

    The Two Higgs Doublet Model at NLO is generated automatically by FeynRules and NLOCT and allows any computation to be performed at NLO in QCD inside MadGraph5_aMC@NLO. The model can handle both four and five massless flavours. Preliminary results of the shape comparison between the two schemes are shown.

  5. Movement of microtubules by single kinesin molecules

    Microsoft Academic Search

    J. Howard; A. J. Hudspeth; R. D. Vale

    1989-01-01

    Kinesin is a motor protein that uses energy derived from ATP hydrolysis to move organelles along microtubules. Using a new technique for measuring the movement produced in vitro by individual kinesin molecules, it is shown that a single kinesin molecule can move a microtubule for several micrometres. New information about the mechanism of force generation by kinesin is presented.

  6. A slow dance for microtubule acetylation.

    PubMed

    Kull, F Jon; Sloboda, Roger D

    2014-06-01

    Microtubules contribute to diverse cellular processes through balancing dynamic, short-lived and stable, long-lived populations. One way in which long-lived microtubules are marked is by posttranslational acetylation of ?-tubulin by tubulin acetyltransferase (TAT). Szyk et al. now provide insight into TAT's mechanism of action and its unique time-stamping ability. PMID:24906144

  7. Parabola-Doublet Aplanat Becomes Anastigmatic when Second Doublet is Inserted Near Focus

    NASA Astrophysics Data System (ADS)

    Blakley, Rick

    2004-08-01

    A doublet of choice glasses may be located in the converging focal cone of the infinity-focused parabola to yield an aplanatic telescope or camera. The resulting angular field is limited by high astigmatism but is significantly larger than that of the coma-limited parabola. The spherical and chromatic aberrations are so well corrected and the coma so well balanced that the doublet may be used unaltered with a parabola of arbitrary focal length and speed with excellent results for the unvignetted rays. A second doublet nearer to the focus and designed independently of the first corrects the system's astigmatism while preserving its aplanaticism. It may also be designed for flattening the field. This arrangement may allow for greater flexibility in the placing of optical elements than does Wynne's triplet for modest-aperture systems. Equations are presented for choosing candidate glasses for the first doublet from the very limited manifold of solving glasses.

  8. Comparison of the motile and enzymatic properties of two microtubule minus-end-directed motors, ncd and cytoplasmic dynein.

    PubMed

    Shimizu, T; Toyoshima, Y Y; Edamatsu, M; Vale, R D

    1995-02-01

    Cytoplasmic dynein and ncd, a kinesin-related protein from Drosophila, are motor proteins that move toward the minus ends of microtubules, while kinesin moves to the microtubule plus end. In previous work, we examined the nucleotide dependence of motility and enzymatic activity by kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F., & Vale, R. D., (1991) J. Cell Biol. 112, 1189-1197]. In this study, we examined these activities of the cytoplasmic dynein from bovine brain and ncd in order to explore what enzymatic features might be shared by these two minus-end-directed motors. Both ncd and cytoplasmic dynein demonstrated an activation of ATPase activity upon the addition of microtubules (30-fold and 6-fold, respectively). A significant difference between ncd and cytoplasmic dynein was their relative sensitivity to vanadate and to aluminum fluoride. In contrast to cytoplasmic dynein, ncd polypeptide was not cleaved by UV-vanadate treatment, and its ATPase and motility were unaffected by vanadate (up to 0.1 mM). When the nucleotide requirement for movement as examined using a battery of 20 nucleotides and nucleotide analogues, cytoplasmic dynein was found to exhibit a specificity very similar to that of axonemal dyneins from Tetrahymena. Surprisingly, however, the nucleotide specificities of in vitro motility produced by ncd or its construct, GST/MC1 (a fusion protein of glutathione S-transferase and 210-700 of the predicted ncd amino acid sequence), were quite distinct from that of kinesin. Thus, the nucleotide specificity profiles of members of the kinesin motor superfamily do not appear to be identical. PMID:7849016

  9. Taccalonolides: Novel microtubule stabilizers with clinical potential.

    PubMed

    Risinger, April L; Mooberry, Susan L

    2010-05-01

    Nature remains an important source for new anticancer drugs. Numerous microtubule-targeting agents currently approved or in clinical development, including paclitaxel, vinblastine, vincristine, colchicine and combretastatin, are plant-derived compounds. The microtubule stabilizing properties of the taccalonolides were discovered as a part of a program to identify new microtubule stabilizers from natural sources. The taccalonolides are unique among all other agents in this class in that they stabilize microtubules through a mechanism of action that does not involve direct tubulin binding. Herein we review the discovery and isolation of the taccalonolides, their biological activities in vitro and in vivo and their potential advantages over clinically used microtubule stabilizers. We also discuss the challenges in formulation and supply that will need to be solved before the taccalonolides can become candidates for clinical development. PMID:19880245

  10. Mechanical and geometrical constraints control kinesin-based microtubule guidance.

    PubMed

    Doodhi, Harinath; Katrukha, Eugene A; Kapitein, Lukas C; Akhmanova, Anna

    2014-02-01

    Proper organization of microtubule networks depends on microtubule-associated proteins and motors that use different spatial cues to guide microtubule growth [1-3]. For example, it has been proposed that the uniform minus-end-out microtubule organization in dendrites of Drosophila neurons is maintained by steering of polymerizing microtubules along the stable ones by kinesin-2 motors bound to growing microtubule plus ends [4]. To explore the mechanics of kinesin-guided microtubule growth, we reconstituted this process in vitro. In the presence of microtubule plus-end tracking EB proteins, a constitutively active kinesin linked to the EB-interacting motif SxIP effectively guided polymerizing microtubules along other microtubules both in cells and in vitro. Experiments combined with modeling revealed that at angles larger than 90°, guidance efficiency is determined by the force needed for microtubule bending. At angles smaller than 90°, guidance requires microtubule growth, and guidance efficiency depends on the ability of kinesins to maintain contact between the two microtubules despite the geometrical constraints imposed by microtubule length and growth rate. Our findings provide a conceptual framework for understanding microtubule guidance during the generation of different types of microtubule arrays. PMID:24462000

  11. Posttranslational regulation of the microtubule cytoskeleton: mechanisms and functions

    Microsoft Academic Search

    Carsten Janke; Jeannette Chloë Bulinski

    2011-01-01

    Half a century of biochemical and biophysical experiments has provided attractive models that may explain the diverse functions of microtubules within cells and organisms. However, the notion of functionally distinct microtubule types has not been explored with similar intensity, mostly because mechanisms for generating divergent microtubule species were not yet known. Cells generate distinct microtubule subtypes through expression of different

  12. EB1–Microtubule Interactions in Xenopus Egg Extracts: Role of EB1 in Microtubule Stabilization and Mechanisms of Targeting to Microtubules

    PubMed Central

    Tirnauer, Jennifer S.; Grego, Sonia; Salmon, E.D.; Mitchison, Timothy J.

    2002-01-01

    EB1 targets to polymerizing microtubule ends, where it is favorably positioned to regulate microtubule polymerization and confer molecular recognition of the microtubule end. In this study, we focus on two aspects of the EB1–microtubule interaction: regulation of microtubule dynamics by EB1 and the mechanism of EB1 association with microtubules. Immunodepletion of EB1 from cytostatic factor-arrested M-phase Xenopus egg extracts dramatically reduced microtubule length; this was complemented by readdition of EB1. By time-lapse microscopy, EB1 increased the frequency of microtubule rescues and decreased catastrophes, resulting in increased polymerization and decreased depolymerization and pausing. Imaging of EB1 fluorescence revealed a novel structure: filamentous extensions on microtubule plus ends that appeared during microtubule pauses; loss of these extensions correlated with the abrupt onset of polymerization. Fluorescent EB1 localized to comets at the polymerizing plus ends of microtubules in cytostatic factor extracts and uniformly along the lengths of microtubules in interphase extracts. The temporal decay of EB1 fluorescence from polymerizing microtubule plus ends predicted a dissociation half-life of seconds. Fluorescence recovery after photobleaching also revealed dissociation and rebinding of EB1 to the microtubule wall with a similar half-life. EB1 targeting to microtubules is thus described by a combination of higher affinity binding to polymerizing ends and lower affinity binding along the wall, with continuous dissociation. The latter is likely to be attenuated in interphase. The highly conserved effect of EB1 on microtubule dynamics suggests it belongs to a core set of regulatory factors conserved in higher organisms, and the complex pattern of EB1 targeting to microtubules could be exploited by the cell for coordinating microtubule behaviors. PMID:12388761

  13. 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 ?1 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

  14. MOVING IN FOR THE KILL: MOTILE MICROTUBULE REGULATORS

    PubMed Central

    Su, Xiaolei; Ohi, Ryoma; Pellman, David

    2012-01-01

    The stereotypical function of kinesin superfamily motors is to transport cargo along microtubules. However, some kinesins also shape the microtubule track by regulating microtubule assembly and disassembly. Recent work has shown that the kinesin-8 family of motors are key regulators of cellular microtubule length. The studied kinesin-8s are highly processive motors that walk towards the microtubule plus-end. Once at plus-ends, they have complex effects on polymer dynamics: kinesin-8s either destabilize or stabilize microtubules, depending on the context. This review will focus on the mechanisms underlying kinesin-8-microtubule interactions and microtubule length control. We will compare and contrast kinesin-8s with the other major microtubule-regulating kinesins (kinesin-4 and kinesin-13), to survey the current understanding of the diverse ways that kinesins control microtubule dynamics. PMID:22959403

  15. Design and realization of an aspherical doublet

    NASA Astrophysics Data System (ADS)

    Melich, Radek; Matela, Milan; Prochaska, Frantisek; Psota, Pavel; Matousek, Ondrej; Tomka, David

    2015-01-01

    We have realized an optical design of air space doublet of 100 mm clear aperture and 520 mm focal length that is optimized with respect to a quality of wavefront error better than 0.07 ? RMS for on-axis imaging at wavelengths of 633 nm and 450 nm. To minimize optical aberrations we have designed one of the four surfaces to be an aspherical. Based on a tolerance analyses those take into account planned spherical and aspherical technologies for surfaces realization and measurement equipment we have realized the doublet. In the paper there is described a technique of the optical design, tolerance analysis, technique of objective realization and results of the optical elements realization.

  16. Dark Matter from the Inert Doublet Model

    E-print Network

    Laura Lopez Honorez

    2007-06-01

    The Inert Doublet Model is an extension of the Standard Model including one extra ``Inert scalar doublet'' and an exact $Z_2$ symmetry. The ``Inert scalar'' provides a new candidate for dark matter. We present a systematic analysis of the dark matter abundance assuming the standard freeze-out mechanism and investigate the potentialities for direct and gamma indirect detection. We show that the dark matter candidate saturates the WMAP dark matter density in two rather separate mass ranges, one between 40 and 80 GeV, the other one over 400 GeV. We also show that the model should be within the range of future experiments, like GLAST and EDELWEISS II or ZEPLIN.

  17. Doublet III beamline: as-built

    SciTech Connect

    Harder, C.R.; Holland, M.M.; Parker, J.W.; Gunn, J.; Resnick, L.

    1980-03-01

    In order to fully exploit Doublet III capabilities and to study new plasma physics regimes, a Neutral Beam Injector System has been constructed. Initially, a two beamline system will supply 7 MW of heat to the plasma. The system is currently being expanded to inject approx. 20 MW of power (6 beamlines). Each beamline is equipped with two Lawrence Berkeley Laboratory type rectangular ion sources with 10 cm x 40 cm extraction grids. These sources will accelerate hydrogen ions to 80 keV, with extracted beam currents in excess of 80 A per source expected. The first completed source is currently being tested and conditioned on the High Voltage Test Stand at Lawrence Livermore Laboratory. This paper pictorially reviews the as-built Doublet III neutral beamline with emphasis on component relation and configuration relative to spatial and source imposed design constraints.

  18. Microtubule array reorientation in response to hormones does not involve changes in microtubule nucleation modes at the periclinal cell surface

    PubMed Central

    Atkinson, Samantha; Kirik, Angela; Kirik, Viktor

    2014-01-01

    Aligned microtubule arrays spatially organize cell division, trafficking, and determine the direction of cell expansion in plant cells. In response to changes in environmental and developmental signals, cells reorganize their microtubule arrays into new configurations. Here, we tested the role of microtubule nucleation during hormone-induced microtubule array reorientation. We have found that in the process of microtubule array reorientation the ratios between branching, parallel, and de-novo nucleations remained constant, suggesting that the microtubule reorientation mechanism does not involve changes in nucleation modes. In the ton2/fass mutant, which has reduced microtubule branching nucleation frequency and decreased nucleation activity of the ?-tubulin complexes, microtubule arrays were able to reorient. Presented data suggest that reorientation of microtubules into transverse arrays in response to hormones does not involve changes in microtubule nucleation at the periclinal cell surface PMID:25135522

  19. Measuring kinetochore-microtubule interaction in vitro

    PubMed Central

    Driver, Jonathan W.; Powers, Andrew F.; Sarangapani, Krishna K.; Biggins, Sue; Asbury, Charles L.

    2014-01-01

    Many proteins and protein complexes perform sophisticated, regulated functions in vivo. Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause-effect relationships, for example between a protein and its phosphorylating kinase. Here, we describe a protocol to purify kinetochores, the protein complexes that attach chromosomes to microtubules during mitosis, and quantitatively assay their microtubule binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of microtubules to kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of kinetochore-microtubule attachments, which is believed to be critical for correcting erroneous kinetochore-spindle attachments and thereby avoiding chromosome mis-segregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology. PMID:24630115

  20. Dynamics of Microtubule Growth and Catastrophe

    NASA Astrophysics Data System (ADS)

    Redner, Sidney; Antal, Tibor; Krapivsky, Paul; Mailman, Mitch; Chakraborty, Bulbul

    2008-03-01

    We investigate a simple dynamical model of a microtubule that evolves by attachment of guanosine triphosphate (GTP) tubulin to its end, irreversible conversion of GTP to guanosine diphosphate (GDP) tubulin by hydrolysis, and detachment of GDP at the end of a microtubule. As a function of rates of these processes, the microtubule can grow steadily or its length can fluctuate wildly. In the regime where detachment can be neglected, we find exact expressions for the tubule and GTP cap length distributions, as well as power-law length distributions of GTP and GDP islands. In the opposite limit of instantaneous detachment, we find the time between catastrophes, where the microtubule shrinks to zero length, scales as e^?. We also determine the size distribution of avalanches (sequence of consecutive GDP detachment events). We obtain the phase diagram for general rates and verify our predictions by numerical simulations.

  1. Tau induces cooperative Taxol binding to microtubules

    E-print Network

    Ross, Jennifer

    are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds of neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, and frontotemporal dementia

  2. Birefringence of Single and Bundled Microtubules

    Microsoft Academic Search

    R. Oldenbourg; E. D. Salmon; P. T. Tran

    1998-01-01

    We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07nm. The peak retardance of a small bundle increased linearly with the number of MTs in the

  3. Using Microtubules to Illustrate Polymer Properties

    NSDL National Science Digital Library

    Hess, Henry

    Microtubules are a biopolymer, which assembles in vitro within minutes via noncovalent interactions from thousands of tubulin proteins at a temperature of 37 degrees Celsius. The large size (25 nm in diameter and several micrometers in length) and stiffness of these tubular, hollow polymers enables the imaging of individual, fluorescently labeled microtubules by fluorescence microscopy. We have utilized microtubules to create a stimulating laboratory, for undergraduate students which illustrates basic polymer concepts using commercially available compounds. By imaging and analyzing a population of microtubules, students can directly determine molecular weight distributions and the degree of polymerization. Polymerization parameters, such as initial monomer concentration, temperature, and polymerization time, as well as postpolymerization processing conditions (such as shearing) can be varied, and their effect on the microtubule population can be directly observed. Based on the assessment of the first group of students conducting this laboratory, we propose that a microtubule-based laboratory is a valuable addition to the curriculum of MSE and BME students specializing in polymers and biomaterials, since it enables striking demonstrations of polymer science and bioengineering principles.

  4. Vibration analysis of a single microtubule surrounded by cytoplasm

    NASA Astrophysics Data System (ADS)

    Ghavanloo, Esmaeal; Daneshmand, Farhang; Amabili, Marco

    2010-11-01

    Microtubules are the central part of the cytoskeleton in eukaryotic cells. The microtubules immersed in the cytoplasm are in contact with water and other cytoplasmic molecules. In this study, a mechanics model of microtubule vibration, considering the coupled effect of the viscoelastic surrounding cytoplasm, is analytically investigated. The microtubule is modeled as a linear elastic cylindrical tube. The cytoplasm is characterized by viscous cytosol and elastic filaments network. The cytosol motion is modeled as the Stokes flow with no-slip condition at the microtubule-cytosol interface. The stress field in the cytosol induced by vibrating microtubule is determined, the effect of the surrounding filament network is considered and the coupled vibrations of the microtubule-cytoplasm system are investigated. The variations of damped-frequencies with the cytosol dynamic viscosity, microtubule bending stiffness, microtubule length and the elastic modulus of the surrounding filament network are also examined.

  5. Design of a dee vacuum vessel for Doublet III

    Microsoft Academic Search

    Larry G. Davis

    1983-01-01

    The Doublet III tokamak is to be modified wherein the original 'doublet' plasma containment vacuum vessel will be exchanged with one of a large dee-shaped cross section. The basic dimensions of the dee vessel will allow plasmas of 1.7-m major radius, 0.7-m minor radius, and a vertical elongation of 1.8. Installation of a large dee vessel in Doublet III is

  6. Microtubule nucleating and severing enzymes for modifying microtubule array organization and cell morphogenesis in response to environmental cues.

    PubMed

    Nakamura, Masayoshi

    2015-02-01

    In higher plants, reorientation of cortical microtubule arrays has been postulated to be of importance for modifying cell growth to adapt to environmental conditions. However, the process of microtubule reorientation is largely unknown. Recent genetic and live cell imaging studies of microtubule dynamics shed light on the regulatory mechanisms of microtubule molecular nucleation and severing apparatuses, which are required for array reorientation in response to blue light signaling. Branching nucleation from ?-tubulin complexes creates a small population of discordant microtubules that are acted on by KATANIN-mediated severing in two ways. KATANIN releases microtubules from nucleation sites and rapidly amplifies discordant microtubules by severing at microtubule crossovers. In this review, I focus on the molecular details of these two enzymes, which enable microtubule array transition. PMID:25729799

  7. Doublet-triplet fermionic dark matter

    NASA Astrophysics Data System (ADS)

    Dedes, Athanasios; Karamitros, Dimitrios

    2014-06-01

    We extend the Standard Model (SM) by adding a pair of fermionic SU(2) doublets with opposite hypercharge and a fermionic SU(2) triplet with zero hypercharge. We impose a discrete Z2 symmetry that distinguishes the SM fermions from the new ones. Then, gauge invariance allows for two renormalizable Yukawa couplings between the new fermions and the SM Higgs field, as well as for direct masses for the doublet (MD) and the triplet (MT). After electroweak symmetry breaking, this model contains, in addition to SM particles, two charged Dirac fermions and a set of three neutral Majorana fermions, the lightest of which contributes to dark matter (DM). We consider a case where the lightest neutral fermion is an equal admixture of the two doublets with mass MD close to the Z-boson mass. This state remains stable under radiative corrections thanks to a custodial SU(2) symmetry and is consistent with the experimental data from oblique electroweak corrections. Moreover, the amplitudes relevant to spin-dependent or spin-independent nucleus-DM particle scattering cross sections both vanish at tree level. They arise at one loop at a level that may be observed in near future DM direct detection experiments. For Yukawa couplings comparable to the top quark, the DM particle relic abundance is consistent with observation, not relying on coannihilation or resonant effects, and has a mass at the electroweak scale. Furthermore, the heavier fermions decay to the DM particle and to electroweak gauge bosons making this model easily testable at the LHC. In the regime of interest, the charged fermions suppress the Higgs decays to diphotons by 45%-75% relative to SM prediction.

  8. Frustrated symmetries in multi-Higgs-doublet models

    E-print Network

    Igor P. Ivanov; Venus Keus

    2010-07-29

    Within multi-Higgs-doublet models, one can impose symmetries on the Higgs potential, either discrete or continuous, that mix several doublets. In two-Higgs-doublet model any such symmetry can be conserved or spontaneously violated after the electroweak symmetry breaking (EWSB), depending on the coefficients of the potential. With more than two doublets, there exist symmetries which are always spontaneously violated after EWSB. We discuss the origin of this phenomenon and show its similarity to geometric frustration in condensed-matter physics.

  9. Dynein axonemal intermediate chain 2 is required for formation of the left-right body axis and kidney in medaka.

    PubMed

    Nagao, Yusuke; Cheng, Jinglei; Kamura, Keiichiro; Seki, Ryoko; Maeda, Aya; Nihei, Daichi; Koshida, Sumito; Wakamatsu, Yuko; Fujimoto, Toyoshi; Hibi, Masahiko; Hashimoto, Hisashi

    2010-11-01

    Ciliary defects lead to various diseases, such as primary ciliary dyskinesia (PCD) and polycystic kidney disease (PKD). We isolated a medaka mutant mii, which exhibits defects in the left-right (LR) polarity of organs, and found that mii encodes dynein axonemal intermediate chain 2a (dnai2a). Ortholog mutations were recently reported to cause PCD in humans. mii mutant embryos exhibited loss of nodal flow in Kupffer's Vesicle (KV), which is equivalent to the mammalian node, and abnormal expression of the left-specific gene. KV cilia in the mii mutant were defective in their outer dynein arms (ODAs), indicating that Dnai2a is required for ODA formation in KV cilia. While the mii mutant retained motility of the renal cilia and failed to show PKD, the loss of dnai2a and another dnai2 ortholog dnai2b led to PKD. These findings demonstrate that Dnai2 proteins control LR polarity and kidney formation through regulation of ciliary motility. PMID:20707998

  10. Kinetochore microtubules in PTK cells

    PubMed Central

    1992-01-01

    We have analyzed the fine structure of 10 chromosomal fibers from mitotic spindles of PtK1 cells in metaphase and anaphase, using electron microscopy of serial thin sections and computer image processing to follow the trajectories of the component microtubules (MTs) in three dimensions. Most of the kinetochore MTs ran from their kinetochore to the vicinity of the pole, retaining a clustered arrangement over their entire length. This MT bundle was invaded by large numbers of other MTs that were not associated with kinetochores. The invading MTs frequently came close to the kinetochore MTs, but a two-dimensional analysis of neighbor density failed to identify any characteristic spacing between the two MT classes. Unlike the results from neighbor density analyses of interzone MTs, the distributions of spacings between kinetochore MTs and other spindle MTs revealed no evidence for strong MT-MT interactions. A three-dimensional analysis of distances of closest approach between kinetochore MTs and other spindle MTs has, however, shown that the most common distances of closest approach were 30-50 nm, suggesting a weak interaction between kinetochore MTs and their neighbors. The data support the ideas that kinetochore MTs form a mechanical connection between the kinetochore and the pericentriolar material that defines the pole, but that the mechanical interactions between kinetochore MTs and other spindle MTs are weak. PMID:1629239

  11. 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

  12. 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

  13. Spindle Microtubule Differentiation and Deployment during Micronuclear Mitosis in Paramecium

    E-print Network

    John B. Tucker; Sally A. Mathews; Kay A. K. Hendry; John B; David L. J. Roche

    ABSTRACT Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of ~24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60 % of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of wellelongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules

  14. Equine platelets contain an anisotropic array of microtubules which reorganise upon activation

    Microsoft Academic Search

    F. Tablin; M. D. Castro

    1992-01-01

    Electron microscopic and immunofluorescence studies of equine platelets reveal a distinctive distribution of microtubules. Resting cells have a crosslinked microtubule coil and an additional array of anisotropic microtubules. Thrombin activation of equine platelets results in platelet shape change accompanied by reorganisation of the microtubule arrays. The microtubule coil remains intact, while short linear microtubules are present within the pseudopodia. To

  15. Dynamics of oscillating erythrocyte doublets after electrofusion.

    PubMed

    Baumann, M

    1999-11-01

    Erythrocytes were electrofused with multiple rectangular voltage pulses to show an oscillatory movement, divided into swell phases and pump events. During each swell phase, which lasted from 0.5 s to more than 180 s, the fused cells' (doublets') volume increased by colloid osmotic swelling, and the membrane area was expanded until rupture. Membrane rupture initiated the pump event, where the doublets' volume and membrane area decreased with an almost exponential time course and time constants between 2 ms and 8 ms. Simultaneously, a portion of cytosolic hemoglobin solution was ejected into extracellular space ("jet"). Pump event time constants and swell phase durations decreased with rising chamber temperature, indicating that both parts of the oscillatory movements were determined by physical properties of membrane and liquids. Relative volume change developments express a gradual loss of membrane elasticity during the oscillation, decreasing the elastic forces stored in the membrane. Evidence is given that the first rupture causes a weakening of the membrane at the rupture site. Heat treatment up to 45 degrees C had a negligible effect on swell times, pump time constants, and relative volume changes. A heat treatment of 50 degrees C prevented oscillatory movements. The rupture location accorded with theories of potential induced membrane electropermeabilization. PMID:10545360

  16. Motor function in interpolar microtubules during metaphase.

    PubMed

    Deutsch, J M; Lewis, Ian P

    2015-04-01

    We analyze experimental motility assays of microtubules undergoing small fluctuations about a "balance point" when mixed in solution of two different kinesin motor proteins, KLP61F and Ncd. It has been proposed that the microtubule movement is due to stochastic variations in the densities of the two species of motor proteins. We test this hypothesis here by showing how it maps onto a one-dimensional random walk in a random environment. Our estimate of the amplitude of the fluctuations agrees with experimental observations. We point out that there is an initial transient in the position of the microtubule where it will typically move of order its own length. We compare the physics of this gliding assay to a recent theory of the role of antagonistic motors on restricting interpolar microtubule sliding of a cell?s mitotic spindle during prometaphase. It is concluded that randomly positioned antagonistic motors can restrict relative movement of microtubules, however they do so imperfectly. A variation in motor concentrations is also analyzed and shown to lead to greater control of spindle length. PMID:25613413

  17. Ferritin associates with marginal band microtubules

    SciTech Connect

    Infante, Anthony A. [Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459 (United States); Infante, Dzintra [Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459 (United States); Chan, M.-C. [Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459 (United States); How, P.-C. [Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459 (United States); Kutschera, Waltraud [Max F. Perutz Laboratories, Department of Molecular Cell Biology, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna (Austria); Linhartova, Irena [Max F. Perutz Laboratories, Department of Molecular Cell Biology, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna (Austria); Muellner, Ernst W. [Max F. Perutz Laboratories, Department of Biochemistry, Medical University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna (Austria); Wiche, Gerhard [Max F. Perutz Laboratories, Department of Molecular Cell Biology, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna (Austria); Propst, Friedrich [Max F. Perutz Laboratories, Department of Molecular Cell Biology, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna (Austria)]. E-mail: friedrich.propst@univie.ac.at

    2007-05-01

    We characterized chicken erythrocyte and human platelet ferritin by biochemical studies and immunofluorescence. Erythrocyte ferritin was found to be a homopolymer of H-ferritin subunits, resistant to proteinase K digestion, heat stable, and contained iron. In mature chicken erythrocytes and human platelets, ferritin was localized at the marginal band, a ring-shaped peripheral microtubule bundle, and displayed properties of bona fide microtubule-associated proteins such as tau. Red blood cell ferritin association with the marginal band was confirmed by temperature-induced disassembly-reassembly of microtubules. During erythrocyte differentiation, ferritin co-localized with coalescing microtubules during marginal band formation. In addition, ferritin was found in the nuclei of mature erythrocytes, but was not detectable in those of bone marrow erythrocyte precursors. These results suggest that ferritin has a function in marginal band formation and possibly in protection of the marginal band from damaging effects of reactive oxygen species by sequestering iron in the mature erythrocyte. Moreover, our data suggest that ferritin and syncolin, a previously identified erythrocyte microtubule-associated protein, are identical. Nuclear ferritin might contribute to transcriptional silencing or, alternatively, constitute a ferritin reservoir.

  18. Contrasting models for kinetochore microtubule attachment in mammalian cells

    PubMed Central

    Dong, Yimin

    2010-01-01

    Kinetochore function is mediated through its interaction with microtubule plus ends embedded in the kinetochore outer plate. Here, we compare and evaluate current models for kinetochore microtubule attachment, beginning with a brief review of the molecular, biochemical, cellular, and structural studies upon which these models are based. The majority of these studies strongly support a model in which the kinetochore outer plate is a network of fibers that form multiple weak attachments to each microtubule, chiefly through the Ndc80 complex. Multiple weak attachments enable kinetochores to remain attached to microtubule plus ends that are continually growing and shrinking. It is unlikely that rings or “kinetochore fibrils” have a significant role in kinetochore microtubule attachment, but such entities could have a role in stabilizing attachment, modifying microtubule dynamics, and harnessing the energy released from microtubule disassembly. It is currently unclear whether kinetochores control and coordinate the dynamics of individual kinetochore microtubules. PMID:20336345

  19. Force fluctuations and polymerization dynamics of intracellular microtubules

    E-print Network

    including cell division, migration, and intracellular transport. Microtubules are very rigid and form, in cultured animal cells, bending is suppressed by the surrounding elastic cytoskeleton, and even large cell divi- sion, migration, and transport. Microtubules exhibit highly dynamic growth behavior

  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. Ethanol impairs microtubule formation via interactions at a microtubule associated protein-sensitive site

    PubMed Central

    Smith, Katherine J.; Butler, Tracy R.; Prendergast, Mark A.

    2014-01-01

    Prolonged ethanol abuse has been associated with brain injury caused by impaired synaptogenesis, cellular migration, neurogenesis, and cell signaling, all of which require proper microtubule functioning. However, the means by which ethanol may impair microtubule formation or function and the role that microtubule-associated proteins (MAPs) have in mediating such effects are not clear. In the present studies, purified MAP-deficient (2 mg/mL) and MAP-rich (pre-conjugated; 1 mg/mL) bovine ?/? tubulin dimer were allowed to polymerize at 37 °C, forming microtubules in the presence or absence of ethanol (25–500 mM). Microtubule formation was assessed in a 96-well format using a turbidity assay, with absorption measured at 340 nm for 45 min. Additional studies co-exposed ?/? tubulin dimers to 50 mM ethanol and purified MAPs (0.1 mg/mL) for 45 min. Polymerization of MAP-deficient tubulin was significantly decreased (at 15–45 min of polymerization) during exposure to ethanol (> 25 mM). In contrast, ethanol exposure did not alter polymerization of ?/? tubulin dimers pre-conjugated to MAPs, at any concentration. Concurrent exposure of MAP-deficient tubulin with purified MAPs and ethanol resulted in significant and time-dependent decreases in tubulin polymerization, with recovery from inhibition at later time points. The present results suggest that ethanol disrupts MAP-independent microtubule formation and MAP-dependent microtubule formation via direct actions at a MAP-sensitive microtubule residue, indicating that disruption of neuronal microtubule formation and function may contribute to the neurodegenerative effects of binge-like ethanol intake. PMID:24055335

  2. 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

  3. GDP-Tubulin Incorporation into Growing Microtubules Modulates Polymer Stability*

    PubMed Central

    Valiron, Odile; Arnal, Isabelle; Caudron, Nicolas; Job, Didier

    2010-01-01

    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.” PMID:20371874

  4. Microtubules in statocytes from roots of cress ( Lepidium sativum L.)

    Microsoft Academic Search

    W. Hensel

    1984-01-01

    Summary Statocytes in root caps ofLepidium sativum L. were examined by means of ultrathin serial sections to evaluate the amount and distribution of cortical microtubules. The microtubules encircle the cell, oriented normal to the root length axis. In the distal cell edges, microtubules form a network, separating the distal complex of endoplasmic reticulum from the plasmalemma. Preprophase bands in meristem

  5. The Zeeman effect in molecular doublet-doublet transitions in the high field-low field limit

    E-print Network

    Paris-Sud XI, Université de

    L-383 The Zeeman effect in molecular doublet-doublet transitions in the « high field-low field state. The respective Zeeman effects are therefore close to the weak-field limit of a strongly coupled state and the strong-field, uncoupled limit (Paschen-Back effect) of the weakly coupled state. Zeeman

  6. Constraining the Doublet Left-Right Model

    E-print Network

    Silva, Luiz Vale

    2015-01-01

    Left-Right Models (LRM) attempt at giving an understanding of the violation of parity (or charge-conjugation) by the weak interactions in the SM through a similar description of left- and right-handed currents at high energies. The spontaneous symmetry breaking of the LRM gauge group is triggered by an enlarged Higgs sector, usually consisting of two triplet fields (left-right symmetry breaking) and a bidoublet (electroweak symmetry breaking). I reconsider an alternative LRM with doublet instead of triplet fields. After explaining some features of this model, I discuss constraints on its parameters using electroweak precision observables (combined using the CKMfitter frequentist statistical framework) and neutral-meson mixing observables.

  7. Analysis of microtubule polymerization dynamics in live cells

    PubMed Central

    Gierke, Sarah; Kumar, Praveen; Wittmann, Torsten

    2012-01-01

    Intracellular microtubule polymerization dynamics are spatiotemporally controlled by numerous microtubule-associated proteins and other mechanisms, and this regulation is central to many cell processes. Here, we give an overview and practical guide on how to acquire and analyze time-lapse sequences of dynamic microtubules in live cells by either fluorescently labeling entire microtubules or by utilizing proteins that specifically associate only with growing microtubule ends, and summarize the strengths and weaknesses of different approaches. We give practical recommendations for imaging conditions, and we also discuss important limitations of such analysis that are dictated by the maximal achievable spatial and temporal sampling frequencies. PMID:20719263

  8. [A functional flagella with a 6 + 0 pattern

    PubMed Central

    1975-01-01

    The male gamete of the Gregarine Lecudina tuzetae has been studied with transmission electron microscopy and microcinematography. It is characterized by a flagellar axoneme of 6 + 0 pattern, a reduction of the chondriome, and the abundance of storage polysaccharide or lipid bodies. The movements of the flagella are of the undulating type and they are performed in the three dimensions of space. They are very slow, with a cycle time of about 2s. The structure of the axoneme components are similar to those of flagella with a 9 + 2 pattern. Each doublet has overall dimensions of 350 x 220 A; the space between the adjacent doublets is about 160 A. The A subfiber bears arms like dynein arms. The diameter of the axoneme is about 1,000 A. The basal body consists of a cylinder of dense material 2,500 A long and 1,300- 1,400 A in diameter; a microtubule 200 A in diameter is present in the axis. This study shows that a 6 + 0 pattern can generate a flagellar movement. The mechanism of the flagellar movement of the male gamete of L. tuzetae does not require the presence of central microtubules and it would include molecular interactions of the dynein-tubulin type between the adjacent peripheric doublets. The slowness of the movements is discussed in terms of the axoneme's structure and its energy supply. Finally, the phylogenetic significance of this flagella is examined on the basis of the morphopoietic potentialities of the centriolar structures. PMID:169268

  9. Microtubule Stabilizing Agents in Clinical Oncology

    Microsoft Academic Search

    Chris H. Takimoto; Muralidhar Beeram

    The taxanes are a versatile and important class of drugs that target microtubules. Paclitaxel and docetaxel are some of the\\u000a most widely used cancer chemofherapeutic agents in clinical oncology. In this chapter, we discuss the most common dose and\\u000a treatment schedules, clinical efficacy, and the toxicity profiles of these agents in detail.

  10. Microtubule Motors in Microfluidics Maruti Uppalapati,

    E-print Network

    Hancock, William O.

    CHAPTER 1 3 Microtubule Motors in Microfluidics Maruti Uppalapati, 1 Ying-Ming Huang, 2 Shankar division. Because emerging microfluidic devices uti- lize channel geometries similar to cellular scales in incorporating biomotor-driven transport into microfluidic devices. Kinesin-driven transport has the advantage

  11. XMAP215 Is a Processive Microtubule Polymerase

    E-print Network

    Harrison, Stephen C.

    , Berlin-Buch, Germany. 5These authors contributed equally to this work. *Correspondence: howard@mpi-cbg.de (J.H.), hyman@mpi-cbg.de (A.A.H.) DOI 10.1016/j.cell.2007.11.043 SUMMARY Fast growth of microtubules

  12. Forces due to curving protofilaments in microtubules

    NASA Astrophysics Data System (ADS)

    Vichare, Shirish; Jain, Ishutesh; Inamdar, Mandar M.; Padinhateeri, Ranjith

    2013-12-01

    Microtubules consist of 13 protofilaments arranged in the form of a cylinder. The protofilaments are composed of longitudinally attached tubulin dimers that can exist in either a less curved state [GTP-bound tubulin (T)] or a more curved state [GDP-bound tubulin (D)]. Hydrolysis of T into D leaves the straight and laterally attached protofilaments of the microtubule in a mechanically stressed state, thus leading to their unzipping. The elastic energy in the unzipping protofilaments can be harnessed by a force transducer such as the Dam1-kinetochore ring complex in order to exert pulling force on chromosomes during cell division. In the present paper we develop a simple continuum model to obtain this pulling force as a function of the mechanical properties of protofilaments and the size of the Dam1-kinetochore ring. We also extend this model to investigate the role played by the T subunits found at the plus end of the microtubule (the T cap) on the mechanical stability of microtubules.

  13. PLANT SCIENCE: Microtubules Make Tracks for Cellulose

    NSDL National Science Digital Library

    Clive Lloyd (John Innes Centre; Department of Cell and Developmental Biology)

    2006-06-09

    Access to the article is free, however registration and sign-in are required. Cellulose fibers encircle plant cells and help support the cell and the whole plant. They are laid down in this pattern as they are spooled out by enzymes that track along microtubules in the cell.

  14. Microtubule dynamics control tail retraction in migrating vascular endothelial cells.

    PubMed

    Ganguly, Anutosh; Yang, Hailing; Zhang, Hong; Cabral, Fernando; Patel, Kamala D

    2013-12-01

    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; however, 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 nondirectional 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, whereas 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

  15. 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.

  16. 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

  17. 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 nanodevices involving microtubules. It will also help in designing and optimizing microtubules for other synthetic uses.

  18. Doublet-mechanical approach to elastic homogenization

    SciTech Connect

    Ferrari, M.; Hanford, D. [Univ. of California, Berkeley, CA (United States)

    1996-10-01

    The process of deducing the overall properties of multi-phase media from phase properties and distributional data is referred to as homogenization. Two prominent homogenization modes are (1) the so-called direct, or concentrator-based approaches; and (2) the so-called mathematical homogenization, or cell-based method. Within the direct method one can classify the Eshelby, the Mori-Tanaka, the Voigt, the Reuss, and the ploy-inclusion approaches. As was proven by one of the authors (MF) in recent publications, none of the existing approaches satisfies even most elementary admissibility criteria for the general bi-phase composite, i.e., the search for general concentrators is still far from complete. The mathematical homogenization method, developed by Tartar and Sanchez-Palencia among others, reduces the overall effective property prediction to the numerical solution of a representative cell problem. In this paper, the methods of the Doublet Mechanics (DM) of V.T. Granik and M. Ferrari are employed to address both the concentrator problem of the direct approach, and the cell problem of mathematical homogenization. In the former, a choice of macroscopic concentrator is determined exactly from the closed-form solution of a micromechanical problem. The latter problem is solved by identifying the representative micro-level volume with an assembly of points with translational regularity, and employing the discrete-continuum transition that underlies DM.

  19. Microtubule Stabilization Leads to Growth Reorientation in Arabidopsis Trichomes

    PubMed Central

    Mathur, Jaideep; Chua, Nam-Hai

    2000-01-01

    The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using transgenic Arabidopsis plants expressing a green fluorescent protein–microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with respect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorientation event, we investigated the effects of microtubule-interacting drugs on branch induction in trichomes. In unbranched trichomes of the mutant stichel, a change in growth directionality, closely simulating branch initiation, could be elicited by a short treatment with paclitaxel, a microtubule-stabilizing drug, but not with microtubule-disrupting drugs. The growth reorientation appeared to be linked to increased microtubule stabilization and to aster formation in the treated trichomes. Taxol-induced microtubule stabilization also led to the initiation of new branch points in the zwichel mutant of Arabidopsis, which is defective in a kinesin-like microtubule motor protein and possesses trichomes that are less branched. Our observations suggest that trichome cell branching in Arabidopsis might be mediated by transiently stabilized microtubular structures, which may form a component of a multiprotein complex required to reorient freshly polymerizing microtubules into new growth directions. PMID:10760237

  20. An assay to image neuronal microtubule dynamics in mice

    PubMed Central

    Kleele, Tatjana; Marinkovi?, Petar; Williams, Philip R.; Stern, Sina; Weigand, Emily E.; Engerer, Peter; Naumann, Ronald; Hartmann, Jana; Karl, Rosa M.; Bradke, Frank; Bishop, Derron; Herms, Jochen; Konnerth, Arthur; Kerschensteiner, Martin; Godinho, Leanne; Misgeld, Thomas

    2014-01-01

    Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease. PMID:25219969

  1. Visualization of microtubules of cells in situ by indirect immunofluorescence.

    PubMed

    Byers, H R; Fujiwara, K; Porter, K R

    1980-11-01

    Microtubule staining patterns can be visualized within cells in situ on the surface of fish scales from the squirrel fish, Holocentrus ascensionis, and the common goldfish, Carassius auratus, after incubation with antibodies to sea urchin tubulin and fluorescein-labeled goat antibodies to rabbit immunoglobulin G. Chromatophores in situ from both species reveal a radial microtubule framework that orients the alignment of pigment granules. Innervating fibers of erythrophores on the H. ascensionis scale can also be observed. In situ, pseudo-epithelial cells called scleroblasts show microtubule patterns with a remarkable degree of similarity within a selected region. Over 90% of the cells have a microtubule framework that is nearly superimposable from cell to adjacent cell. The microtubules in scleroblasts are few and form a simple radial framework with a localized microtubule organizing center (MTOC). Microtubules in scleroblasts in vitro emanate from localized MTOCs but are much less radially organized than in situ. Scleroblasts in situ on the scale of C. auratus show microtubules that curve abruptly into coalignment with phase striations on the fibrillary plate. The phase striations arise from the orthogonal plies of collagen in intimate association with the scleroblasts. The role of microtubules in scleroblasts may thus be to provide orientation for collagen fibrillogenesis, analogous to their role in orientation of cellulose fibers in plants. That cells in situ exhibit highly related and coordinated microtubule staining patterns reaffirms that the cytoskeleton plays an important role in the organization of differentiated tissues. PMID:6935678

  2. Regulation of microtubule assembly by human EB1 family proteins.

    PubMed

    Bu, W; Su, L K

    2001-05-31

    The EB1 family proteins are highly conserved microtubule-associated proteins. The EB1 protein in yeast has been shown to play an important role in regulating microtubule dynamics and chromosome segregation. Human EB1 family proteins include EB1, RP1 and EBF3. Although EB1 and RP1 have been shown to associate with microtubules, the subcellular localization of endogenous EBF3 had not been characterized. The function of human EB1 family proteins was also not clear. We therefore investigated the cellular localization of EBF3 and the regulation of microtubule organization by EB1 family proteins. As do EB1 and RP1, EBF3 was found to colocalize with microtubules, preferentially at their plus ends, throughout the cell cycle. Moreover, there was a very strong EBF3 signal at the centrosome in interphase cells and at the spindle poles in mitotic cells. When EB1 family proteins were overexpressed, they associated with the entire microtubule cytoskeleton. In addition, EB1 and EBF3 induced microtubule bundling in some cells overexpressing these proteins. These microtubule bundles were more resistant to nocodazole and were more acetylated than regular microtubules. Our results demonstrate for the first time that human EB1 family proteins could regulate microtubule assembly and stability. PMID:11423968

  3. INF1 Is a Novel Microtubule-associated Formin

    PubMed Central

    Young, Kevin G.; Thurston, Susan F.; Copeland, Sarah; Smallwood, Chelsea

    2008-01-01

    Formin proteins, characterized by the presence of conserved formin homology (FH) domains, play important roles in cytoskeletal regulation via their abilities to nucleate actin filament formation and to interact with multiple other proteins involved in cytoskeletal regulation. The C-terminal FH2 domain of formins is key for actin filament interactions and has been implicated in playing a role in interactions with microtubules. Inverted formin 1 (INF1) is unusual among the formin family in having the conserved FH1 and FH2 domains in its N-terminal half, with its C-terminal half being composed of a unique polypeptide sequence. In this study, we have examined a potential role for INF1 in regulating microtubule structure. INF1 associates discretely with microtubules, and this association is dependent on a novel C-terminal microtubule-binding domain. INF1 expressed in fibroblast cells induced actin stress fiber formation, coalignment of microtubules with actin filaments, and the formation of bundled, acetylated microtubules. Endogenous INF1 showed an association with acetylated microtubules, and knockdown of INF1 resulted in decreased levels of acetylated microtubules. Our data suggests a role for INF1 in microtubule modification and potentially in coordinating microtubule and F-actin structure. PMID:18815276

  4. Hyperphosphorylation of microtubule-associated tau protein plays dual role in neurodegeneration and neuroprotection

    Microsoft Academic Search

    Yao Zhang; Qing Tian; Qi Zhang; Xinwen Zhou; Shijie Liu; Jian-Zhi Wang

    2009-01-01

    The microtubule-associated protein tau plays a major role in maintaining the normal morphology of the neurons. The major biological activity of tau is to promote microtubule assembly and stabilize the microtubules. In the brain of Alzheimer's disease (AD) patients, tau protein is abnormally hyperphosphorylated and thus become incompetent in promoting microtubule assemble and maintaining the stability of the microtubules. These

  5. Statistical case for specifying tolerances of doublet lenses jointly

    NASA Astrophysics Data System (ADS)

    Kehoe, Michael

    2014-12-01

    The interactions between errors in manufacturing are examined for ten double Gauss lens specifications drawn from U.S. patents. The particular focus is on center thickness and radius tolerances of doublet lenses in these specifications and on the possibility of specifying these tolerances jointly. A procedure for rapid identification of lenses whose performance would be improved by joint tolerance specification is described. Then benefits of specifying thickness and radius tolerances of doublet lenses jointly are demonstrated using Monte Carlo analysis.

  6. The inert doublet model of dark matter revisited

    Microsoft Academic Search

    Laura Lopez Honorez; Carlos E. Yaguna

    2010-01-01

    The inert doublet model, a minimal extension of the Standard Model by a second higgs doublet with no direct couplings to quarks\\u000a or leptons, is one of the simplest scenarios that can explain the dark matter. In this paper, we study in detail the impact\\u000a of dark matter annihilation into the three-body final state on the phenomenology of the inert

  7. The inert doublet model of dark matter revisited

    Microsoft Academic Search

    Laura Lopez Honorez; Carlos E. Yaguna

    2010-01-01

    The inert doublet model, a minimal extension of the Standard Model by a second higgs doublet with no direct couplings to quarks or leptons, is one of the simplest scenarios that can explain the dark matter. In this paper, we study in detail the impact of dark matter annihilation into the three-body final state W{W^*}left( { to Wfbar{f}'} right) on

  8. Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration

    PubMed Central

    Tang-Schomer, Min D.; Patel, Ankur R.; Baas, Peter W.; Smith, Douglas H.

    2010-01-01

    Little is known about which components of the axonal cytoskeleton might break during rapid mechanical deformation, such as occurs in traumatic brain injury. Here, we micropatterned neuronal cell cultures on silicone membranes to induce dynamic stretch exclusively of axon fascicles. After stretch, undulating distortions formed along the axons that gradually relaxed back to a straight orientation, demonstrating a delayed elastic response. Subsequently, swellings developed, leading to degeneration of almost all axons by 24 h. Stabilizing the microtubules with taxol maintained the undulating geometry after injury but greatly reduced axon degeneration. Conversely, destabilizing microtubules with nocodazole prevented undulations but greatly increased the rate of axon loss. Ultrastructural analyses of axons postinjury revealed immediate breakage and buckling of microtubules in axon undulations and progressive loss of microtubules. Collectively, these data suggest that dynamic stretch of axons induces direct mechanical failure at specific points along microtubules. This microtubule disorganization impedes normal relaxation of the axons, resulting in undulations. However, this physical damage also triggers progressive disassembly of the microtubules around the breakage points. While the disintegration of microtubules allows delayed recovery of the “normal” straight axon morphology, it comes at a great cost by interrupting axonal transport, leading to axonal swelling and degeneration.—Tang-Schomer, M. D., Patel, A. R,, Baas, P. W., Smith, D. H. Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration. PMID:20019243

  9. The Arabidopsis Microtubule-Associated Protein AtMAP65-1: Molecular Analysis of Its Microtubule Bundling Activity

    PubMed Central

    Smertenko, Andrei P.; Chang, Hsin-Yu; Wagner, Vera; Kaloriti, Despina; Fenyk, Stepan; Sonobe, Seiji; Lloyd, Clive; Hauser, Marie-Theres; Hussey, Patrick J.

    2004-01-01

    The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle. PMID:15273298

  10. Inhibition of HDAC6 deacetylase activity increases its binding with microtubules and suppresses microtubule dynamic instability in MCF-7 cells.

    PubMed

    Asthana, Jayant; Kapoor, Sonia; Mohan, Renu; Panda, Dulal

    2013-08-01

    The post-translational modification of tubulin appears to be a highly controlled mechanism that regulates microtubule functioning. Acetylation of the ?-amino group of Lys-40 of ?-tubulin marks stable microtubules, although the causal relationship between tubulin acetylation and microtubule stability has remained poorly understood. HDAC6, the tubulin deacetylase, plays a key role in maintaining typical distribution of acetylated microtubules in cells. Here, by using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of HDAC6, we have explored whether tubulin acetylation has a role in regulating microtubule stability. We found that whereas both pharmacological inhibition of HDAC6 as well as its depletion enhance microtubule acetylation, only pharmacological inhibition of HDAC6 activity leads to an increase in microtubule stability against cold and nocodazole-induced depolymerizing conditions. Tubastatin A treatment suppressed the dynamics of individual microtubules in MCF-7 cells and delayed the reassembly of depolymerized microtubules. Interestingly, both the localization of HDAC6 on microtubules and the amount of HDAC6 associated with polymeric fraction of tubulin were found to increase in the tubastatin A-treated cells compared with the control cells, suggesting that the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to microtubules. The evidence presented in this study indicated that the increased binding of HDAC6, rather than the acetylation per se, causes microtubule stability. The results are in support of a hypothesis that in addition to its deacetylase function, HDAC6 might function as a MAP that regulates microtubule dynamics under certain conditions. PMID:23798680

  11. MICROBIOLOGY: Bacterial Bushwacking Through a Microtubule Jungle

    NSDL National Science Digital Library

    Jean-Pierre Gorvel (INSERM-CNRSUniversité de la Méditerranée Parc Scientifique de Luminy Case 906; Centre d'Immunologie)

    2006-11-10

    Access to the article is free, however registration and sign-in are required. Our view of the cell's cytoplasm has come a long way. Once considered static "free space" between the nucleus and plasma membrane, it is now known to be a highly dynamic cellular entity with limited space for free movement. It is a dense, organized, tightly regulated, and dynamic network of organelles, cytoskeleton (including microtubules, actin, and intermediate filaments), and vesicles that shuttle between organelles. Yet, some pathogenic bacteria move quite efficiently through this cytoplasmic jungle, invading one cell to the next. Yoshida et al. report that Shigella, the bacteria responsible for dysentary, hacks its way through microtubules by wielding a tubulin- specific protease.

  12. Fluorescent microtubules break up under illumination

    Microsoft Academic Search

    G. P. A. Vigers; M. Coue; J. R. McIntosh

    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 an- alogues. We have also further characterized the tubulin derivatized with dichlorotriazinyl-aminofluorescein that has previously been used in this and other laborato- ries. Our results show that all four analogues assemble into microtubules which

  13. Dynamic instability of microtubules: effect of catastrophe-suppressing drugs

    E-print Network

    Pankaj Kumar Mishra; Ambarish Kunwar; Sutapa Mukherji; Debashish Chowdhury

    2007-02-21

    Microtubules are stiff filamentary proteins that constitute an important component of the cytoskeleton of cells. These are known to exhibit a dynamic instability. A steadily growing microtubule can suddenly start depolymerizing very rapidly; this phenomenon is known as ``catastrophe''. However, often a shrinking microtubule is ``rescued'' and starts polymerizing again. Here we develope a model for the polymerization-depolymerization dynamics of microtubules in the presence of {\\it catastrophe-suppressing drugs}. Solving the dynamical equations in the steady-state, we derive exact analytical expressions for the length distributions of the microtubules tipped with drug-bound tubulin subunits as well as those of the microtubules, in the growing and shrinking phases, tipped with drug-free pure tubulin subunits. We also examine the stability of the steady-state solutions.

  14. Microtubules stabilize cell polarity by localizing rear signals.

    PubMed

    Zhang, Jian; Guo, Wei-Hui; Wang, Yu-Li

    2014-11-18

    Microtubules are known to play an important role in cell polarity; however, the mechanism remains unclear. Using cells migrating persistently on micropatterned strips, we found that depolymerization of microtubules caused cells to change from persistent to oscillatory migration. Mathematical modeling in the context of a local-excitation-global-inhibition control mechanism indicated that this mechanism can account for microtubule-dependent oscillation, assuming that microtubules remove inhibitory signals from the front after a delayed generation. Experiments further supported model predictions that the period of oscillation positively correlates with cell length and that oscillation may be induced by inhibiting retrograde motors. We suggest that microtubules are required not for the generation but for the maintenance of cell polarity, by mediating the global distribution of inhibitory signals. Disassembly of microtubules induces cell oscillation by allowing inhibitory signals to accumulate at the front, which stops frontal protrusion and allows the polarity to reverse. PMID:25368191

  15. Drugs That Target Dynamic Microtubules: A New Molecular Perspective

    PubMed Central

    Stanton, Richard A.; Gernert, Kim M.; Nettles, James H.; Aneja, Ritu

    2011-01-01

    Microtubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. As such, there is a wide variety of compounds currently in clinical use and in development that act as antimitotic agents by altering microtubule dynamics. Although these diverse molecules are known to affect microtubule dynamics upon binding to one of the three established drug domains (taxane, vinca alkaloid, or colchicine site), the exact mechanism by which each drug works is still an area of intense speculation and research. In this study, we review the effects of microtubule-binding chemotherapeutic agents from a new perspective, considering how their mode of binding induces conformational changes and alters biological function relative to the molecular vectors of microtubule assembly or disassembly. These “biological vectors” can thus be used as a spatiotemporal context to describe molecular mechanisms by which microtubule-targeting drugs work. PMID:21381049

  16. 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.

  17. Novel synthetic pharmacophores inducing a stabilization of cellular microtubules.

    PubMed

    Martinez, Anne; Soleilhac, Emmanuelle; Barette, Caroline; Prudent, Renaud; Gozzi, Gustavo J; Vassal-Stermann, Emilie; Pillet, Catherine; Di Pietro, Attilio; Fauvarque, Marie-Odile; Lafanechere, Laurence

    2015-01-01

    Microtubule drugs have been widely used in cancer chemotherapies. Although microtubules are subject to regulation by signal transduction mechanisms, their pharmacological modulation has so far relied on compounds that bind to the tubulin subunit. Using a cell-based assay designed to probe the microtubule polymerization status, we identified two pharmacophores, CM09 and CM10, as cell-permeable microtubule stabilizing agents. These synthetic compounds do not affect the assembly state of purified microtubules in vitro but they profoundly suppress microtubule dynamics in vivo. Moreover, they exert cytotoxic effects on several cancer cell lines including multidrug resistant cell lines. Therefore, these classes of compounds represent novel attractive leads for cancer chemotherapy. PMID:25543663

  18. 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.

  19. Direct observation of single kinesin molecules moving along microtubules

    Microsoft Academic Search

    Ronald D. Vale; Takashi Funatsu; Daniel W. Pierce; Laura Romberg; Yoshie Harada; Toshio Yanagida

    1996-01-01

    KINESIN is a two-headed motor protein that powers organelle transport along microtubules1. Many ATP molecules are hydro-lysed by kinesin for each diffusional encounter with the micro-tubule2,3. Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total

  20. Gibberellin stabilizes microtubules in onion leaf sheath cells

    Microsoft Academic Search

    T. Mita; H. Shibaoka

    1984-01-01

    Summary Colchicine and cremart (O-ethyl O-(3-methyl-6-nitrophenyl) N-sec-butylphosphorothioamidate) disrupt microtubules in leaf sheath cells of onion plants (Allium cepa L. cv. Senshu-Chuko) and cause cell swelling to make the basal parts of the plants bulbous. Gibberellin A3(GA3) protects microtubules from disruption by colchicine and cremart and suppresses the swelling caused by them. GA3 also protects microtubules from disruption by low temperature.

  1. ARRANGEMENT OF SUBUNITS IN FLAGELLAR MICROTUBULES

    Microsoft Academic Search

    LINDA A. AMOS; A. KLUG

    SUMMARY Electron micrographs of outer doublet tubules from flagella have been analysed by methods which make use of the computed diffraction patterns of electron-microscope images. Analysis of singlet A-tubules in the tips of flagella has led to a determination of the helical surface lattice of the A-subfibre, confirming that there are 13 longitudinal protofilaments in the tubule wall and that

  2. Identification of cell cycle-regulated Drosophila microtubule-associated proteins using quantitative mass spectrometry 

    E-print Network

    Syred, Heather

    2011-01-01

    The microtubule network is the central framework in multiple cellular processes. The microtubule array undergoes dramatic changes as cells progress through the cell cycle. In mitosis the interphase microtubule array is ...

  3. The Human Kinetochore Ska1 Complex Facilitates Microtubule Depolymerization-Coupled Motility

    E-print Network

    Cheeseman, Iain McPherson

    Mitotic chromosome segregation requires that kinetochores attach to microtubule polymers and harness microtubule dynamics to drive chromosome movement. In budding yeast, the Dam1 complex couples kinetochores with microtubule ...

  4. ?-Tubulin controls neuronal microtubule polarity independently of Golgi outposts

    PubMed Central

    Nguyen, Michelle M.; McCracken, Christie J.; Milner, E. S.; Goetschius, Daniel J.; Weiner, Alexis T.; Long, Melissa K.; Michael, Nick L.; Munro, Sean; Rolls, Melissa M.

    2014-01-01

    Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by ?-tubulin might contribute to polarity, we analyzed neuronal microtubules in Drosophila containing gain- or loss-of-function alleles of ?-tubulin. Both increased and decreased activity of ?-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, we examined the distribution of ?-tubulin. Consistent with local nucleation, tagged and endogenous ?-tubulins were found in specific positions in dendrites and axons. Because the Golgi complex can house nucleation sites, we explored whether microtubule nucleation might occur at dendritic Golgi outposts. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when we dragged the Golgi out of dendrites with an activated kinesin, ?-tubulin remained in dendrites. We conclude that regulated microtubule nucleation controls neuronal microtubule polarity but that the Golgi complex is not directly involved in housing nucleation sites. PMID:24807906

  5. 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).

  6. Dynein and kinesin share an overlapping microtubule-binding site

    PubMed Central

    Mizuno, Naoko; Toba, Shiori; Edamatsu, Masaki; Watai-Nishii, Junko; Hirokawa, Nobutaka; Toyoshima, Yoko Y; Kikkawa, Masahide

    2004-01-01

    Dyneins and kinesins move in opposite directions on microtubules. The question of how the same-track microtubules are able to support movement in two directions remains unanswered due to the absence of details on dynein–microtubule interactions. To address this issue, we studied dynein–microtubule interactions using the tip of the microtubule-binding stalk, the dynein stalk head (DSH), which directly interacts with microtubules upon receiving conformational change from the ATPase domain. Biochemical and cryo-electron microscopic studies revealed that DSH bound to tubulin dimers with a periodicity of 80 Å, corresponding to the step size of dyneins. The DSH molecule was observed as a globular corn grain-like shape that bound the same region as kinesin. Biochemical crosslinking experiments and image analyses of the DSH–kinesin head–microtubule complex revealed competition between DSH and the kinesin head for microtubule binding. Our results demonstrate that dynein and kinesin share an overlapping microtubule-binding site, and imply that binding at this site has an essential role for these motor proteins. PMID:15175652

  7. Dynein and kinesin share an overlapping microtubule-binding site.

    PubMed

    Mizuno, Naoko; Toba, Shiori; Edamatsu, Masaki; Watai-Nishii, Junko; Hirokawa, Nobutaka; Toyoshima, Yoko Y; Kikkawa, Masahide

    2004-07-01

    Dyneins and kinesins move in opposite directions on microtubules. The question of how the same-track microtubules are able to support movement in two directions remains unanswered due to the absence of details on dynein-microtubule interactions. To address this issue, we studied dynein-microtubule interactions using the tip of the microtubule-binding stalk, the dynein stalk head (DSH), which directly interacts with microtubules upon receiving conformational change from the ATPase domain. Biochemical and cryo-electron microscopic studies revealed that DSH bound to tubulin dimers with a periodicity of 80 A, corresponding to the step size of dyneins. The DSH molecule was observed as a globular corn grain-like shape that bound the same region as kinesin. Biochemical crosslinking experiments and image analyses of the DSH-kinesin head-microtubule complex revealed competition between DSH and the kinesin head for microtubule binding. Our results demonstrate that dynein and kinesin share an overlapping microtubule-binding site, and imply that binding at this site has an essential role for these motor proteins. PMID:15175652

  8. ?-tubulin controls neuronal microtubule polarity independently of Golgi outposts.

    PubMed

    Nguyen, Michelle M; McCracken, Christie J; Milner, E S; Goetschius, Daniel J; Weiner, Alexis T; Long, Melissa K; Michael, Nick L; Munro, Sean; Rolls, Melissa M

    2014-07-01

    Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by ?-tubulin might contribute to polarity, we analyzed neuronal microtubules in Drosophila containing gain- or loss-of-function alleles of ?-tubulin. Both increased and decreased activity of ?-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, we examined the distribution of ?-tubulin. Consistent with local nucleation, tagged and endogenous ?-tubulins were found in specific positions in dendrites and axons. Because the Golgi complex can house nucleation sites, we explored whether microtubule nucleation might occur at dendritic Golgi outposts. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when we dragged the Golgi out of dendrites with an activated kinesin, ?-tubulin remained in dendrites. We conclude that regulated microtubule nucleation controls neuronal microtubule polarity but that the Golgi complex is not directly involved in housing nucleation sites. PMID:24807906

  9. Disruption of the A-Kinase Anchoring Domain in Flagellar Radial Spoke Protein 3 Results in Unregulated Axonemal cAMP-dependent Protein Kinase Activity and Abnormal Flagellar Motility

    PubMed Central

    Gaillard, Anne R.; Fox, Laura A.; Rhea, Jeanne M.; Craige, Branch

    2006-01-01

    Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility. PMID:16571668

  10. CSAP localizes to polyglutamylated microtubules and promotes proper cilia function and zebrafish development

    E-print Network

    Backer, Chelsea B.

    The diverse populations of microtubule polymers in cells are functionally distinguished by different posttranslational modifications, including polyglutamylation. Polyglutamylation is enriched on subsets of microtubules ...

  11. Axonemal Positioning and Orientation in 3-D Space for Primary Cilia: What is Known, What is Assumed, and What Needs Clarification

    PubMed Central

    Farnum, Cornelia E.; Wilsman, Norman J.

    2012-01-01

    Two positional characteristics of the ciliary axoneme – its location on the plasma membrane as it emerges from the cell, and its orientation in three-dimensional space – are known to be critical for optimal function of actively motile cilia (including nodal cilia), as well as for modified cilia associated with special senses. However, these positional characteristics have not been analyzed to any significant extent for primary cilia. This review briefly summarizes the history of knowledge of these two positional characteristics across a wide spectrum of cilia, emphasizing their importance for proper function. Then the review focuses what is known about these same positional characteristics for primary cilia in all major tissue types where they have been reported. The review emphasizes major areas that would be productive for future research for understanding how positioning and 3-D orientation of primary cilia may be related to their hypothesized signaling roles within different cellular populations. PMID:22012592

  12. Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2

    PubMed Central

    Petry, Sabine; Groen, Aaron C.; Ishihara, Keisuke; Mitchison, Timothy J.; Vale, Ronald D.

    2013-01-01

    Summary The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence also has suggested that microtubules might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires gamma-tubulin and augmin and is stimulated by GTP-bound Ran and its effector TPX2, factors previously implicated in chromatin-stimulated nucleation. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance. PMID:23415226

  13. Determination of drug binding to microtubules in vitro.

    PubMed

    Smith, Jennifer A; Jordan, Mary Ann

    2010-01-01

    Many naturally occurring compounds and their synthetic analogs bind to soluble tubulin or to tubulin in microtubules. These compounds are often important drugs or drug candidates. They can potently alter both the dynamics and the polymer mass of microtubules. The binding affinity of a drug for soluble tubulin heterodimers is a common and relatively readily determined biochemical characteristic of a tubulin-targeted drug. However, it is only one important aspect of the drug-tubulin interaction. In solution and in cells, soluble tubulin is in equilibrium with polymerized microtubules. It is as important to determine drug binding to microtubules as it is to determine binding to soluble tubulin, since drug binding to microtubules frequently alters their function. The affinity of a compound for microtubules often differs vastly from its affinity for soluble tubulin. Here, we present detailed instructions for assessing binding stoichiometry and affinity to assembled unstabilized microtubules using radiolabeled drug. In addition, using examples from binding results with several important drugs including vinca alkaloids, colchicine, eribulin, and taxanes, we discuss aspects of the interactions with microtubules that may alter the experimental design of the drug-binding experiments. PMID:20466141

  14. Jupiter, a New Drosophila Protein Associated With Microtubules

    E-print Network

    Villefranche sur mer

    Jupiter, a New Drosophila Protein Associated With Microtubules Nina Karpova,* Yves Bobinnec Drosophila protein Jupiter, which shares proper- ties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net pos- itive

  15. Cytoplasmic microtubule proteins of the embryo of Drosophila melanogaster.

    PubMed

    Green, L H; Brandis, J W; Turner, F R; Raff, R A

    1975-10-01

    We have been able to purify, in bulk, the cytoplasmic microtubule proteins of eggs and embryos of Drosophila melanogaster by means of in vitro self-assembly of microtubules from subunits present in a high-speed supernatant fraction of eggs or embryos. This provides the first successful application of this method to purification of microtubule protein from a source other than vertebrate brain, and the first purification of insect microtubule proteins. Our electron micrographs show that the in vitro assembled microtubules are morphologically typical and apparently are comprised of the expected 13 protofilaments. The protein we obtain from such preparations binds [3H]colchicine and has a sedimentation value of 6.4 S-6.9 S which is close to the predicted value for microtubule protein dimer. Both alpha- and beta-microtubule proteins are evident in sodium dodecyl sulfate polyacrylamide electropherograms of the isolated proteins. The apparent molecular weights of these species on dodecyl sulfate polyacrylamide gels are 54,000 and 52,000, respectively. These values as well as the amino acid composition and N-terminal methionine of the Drosophilia proteins are very closely comparable to microtubule proteins from other, unrelated organisms. PMID:809055

  16. Mathematics and biophysics of cortical microtubules in plants

    E-print Network

    Allard, Jun

    Mathematics and biophysics of cortical microtubules in plants by Jun Allard A THESIS SUBMITTED;Abstract Microtubules confined to the two-dimensional cortex of elongating plant cells must form a parallel membrane-anchor densities in different plants, including Arabidopsis cells and Tobacco cells. ii #12;Table

  17. 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.

  18. Neuronal Osmotransduction: Push-Activating TRPV1 with Microtubules

    E-print Network

    Gollisch, Tim

    Neuronal Osmotransduction: Push-Activating TRPV1 with Microtubules Marta Andre´ s1 and Martin C. Go channel TRPV1 and microtubules, which seem to directly push open the channel. Mammals employ sophisticated in ONs is medi- ated by the TRPV1 channel (Sharif Naeini et al., 2006), whose carboxy terminus was found

  19. Conserved microtubule–actin interactions in cell movement and morphogenesis

    Microsoft Academic Search

    Olga C. Rodriguez; Andrew W. Schaefer; Craig A. Mandato; William M. Bement; Clare M. Waterman-Storer

    2003-01-01

    Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions — regulatory and structural. These interactions comprise at

  20. A new directionality tool for assessing microtubule pattern alterations

    PubMed Central

    Liu, Wenhua; Ralston, Evelyn

    2014-01-01

    The cytoskeleton (microtubules, actin and intermediate filaments) has a cell type-specific spatial organization that is essential and reflects cell health. We are interested in understanding how changes in the organization of microtubules contribute to muscle diseases such as Duchenne muscular dystrophy (DMD). The grid-like immunofluorescence microtubule pattern of fast-twitch muscle fibers lends itself well to visual assessment. The more complicated pattern of other fibers does not. Furthermore, visual assessment is not quantitative. Therefore we have developed a robust software program for detecting and quantitating microtubule directionality. Such a tool was necessary because existing methods focus mainly on local image features and are not well suited for microtubules. Our tool, TeDT, is based on the Haralick texture method and takes into account both local and global features with more weight on the latter. The results are expressed in a graphic form responsive to subtle variations in microtubule distribution, while a numerical score allows quantitation of directionality. Furthermore, the results are not affected by imaging conditions or post-imaging procedures. TeDT successfully assesses test images and microtubules in fast-twitch fibers of wild-type and mdx mice (a model for DMD); TeDT also identifies and quantitates microtubule directionality in slow-twitch fibers, in the fibers of young animals, and in other mouse models which could not be assessed visually. TeDT might also contribute to directionality assessments of other cytoskeletal components. PMID:24497496

  1. Microtubules of the mouse testis exhibit differential sensitivity to the microtubule disruptors Carbendazim and colchicine.

    PubMed

    Correa, Liane M; Nakai, Masaaki; Strandgaard, Christina S; Hess, Rex A; Miller, Marion G

    2002-09-01

    The testicular toxicant benomyl and its metabolite, carbendazim cause reproductive damage to the rat, an early sign of which is sloughing of germ cells with associated Sertoli cell fragments. However, the sensitivity of other mammalian species to these benzimidazole compounds is not clear. In this study, the effects of carbendazim and colchicine, a known microtubule disruptor, on the mouse seminiferous epithelium were characterized, and the amount of carbendazim reaching the mouse testis was measured. Testes were assessed for histological effects 3 h and 6 h after administration of carbendazim (2000 mg/kg, ip), and 6 h after intratesticular administration of either a low or high dose (5.3 or 117.6 micro g/g testis) of colchicine. Carbendazim caused no signs of histological damage to the mouse testis, and the microtubule cytoskeleton was intact and identical to controls based on immunostaining with tyrosinated alpha tubulin and beta tubulin antibodies. Similarly, the seminiferous epithelium of mouse testis was undamaged and the microtubule cytoskeleton was intact after a low dose of colchicine, while a comparable dose of colchicine injected into rat testis caused marked toxicity. However, mouse testes did show microtubule disruption and severe germ cell sloughing after administration of a high dose of colchicine. The amount of carbendazim measured in mouse testis was 375 nmol/g testis, which is higher than the value measured in rat testis after a toxic dose of carbendazim. Therefore, carbendazim reaches the mouse testis at or above levels measured in the rat, yet the mouse is apparently insensitive to this microtubule disrupting agent. PMID:12215672

  2. Singlet scalar Dark Matter in Dark Two Higgs Doublet Model

    E-print Network

    R. Gaitan; E. A. Garces; J. H. Montes de Oca

    2014-10-20

    We consider the case of the Dark Two Higgs Doublet Model (D2HDM) where a $U(1)'$ symmetry group and an extra Higgs doublet are added to the Standard Model. This model leads to a gauge singlet particle as an interesting Dark Matter (DM) candidate. We obtain phenomenological constraints to the parameter space of the model considering the one necessary to produce the correct density of thermal relic dark matter $\\Omega h^2$. We find a relation between the masses of the DM matter candidate $m_S$ and $m_{Z'}$ that satisfy the relic density for given values of $\\tan\\beta$.

  3. Singlet Dark Matter in Type II Two Higgs Doublet Model

    E-print Network

    Yi Cai; Tong Li

    2013-08-24

    Inspired by the dark matter searches in the low mass region, we study the Type II two Higgs doublet model with a light gauge singlet WIMP stabilized by a Z_2 symmetry. The real singlet is required to only couple to the non-Standard Model Higgs. We investigate singlet candidates with different spins as well as isospin violating effect. The parameter space favored by LHC data in two Higgs doublet model and hadronic uncertainties in WIMP-nucleon elastic scattering are also taken into account. We find only the scalar singlet in the isospin conserving case leads to a major overlap with the region of interests of most direct detection experiments.

  4. Intrinsically disordered tubulin tails: complex tuners of microtubule functions?

    PubMed

    Roll-Mecak, Antonina

    2015-01-01

    Microtubules are essential cellular polymers assembled from tubulin heterodimers. The tubulin dimer consists of a compact folded globular core and intrinsically disordered C-terminal tails. The tubulin tails form a lawn of densely grafted, negatively charged, flexible peptides on the exterior of the microtubule, potentially akin to brush polymers in the field of synthetic materials. These tails are hotspots for conserved, chemically complex posttranslational modifications that have the potential to act in a combinatorial fashion to regulate microtubule polymer dynamics and interactions with microtubule effectors, giving rise to a "tubulin code". In this review, I summarize our current knowledge of the enzymes that generate the astonishing tubulin chemical diversity observed in cells and describe recent advances in deciphering the roles of tubulin C-terminal tails and their posttranslational modifications in regulating the activity of molecular motors and microtubule associated proteins. Lastly, I outline the promises, challenges and potential pitfalls of deciphering the tubulin code. PMID:25307498

  5. Microtubule-binding natural products for cancer therapy.

    PubMed

    Yue, Qing-Xi; Liu, Xuan; Guo, De-An

    2010-08-01

    Natural products, especially microtubule-binding natural products, play important roles in the war against cancer. From the clinical use of vinblastine in 1961, paclitaxel in 1992, to ixabepilone in 2007, microtubule-binding natural products have continually contributed to the development of cancer therapy. The present review summarizes the development of representative microtubule-binding natural products including agents binding to the colchicine-binding site, the VINCA alkaloid-binding site, the taxane-binding site and other binding sites. Future directions for the development of new anticancer microtubule-binding natural products are discussed. Finding new formulations, new targets and new sources of microtubule-binding natural products may enable more members of this kind of agent to be introduced into the clinic for cancer therapy. PMID:20577942

  6. Measuring Microtubule Polarity in Spindles with Second-Harmonic Generation

    PubMed Central

    Yu, Che-Hang; Langowitz, Noah; Wu, Hai-Yin; Farhadifar, Reza; Brugues, Jan; Yoo, Tae Yeon; Needleman, Daniel

    2014-01-01

    The spatial organization of microtubule polarity, and the interplay between microtubule polarity and protein localization, is thought to be crucial for spindle assembly, anaphase, and cytokinesis, but these phenomena remain poorly understood, in part due to the difficulty of measuring microtubule polarity in spindles. We develop and implement a method to nonperturbatively and quantitatively measure microtubule polarity throughout spindles using a combination of second-harmonic generation and two-photon fluorescence. We validate this method using computer simulations and by comparison to structural data on spindles obtained from electron tomography and laser ablation. This method should provide a powerful tool for studying spindle organization and function, and may be applicable for investigating microtubule polarity in other systems. PMID:24739157

  7. Anisotropic Elastic Network Modeling of Entire Microtubules

    PubMed Central

    Deriu, Marco A.; Soncini, Monica; Orsi, Mario; Patel, Mishal; Essex, Jonathan W.; Montevecchi, Franco M.; Redaelli, Alberto

    2010-01-01

    Microtubules are supramolecular structures that make up the cytoskeleton and strongly affect the mechanical properties of the cell. Within the cytoskeleton filaments, the microtubule (MT) exhibits by far the highest bending stiffness. Bending stiffness depends on the mechanical properties and intermolecular interactions of the tubulin dimers (the MT building blocks). Computational molecular modeling has the potential for obtaining quantitative insights into this area. However, to our knowledge, standard molecular modeling techniques, such as molecular dynamics (MD) and normal mode analysis (NMA), are not yet able to simulate large molecular structures like the MTs; in fact, their possibilities are normally limited to much smaller protein complexes. In this work, we developed a multiscale approach by merging the modeling contribution from MD and NMA. In particular, MD simulations were used to refine the molecular conformation and arrangement of the tubulin dimers inside the MT lattice. Subsequently, NMA was used to investigate the vibrational properties of MTs modeled as an elastic network. The coarse-grain model here developed can describe systems of hundreds of interacting tubulin monomers (corresponding to up to 1,000,000 atoms). In particular, we were able to simulate coarse-grain models of entire MTs, with lengths up to 350 nm. A quantitative mechanical investigation was performed; from the bending and stretching modes, we estimated MT macroscopic properties such as bending stiffness, Young modulus, and persistence length, thus allowing a direct comparison with experimental data. PMID:20923653

  8. Anisotropic elastic network modeling of entire microtubules.

    PubMed

    Deriu, Marco A; Soncini, Monica; Orsi, Mario; Patel, Mishal; Essex, Jonathan W; Montevecchi, Franco M; Redaelli, Alberto

    2010-10-01

    Microtubules are supramolecular structures that make up the cytoskeleton and strongly affect the mechanical properties of the cell. Within the cytoskeleton filaments, the microtubule (MT) exhibits by far the highest bending stiffness. Bending stiffness depends on the mechanical properties and intermolecular interactions of the tubulin dimers (the MT building blocks). Computational molecular modeling has the potential for obtaining quantitative insights into this area. However, to our knowledge, standard molecular modeling techniques, such as molecular dynamics (MD) and normal mode analysis (NMA), are not yet able to simulate large molecular structures like the MTs; in fact, their possibilities are normally limited to much smaller protein complexes. In this work, we developed a multiscale approach by merging the modeling contribution from MD and NMA. In particular, MD simulations were used to refine the molecular conformation and arrangement of the tubulin dimers inside the MT lattice. Subsequently, NMA was used to investigate the vibrational properties of MTs modeled as an elastic network. The coarse-grain model here developed can describe systems of hundreds of interacting tubulin monomers (corresponding to up to 1,000,000 atoms). In particular, we were able to simulate coarse-grain models of entire MTs, with lengths up to 350 nm. A quantitative mechanical investigation was performed; from the bending and stretching modes, we estimated MT macroscopic properties such as bending stiffness, Young modulus, and persistence length, thus allowing a direct comparison with experimental data. PMID:20923653

  9. Dynamics and length distribution of microtubules under force and confinement

    NASA Astrophysics Data System (ADS)

    Zelinski, Björn; Müller, Nina; Kierfeld, Jan

    2012-10-01

    We investigate the microtubule polymerization dynamics with catastrophe and rescue events for three different confinement scenarios, which mimic typical cellular environments: (i) The microtubule is confined by rigid and fixed walls, (ii) it grows under constant force, and (iii) it grows against an elastic obstacle with a linearly increasing force. We use realistic catastrophe models and analyze the microtubule dynamics, the resulting microtubule length distributions, and force generation by stochastic and mean field calculations; in addition, we perform stochastic simulations. Freely growing microtubules exhibit a phase of bounded growth with finite microtubule length and a phase of unbounded growth. The main results for the three confinement scenarios are as follows: (i) In confinement by fixed rigid walls, we find exponentially decreasing or increasing stationary microtubule length distributions instead of bounded or unbounded phases, respectively. We introduce a realistic model for wall-induced catastrophes and investigate the behavior of the average length as a function of microtubule growth parameters. (ii) Under a constant force, the boundary between bounded and unbounded growth is shifted to higher tubulin concentrations and rescue rates. The critical force fc for the transition from unbounded to bounded growth increases logarithmically with tubulin concentration and the rescue rate, and it is smaller than the stall force. (iii) For microtubule growth against an elastic obstacle, the microtubule length and polymerization force can be regulated by microtubule growth parameters. For zero rescue rate, we find that the average polymerization force depends logarithmically on the tubulin concentration and is always smaller than the stall force in the absence of catastrophes and rescues. For a nonzero rescue rate, we find a sharply peaked steady-state length distribution, which is tightly controlled by microtubule growth parameters. The corresponding average microtubule length self-organizes such that the average polymerization force equals the critical force fc for the transition from unbounded to bounded growth. We also investigate the force dynamics if growth parameters are perturbed in dilution experiments. Finally, we show the robustness of our results against changes of catastrophe models and load distribution factors.

  10. Intrinsic microtubule GTP-cap dynamics in semi-confined systems: kinetochore-microtubule interface.

    PubMed

    Buljan, Vlado A; Damian Holsinger, R M; Hambly, Brett D; Banati, Richard B; Ivanova, Elena P

    2013-01-01

    In order to quantify the intrinsic dynamics associated with the tip of a GTP-cap under semi-confined conditions, such as those within a neuronal cone and at a kinetochore-microtubule interface, we propose a novel quantitative concept of critical nano local GTP-tubulin concentration (CNLC). A simulation of a rate constant of GTP-tubulin hydrolysis, under varying conditions based on this concept, generates results in the range of 0-420 s(-1). These results are in agreement with published experimental data, validating our model. The major outcome of this model is the prediction of 11 random and distinct outbursts of GTP hydrolysis per single layer of a GTP-cap. GTP hydrolysis is accompanied by an energy release and the formation of discrete expanding zones, built by less-stable, skewed GDP-tubulin subunits. We suggest that the front of these expanding zones within the walls of the microtubule represent soliton-like movements of local deformation triggered by energy released from an outburst of hydrolysis. We propose that these solitons might be helpful in addressing a long-standing question relating to the mechanism underlying how GTP-tubulin hydrolysis controls dynamic instability. This result strongly supports the prediction that large conformational movements in tubulin subunits, termed dynamic transitions, occur as a result of the conversion of chemical energy that is triggered by GTP hydrolysis (Satari? et al., Electromagn Biol Med 24:255-264, 2005). Although simple, the concept of CNLC enables the formulation of a rationale to explain the intrinsic nature of the "push-and-pull" mechanism associated with a kinetochore-microtubule complex. In addition, the capacity of the microtubule wall to produce and mediate localized spatio-temporal excitations, i.e., soliton-like bursts of energy coupled with an abundance of microtubules in dendritic spines supports the hypothesis that microtubule dynamics may underlie neural information processing including neurocomputation (Hameroff, J Biol Phys 36:71-93, 2010; Hameroff, Cognit Sci 31:1035-1045, 2007; Hameroff and Watt, J Theor Biol 98:549-561, 1982). PMID:23860835

  11. Repetitive doublet firing of motor units: evidence for plateau potentials in human motoneurones?

    Microsoft Academic Search

    Lydia P. Kudina; Regina E. Andreeva

    2010-01-01

    During voluntary muscle contraction, human motoneurones can exhibit specific discharge patterns: single and repetitive doublets.\\u000a Delayed depolarization has been accepted as the mechanism underlying single doublets. Repetitive doublet firing has been studied\\u000a much less and its controlling mechanisms remain obscure. The aim of the present study was to examine properties of repetitive\\u000a doublets in human motoneurones and to consider their

  12. Electroweak baryogenesis in the two-doublet model

    Microsoft Academic Search

    Neil Turok; John Zadrozny

    1991-01-01

    A new mechanism through which a net baryon asymmetry could be generated at the electroweak phase transition is discussed. It works efficiently in minimal extensions of the standard model such as occur in supersymmetric theories, where the Higgs sector involves more than one doublet.

  13. CP violation conditions in N-Higgs-doublet potentials

    E-print Network

    C. C. Nishi

    2007-10-26

    Conditions for CP violation in the scalar potential sector of general N-Higgs-doublet models (NHDMs) are analyzed from a group theoretical perspective. For the simplest two-Higgs-doublet model (2HDM) potential, a minimum set of conditions for explicit and spontaneous CP violation is presented. The conditions can be given a clear geometrical interpretation in terms of quantities in the adjoint representation of the basis transformation group for the two doublets. Such conditions depend on CP-odd pseudoscalar invariants. When the potential is CP invariant, the explicit procedure to reach the real CP-basis and the explicit CP transformation can also be obtained. The procedure to find the real basis and the conditions for CP violation are then extended to general NHDM potentials. The analysis becomes more involved and only a formal procedure to reach the real basis is found. Necessary conditions for CP invariance can still be formulated in terms of group invariants: the CP-odd generalized pseudoscalars. The problem can be completely solved for three Higgs-doublets.

  14. The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae

    Microsoft Academic Search

    Kerry S. Bloom; E. D. Salmon; Paul S. Maddox

    1999-01-01

    Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus

  15. Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy

    Microsoft Academic Search

    Christopher Gell; Volker Bormuth; Gary J. Brouhard; Daniel N. Cohen; Stefan Diez; Claire T. Friel; Jonne Helenius; Bert Nitzsche; Heike Petzold; Jan Ribbe; Erik Schäffer; Jeffrey H. Stear; Anastasiya Trushko; Vladimir Varga; Per O. Widlund; Marija Zanic; Jonathon Howard

    2010-01-01

    In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present

  16. Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast

    Microsoft Academic Search

    Isabelle Loiodice; Jayme Staub; Thanuja Gangi Setty; Nam-Phuong T. Nguyen; Anne Paoletti; P. T. Tran

    2005-01-01

    Abstract: 159 words Manuscript: 11,250 words Running title: ase1p bundles plus and minus ends of microtubules Key words: ase1p, microtubule bundling, microtubule organization, nuclear positioning Loiodice et al., ase1p bundles microtubules E04-10-0899R; final 1\\/25\\/05

  17. Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes

    PubMed Central

    Li, Shundai; Lei, Lei; Somerville, Chris R.; Gu, Ying

    2012-01-01

    Cellulose synthase (CESA) complexes can be observed by live-cell imaging to move with trajectories that parallel the underlying cortical microtubules. Here we report that CESA interactive protein 1 (CSI1) is a microtubule-associated protein that bridges CESA complexes and cortical microtubules. Simultaneous in vivo imaging of CSI1, CESA complexes, and microtubules demonstrates that the association of CESA complexes and cortical microtubules is dependent on CSI1. CSI1 directly binds to microtubules as demonstrated by in vitro microtubule-binding assay. PMID:22190487

  18. An Improved Quantitative Analysis Method for Plant Cortical Microtubules

    PubMed Central

    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. PMID:24744684

  19. 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. PMID:24744684

  20. Centrosomal nucleolin is required for microtubule network organization.

    PubMed

    Gaume, Xavier; Tassin, Anne-Marie; Ugrinova, Iva; Mongelard, Fabien; Monier, Karine; Bouvet, Philippe

    2015-03-19

    Nucleolin is a pleiotropic protein involved in a variety of cellular processes. Although multipolar spindle formation has been observed after nucleolin depletion, the roles of nucleolin in centrosome regulation and functions have not been addressed. Here we report using immunofluorescence and biochemically purified centrosomes that nucleolin co-localized only with one of the centrioles during interphase which was further identified as the mature centriole. Upon nucleolin depletion, cells exhibited an amplification of immature centriole markers surrounded by irregular pericentrin staining; these structures were exempt from maturation markers and unable to nucleate microtubules. Furthermore, the microtubule network was disorganized in these cells, exhibiting frequent non-centrosomal microtubules. At the mature centriole a reduced kinetics in the centrosomal microtubule nucleation phase was observed in live silenced cells, as well as a perturbation of microtubule anchoring. Immunoprecipitation experiments showed that nucleolin belongs to protein complexes containing 2 key centrosomal proteins, ?-tubulin and ninein, involved in microtubule nucleation and anchoring steps. Altogether, our study uncovered a new role for nucleolin in restricting microtubule nucleation and anchoring at centrosomes in interphase cells. PMID:25590348

  1. Model of ionic currents through microtubule nanopores and the lumen.

    PubMed

    Freedman, Holly; Rezania, Vahid; Priel, Avner; Carpenter, Eric; Noskov, Sergei Y; Tuszynski, Jack A

    2010-05-01

    It has been suggested that microtubules and other cytoskeletal filaments may act as electrical transmission lines. An electrical circuit model of the microtubule is constructed incorporating features of its cylindrical structure with nanopores in its walls. This model is used to study how ionic conductance along the lumen is affected by flux through the nanopores, both with and without an external potential applied across its two ends. Based on the results of Brownian dynamics simulations, the nanopores were found to have asymmetric inner and outer conductances, manifested as nonlinear IV curves. Our simulations indicate that a combination of this asymmetry and an internal voltage source arising from the motion of the C-terminal tails causes cations to be pumped across the microtubule wall and propagate in both directions down the microtubule through the lumen, returning to the bulk solution through its open ends. This effect is demonstrated to add directly to the longitudinal current through the lumen resulting from an external voltage source applied across the two ends of the microtubule. The predicted persistent currents directed through the microtubule wall and along the lumen could be significant in directing the dissipation of weak, endogenous potential gradients toward one end of the microtubule within the cellular environment. PMID:20866266

  2. Analysis of microtubules in isolated axoplasm from the squid giant axon.

    PubMed

    Song, Yuyu; Brady, Scott T

    2013-01-01

    Biochemical specialization of cellular microtubules has emerged as a primary mechanism in specifying microtubule dynamics and function. However, study of specific subcellular populations of cytoplasmic microtubules has been limited, particularly in the nervous system. The complexity of nervous tissue makes it difficult to distinguish neuronal microtubules from glial microtubules, and axonal microtubules from dendritic and cell body microtubules. The problem is further compounded by the finding that a large fraction of neuronal tubulin is lost during standard preparations of brain tubulin, and this population of stable microtubules is enriched in axons. Here, we consider a unique biological model that provides a unique opportunity to study axonal microtubules both in situ and in vitro: isolated axoplasm from the squid giant axon. The axoplasm model represents a powerful system for addressing fundamental questions of microtubule structure and function in the axon. PMID:23973070

  3. Centrosome and microtubule instability in aging Drosophila cells

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Chakrabarti, A.; Hedrick, J.

    1999-01-01

    Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

  4. Centering of a radial microtubule array by translocation along microtubules spontaneously nucleated in the cytoplasm

    Microsoft Academic Search

    Viacheslav Malikov; Eric N. Cytrynbaum; Anna Kashina; Alexander Mogilner; Vladimir Rodionov

    2005-01-01

    Positioning of a radial array of microtubules (MTs) in the cell centre is crucial for cytoplasmic organization, but the mechanisms of such centering are difficult to study in intact cells that have pre-formed radial arrays. Here, we use cytoplasmic fragments of melanophores, and cytoplasts of BS-C-1 cells to study MT centering mechanisms. Using live imaging and computer modelling, we show

  5. Unleashing formins to remodel the actin and microtubule cytoskeletons.

    PubMed

    Chesarone, Melissa A; DuPage, Amy Grace; Goode, Bruce L

    2010-01-01

    Formins are highly conserved proteins that have essential roles in remodelling the actin and microtubule cytoskeletons to influence eukaryotic cell shape and behaviour. Recent work has identified numerous cellular factors that locally recruit, activate or inactivate formins to bridle and unleash their potent effects on actin nucleation and elongation. The effects of formins on microtubules have also begun to be described, which places formins in a prime position to coordinate actin and microtubule dynamics. The emerging complexity in the mechanisms governing formins mirrors the wide range of essential functions that they perform in cell motility, cell division and cell and tissue morphogenesis. PMID:19997130

  6. Kinesin-8 Motors Improve Nuclear Centering by Promoting Microtubule Catastrophe

    NASA Astrophysics Data System (ADS)

    Glun?i?, Matko; Maghelli, Nicola; Krull, Alexander; Krsti?, Vladimir; Ramunno-Johnson, Damien; Pavin, Nenad; Toli?, Iva M.

    2015-02-01

    In fission yeast, microtubules push against the cell edge, thereby positioning the nucleus in the cell center. Kinesin-8 motors regulate microtubule catastrophe; however, their role in nuclear positioning is not known. Here we develop a physical model that describes how kinesin-8 motors affect nuclear centering by promoting a microtubule catastrophe. Our model predicts the improved centering of the nucleus in the presence of motors, which we confirmed experimentally in living cells. The model also predicts a characteristic time for the recentering of a displaced nucleus, which is supported by our experiments where we displaced the nucleus using optical tweezers.

  7. Mobility, Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae of Ashbya gossypii

    PubMed Central

    Lang, Claudia; Grava, Sandrine; van den Hoorn, Tineke; Trimble, Rhonda; Philippsen, Peter

    2010-01-01

    We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 ?m/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 ?m/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae. PMID:19910487

  8. Yeast Bim1p Promotes the G1-specific Dynamics of Microtubules

    Microsoft Academic Search

    Jennifer S. Tirnauer; Eileen O'Toole; Lisbeth Berrueta; Barbara E. Bierer; David Pellman

    1999-01-01

    Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro. Proteins that promote dynamic insta- bility are therefore central to microtubule behavior in living cells. Here, we report that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cyto- plasmic microtubule dynamics specifically during G1. During G1, microtubules in

  9. The spectraplakin Short stop is an actin-microtubule cross-linker that contributes to organization of the microtubule network.

    PubMed

    Applewhite, Derek A; Grode, Kyle D; Keller, Darby; Zadeh, Alireza Dehghani; Zadeh, Alireza; Slep, Kevin C; Rogers, Stephen L

    2010-05-15

    The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes; however, little is known about the molecules mediating this cytoskeletal cross-talk. We are studying Short stop (Shot), the sole Drosophila spectraplakin, as a model actin-microtubule cross-linking protein. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions; yet, we know little about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. In this study we describe the intracellular dynamics of Shot and a structure-function analysis of its role as a cytoskeletal cross-linker. We find that Shot interacts with microtubules using two different mechanisms. In the cell interior, Shot binds growing plus ends through an interaction with EB1. In the cell periphery, Shot associates with the microtubule lattice via its GAS2 domain, and this pool of Shot is actively engaged as a cross-linker via its NH(2)-terminal actin-binding calponin homology domains. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. Our results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk. PMID:20335501

  10. Crowding of molecular motors determines microtubule depolymerization

    E-print Network

    Reese, Louis; Frey, Erwin

    2011-01-01

    Assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT associated proteins. Here, we investigate how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. Employing an individual-based model, we reproduce experimental findings. Moreover, crowding is identified as the key regulatory mechanism of depolymerization dynamics. Our analysis gives two qualitatively distinct regimes. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise which cancel out the effect of the depolymerization kinetics such that the depolymerization speed is solely determined by the motor density. Because this density changes over the MT length, length-dependent regulation is possible. Remarkably, motor cooperativity does not affect the depolymerization speed but only the end-residence time of depo...

  11. Synthesis of neolignans as microtubule stabilisers.

    PubMed

    Sathish Kumar, B; Singh, Aastha; Kumar, Amit; Singh, Jyotsna; Hasanain, Mohammad; Singh, Arjun; Masood, Nusrat; Yadav, Dharmendra K; Konwar, Rituraj; Mitra, Kalyan; Sarkar, Jayanta; Luqman, Suaib; Pal, Anirban; Khan, Feroz; Chanda, Debabrata; Negi, Arvind S

    2014-02-15

    Tubulin is a well established target for anticancer drug development. Lignans and neolignans were synthesized as tubulin interacting agents. Neolignans 10 and 19 exhibited significant anticancer activity against MCF-7 and MDAMB-231 human breast cancer cell lines. Both the compounds effectively induced stabilization of microtubule at 4 and 20 ?M concentrations respectively. Neolignan 10 induced G2/M phase arrest in MCF-7 cells. Docking experiments raveled that 10 and 19 occupied the same binding pocket of paclitaxel with some difference in active site amino acids and good bioavailability of both the compounds. In in vivo acute oral toxicity 10 was well tolerated up to 300 mg/kg dose in Swiss-albino mice. PMID:24457094

  12. The microtubule-binding protein Cep170 promotes the targeting of the kinesin-13 depolymerase Kif2b to the mitotic spindle

    E-print Network

    Welburn, Julie P. I.

    Microtubule dynamics are essential throughout mitosis to ensure correct chromosome segregation. Microtubule depolymerization is controlled in part by microtubule depolymerases, including the kinesin-13 family of proteins. ...

  13. Association between endocrine pancreatic secretory granules and in-vitro-assembled microtubules is dependent upon microtubule-associated proteins

    E-print Network

    Suprenant, Kathy A.; Dentler, William L., Jr

    1982-04-01

    -microtubule/actin attachments . Sherline et al . (49) studied the associations between microtu- THE JOURNAL OF CELL BIOLOGY " VOLUME 93 APRIL 1982 164-174 ©The Rockefeller University Press " 0021-9525/82/04/0164/11 $1 .00 o n Septem ber 10, 2014 jcb.rupress.org D ow nloaded...M in GTPand l mM in MgSO4. The tubulin-containing fractions were pooled and made 10% in dimethyl sulfoxide (DMSO) (24), and microtubules were assembled by warming the preparations to 37°C. Microtubules were pelleted, resuspended at a concentration of 10-15 mg...

  14. Cortical microtubules in sweet clover columella cells developed in microgravity

    NASA Technical Reports Server (NTRS)

    Hilaire, E.; Paulsen, A. Q.; Brown, C. S.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Electron micrographs of columella cells from sweet clover seedlings grown and fixed in microgravity revealed longitudinal and cross sectioned cortical microtubules. This is the first report demonstrating the presence and stability of this network in plants in microgravity.

  15. Nanotube interactions with microtubules: implications for cancer medicine.

    PubMed

    García-Hevia, Lorena; Fernández, Fidel; Grávalos, Cristina; García, Almudena; Villegas, Juan C; Fanarraga, Mónica L

    2014-07-01

    Carbon nanotubes (CNTs) and microtubules are both hollow nanofibers and have similar dimensions; they both self-assemble and form bundles. These common features prompt their association into biosynthetic polymers in vitro and in vivo. Unlike CNTs, microtubules are highly dynamic protein polymers essential for cell proliferation and migration. Interaction between these filaments inside live cells leads to microtubule dysfunction, mitotic arrest and cell death. Thus, CNTs behave as spindle poisons, same as taxanes, vinca alkaloids or epotilones. Recent findings support the idea that CNTs represent a ground-breaking type of synthetic microtubule-stabilizing agents that could play a pivotal role in future cancer treatments in combination to traditional antineoplastic drugs. Here we review the potential use of CNTs in cancer medicine. PMID:25253503

  16. Motion observation and SPR measurements of kinesin motility on microtubules

    NASA Astrophysics Data System (ADS)

    Sikora, A.; Oliveira, D.; Kim, K.; Liao, A. L.; Umetsu, M.; Adschiri, T.; Hwang, W.; Teizer, W.

    2012-02-01

    Motor proteins convert chemical energy directly into mechanical work with high efficiency (˜50%). One of these proteins, kinesin, is used in the cell to transport organelles. It ``walks'' along biopolymer tracks called microtubules and, depending on the type, can reach speeds of a few micrometers per second. Kinesin can carry intracellular cargo over long distances against several piconewtons of loads and is barely limited by the cargo size. Motion of streptavidin-coated quantum dots carried by kinesin on microtubules will be presented. We have expressed biotinylated Kinesin-1 using Escherichia coli. Attachment to quantum dots was performed using the strong binding affinity between streptavidin and biotin. Microtubules, labeled with rhodamine, allow visualization by fluorescence microscopy. The measured speed of our kinesin fits well with results found in the literature. Surface Plasmon Resonance (SPR) measurements allow the identification and strength evaluation of bonding. Using this technique, we will present results on the binding between our expressed kinesin and microtubule.

  17. Mechanical Models of Microtubule Bundle Collapse in Alzheimer's Disease

    NASA Astrophysics Data System (ADS)

    Sendek, Austin; Singh, Rajiv; Cox, Daniel

    2013-03-01

    Amyloid-beta aggregates initiate Alzheimer's disease, and downstream trigger degradation of tau proteins that act as microtubule bundle stabilizers and mechanical spacers. Currently it is unclear which of tau cutting by proteases, tau phosphorylation, or tau aggregation are responsible for cytoskeleton degradation., We construct a percolation simulation of the microtubule bundle using a molecular spring model for the taus and including depletion force attraction between microtubules and membrane/actin cytoskeletal surface tension. The simulation uses a fictive molecular dynamics to model the motion of the individual microtubules within the bundle as a result of random tau removal, and calculates the elastic modulus of the bundle as the tau concentration falls. We link the tau removal steps to kinetic tau steps in various models of tau degradation.

  18. Molecular Cell Structural Basis of Microtubule Plus End

    E-print Network

    Vale, Ronald D.

    they act upon microtubule plus ends through a similar mechanism has not been resolved. Here, we re- port (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces

  19. 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. PMID:25679909

  20. Mechanism and dynamics of breakage of fluorescent microtubules.

    PubMed

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

    2006-03-15

    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

  1. Dynamics of an idealized model of microtubule growth and catastrophe

    NASA Astrophysics Data System (ADS)

    Antal, T.; Krapivsky, P. L.; Redner, S.; Mailman, M.; Chakraborty, B.

    2007-10-01

    We investigate a simple dynamical model of a microtubule that evolves by attachment of guanosine triphosphate (GTP) tubulin to its end, irreversible conversion of GTP to guanosine diphosphate (GDP) tubulin by hydrolysis, and detachment of GDP at the end of a microtubule. As a function of rates of these processes, the microtubule can grow steadily or its length can fluctuate wildly. In the regime where detachment can be neglected, we find exact expressions for the tubule and GTP cap length distributions, as well as power-law length distributions of GTP and GDP islands. In the opposite limit of instantaneous detachment, we find the time between catastrophes, where the microtubule shrinks to zero length, and determine the size distribution of avalanches (sequence of consecutive GDP detachment events). We obtain the phase diagram for general rates and verify our predictions by numerical simulations.

  2. Crosstalk Between Microtubule Attachment Complexes Ensures Accurate Chromosome Segregation*

    PubMed Central

    Cheerambathur, Dhanya K.; Gassmann, Reto; Cook, Brian; Oegema, Karen; Desai, Arshad

    2013-01-01

    The microtubule-based mitotic spindle segregates chromosomes during cell division. During chromosome segregation, the centromeric regions of chromosomes build kinetochores that establish end-coupled attachments to spindle microtubules. Here, we used the C. elegans embryo as a model system to examine the crosstalk between two kinetochore protein complexes implicated in temporally distinct stages of attachment formation. The kinetochore dynein module, which mediates initial lateral microtubule capture, inhibited microtubule binding by the Ndc80 complex, which ultimately forms the end-coupled attachments that segregate chromosomes. The kinetochore dynein module directly regulated Ndc80, independently of phosphorylation by Aurora B kinase, and this regulation was required for accurate segregation. Thus, the conversion from initial dynein-mediated, lateral attachments to correctly oriented, Ndc80-mediated end-coupled attachments is actively controlled. PMID:24231804

  3. Nek4 Status Differentially Alters Sensitivity to Microtubule Poisons

    E-print Network

    Doles, Jason D.

    Microtubule poisons are widely used in cancer treatment, but the factors determining the relative efficacy of different drugs in this class remain obscure. In this study, we identified the NIMA kinase Nek4 in a genetic ...

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

    E-print Network

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

    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 crosslinking motors allow us to study microscopic organization and stresses. Polarity sorting and crosslink 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.

  5. Site-Specific Chemistry on the Microtubule Polymer

    PubMed Central

    Kleiner, Ralph E.; Ti, Shih-Chieh; Kapoor, Tarun M.

    2013-01-01

    Microtubules are hollow tube-like biological polymers required for transport in diverse cellular contexts and are important drug targets. Microtubule function depends on interactions with associated proteins and post-translational modifications at specific sites located on its interior and exterior surfaces. However, we lack strategies to selectively perturb or probe these basic biochemical mechanisms. In this work, by combining amber suppression-mediated non-natural amino acid incorporation and tubulin overexpression in budding yeast, we demonstrate, for the first time, a general strategy for site-specific chemistry on microtubules. Probes and labels targeted to precise sites on the interior and exterior surfaces of microtubules will allow analysis and modulation of interactions with proteins and drugs, and elucidation of the functions of post-translational modifications. PMID:23930594

  6. Disruption of microtubule network by Alzheimer abnormally hyperphosphorylated tau

    Microsoft Academic Search

    Bin Li; Muhammad Omar Chohan; Inge Grundke-Iqbal; Khalid Iqbal

    2007-01-01

    Hyperphosphorylated tau has long been proposed as the key molecule disrupting normal neuronal microtubule dynamics and leading\\u000a to neurofibrillary degeneration in Alzheimer disease. Here we provide a direct evidence of hyperphosphorylated tau-induced\\u000a disruption of microtubule network. Using Nocodozole-treated and detergent-extracted cells, we created a neuronal environment\\u000a in mouse embryonic fibroblasts, 3T3 cells, by replacing their cytoplasm with adult rat brain

  7. Microtubule reorganization during pollen development of rice ( Oryza sativa L.)

    Microsoft Academic Search

    S. Y. Zee; X. L. Ye

    2000-01-01

    Summary Anthers of rice (Oryza sativa L.) at different stages of development were cryofixed, freeze-substituted, and embedded in methacrylate. Sections were then cut and immuno-labeled with anti-tubulin to localize microspore microtubules. Changes in microtubule distribution pattern were followed by confocal fluorescence microscopy. To facilitate description, pollen development has been divided into four developmental stages (twenty-four phases). (i) The young-microspore stage

  8. Microtubules and control of macronuclear 'amitosis' in Paramecium.

    PubMed

    Tucker, J B; Beisson, J; Roche, D L; Cohen, J

    1980-08-01

    The 'amitotic' division of the macronucleus during binary fission in P. tetraurelia includes a detailed sequence of shape changes that are temporally coordinated with the adoption of a series of well-defined positions and orientations inside the cell. The deployment of nucleoplasmic microtubules that is spatially correlated with the shaping ritual is more complex and precise than has been reported previously. Macronuclear division is not amitotic. It is not a simple constriction into two halves. As a dividing macronucleus starts to elongate it becomes dorsoventrally flattened against the dorsal cortex of the organism and assumes an elliptical shape. Concurrently, an elliptical marginal band of intranuclear microtubules assembles that has the same spatial relationship to nuclear shape as the marginal microtubules assembles that has the same spatial relationship to nuclear shape as the marginal microtubule bands of certain elliptical vertebrate blood cells have to cell shape. The band breaks down as further elongation occurs and the nucleus adopts the shape of a straight and slender sausage. Most of the intranuclear microtubules assemble as elongation starts and break down shortly after elongation is completed; the majority are oriented parallel to the longitudinal axis of the nucleus throughout elongation. Some of them are attached to nucleoli and are coated with granules which are almost certainly derived from the cortices of nucleoli. The peripheral concentration, interconnexion, orientation, and overlapping arrangement of microtubules, and the reduction in microtubule number per nuclear cross-section as elongation proceeds at a rate of about 40 micrometers min-1, are all compatible with the provision of a microtubule sliding mechanism as the main skeletal basis for elongation. There are indications that this mechanism is augmented by anchorage and/or active propulsion of nucleoli that may perhaps facilitate fairly equitable segregation of chromosomal material to daughter nuclei. PMID:7440651

  9. Structure of cortical microtubule arrays in plant cells

    Microsoft Academic Search

    A. R. Hardham; B. E. S. Gunning

    1978-01-01

    ABSTRACT Serial sectioning was,used,to track the position and,measure,the lengths of cortical microtubules,in glutaraldehyde-osmium,tetroxide-fixed root tip cells. Microtubules lying against the longitudinal walls during interphase, those overly- ing developing xylem thickenings, and those in pre-prophase bands are oriented circumferentially but on average are only about one-eighth of the cell circumfer- ence in length, i.e., 2-4 \\/~m. The arrays consist of overlapping

  10. 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.

  11. New Description of the Doublet Bands in Doubly Odd Nuclei

    E-print Network

    H. G. Ganev; A. I. Georgieva; S. Brant; A. Ventura

    2009-01-22

    The experimentally observed $\\Delta I = 1$ doublet bands in some odd-odd nuclei are analyzed within the orthosymplectic extension of the Interacting Vector Boson Model (IVBM). A new, purely collective interpretation of these bands is given on the basis of the obtained boson-fermion dynamical symmetry of the model. It is illustrated by its application to three odd-odd nuclei from the $A\\sim 130$ region, namely $^{126}Pr$, $^{134}Pr$ and $^{132}La$. The theoretical predictions for the energy levels of the doublet bands as well as $E2$ and $M1$ transition probabilities between the states of the yrast band in the last two nuclei are compared with experiment and the results of other theoretical approaches. The obtained results reveal the applicability of the orthosymplectic extension of the IVBM.

  12. New description of the doublet bands in doubly odd nuclei

    SciTech Connect

    Ganev, H. G.; Georgieva, A. I. [Institute of Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences, Sofia 1784 (Bulgaria); Brant, S. [Department of Physics, Faculty of Science, University of Zagreb, 10000 Zagreb (Croatia); Ventura, A. [Ente per le Nuove tecnologie, l'Energia e l'Ambiente, I-40129 Bologna and Istituto Nazionale di Fisica Nucleare, Sezione di Bologna (Italy)

    2009-04-15

    The experimentally observed {delta}I=1 doublet bands in some odd-odd nuclei are analyzed within the orthosymplectic extension of the interacting vector boson model (IVBM). A new, purely collective interpretation of these bands is given on the basis of the obtained boson-fermion dynamical symmetry of the model. It is illustrated by its application to three odd-odd nuclei from the A{approx}130 region, namely {sup 126}Pr, {sup 134}Pr, and {sup 132}La. The theoretical predictions for the energy levels of the doublet bands as well as E2 and M1 transition probabilities between the states of the yrast band in the last two nuclei are compared with experiment and the results of other theoretical approaches. The obtained results reveal the applicability of the orthosymplectic extension of the IVBM.

  13. A doublet microlens array for imaging micron-sized objects

    PubMed Central

    Tripathi, A; Chronis, N

    2011-01-01

    We present a high-numerical aperture, doublet microlens array for imaging micron-sized objects. The proposed doublet architecture consists of glass microspheres trapped on a predefined array of silicon microholes and covered with a thin polymer layer. A standard silicon microfabrication process and a novel fluidic assembly technique were combined to obtain an array of 56 ?m diameter microlenses with a numerical aperture of ~0.5. Using such an array, we demonstrated brightfield and fluorescent image formation of objects directly on a CCD sensor without the use of intermediate lenses. The proposed technology is a significant advancement toward the unmet need of inexpensive, miniaturized optical modules which can be further integrated with lab-on-chip microfluidic devices and photonic chips for a variety of high-end imaging/detection applications. PMID:22003271

  14. Tau decay in the two Higgs doublet model

    NASA Astrophysics Data System (ADS)

    Guth, R. J.; Hoang, A. H.; Kühn, J. H.

    1992-07-01

    Electroweak radiative corrections for ?(?? eoverline?e? ?) are calculated in the two Higgs doublet model. For a large ratio of vacuum expectation values and moderate mass splitting in the Higgs sector the ratio ?(?? eoverline?e? ?)m ?5/? ? m ?5 is smaller than the result obtained in the minimal standard model by up to half a percent. The effect goes into the right direction, but is too small to explain the 2.5? discrepancy between present experimental results for the ? lifetime and the predictions within the standard model. However, future experiments at a charm -? or B meson factory may be sufficiently accurate to determine the ? lifetime to a precision of (2-3)%. This would allow to observe deviations from the predictions of the standard model induced by the two Higgs doublet model.

  15. 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.

  16. 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 [1] 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.[4pt] [1] Rosenberg, K. J. Ross, J. L. Feinstein, H. E., Feinstein, S. C. Israelachvili, J., PNAS (USA) 105, 2008, 7445-50.

  17. Structural microtubule cap: stability, catastrophe, rescue, and third state.

    PubMed Central

    Jánosi, Imre M; Chrétien, Denis; Flyvbjerg, Henrik

    2002-01-01

    Microtubules polymerize from GTP-liganded tubulin dimers, but are essentially made of GDP-liganded tubulin. We investigate the tug-of-war resulting from the fact that GDP-liganded tubulin favors a curved configuration, but is forced to remain in a straight one when part of a microtubule. We point out that near the end of a microtubule, the proximity of the end shifts the balance in this tug-of-war, with some protofilament bending as result. This somewhat relaxes the microtubule lattice near its end, resulting in a structural cap. This structural cap thus is a simple mechanical consequence of two well-established facts: protofilaments made of GDP-liganded tubulin have intrinsic curvature, and microtubules are elastic, made from material that can yield to forces, in casu its own intrinsic forces. We explore possible properties of this structural cap, and demonstrate 1) how it allows both polymerization from GTP-liganded tubulin and rapid depolymerization in its absence; 2) how rescue can occur; 3) how a third, meta-stable intermediate state is possible and can explain some experimental results; and 4) how the tapered tips observed at polymerizing microtubule ends are stabilized during growth, though unable to accommodate a lateral cap. This scenario thus supports the widely accepted GTP-cap model by suggesting a stabilizing mechanism that explains the many aspects of dynamic instability. PMID:12202357

  18. A dynamical model of kinesin-microtubule motility assays.

    PubMed Central

    Gibbons, F; Chauwin, J F; Despósito, M; José, J V

    2001-01-01

    A two-dimensional stochastic model for the dynamics of microtubules in gliding-assay experiments is presented here, which includes the viscous drag acting on the moving fiber and the interaction with the kinesins. For this purpose, we model kinesin as a spring, and explicitly use parameter values to characterize the model from experimental data. We numerically compute the mean attachment lifetimes of all motors, the total force exerted on the microtubules at all times, the effects of a distribution in the motor speeds, and also the mean velocity of a microtubule in a gliding assay. We find quantitative agreement with the results of J. Howard, A. J. Hudspeth, and R. D. Vale, Nature. 342:154-158. We perform additional numerical analysis of the individual motors, and show how cancellation of the forces exerted by the many motors creates a resultant longitudinal force much smaller than the maximum force that could be exerted by a single motor. We also examine the effects of inhomogeneities in the motor-speeds. Finally, we present a simple theoretical model for microtubules dynamics in gliding assays. We show that the model can be analytically solved in the limit of few motors attached to the microtubule and in the opposite limit of high motor density. We find that the speed of the microtubule goes like the mean speed of the motors in good quantitative agreement with the experimental and numerical results. PMID:11371430

  19. Fate of microtubule-organizing centers during myogenesis in vitro

    PubMed Central

    1985-01-01

    Microtubule organization and nucleation were studied during in vitro human myogenesis by immunocytology that used monoclonal and polyclonal antitubulin antibodies and a rabbit nonimmune serum that reacts with human centrosomes. In myoblasts, we observed a classical microtubule network centered on juxtanuclear centrosomes. Myotubes possessed numerous microtubules organized in parallel without any apparent nucleation centers. Centrosomes in these cells were not associated one to each nucleus but were often clustered in the vicinity of nuclei groups. They were significantly smaller than those of the mononucleated cells. The periphery of each nucleus in myotubes was labeled with the serum that labels centrosomes suggesting a profound reorganization of microtubule-nucleating material. Regrowth experiments after Nocodazole treatment established that microtubules were growing from the periphery of the nuclei. The redistribution of nucleating material was shown to take place early after myoblast fusion. Such a phenomenon appears to be specific to myogenic differentiation in that artificially induced polykaryons behaved differently: the centrosomes aggregated to form only one or a few giant nucleating centers and the nuclei did not participate directly in the nucleation of microtubules. The significance of these results is discussed in relation to the possible role of the centrosome in establishing cell polarity. PMID:3880758

  20. Modulation of host microtubule dynamics by pathogenic bacteria

    PubMed Central

    Radhakrishnan, Girish K.; Splitter, Gary A.

    2013-01-01

    The eukaryotic cytoskeleton is a vulnerable target of many microbial pathogens during the course of infection. Rearrangements of host cytoskeleton benefit microbes in various stages of their infection cycle such as invasion, motility, and persistence. Bacterial pathogens deliver a number of effector proteins into host cells for modulating the dynamics of actin and microtubule cytoskeleton. Alteration of the actin cytoskeleton is generally achieved by bacterial effectors that target the small GTPases of the host. Modulation of microtubule dynamics involves direct interaction of effector proteins with the subunits of microtubules or recruiting cellular proteins that affect microtubule dynamics. This review will discuss effector proteins from animal and human bacterial pathogens that either destabilize or stabilize host micro-tubules to advance the infectious process. A compilation of these research findings will provide an overview of known and unknown strategies used by various bacterial effectors to modulate the host microtubule dynamics. The present review will undoubtedly help direct future research to determine the mechanisms of action of many bacterial effector proteins and contribute to understanding the survival strategies of diverse adherent and invasive bacterial pathogens. PMID:23585820

  1. Isolation of a Murine Homologue of the Drosophila neuralized Gene, a Gene Required for Axonemal Integrity in Spermatozoa and Terminal Maturation of the Mammary Gland

    PubMed Central

    Vollrath, Benedikt; Pudney, Jeffrey; Asa, Sylvia; Leder, Philip; Fitzgerald, Kevin

    2001-01-01

    The Drosophila neuralized gene shows genetic interactions with Notch, Enhancer of split, and other neurogenic genes and is thought to be involved in cell fate specification in the central nervous system and the mesoderm. In addition, a human homologue of the Drosophila neuralized gene has been described as a potential tumor suppressor gene in malignant astrocytomas. We have isolated a murine homologue of the Drosophila and human Neuralized genes and, in an effort to understand its physiological function, derived mice with a targeted deletion of this gene. Surprisingly, mice homozygous for the introduced mutation do not show aberrant cell fate specifications in the central nervous system or in the developing mesoderm. This is in contrast to mice with targeted deletions in other vertebrate homologues of neurogenic genes such as Notch, Delta, and Cbf-1. Male Neuralized null mice, however, are sterile due to a defect in axoneme organization in the spermatozoa that leads to highly compromised tail movement and sperm immotility. In addition, female Neuralized null animals are defective in the final stages of mammary gland maturation during pregnancy. PMID:11585928

  2. A simple description of doublet bands in mass around 100

    SciTech Connect

    Yoshinaga, N. [Department of Physics, Saitama University, Saitama City 338-8570 (Japan); Higashiyama, K. [Department of Physics, Chiba Institute of Technology, Narashino, Chiba 275-0023 (Japan)

    2009-05-04

    The structure of doublet bands associated with neutron 0h{sub 11/2} and proton 0g{sub 9/2} orbital in the doubly-odd nuclei, {sup 98-104}Tc and {sup 100-106}Rh is studied theoretically using the quadrupole coupling model. The calculated energy levels and electromagnetic transitions are in excellent agreement with experimental data. The internal structure of the yrast states is discussed in terms of the QCM wave functions.

  3. Baryogenesis in the two-Higgs doublet model

    Microsoft Academic Search

    Lars Fromme; Stephan J. Huber; Michael Seniuch

    2006-01-01

    We consider the generation of the baryon asymmetry in the two-Higgs doublet model. Investigating the thermal potential in the presence of CP violation, as relevant for baryogenesis, we find a strong first-order phase transition if the extra Higgs states are heavier than about 300 GeV. The mass of the lightest Higgs can be as large as about 200 GeV. We

  4. Superconducting Quadrupole Doublet for the Los Alamos Meson Physics Facility

    Microsoft Academic Search

    J. D. Rogers; W. V. Hassenzahl; H. L. Laquer; J. K. Novak; R. W. Stokes

    1971-01-01

    A superconducting quadrupole doublet for beam focusing has been operated with a 30-kG field at the center of the 30-cm-long straight section of the magnets and a 3-kG?cm field gradient. The magnets used twisted multifilament Nb&sngbnd;Ti superconductor imbedded in a Cu matrix of 0.050-in. diameter and operate at currents up to 500 A. Each magnet is equipped with a persistent

  5. Candidates for chiral doublet bands in 136 Nd

    Microsoft Academic Search

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

    2002-01-01

    :   The even-even nucleus 136Nd was studied via in-beam ?-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\\u000a band, whose position in the level scheme is revised, the new band forms a doublet structure similar to the recently observed\\u000a chiral bands in

  6. The Triplet Genetic Code had a Doublet Predecessor

    E-print Network

    Apoorva Patel

    2004-10-28

    Information theoretic analysis of genetic languages indicates that the naturally occurring 20 amino acids and the triplet genetic code arose by duplication of 10 amino acids of class-II and a doublet genetic code having codons NNY and anticodons $\\overleftarrow{\\rm GNN}$. Evidence for this scenario is presented based on the properties of aminoacyl-tRNA synthetases, amino acids and nucleotide bases.

  7. Evidence for Multiple Chiral Doublet Bands in Ce133

    NASA Astrophysics Data System (ADS)

    Ayangeakaa, A. D.; Garg, U.; Anthony, M. D.; Frauendorf, S.; Matta, J. T.; Nayak, B. K.; Patel, D.; Chen, Q. B.; Zhang, S. Q.; Zhao, P. W.; Qi, B.; Meng, J.; Janssens, R. V. F.; Carpenter, M. P.; Chiara, C. J.; Kondev, F. G.; Lauritsen, T.; Seweryniak, D.; Zhu, S.; Ghugre, S. S.; Palit, R.

    2013-04-01

    Two distinct sets of chiral-partner bands have been identified in the nucleus Ce133. They constitute a multiple chiral doublet, a phenomenon predicted by relativistic mean field (RMF) calculations and observed experimentally here for the first time. The properties of these chiral bands are in good agreement with results of calculations based on a combination of the constrained triaxial RMF theory and the particle-rotor model.

  8. Scalar Dark Matter Candidates in Two Inert Higgs Doublet Model

    E-print Network

    E. C. F. S. Fortes; A. C. B. Machado; J. Montaño; V. Pleitez

    2014-07-17

    We study a two scalar inert doublet model (IDMS$_3$) which is stabilized by a $S_3$ symmetry. We consider two scenarios: i) two of the scalars in each charged sector are mass degenerated due to a residual $Z_2$ symmetry, ii) there is no mass degeneracy because of the introduction of soft terms that break the $Z_2$ symmetry. We show that both scenarios provide good dark matter candidates for some range of parameters.

  9. Fast Microtubule Dynamics in Meiotic Spindles Measured by Single Imaging: Evidence that the Spindle Environment does not Stabilize Microtubules

    E-print Network

    Mirny, Leonid A.

    Metaphase spindles are steady-state ensembles of microtubules that turn over rapidly and slide poleward in some systems. Since the discovery of dynamic instability in the mid-1980s, models for spindle morphogenesis have ...

  10. The dual specificity phosphatase Cdc14B bundles and stabilizes microtubules

    SciTech Connect

    Plumley, Hyekyung [ORNL; Liu, Yie [ORNL; Gomez, Marla V [ORNL; Wang, Yisong [ORNL

    2005-01-01

    The Cdc14 dual-specificity phosphatases regulate key events in the eukaryotic cell cycle. However, little is known about the function of mammalian CDC14B family members. Here, we demonstrate that subcellular localization of CDC14B protein is cell cycle regulated. CDC14B can bind, bundle, and stabilize microtubules in vitro independently of its catalytic activity. Basic amino acid residues within the nucleolar targeting domain are important for both retaining CDC14B in the nucleolus and preventing microtubule bundling. Overexpression of CDC14B resulted in the formation of cytoplasmic CDC14B and microtubule bundles in interphase cells. These microtubule bundles were resistant to microtubule depolymerization reagents and enriched in acetylated -tubulin. Expression of cytoplasmic forms of CDC14B impaired microtubule nucleation from the microtubule organization center. CDC14B is thus a novel microtubule-bundling and -stabilizing protein, whose regulated subcellular localization may help modulate spindle and microtubule dynamics in mitosis.

  11. Dark Matter with Topological Defects in the Inert Doublet Model

    E-print Network

    Hindmarsh, Mark; No, Jose Miguel; West, Stephen M

    2014-01-01

    We examine the production of dark matter by decaying topological defects in the high mass region $m_{\\mathrm{DM}} \\gg m_W$ 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 $\\gamma$-ray background and Big Bang Nucleosynthesis, together with constraints on the dark matter properties coming from direct and indirect detectio...

  12. An EB1-kinesin complex is sufficient to steer microtubule growth in vitro.

    PubMed

    Chen, Yalei; Rolls, Melissa M; Hancock, William O

    2014-02-01

    Proper microtubule polarity underlies overall neuronal polarity, but mechanisms for maintaining microtubule polarity are not well understood. Previous live imaging in Drosophila dendritic arborization neurons showed that while microtubules are uniformly plus-end out in axons, dendrites possess uniformly minus-end-out microtubules [1]. Thus, maintaining uniform microtubule polarity in dendrites requires that growing microtubule plus ends entering branch points be actively directed toward the cell body. A model was proposed in which EB1 tracks the plus ends of microtubules growing into a branch and an associated kinesin-2 motor walks along a static microtubule to steer the plus end toward the cell body. However, the fast plus-end binding dynamics of EB1 [2-5] appear to be at odds with this proposed mechanical function. To test this model in vitro, we reconstituted the system by artificially dimerizing EB1 to kinesin, growing microtubules from immobilized seeds, and imaging encounters between growing microtubule plus ends and static microtubules. Consistent with in vivo observations, the EB1-kinesin complex actively steered growing microtubules. Thus, EB1 kinetics and mechanics are sufficient to bend microtubules for several seconds. Other kinesins also demonstrated this activity, suggesting this is a general mechanism for organizing and maintaining proper microtubule polarity in cells. PMID:24462004

  13. Lessons from in vitro reconstitution analyses of plant microtubule-associated proteins

    PubMed Central

    Hamada, Takahiro

    2014-01-01

    Plant microtubules, composed of tubulin GTPase, are irreplaceable cellular components that regulate the directions of cell expansion and cell division, chromosome segregation and cell plate formation. To accomplish these functions, plant cells organize microtubule structures by regulating microtubule dynamics. Each microtubule localizes to the proper position with repeated growth and shortening. Although it is possible to reconstitute microtubule dynamics with pure tubulin solution in vitro, many microtubule-associated proteins (MAPs) govern microtubule dynamics in cells. In plants, major MAPs are identified as microtubule stabilizers (CLASP and MAP65 etc.), microtubule destabilizers (kinesin-13, katanin, MAP18 and MDP25), and microtubule dynamics promoters (EB1, MAP215, MOR1, MAP200, SPR2). Mutant analyses with forward and reverse genetics have shown the importance of microtubules and individual MAPs in plants. However, it is difficult to understand how each MAP regulates microtubule dynamics, such as growth and shortening, through mutant analyses. In vitro reconstitution analyses with individual purified MAPs and tubulin are powerful tools to reveal how each MAP regulates microtubule dynamics at the molecular level. In this review, I summarize the results of in vitro reconstitution analyses and introduce current models of how each MAP regulates microtubule dynamic instability. PMID:25202315

  14. An EB1-kinesin complex is sufficient to steer microtubule growth in vitro

    PubMed Central

    Chen, Yalei; Rolls, Melissa M.; Hancock, William O.

    2013-01-01

    Summary Proper microtubule polarity underlies overall neuronal polarity, but mechanisms for maintaining microtubule polarity are not well understood. Previous live imaging in Drosophila dendritic arborization (da) neurons showed that, while microtubules are uniformly plus-end out in axons, dendrites possess uniformly minus-end-out microtubules [1]. Thus, maintaining uniform microtubule polarity in dendrites requires that growing microtubule plus-ends entering branch points must be actively directed towards the cell body. A model was proposed in which EB1 tracks the plus-ends of microtubules growing into a branches and an associated kinesin-2 motor walks along a static microtubule to steer the plus-end toward the cell body. However, the fast plus-end binding dynamics of EB1 [2–5] appear at odds with this proposed mechanical function. To test this model in vitro, we reconstituted the system by artificially dimerizing EB1 to kinesin, growing microtubules from immobilized seeds, and imaging encounters between growing microtubule plus-ends and static microtubules. Consistent with in vivo observations, the EB1-kinesin complex actively steered growing microtubules. Thus EB1 kinetics and mechanics are sufficient to bend microtubules for several seconds. Other kinesins also demonstrated this activity, suggesting this is a general mechanism for organizing and maintaining proper microtubule polarity in cells. PMID:24462004

  15. The chemical complexity of cellular microtubules: tubulin post-translational modification enzymes and their roles in tuning microtubule functions

    PubMed Central

    Garnham, Christopher P.; Roll-Mecak, Antonina

    2012-01-01

    Cellular microtubules are marked by abundant and evolutionarily conserved post-translational modifications that have the potential to tune their functions. This review focuses on the astonishing chemical complexity introduced in the tubulin heterodimer at the post-translational level and summarizes the recent advances in identifying the enzymes responsible for these modifications and deciphering the consequences of tubulin’s chemical diversity on the function of molecular motors and microtubule associated proteins. PMID:22422711

  16. Essential role of tubulin-folding cofactor D in microtubule assembly and its association with microtubules in fission yeast.

    PubMed

    Hirata, D; Masuda, H; Eddison, M; Toda, T

    1998-02-01

    The main structural components of microtubules are alpha- and beta-tubulins. A group of proteins called cofactors are crucial in the formation of assembly-competent tubulin molecules in vitro. Whilst an in vitro role is emerging for these cofactors, their biological functions in vivo remain to be established. In order to understand the fundamental mechanisms that determine cell polarity, we have screened for fission yeast mutants with altered polarity. Here we show that alp1+ encodes a homologue of cofactor D and executes a function essential for cell viability. A temperature-sensitive alp1 mutant shows a variety of defects including abnormal mitoses, loss of microtubule structures, displacement of the nucleus, altered growth polarity and asymmetrical cell division. Overexpression of Alp1 is lethal in wild-type cells, resulting in altered cell shape, but is rescued by co-overexpression of beta-tubulin. Alp1 co-localizes with microtubules, both interphase arrays and mitotic spindles. Furthermore, Alp1 binds to and co-sediments with taxol (paclitaxel)-stabilized porcine microtubules. Our results suggest that, in addition to a function in the folding of beta-tubulin, cofactor D may play a vital role in microtubule-dependent processes as a microtubule-associated protein. PMID:9450991

  17. 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.

  18. Hooks and Comets: The Story of Microtubule Polarity Orientation in the Neuron

    PubMed Central

    Baas, Peter W.; Lin, Shen

    2011-01-01

    It is widely believed that signature patterns of microtubule polarity orientation within axons and dendrites underlie compositional and morphological differences that distinguish these neuronal processes from one another. Axons of vertebrate neurons display uniformly plus-end-distal microtubules, whereas their dendrites display non-uniformly oriented microtubules. Fly axons also display uniformly plus-end-distal microtubules, but their dendritic microtubules are nearly uniformly minus-end-distal. Discussed in this article are the history of these findings, their implications for the regulation of neuronal polarity across the animal kingdom, and potential mechanisms by which neurons establish the distinct microtubule polarity patterns that define axons and dendrites. PMID:21557497

  19. Dynamical Length-Regulation of Microtubules

    NASA Astrophysics Data System (ADS)

    Melbinger, Anna; Reese, Louis; Frey, Erwin

    2012-02-01

    Microtubules (MTs) are vital constituents of the cytoskeleton. These stiff filaments are not only needed for mechanical support. They also fulfill highly dynamic tasks. For instance MTs build the mitotic spindle, which pulls the doubled set of chromosomes apart during mitosis. Hence, a well-regulated and adjustable MT length is essential for cell division. Extending a recently introduced model [1], we here study length-regulation of MTs. Thereby we account for both spontaneous polymerization and depolymerization triggered by motor proteins. In contrast to the polymerization rate, the effective depolymerization rate depends on the presence of molecular motors at the tip and thereby on crowding effects which in turn depend on the MT length. We show that these antagonistic effects result in a well-defined MT length. Stochastic simulations and analytic calculations reveal the exact regimes where regulation is feasible. Furthermore, the adjusted MT length and the ensuing strength of fluctuations are analyzed. Taken together, we make quantitative predictions which can be tested experimentally. These results should help to obtain deeper insights in the microscopic mechanisms underlying length-regulation. [4pt] [1] L.Reese, A.Melbinger, E.Frey, Biophys. J., 101, 9, 2190 (2011)

  20. MAP65/Ase1 promote microtubule flexibility

    PubMed Central

    Portran, D.; Zoccoler, M.; Gaillard, J.; Stoppin-Mellet, V.; Neumann, E.; Arnal, I.; Martiel, J. L.; Vantard, M.

    2013-01-01

    Microtubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. Although they are highly rigid polymers with a persistence length of 1–8 mm, they may exhibit a curved shape at a scale of few micrometers within cells, depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poorly understood. Here we ask whether MT-associated proteins (MAPs) could locally control the mechanical properties of MTs. We show that two major cross-linkers of the conserved MAP65/PRC1/Ase1 family drastically decrease MT rigidity. Their MT-binding domain mediates this effect. Remarkably, the softening effect of MAP65 observed on single MTs is maintained when MTs are cross-linked. By reconstituting physical collisions between growing MTs/MT bundles, we further show that the decrease in MT stiffness induced by MAP65 proteins is responsible for the sharp bending deformations observed in cells when they coalign at a steep angle to create bundles. Taken together, these data provide new insights into how MAP65, by modifying MT mechanical properties, may regulate the formation of complex MT arrays. PMID:23615441

  1. Role of microtubules in the contractile dysfunction of hypertrophied myocardium

    NASA Technical Reports Server (NTRS)

    Zile, M. R.; Koide, M.; Sato, H.; Ishiguro, Y.; Conrad, C. H.; Buckley, J. M.; Morgan, J. P.; Cooper, G. 4th

    1999-01-01

    OBJECTIVES: We sought to determine whether the ameliorative effects of microtubule depolymerization on cellular contractile dysfunction in pressure overload cardiac hypertrophy apply at the tissue level. BACKGROUND: A selective and persistent increase in microtubule density causes decreased contractile function of cardiocytes from cats with hypertrophy produced by chronic right ventricular (RV) pressure overloading. Microtubule depolymerization by colchicine normalizes contractility in these isolated cardiocytes. However, whether these changes in cellular function might contribute to changes in function at the more highly integrated and complex cardiac tissue level was unknown. METHODS: Accordingly, RV papillary muscles were isolated from 25 cats with RV pressure overload hypertrophy induced by pulmonary artery banding (PAB) for 4 weeks and 25 control cats. Contractile state was measured using physiologically sequenced contractions before and 90 min after treatment with 10(-5) mol/liter colchicine. RESULTS: The PAB significantly increased RV systolic pressure and the RV weight/body weight ratio in PAB; it significantly decreased developed tension from 59+/-3 mN/mm2 in control to 25+/-4 mN/mm2 in PAB, shortening extent from 0.21+/-0.01 muscle lengths (ML) in control to 0.12+/-0.01 ML in PAB, and shortening rate from 1.12+/-0.07 ML/s in control to 0.55+/-0.03 ML/s in PAB. Indirect immunofluorescence confocal microscopy showed that PAB muscles had a selective increase in microtubule density and that colchicine caused complete microtubule depolymerization in both control and PAB papillary muscles. Microtubule depolymerization normalized myocardial contractility in papillary muscles of PAB cats but did not alter contractility in control muscles. CONCLUSIONS: Excess microtubule density, therefore, is equally important to both cellular and to myocardial contractile dysfunction caused by chronic, severe pressure-overload cardiac hypertrophy.

  2. Automated Stitching of Microtubule Centerlines across Serial Electron Tomograms

    PubMed Central

    Weber, Britta; Tranfield, Erin M.; Höög, Johanna L.; Baum, Daniel; Antony, Claude; Hyman, Tony; Verbavatz, Jean-Marc; Prohaska, Steffen

    2014-01-01

    Tracing microtubule centerlines in serial section electron tomography requires microtubules to be stitched across sections, that is lines from different sections need to be aligned, endpoints need to be matched at section boundaries to establish a correspondence between neighboring sections, and corresponding lines need to be connected across multiple sections. We present computational methods for these tasks: 1) An initial alignment is computed using a distance compatibility graph. 2) A fine alignment is then computed with a probabilistic variant of the iterative closest points algorithm, which we extended to handle the orientation of lines by introducing a periodic random variable to the probabilistic formulation. 3) Endpoint correspondence is established by formulating a matching problem in terms of a Markov random field and computing the best matching with belief propagation. Belief propagation is not generally guaranteed to converge to a minimum. We show how convergence can be achieved, nonetheless, with minimal manual input. In addition to stitching microtubule centerlines, the correspondence is also applied to transform and merge the electron tomograms. We applied the proposed methods to samples from the mitotic spindle in C. elegans, the meiotic spindle in X. laevis, and sub-pellicular microtubule arrays in T. brucei. The methods were able to stitch microtubules across section boundaries in good agreement with experts' opinions for the spindle samples. Results, however, were not satisfactory for the microtubule arrays. For certain experiments, such as an analysis of the spindle, the proposed methods can replace manual expert tracing and thus enable the analysis of microtubules over long distances with reasonable manual effort. PMID:25438148

  3. The Ability to Induce Microtubule Acetylation Is a General Feature of Formin Proteins

    PubMed Central

    Thurston, Susan F.; Kulacz, Wojciech A.; Shaikh, Sahir; Lee, Jonathan M.; Copeland, John W.

    2012-01-01

    Cytoplasmic microtubules exist as distinct dynamic and stable populations within the cell. Stable microtubules direct and maintain cell polarity and it is thought that their stabilization is dependent on coordinative organization between the microtubule network and the actin cytoskeleton. A growing body of work suggests that some members of the formin family of actin remodeling proteins also regulate microtubule organization and stability. For example, we showed previously that expression of the novel formin INF1 is sufficient to induce microtubule stabilization and tubulin acetylation, but not tubulin detyrosination. An important issue with respect to the relationship between formins and microtubules is the determination of which formin domains mediate microtubule stabilization. INF1 has a distinct microtubule-binding domain at its C-terminus and the endogenous INF1 protein is associated with the microtubule network. Surprisingly, the INF1 microtubule-binding domain is not essential for INF1-induced microtubule acetylation. We show here that expression of the isolated FH1 + FH2 functional unit of INF1 is sufficient to induce microtubule acetylation independent of the INF1 microtubule-binding domain. It is not yet clear whether or not microtubule stabilization is a general property of all mammalian formins; therefore we expressed constitutively active derivatives of thirteen of the fifteen mammalian formin proteins in HeLa and NIH3T3 cells and measured their effects on stress fiber formation, MT organization and MT acetylation. We found that expression of the FH1 + FH2 unit of the majority of mammalian formins is sufficient to induce microtubule acetylation. Our results suggest that the regulation of microtubule acetylation is likely a general formin activity and that the FH2 should be thought of as a dual-function domain capable of regulating both actin and microtubule networks. PMID:23110170

  4. Single molecule imaging reveals differences in microtubule track selection between Kinesin motors.

    PubMed

    Cai, Dawen; McEwen, Dyke P; Martens, Jeffery R; Meyhofer, Edgar; Verhey, Kristen J

    2009-10-01

    Cells generate diverse microtubule populations by polymerization of a common alpha/beta-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations. PMID:19823565

  5. Fission yeast mtr1p regulates interphase microtubule cortical dwell-time

    PubMed Central

    Carlier-Grynkorn, Frédérique; Ji, Liang; Fraisier, Vincent; Lombard, Berangère; Dingli, Florent; Loew, Damarys; Paoletti, Anne; Ronot, Xavier; Tran, Phong T.

    2014-01-01

    ABSTRACT The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs) and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1+, a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1?) primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3? results in short microtubules, but can be partially rescued by mtr1?, as the double mutant mal3? mtr1? exhibits longer microtubules than mal3? single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1?. The double-mutant mal3? rps1801? also exhibits longer microtubules than mal3? single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics. PMID:24928430

  6. Fission yeast mtr1p regulates interphase microtubule cortical dwell-time.

    PubMed

    Carlier-Grynkorn, Frédérique; Ji, Liang; Fraisier, Vincent; Lombard, Berangère; Dingli, Florent; Loew, Damarys; Paoletti, Anne; Ronot, Xavier; Tran, Phong T

    2014-01-01

    The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs) and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1(+), a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1?) primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3? results in short microtubules, but can be partially rescued by mtr1?, as the double mutant mal3? mtr1? exhibits longer microtubules than mal3? single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1?. The double-mutant mal3? rps1801? also exhibits longer microtubules than mal3? single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics. PMID:24928430

  7. Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling

    Microsoft Academic Search

    Clare M. Waterman-Storer; E. D. Salmon

    1997-01-01

    We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluores- cently labeled, microinjected tubulin. These cells ex- hibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge un- til they reach the base

  8. Microtubules and Their Role in Cellular Stress in Cancer

    PubMed Central

    Parker, Amelia L.; Kavallaris, Maria; McCarroll, Joshua A.

    2014-01-01

    Microtubules are highly dynamic structures, which consist of ?- and ?-tubulin heterodimers, and are involved in cell movement, intracellular trafficking, and mitosis. In the context of cancer, the tubulin family of proteins is recognized as the target of the tubulin-binding chemotherapeutics, which suppress the dynamics of the mitotic spindle to cause mitotic arrest and cell death. Importantly, changes in microtubule stability and the expression of different tubulin isotypes as well as altered post-translational modifications have been reported for a range of cancers. These changes have been correlated with poor prognosis and chemotherapy resistance in solid and hematological cancers. However, the mechanisms underlying these observations have remained poorly understood. Emerging evidence suggests that tubulins and microtubule-associated proteins may play a role in a range of cellular stress responses, thus conferring survival advantage to cancer cells. This review will focus on the importance of the microtubule–protein network in regulating critical cellular processes in response to stress. Understanding the role of microtubules in this context may offer novel therapeutic approaches for the treatment of cancer. PMID:24995158

  9. Random Hydrolysis Controls the Dynamic Instability of Microtubules

    PubMed Central

    Padinhateeri, Ranjith; Kolomeisky, Anatoly B.; Lacoste, David

    2012-01-01

    Uncovering mechanisms that control the dynamics of microtubules is fundamental for our understanding of multiple cellular processes such as chromosome separation and cell motility. Building on previous theoretical work on the dynamic instability of microtubules, we propose here a stochastic model that includes all relevant biochemical processes that affect the dynamics of microtubule plus-end, namely, the binding of GTP-bound monomers, unbinding of GTP- and GDP-bound monomers, and hydrolysis of GTP monomers. The inclusion of dissociation processes, present in our approach but absent from many previous studies, is essential to guarantee the thermodynamic consistency of the model. Our theoretical method allows us to compute all dynamic properties of microtubules explicitly. Using experimentally determined rates, it is found that the cap size is ?3.6 layers, an estimate that is compatible with several experimental observations. In the end, our model provides a comprehensive description of the dynamic instability of microtubules that includes not only the statistics of catastrophes but also the statistics of rescues. PMID:22455910

  10. 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 ?-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 ?-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 ?-tubulin and kinesin-8 interaction. Generally, presented data suggest that plant ?-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

  11. Quantitative Analysis of Tau-Microtubule Interaction Using FRET

    PubMed Central

    Di Maïo, Isabelle L.; Barbier, Pascale; Allegro, Diane; Brault, Cédric; Peyrot, Vincent

    2014-01-01

    The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET) assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using microtubules stabilized with Flutax-2 as donor and tau labeled with rhodamine as acceptor, a donor-to-acceptor distance of 54 ± 1 Å was found. A molecular model is proposed in which Flutax-2 is directly accessible to tau-rhodamine molecules for energy transfer. By titration, we calculated the stoichiometric dissociation constant to be equal to 1.0 ± 0.5 µM. The influence of the C-terminal tails of ??-tubulin on the tau-microtubule interaction is presented once a procedure to form homogeneous solution of cleaved tubulin has been determined. The results indicate that the C-terminal tails of ?- and ?-tubulin by electrostatic effects and of recruitment seem to be involved in the binding mechanism of tau. PMID:25196605

  12. Diffusible crosslinkers generate directed forces in microtubule networks.

    PubMed

    Lansky, Zdenek; Braun, Marcus; Lüdecke, Annemarie; Schlierf, Michael; Ten Wolde, Pieter Rein; Janson, Marcel E; Diez, Stefan

    2015-03-12

    Cytoskeletal remodeling is essential to eukaryotic cell division and morphogenesis. The mechanical forces driving the restructuring are attributed to the action of molecular motors and the dynamics of cytoskeletal filaments, which both consume chemical energy. By contrast, non-enzymatic filament crosslinkers are regarded as mere friction-generating entities. Here, we experimentally demonstrate that diffusible microtubule crosslinkers of the Ase1/PRC1/Map65 family generate directed microtubule sliding when confined between partially overlapping microtubules. The Ase1-generated forces, directly measured by optical tweezers to be in the piconewton-range, were sufficient to antagonize motor-protein driven microtubule sliding. Force generation is quantitatively explained by the entropic expansion of confined Ase1 molecules diffusing within the microtubule overlaps. The thermal motion of crosslinkers is thus harnessed to generate mechanical work analogous to compressed gas propelling a piston in a cylinder. As confinement of diffusible proteins is ubiquitous in cells, the associated entropic forces are likely of importance for cellular mechanics beyond cytoskeletal networks. PMID:25748652

  13. Intracellular Spatial Localization Regulated by the Microtubule Network

    PubMed Central

    Chen, Jing; Lippincott-Schwartz, Jennifer; Liu, Jian

    2012-01-01

    The commonly recognized mechanisms for spatial regulation inside the cell are membrane-bounded compartmentalization and biochemical association with subcellular organelles. We use computational modeling to investigate another spatial regulation mechanism mediated by the microtubule network in the cell. Our results demonstrate that the mitotic spindle can impose strong sequestration and concentration effects on molecules with binding affinity for microtubules, especially dynein-directed cargoes. The model can recapitulate the essence of three experimental observations on distinct microtubule network morphologies: the sequestration of germ plasm components by the mitotic spindles in the Drosophila syncytial embryo, the asymmetric cell division initiated by the time delay in centrosome maturation in the Drosophila neuroblast, and the diffusional block between neighboring energids in the Drosophila syncytial embryo. Our model thus suggests that the cell cycle-dependent changes in the microtubule network are critical for achieving different spatial regulation effects. The microtubule network provides a spatially extensive docking platform for molecules and gives rise to a “structured cytoplasm”, in contrast to a free and fluid environment. PMID:22532834

  14. Diffusive movement of processive kinesin-1 on microtubules.

    PubMed

    Lu, Hailong; Ali, M Yusuf; Bookwalter, Carol S; Warshaw, David M; Trybus, Kathleen M

    2009-10-01

    The processive motor kinesin-1 moves unidirectionally toward the plus end of microtubules. This process can be visualized by total internal reflection fluorescence microscopy of kinesin bound to a carboxylated quantum dot (Qdot), which acts both as cargo and label. Surprisingly, when kinesin is bound to an anti-HIS Qdot, it shows diffusive movement on microtubules, which decreased in favor of processive runs with increasing salt concentration. This observation implies that kinesin movement on microtubules is governed by its conformation, as it is well established that kinesin undergoes a salt-dependent transition from a folded (inactive) to an extended (active) molecule. A truncated kinesin lacking the last 75 amino acids (kinesin-Delta C) showed both processive and diffusive movement on microtubules. The extent of each behavior depends on the relative amounts of ADP and ATP, with purely diffusive movement occurring in ADP alone. Taken together, these data imply that folded kinesin.ADP can exist in a state that diffuses along the microtubule lattice without expending energy. This mechanism may facilitate the ability of kinesin to pick up cargo, and/or allow the kinesin/cargo complex to stay bound after encountering obstacles. PMID:19682327

  15. Repetitive doublet firing of motor units: evidence for plateau potentials in human motoneurones?

    PubMed

    Kudina, Lydia P; Andreeva, Regina E

    2010-07-01

    During voluntary muscle contraction, human motoneurones can exhibit specific discharge patterns: single and repetitive doublets. Delayed depolarization has been accepted as the mechanism underlying single doublets. Repetitive doublet firing has been studied much less and its controlling mechanisms remain obscure. The aim of the present study was to examine properties of repetitive doublets in human motoneurones and to consider their underlying potential mechanisms. It was found that 22 of 41 (53.7%) lower-threshold motor units (MUs) in the trapezius and 15 of 42 (35.7%) MUs in triceps brachii displayed repetitive doublets with the mean interspike intervals (ISIs) of 5.5 +/- 1.1 and 6.4 +/- 2.6 ms, respectively. Each doublet was followed by a prolonged post-doublet ISI. The analysis of properties of repetitive doublets showed that they were typically initiated in quiescent motoneurones rather than in firing ones (appearing just at recruitment in an all-or-none manner) and could only be maintained at a certain level of muscle contraction. Repetitive doublets were interrupted either voluntarily (by the subject), or spontaneously with sudden transition from doublet firing to single discharges-the firing behaviour that may be referred to as a firing-pattern "jump". The properties of doublet firing seem to be consistent with traits of motoneurone firing in the presence of plateau potentials reported in animal studies. It was suggested that the potential mechanisms underlying repetitive doublet firing could include a delayed depolarization as the primary determinant, which likely could become persistent probably due to a plateau potential activated in parallel with a common synaptic input. PMID:20508919

  16. Flexural Rigidity of Individual Microtubules Measured by a Buckling Force with Optical Traps

    PubMed Central

    Kikumoto, Mahito; Kurachi, Masashi; Tosa, Valer; Tashiro, Hideo

    2006-01-01

    We used direct buckling force measurements with optical traps to determine the flexural rigidity of individual microtubules bound to polystyrene beads. To optimize the accuracy of the measurement, we used two optical traps and antibody-coated beads to manipulate each microtubule. We then applied a new analytical model assuming nonaxial buckling. Paclitaxel-stabilized microtubules were polymerized from purified tubulin, and the average microtubule rigidity was calculated as 2.0 × 10?24 Nm2 using this novel microtubule buckling system. This value was not dependent on microtubule length. We also measured the rigidity of paclitaxel-free microtubules, and obtained the value of 7.9 × 10?24 Nm2, which is nearly four times that measured for paclitaxel-stabilized microtubules. PMID:16339879

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

    PubMed Central

    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.

  18. Linked In: Formation and Regulation of Microtubule Attachments During Chromosome Segregation

    PubMed Central

    Cheerambathur, Dhanya K.; Desai, Arshad

    2014-01-01

    Accurate segregation of the replicated genome during cell division depends on dynamic attachments formed between chromosomes and the microtubule polymers of the spindle. Here we review recent advances in mechanistic analysis of microtubule attachment formation and regulation. PMID:24529253

  19. Nearest-neighbor doublets in protein-coding regions of MS2 RNA. [coliphage virus

    NASA Technical Reports Server (NTRS)

    Jukes, T. H.

    1977-01-01

    'Nearest neighbor' base pairs ('doublets') in the protein-coding regions of MS2 RNA have been tabulated with respect to their positions in the first two bases of amino acid codons, in the second two bases, or paired by contact between adjoining codons. Considerable variation is evident between numbers of doublets in each of these three possible positions, but the totals of each of the 16 doublets in the coding regions of the MS2 RNA molecule show much less variation. Compilations of doublets in nucleic acid strands have no predictive value for the amino acid composition of proteins coded by such strands.

  20. Electroweak Baryogenesis in a Two-Higgs Doublet Model

    E-print Network

    Stephan J. Huber

    2006-06-01

    Electroweak baryogenesis fails in the SM because of too small CP violation and the lack of a strong first-order phase transition. It has been shown that supersymmetric models allow for successful baryogenesis, where the Higgsinos play an important role in the transport processes that generate the asymmetry. I will demonstrate that also non-supersymmetric models can provide the observed baryon asymmetry. The top quark takes the role of the Higgsinos. Focusing on the two-Higgs doublet model, I will discuss details of the phase transition and consequences for Higgs physics and EDM searches.

  1. Nuclear Matter and Neutron stars in a Parity Doublet Model

    E-print Network

    V. Dexheimer; S. Schramm; D. Zschiesche

    2007-10-23

    We investigate the properties of isospin-symmetric nuclear matter and neutron stars in a chiral model approach adopting the SU(2) parity doublet formulation. This ansatz explicitly incorporates chiral symmetry restoration with the limit of degenerate masses of the nucleons and their parity partners. Instead of searching for an optimized parameter set we explore the general parameter dependence of nuclear matter and star properties in the model. We are able to get a good description of ground state nuclear matter as well as large values of mass for neutron stars in agreement with observation.

  2. 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

  3. Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules.

    PubMed

    Lopus, Manu; Oroudjev, Emin; Wilson, Leslie; Wilhelm, Sharon; Widdison, Wayne; Chari, Ravi; Jordan, Mary Ann

    2010-10-01

    Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at subnanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. Recently, antibody-conjugated maytansine derivatives have been developed to overcome these drawbacks. Several conjugates show promising early clinical results. We evaluated the effects on microtubule polymerization and dynamic instability of maytansine and two cellular metabolites (S-methyl-DM1 and S-methyl-DM4) of antibody-maytansinoid conjugates that are potent in cells at picomolar levels and that are active in tumor-bearing mice. Although S-methyl-DM1 and S-methyl-DM4 inhibited polymerization more weakly than maytansine, at 100 nmol/L they suppressed dynamic instability more strongly than maytansine (by 84% and 73%, respectively, compared with 45% for maytansine). However, unlike maytansine, S-methyl-DM1 and S-methyl-DM4 induced tubulin aggregates detectable by electron microscopy at concentrations ?2 ?mol/L, with S-methyl-DM4 showing more extensive aggregate formation than S-methyl-DM1. Both maytansine and S-methyl-DM1 bound to tubulin with similar K(D) values (0.86 ± 0.2 and 0.93 ± 0.2 ?mol/L, respectively). Tritiated S-methyl-DM1 bound to 37 high-affinity sites per microtubule (K(D), 0.1 ± 0.05 ?mol/L). Thus, S-methyl-DM1 binds to high-affinity sites on microtubules 20-fold more strongly than vinblastine. The high-affinity binding is likely at microtubule ends and is responsible for suppression of microtubule dynamic instability. Also, at higher concentrations, S-methyl-DM1 showed low-affinity binding either to a larger number of sites on microtubules or to sedimentable tubulin aggregates. Overall, the maytansine derivatives that result from cellular metabolism of the antibody conjugates are themselves potent microtubule poisons, interacting with microtubules as effectively as or more effectively than the parent molecule. PMID:20937594

  4. S. pombe Kinesins-8 Promote Both Nucleation and Catastrophe of Microtubules

    PubMed Central

    Erent, Muriel; Drummond, Douglas R.; Cross, Robert A.

    2012-01-01

    The kinesins-8 were originally thought to be microtubule depolymerases, but are now emerging as more versatile catalysts of microtubule dynamics. We show here that S. pombe Klp5-436 and Klp6-440 are non-processive plus-end-directed motors whose in vitro velocities on S. pombe microtubules at 7 and 23 nm s?1 are too slow to keep pace with the growing tips of dynamic interphase microtubules in living S. pombe. In vitro, Klp5 and 6 dimers exhibit a hitherto-undescribed combination of strong enhancement of microtubule nucleation with no effect on growth rate or catastrophe frequency. By contrast in vivo, both Klp5 and Klp6 promote microtubule catastrophe at cell ends whilst Klp6 also increases the number of interphase microtubule arrays (IMAs). Our data support a model in which Klp5/6 bind tightly to free tubulin heterodimers, strongly promoting the nucleation of new microtubules, and then continue to land as a tubulin-motor complex on the tips of growing microtubules, with the motors then dissociating after a few seconds residence on the lattice. In vivo, we predict that only at cell ends, when growing microtubule tips become lodged and their growth slows down, will Klp5/6 motor activity succeed in tracking growing microtubule tips. This mechanism would allow Klp5/6 to detect the arrival of microtubule tips at cells ends and to amplify the intrinsic tendency for microtubules to catastrophise in compression at cell ends. Our evidence identifies Klp5 and 6 as spatial regulators of microtubule dynamics that enhance both microtubule nucleation at the cell centre and microtubule catastrophe at the cell ends. PMID:22363481

  5. Microtubule acetylation regulates dynamics of KIF1C-powered vesicles and contact of microtubule plus ends with podosomes.

    PubMed

    Bhuwania, Ridhirama; Castro-Castro, Antonio; Linder, Stefan

    2014-10-01

    Microtubule dynamics are important for a variety of key cellular functions such as intracellular trafficking, adjustment of the cell surface proteome, or adhesion structure turnover. In the current study, we investigate the effects of altered microtubule acetylation levels on the subcellular distribution of kinesins and actin cytoskeletal architecture in primary human macrophages. Microtubule acetylation was altered by overexpression or siRNA-induced depletion of the acetylase MEC-17, or by blocking ?-tubulin deacetylation by addition of the inhibitor tubacin. We show that microtubule acetylation influences the subcellular distribution of vesicles associated with the kinesin KIF1C, as well as their directionality, velocity and run length. Moreover, tubulin acetylation alters the targeting frequency of microtubule plus ends on podosomes and influences the number of podosomes per cell and thus the matrix-degrading capacity of macrophages. Collectively, our results point to ?-tubulin acetylation as an important modification that impacts on kinesin vesicle dynamics, actin cytoskeletal architecture and cellular function of macrophages. PMID:25151635

  6. CENTROSOMES AND MICROTUBULES DURING MEIOSIS IN THE MUSHROOM BOLETUS RUBINELLUS

    PubMed Central

    McLaughlin, David J.

    1971-01-01

    The double centrosome in the basidium of Boletus rubinellus has been observed in three planes with the electron microscope at interphase preceding nuclear fusion, at prophase I, and at interphase I. It is composed of two components connected by a band-shaped middle part. At anaphase I a single, enlarged centrosome is found at the spindle pole, which is attached to the cell membrane. Microtubules mainly oriented parallel to the longitudinal axis of the basidium are present at prefusion, prophase I and interphase I. Cytoplasmic microtubules are absent when the spindle is present. The relationship of the centrosome in B. rubinellus to that in other organisms and the role of the cytoplasmic microtubules are discussed. PMID:4329156

  7. Tunable dynamics of microtubule based active isotropic gels

    E-print Network

    Gil Henkin; Stephen J. DeCamp; Daniel TN Chen; Tim Sanchez; Zvonimir Dogic

    2014-09-26

    We investigate the dynamics of an active gel of bundled microtubules that is driven by clusters of kinesin molecular motors. Upon the addition of ATP, the coordinated action of thousands of molecular motors drives the gel to a highly dynamical turbulent-like state that persists for hours and is only limited by the stability of constituent proteins and the availability of the chemical fuel. We characterize how enhanced transport and emergent macroscopic flows of active gels depend on relevant molecular parameters, including ATP, kinesin motor, and depletant concentrations, microtubule volume fraction, as well as the stoichiometry of the constituent motor clusters. Our results show that the dynamical and structural properties of microtubule based active gels are highly tunable. They also indicate existence of an optimal concentration of molecular motors that maximize far-from-equilibrium activity of active isotropic MT gels.

  8. Disruption of Microtubule Integrity Initiates Mitosis during CNS Repair

    PubMed Central

    Bossing, Torsten; Barros, Claudia S.; Fischer, Bettina; Russell, Steven; Shepherd, David

    2012-01-01

    Summary Mechanisms of CNS repair have vital medical implications. We show that traumatic injury to the ventral midline of the embryonic Drosophila CNS activates cell divisions to replace lost cells. A pilot screen analyzing transcriptomes of single cells during repair pointed to downregulation of the microtubule-stabilizing GTPase mitochondrial Rho (Miro) and upregulation of the Jun transcription factor Jun-related antigen (Jra). Ectopic Miro expression can prevent midline divisions after damage, whereas Miro depletion destabilizes cortical ?-tubulin and increases divisions. Disruption of cortical microtubules, either by chemical depolymerization or by overexpression of monomeric tubulin, triggers ectopic mitosis in the midline and induces Jra expression. Conversely, loss of Jra renders midline cells unable to replace damaged siblings. Our data indicate that upon injury, the integrity of the microtubule cytoskeleton controls cell division in the CNS midline, triggering extra mitosis to replace lost cells. The conservation of the identified molecules suggests that similar mechanisms may operate in vertebrates. PMID:22841498

  9. Microtubule Motors in Eukaryotic Spindle Assembly and Maintenance

    PubMed Central

    Bloom, Kerry

    2010-01-01

    The main function of the mitotic spindle is to accurately segregate replicated chromosomes during cell division. This dynamic, microtubule-based structure is assembled by a dividing cell and facilitates the orchestrated movement of chromosomes that is the hallmark of mitosis. Steady-state spindle size and morphology are relatively constant for cells of a specified type but vary considerably from one cell type to the next. Despite these differences, all eukaryotic spindles share basic architectural similarities, perhaps the most important of which is bipolar symmetry. At its core, assembling a bipolar spindle is a mechanical process that requires dynamic microtubules be moved and arranged to realize some ultimate functional form. These movements are the result of forces generated either by microtubule polymer dynamics or molecular motors. In this review we focus specifically on the motor-dependent mechanisms that shape the spindle and defer a more comprehensive treatment of spindle assembly and other motor functions during mitosis to others [1]. PMID:20109569

  10. 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

  11. Tau proteins: the molecular structure and mode of binding on microtubules

    Microsoft Academic Search

    Nobutaka Hirokawa; Yoko Shiomura; Shigeo Okabe

    1988-01-01

    Tau is a family of closely related proteins (55,000-62,000 mol wt) which are contained in the nerve cells and copolymerize with tubulin to induce the formation of microtubules in vitro. All information so far has indicated that tau is closely apposed to the microtubule lattice, and there was no indication of do- mains projecting from the microtubule polymer lattice. We

  12. Tau Is Enriched on Dynamic Microtubules in the Distal Region of Growing Axons

    E-print Network

    Fischer, Itzhak

    Tau Is Enriched on Dynamic Microtubules in the Distal Region of Growing Axons Mark M. Black,1 19129 It is widely held that tau determines the stability of microtubules in growing axons, although reasoned that if the stability of axonal microtubules is directly related to their content of tau

  13. Microtubule-associated protein tau in development, degeneration and protection of neurons

    Microsoft Academic Search

    Jian-Zhi Wang; Fei Liu

    2008-01-01

    As a principal neuronal microtubule-associated protein, tau has been recognized to play major roles in promoting microtubule assembly and stabilizing the microtubules and to maintain the normal morphology of the neurons. Recent studies suggest that tau, upon alternative mRNA splicing and multiple posttranslational modifications, may participate in the regulations of intracellular signal transduction, development and viability of the neurons. Furthermore,

  14. Aurora B Inhibits MCAK Activity Through a Phospho-conformational Switch that Reduces Microtubule Association

    PubMed Central

    Ems-McClung, Stephanie C.; Hainline, Sarah G.; Devare, Jenna; Zong, Hailing; Cai, Shang; Carnes, Stephanie K.; Shaw, Sidney L.; Walczak, Claire E.

    2014-01-01

    SUMMARY Background Proper spindle assembly and chromosome segregation relies on precise microtubule dynamics, which are governed in part by the Kinesin-13 MCAK. MCAK microtubule depolymerization activity is inhibited by Aurora B-dependent phosphorylation, but the mechanism of this inhibition is not understood. Results Here we develop the first FRET-based biosensor for MCAK and show that MCAK in solution exists in a closed conformation mediated by an interaction between the C-terminal domain (CT) and the neck. Using fluorescence lifetime imaging (FLIM) we show that MCAK bound to microtubule ends is closed relative to MCAK associated with the microtubule lattice. Aurora B phosphorylation at S196 in the neck opens MCAK conformation and diminishes the interaction between the CT and the neck. Using FLIM and TIRF imaging we found that changes in MCAK conformation are associated with a decrease in MCAK affinity for the microtubule. Conclusions Unlike motile kinesins, which are open when doing work, the high affinity binding state for microtubule depolymerizing kinesins is in a closed conformation. Phosphorylation switches MCAK conformation, which inhibits its ability to interact with microtubules and reduces its microtubule depolymerization activity. This work shows that the conformational model proposed for regulating kinesin activity is not universal and that microtubule depolymerizing kinesins utilize a distinct conformational mode to regulate affinity for the microtubule, thus controlling their catalytic efficiency. Furthermore, our work provides a mechanism by which the robust microtubule depolymerization activity of Kinesin-13s can be rapidly modulated to control cellular microtubule dynamics. PMID:24291095

  15. Long Astral Microtubules and RACK-1 Stabilize Polarity Domains during Maintenance Phase in Caenorhabditis

    E-print Network

    Skop, Ahna

    Long Astral Microtubules and RACK-1 Stabilize Polarity Domains during Maintenance Phase. In this study, we show that depletion of Caenorhabditis elegans RACK-1, which leads to short astral microtubules, Skop AR (2011) Long Astral Microtubules and RACK-1 Stabilize Polarity Domains during Maintenance Phase

  16. Microtubule Dynamics in Living Root Hairs: Transient Slowing by Lipochitin Oligosaccharide Nodulation Signals

    Microsoft Academic Search

    Valya N. Vassileva; Hiroshi Kouchi; Robert W. Ridge

    2005-01-01

    The incorporation of a fusion of green fluorescent protein and tubulin-a 6 from Arabidopsis thaliana in root hairs of Lotus japonicus has allowed us to visualize and quantify the dynamic parameters of the cortical microtubules in living root hairs. Analysis of individual microtubule turnover in real time showed that only plus polymer ends contributed to overall microtubule dynamicity, exhibiting dynamic

  17. A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

    PubMed Central

    Marc, J; Granger, CL; Brincat, J; Fisher, DD; Kao, Th; McCubbin, AG; Cyr, RJ

    1998-01-01

    Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation. PMID:9811799

  18. Recovery of Microtubules on the Blepharoplast of Ceratopteris Spermatogenous Cells after Oryzalin Treatment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most land plants have ill-defined microtubule-organizing centers (MTOC’s), consisting of sites on the nuclear envelope or even along microtubules. In contrast, spermatogenous cells of the pteridophyte Ceratopteris richardii have a well-defined MTOC, the blepharoplast, which organizes microtubules th...

  19. Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcement

    Microsoft Academic Search

    Clifford P. Brangwynne; Frederick C. MacKintosh; Sanjay Kumar; Nicholas A. Geisse; Jennifer Talbot; L. Mahadevan; Kevin K. Parker; Donald E. Ingber; David A. Weitz

    2006-01-01

    Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of compressive loads. However, the results of in vitro studies suggest that microtubules should buckle at much larger length scales, withstanding

  20. Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy

    Microsoft Academic Search

    KEITH SUMMERS; MARC W. KIRSCHNER

    1979-01-01

    We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy . In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine

  1. The Fission Yeast XMAP215 Homolog Dis1p Is Involved in Microtubule Bundle Organization

    PubMed Central

    Roque, Hélio; Ward, Jonathan J.; Murrells, Lindsay; Brunner, Damian; Antony, Claude

    2010-01-01

    Microtubules are essential for a variety of fundamental cellular processes such as organelle positioning and control of cell shape. Schizosaccharomyces pombe is an ideal organism for studying the function and organization of microtubules into bundles in interphase cells. Using light microscopy and electron tomography we analyzed the bundle organization of interphase microtubules in S. pombe. We show that cells lacking ase1p and klp2p still contain microtubule bundles. In addition, we show that ase1p is the major determinant of inter-microtubule spacing in interphase bundles since ase1 deleted cells have an inter-microtubule spacing that differs from that observed in wild-type cells. We then identified dis1p, a XMAP215 homologue, as factor that promotes the stabilization of microtubule bundles. In wild-type cells dis1p partially co-localized with ase1p at regions of microtubule overlap. In cells deleted for ase1 and klp2, dis1p accumulated at the overlap regions of interphase microtubule bundles. In cells lacking all three proteins, both microtubule bundling and inter-microtubule spacing were further reduced, suggesting that Dis1p contributes to interphase microtubule bundling. PMID:21151990

  2. Effects of the principal hydroxy-metabolites of benzene on microtubule polymerization

    Microsoft Academic Search

    Richard D. Irons; Douglas A. Neptun

    1980-01-01

    The principal hydroxy-metabolites of benzene — phenol, catechol and hydroquinone — possess characteristics and produce toxicity similar to those reported for certain inhibitors of microtubule polymerization. In this study we examined the effects of phenol, catechol and hydroquinone on purified microtubule polymerization and the decay of tubulin-colchicine binding activity. Hydroquinone, but not catechol or phenol, inhibited microtubule polymerization and accelerated

  3. Rectified Brownian motion and kinesin motion along microtubules

    NASA Astrophysics Data System (ADS)

    Fox, Ronald F.; Choi, Mee Hyang

    2001-05-01

    The mechanism of rectified Brownian movement is used to analyze measured data for kinesin motion along microtubules. A key component of the mechanism is the diffusive movement of the microtubule binding heads of kinesin during the adenosine triphosphate (ATP) cycle. The first-passage time distribution for this step is analyzed in detail and is shown to be responsible for observed load-velocity profiles. The ATPase activity of the kinesin heads is that of a nucleotide switch and not that of a direct chemomechanical energy converter. Experimental data acquisition, rate constants, and alternative explanations are discussed. The mechanism described in this paper is fundamental to the nanobiology of intracellular processes.

  4. Three-Higgs-doublet model with $A_4$ symmetry

    E-print Network

    A. C. B. Machado; J. C. Montero; V. Pleitez

    2011-01-05

    We worked out in detail the three-Higgs-doublet extension of the standard model when the $A_4$ symmetry, which is imposed to solve the flavor problem, is extended to the scalar sector. The three doublets may be related to the fermion mass generation and, in particular, they may be the unique responsible for the generation of the neutrino masses. If this is the case, the respective VEVs have to be quite smaller than the electroweak scale if no fine tuning in the Yukawa couplings is assumed. We consider here the mass spectra in the scalar sector in three different situations. In one of them there are no light scalars at all, but in the other ones a light or two massless scalars, at the tree level, may survive. The later fields are safe, from the phenomenological point of view, since it couples mainly with neutrinos and/or becomes enough massive at the tree level if there exist trilinear interactions. Quantum effects may be important too.

  5. Next-to-Minimal Two Higgs Doublet Model

    DOE PAGESBeta

    Chen, Chien-Yi [BNL; Freid, Michael [W&M, JLAB; Sher, Marc [W&M

    2014-04-01

    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. 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.

  6. Spinning Janus doublets driven in uniform ac electric fields

    NASA Astrophysics Data System (ADS)

    Boymelgreen, Alicia; Yossifon, Gilad; Park, Sinwook; Miloh, Touvia

    2014-01-01

    We provide an experimental proof of concept for a robust, continuously rotating microstructure—consisting of two metallodielectric (gold-polystyrene) Janus particles rigidly attached to each other—which is driven in uniform ac fields by asymmetric induced-charge electro-osmosis. The pairs (doublets) are stabilized on the substrate surface which is parallel to the plane of view and normal to the direction of the applied electric field. We find that the radius of orbit and angular velocity of the pair are predominantly dependent on the relative orientations of the interfaces between the metallic and dielectric hemispheres and that the electrohydrodynamic particle-particle interactions are small. Additionally, we verify that both the angular and linear velocities of the pair are proportional to the square of the applied field which is consistent with the theory for nonlinear electrokinetics. A simple kinematic rigid body model is used to predict the paths and doublet velocities (angular and linear) based on their relative orientations with good agreement.

  7. Emulsion sheet doublets as interface trackers for the OPERA experiment

    NASA Astrophysics Data System (ADS)

    Anokhina, A.; Aoki, S.; Ariga, A.; Arrabito, L.; Autiero, D.; Badertscher, A.; Bay, F.; Bersani Greggio, F.; Bertolin, A.; Besnier, M.; Bick, D.; Bozza, C.; Brugiere, T.; Brugnera, R.; Brunetti, G.; Buontempo, S.; Carrara, E.; Cazes, A.; Chaussard, L.; Chernyavsky, M.; Chiarella, V.; Chon-Sen, N.; Chukanov, A.; Consiglio, L.; Cozzi, M.; Cuha, V.; Dal Corso, F.; D'Amato, G.; D'Ambrosio, N.; DeLellis, G.; Déclais, Y.; DeSerio, M.; Di Capua, F.; Di Ferdinando, D.; Di Giovanni, A.; Di Marco, N.; Di Troia, C.; Dmitrievski, S.; Dominjon, A.; Dracos, M.; Duchesneau, D.; Dusini, S.; Ebert, J.; Egorov, O.; Enikeev, R.; Ereditato, A.; Esposito, L. S.; Favier, J.; Felici, G.; Ferber, T.; Fini, R.; Frekers, D.; Fukuda, T.; Galkin, V. I.; Galkin, V. A.; Garfagnini, A.; Giacomelli, G.; Giorgini, M.; Goellnitz, C.; Goldberg, J.; Golubkov, D.; Gornushkin, Y.; Grella, G.; Grianti, F.; Guler, M.; Gusev, G.; Gustavino, C.; Hagner, C.; Hara, T.; Hierholzer, M.; Hiramatsu, S.; Hoshino, K.; Ieva, M.; Jakovcic, K.; Janicsko Csathy, J.; Janutta, B.; Jollet, C.; Juget, F.; Kawai, T.; Kazuyama, M.; Kim, S. H.; Knuesel, J.; Kodama, K.; Komatsu, M.; Kose, U.; Kreslo, I.; Laktineh, I.; Lazzaro, C.; Lenkeit, J.; Ljubicic, A.; Longhin, A.; Lutter, G.; Manai, K.; Mandrioli, G.; Marotta, A.; Marteau, J.; Matsuo, T.; Matsuoka, H.; Mauri, N.; Meisel, F.; Meregaglia, A.; Messina, M.; Migliozzi, P.; Mikado, S.; Miyamoto, S.; Monacelli, P.; Morishima, K.; Moser, U.; Muciaccia, M. T.; Naganawa, N.; Naka, T.; Nakamura, M.; Nakamura, T.; Nakano, T.; Nikitina, V.; Niwa, K.; Nonoyama, Y.; Ogawa, S.; Osedlo, V.; Ossetski, D.; Paoloni, A.; Park, B. D.; Park, I. G.; Pastore, A.; Patrizii, L.; Pennacchio, E.; Pessard, H.; Pilipenko, V.; Pistillo, C.; Polukhina, N.; Pozzato, M.; Pretzl, K.; Publichenko, P.; Pupilli, F.; Roganova, T.; Rosa, G.; Rostovtseva, I.; Rubbia, A.; Russo, A.; Ryazhskaya, O.; Ryzhikov, D.; Sato, O.; Sato, Y.; Saveliev, V.; Sazhina, G.; Schembri, A.; Scotto Lavina, L.; Shibuya, H.; Simone, S.; Sioli, M.; Sirignano, C.; Sirri, G.; Song, J. S.; Spinetti, M.; Stanco, L.; Starkov, N.; Stipcevic, M.; Strauss, T.; Strolin, P.; Sugonyaev, V.; Taira, Y.; Takahashi, S.; Tenti, M.; Terranova, F.; Tezuka, I.; Tioukov, V.; Tolun, P.; Tsarev, V.; Tufanli, S.; Ushida, N.; Vilain, P.; Vladimirov, M.; Votano, L.; Vuilleumier, J. L.; Wilquet, G.; Wonsak, B.; Yoon, C. S.; Yoshida, J.; Zaitsev, Y.; Zemskova, S.; Zghiche, A.; Zimmermann, R.

    2008-07-01

    New methods for efficient and unambiguous interconnection between electronic position sensitive detectors and target units based on nuclear photographic emulsion films have been developed. The application to the OPERA experiment, that aims at detecting ??rightleftharpoons?? oscillations in the CNGS neutrino beam, is reported in this paper. In order to reduce background due to latent tracks collected before installation in the detector, on-site large-scale treatments of the emulsions (''refreshing'') have been applied. Changeable Sheet (CSd) packages, each made of a doublet of emulsion films, have been designed, assembled and coupled to the OPERA target units (''ECC bricks''). A device has been built to print X-ray spots for accurate interconnection both within the CSd and between the CSd and the related ECC brick. Sample emulsion films have been extensively scanned with state-of-the-art automated optical microscopes. Efficient track-matching and powerful background rejection have been achieved in tests with electronically tagged penetrating muons. Further improvement of in-doublet film alignment was obtained by matching the pattern of low-energy electron tracks. The commissioning of the overall OPERA alignment procedure is in progress.

  8. Natural Standard Model Alignment in the Two Higgs Doublet Model

    E-print Network

    Dev, P S Bhupal

    2015-01-01

    The current LHC Higgs data provide strong constraints on possible deviations of the couplings of the observed 125 GeV Higgs boson from the Standard Model (SM) expectations. Therefore, it now becomes compelling that any extended Higgs sector must comply with the so-called SM alignment limit. In the context of the Two Higgs Doublet Model (2HDM), this alignment is often associated with either decoupling of the heavy Higgs sector or accidental cancellations in the 2HDM potential. Here we present a new solution realizing natural alignment based on symmetries, without decoupling or fine-tuning. In particular, we show that in 2HDMs where both Higgs doublets acquire vacuum expectation values, there exist only three different symmetry realizations leading to natural alignment. We discuss some phenomenological implications of the Maximally-Symmetric 2HDM based on SO(5) symmetry group and analyze new collider signals for the heavy Higgs sector, involving third-generation quarks, which can be a useful observational tool ...

  9. Natural Standard Model Alignment in the Two Higgs Doublet Model

    E-print Network

    P. S. Bhupal Dev; Apostolos Pilaftsis

    2015-03-31

    The current LHC Higgs data provide strong constraints on possible deviations of the couplings of the observed 125 GeV Higgs boson from the Standard Model (SM) expectations. Therefore, it now becomes compelling that any extended Higgs sector must comply with the so-called SM alignment limit. In the context of the Two Higgs Doublet Model (2HDM), this alignment is often associated with either decoupling of the heavy Higgs sector or accidental cancellations in the 2HDM potential. Here we present a new solution realizing natural alignment based on symmetries, without decoupling or fine-tuning. In particular, we show that in 2HDMs where both Higgs doublets acquire vacuum expectation values, there exist only three different symmetry realizations leading to natural alignment. We discuss some phenomenological implications of the Maximally-Symmetric 2HDM based on SO(5) symmetry group and analyze new collider signals for the heavy Higgs sector, involving third-generation quarks, which can be a useful observational tool during the Run-II phase of the LHC.

  10. Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

    PubMed Central

    Bicek, Andrew D.; Tüzel, Erkan; Demtchouk, Aleksey; Uppalapati, Maruti; Hancock, William O.; Kroll, Daniel M.

    2009-01-01

    Microtubules (MTs) have been proposed to act mechanically as compressive struts that resist both actomyosin contractile forces and their own polymerization forces to mechanically stabilize cell shape. To identify the origin of MT bending, we directly observed MT bending and F-actin transport dynamics in the periphery of LLC-PK1 epithelial cells. We found that F-actin is nearly stationary in these cells even as MTs are deformed, demonstrating that MT bending is not driven by actomyosin contractility. Furthermore, the inhibition of myosin II activity through the use of blebbistatin results in microtubules that are still dynamically bending. In addition, as determined by fluorescent speckle microscopy, MT polymerization rarely results, if ever, in bending. We suppressed dynamic instability using nocodazole, and we observed no qualitative change in the MT bending dynamics. Bending most often results from anterograde transport of proximal portions of the MT toward a nearly stationary distal tip. Interestingly, we found that in an in vitro kinesin-MT gliding assay, MTs buckle in a similar manner. To make quantitative comparisons, we measured curvature distributions of observed MTs and found that the in vivo and in vitro curvature distributions agree quantitatively. In addition, the measured MT curvature distribution is not Gaussian, as expected for a thermally driven semiflexible polymer, indicating that thermal forces play a minor role in MT bending. We conclude that many of the known mechanisms of MT deformation, such as polymerization and acto-myosin contractility, play an inconsequential role in mediating MT bending in LLC-PK1 cells and that MT-based molecular motors likely generate most of the strain energy stored in the MT lattice. The results argue against models in which MTs play a major mechanical role in LLC-PK1 cells and instead favor a model in which mechanical forces control the spatial distribution of the MT array. PMID:19403700

  11. EB1 Accelerates Two Conformational Transitions Important for Microtubule Maturation and Dynamics

    PubMed Central

    Maurer, Sebastian P.; Cade, Nicholas I.; Bohner, Gerg?; Gustafsson, Nils; Boutant, Emmanuel; Surrey, Thomas

    2014-01-01

    Summary Background The dynamic properties of microtubules depend on complex nanoscale structural rearrangements in their end regions. Members of the EB1 and XMAP215 protein families interact autonomously with microtubule ends. EB1 recruits several other proteins to growing microtubule ends and has seemingly antagonistic effects on microtubule dynamics: it induces catastrophes, and it increases growth velocity, as does the polymerase XMAP215. Results Using a combination of in vitro reconstitution, time-lapse fluorescence microscopy, and subpixel-precision image analysis and convolved model fitting, we have studied the effects of EB1 on conformational transitions in growing microtubule ends and on the time course of catastrophes. EB1 density distributions at growing microtubule ends reveal two consecutive conformational transitions in the microtubule end region, which have growth-velocity-independent kinetics. EB1 binds to the microtubule after the first and before the second conformational transition has occurred, positioning it several tens of nanometers behind XMAP215, which binds to the extreme microtubule end. EB1 binding accelerates conformational maturation in the microtubule, most likely by promoting lateral protofilament interactions and by accelerating reactions of the guanosine triphosphate (GTP) hydrolysis cycle. The microtubule maturation time is directly linked to the duration of a growth pause just before microtubule depolymerization, indicating an important role of the maturation time for the control of dynamic instability. Conclusions These activities establish EB1 as a microtubule maturation factor and provide a mechanistic explanation for its effects on microtubule growth and catastrophe frequency, which cause microtubules to be more dynamic. PMID:24508171

  12. 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…

  13. Stability and symmetry breaking in the general three-Higgs-doublet model

    NASA Astrophysics Data System (ADS)

    Maniatis, M.; Nachtmann, O.

    2015-02-01

    Stability, electroweak symmetry breaking, and the stationarity equations of the general three-Higgs doublet model (3HDM) where all doublets carry the same hypercharge are discussed in detail. Employing the bilinear formalism the study of the 3HDM potential turns out to be straightforward.

  14. Multiple chiral doublet candidate nucleus $^{105}$Rh in a relativistic mean-field approach

    E-print Network

    Jian Li; S. Q. Zhang; J. Meng

    2011-03-25

    Following the reports of two pairs of chiral doublet bands observed in $^{105}$Rh, the adiabatic and configuration-fixed constrained triaxial relativistic mean-field (RMF) calculations are performed to investigate their triaxial deformations with the corresponding configuration and the possible multiple chiral doublet (M$\\chi$D) phenomenon. The existence of M$\\chi$D phenomenon in $^{105}$Rh is highly expected.

  15. Inert doublet dark matter and mirror\\/extra families after Xenon100

    Microsoft Academic Search

    Alejandra Melfo; Miha Nemevsek; Fabrizio Nesti; Goran Senjanovic; Yue Zhang

    2011-01-01

    It was shown recently that mirror fermions, naturally present in a number of directions for new physics, seem to require an inert scalar doublet in order to pass the electroweak precision tests. This provides a further motivation for considering the inert doublet as a dark matter candidate. Moreover, the presence of extra families enhances the standard model Higgs-nucleon coupling, which

  16. Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones.

    PubMed

    Burnette, Dylan T; Schaefer, Andrew W; Ji, Lin; Danuser, Gaudenz; Forscher, Paul

    2007-12-01

    Filopodial actin bundles guide microtubule assembly in the growth cone peripheral (P) domain and retrograde actin-network flow simultaneously transports microtubules rearward. Therefore, microtubule-end position is determined by the sum of microtubule assembly and retrograde transport rates. However, how filopodia actually affect microtubule assembly dynamics is unknown. To address this issue we quantitatively assessed microtubule and actin dynamics before and after selective removal of filopodia. Filopodium removal had surprisingly little effect on retrograde actin-flow rates or underlying network structures, but resulted in an approximate doubling of peripheral microtubule density and deeper penetration of microtubules into the P domain. The latter stemmed from less efficient coupling of microtubules to remaining actin networks and not from a change in microtubule polymer dynamics. Loss of filopodia also resulted in increased lateral microtubule movements and a more randomized microtubule distribution in the P domain. In summary, filopodia do not seem to be formally required for microtubule advance; however, their presence ensures radial distribution of microtubules in the P domain and facilitates microtubule transport by retrograde flow. The resulting dynamic steady state has interesting implications for rapid microtubule-positioning responses in the P domain. PMID:18026092

  17. Functional Architecture of the Outer Arm Dynein Conformational Switch*

    PubMed Central

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

    2012-01-01

    Dynein light chain 1 (LC1/DNAL1) is one of the most highly conserved components of ciliary axonemal outer arm dyneins, and it associates with both a heavy chain motor unit and tubulin located within the A-tubule of the axonemal outer doublet microtubules. In a variety of model systems, lack of LC1 or expression of mutant forms leads to profound defects in ciliary motility, including the failure of the hydrodynamic coupling needed for ciliary metachronal synchrony, random stalling during the power/recovery stroke transition, an aberrant response to imposed viscous load, and in some cases partial failure of motor assembly. These phenotypes have led to the proposal that LC1 acts as part of a mechanical switch to control motor function in response to alterations in axonemal curvature. Here we have used NMR chemical shift mapping to define the regions perturbed by a series of mutations in the C-terminal domain that yield a range of phenotypic effects on motility. In addition, we have identified the subdomain of LC1 involved in binding microtubules and characterized the consequences of an Asn ? Ser alteration within the terminal leucine-rich repeat that in humans causes primary ciliary dyskinesia. Together, these data define a series of functional subdomains within LC1 and allow us to propose a structural model for the organization of the dynein heavy chain-LC1-microtubule ternary complex that is required for the coordinated activity of dynein motors in cilia. PMID:22157010

  18. Ultrastructural characters of the spermatozoa in Digeneans of the genus Lecithochirium Lühe, 1901 (Digenea, Hemiuridae), parasites of fishes: comparative study of L. microstomum and L. musculus

    PubMed Central

    Ndiaye, Papa Ibnou; Quilichini, Yann; Sène, Aminata; Tkach, Vasyl V.; Bâ, Cheikh Tidiane; Marchand, Bernard

    2014-01-01

    This study provides the first ultrastructural data of spermatozoa in the genus Lecithochirium. The spermatozoa of L. microstomum (from Trichiurus lepturus in Senegal) and L. musculus (from Anguilla anguilla in Corsica) exhibit the general pattern described in the great majority of the Digenea, namely two axonemes with the 9 + “1” pattern typical of the Trepaxonemata, one mitochondrion, a nucleus, parallel cortical microtubules and external ornamentation of the plasma membrane. Spermatozoa of L. microstomum and L. musculus have some specific features such as the presence of a reduced number of cortical microtubules arranged on only one side of the spermatozoon, the lack of spine-like bodies and expansion of the plasma membrane. The external ornamentation of the plasma membrane entirely covers the anterior extremity of the spermatozoa. The ultrastructure of the posterior extremity of the spermatozoa corresponds to the pattern previously described in the Hemiuridae, characterized by only singlets of the second axoneme. A particularity of these spermatozoa is the organization of the microtubule doublets of the second axoneme around the nucleus in the posterior part of the spermatozoon. PMID:25275216

  19. Ultrastructural characters of the spermatozoa in Digeneans of the genus Lecithochirium Lühe, 1901 (Digenea, Hemiuridae), parasites of fishes: comparative study of L. microstomum and L. musculus.

    PubMed

    Ndiaye, Papa Ibnou; Quilichini, Yann; Sène, Aminata; Tkach, Vasyl V; Bâ, Cheikh Tidiane; Marchand, Bernard

    2014-01-01

    This study provides the first ultrastructural data of spermatozoa in the genus Lecithochirium. The spermatozoa of L. microstomum (from Trichiurus lepturus in Senegal) and L. musculus (from Anguilla anguilla in Corsica) exhibit the general pattern described in the great majority of the Digenea, namely two axonemes with the 9 + "1" pattern typical of the Trepaxonemata, one mitochondrion, a nucleus, parallel cortical microtubules and external ornamentation of the plasma membrane. Spermatozoa of L. microstomum and L. musculus have some specific features such as the presence of a reduced number of cortical microtubules arranged on only one side of the spermatozoon, the lack of spine-like bodies and expansion of the plasma membrane. The external ornamentation of the plasma membrane entirely covers the anterior extremity of the spermatozoa. The ultrastructure of the posterior extremity of the spermatozoa corresponds to the pattern previously described in the Hemiuridae, characterized by only singlets of the second axoneme. A particularity of these spermatozoa is the organization of the microtubule doublets of the second axoneme around the nucleus in the posterior part of the spermatozoon. PMID:25275216

  20. Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process.

    PubMed

    Hutchins, B Ian; Wray, Susan

    2014-01-01

    Microtubules are a critical part of neuronal polarity and leading process extension, thus microtubule movement plays an important role in neuronal migration. However, the dynamics of microtubules during the forward movement of the nucleus into the leading process (nucleokinesis) is unclear and may be dependent on the cell type and mode of migration used. In particular, little is known about cytoskeletal changes during axophilic migration, commonly used in anteroposterior neuronal migration. We recently showed that leading process actin flow in migrating GnRH neurons is controlled by a signaling cascade involving IP3 receptors, CaMKK, AMPK, and RhoA. In the present study, microtubule dynamics were examined in GnRH neurons. Failure of the migration of these cells leads to the neuroendocrine disorder Kallmann Syndrome. Microtubules translocated forward along the leading process shaft during migration, but reversed direction and moved toward the nucleus when migration stalled. Blocking calcium release through IP3 receptors halted migration and induced the same reversal of microtubule translocation, while blocking cortical actin flow prevented microtubules from translocating toward the distal leading process. Super-resolution imaging revealed that microtubule plus-end tips are captured at the actin cortex through calcium-dependent mechanisms. This work shows that cortical actin flow draws the microtubule network forward through calcium-dependent capture in order to promote nucleokinesis, revealing a novel mechanism engaged by migrating neurons to facilitate movement. PMID:25505874

  1. Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua

    PubMed Central

    1991-01-01

    Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin. PMID:2010465

  2. High Rectifying Efficiencies of Microtubule Motility on Kinesin-Coated

    E-print Network

    Dekker, Cees

    with an outer diameter of 25 nm and lengths up to tens of micrometers. Integration of biomolecular motors of molecular matter may become possible.2 In the inverted gliding assay, kinesin motor proteins are adsorbed to 97% of the microtubules move unidirectionally. Introduction. Biomolecular motors are complex

  3. Regulation of Tau Phosphorylation in Microtubule Fractions by Apolipoprotein E

    E-print Network

    Wood, John G.

    In Alzheimer's disease (AD), a family of proteins collectively named tau are displaced from their nor- malRegulation of Tau Phosphorylation in Microtubule Fractions by Apolipoprotein E Denise Flaherty,1 an interaction and potentially modulatory effects on tau hyperphosphorylation by the different apoE isoforms

  4. The physiology and pathology of microtubule-associated protein tau.

    PubMed

    Wang, Jian-Zhi; Gao, Xinya; Wang, Zhi-Hao

    2014-01-01

    Tau belongs to the family of microtubule-associated proteins predominantly expressed in neurons where they play an important role in promoting microtubule assembly and stabilizing microtubules. In addition, tau proteins interact with other cytoskeletal elements to allow spacing between microtubules. Recent studies have shown that tau is also actively involved in regulating cell viability and activity. Translated from a single gene located on chromosome 17q21, six isoforms of tau are produced by alternative splicing in adult human brain. Due to multiple post-translational modifications, heterogeneous tau species with a wide range of apparent molecular masses have been observed by denaturing polyacrylamide-gel electrophoresis. Since tau gene mutations and abnormal post-translational modifications have been detected in over 20 neurodegenerative disorders, namely the tauopathies, tau has gained widespread attention as a target protein in Alzheimer's disease and other neurodegenerative disorders. In the present chapter, research progress regarding physiology and pathology of tau is reviewed, particularly in terms of the role of post-translational modification. PMID:25131590

  5. Fission Yeast Scp3 Potentially Maintains Microtubule Orientation through Bundling

    PubMed Central

    Ozaki, Kanako; Chikashige, Yuji; Hiraoka, Yasushi; Matsumoto, Tomohiro

    2015-01-01

    Microtubules play important roles in organelle transport, the maintenance of cell polarity and chromosome segregation and generally form bundles during these processes. The fission yeast gene scp3+ was identified as a multicopy suppressor of the cps3-81 mutant, which is hypersensitive to isopropyl N-3-chlorophenylcarbamate (CIPC), a poison that induces abnormal multipolar spindle formation in higher eukaryotes. In this study, we investigated the function of Scp3 along with the effect of CIPC in the fission yeast Schizosaccharomyces pombe. Microscopic observation revealed that treatment with CIPC, cps3-81 mutation and scp3+ gene deletion disturbed the orientation of microtubules in interphase cells. Overexpression of scp3+ suppressed the abnormal orientation of microtubules by promoting bundling. Functional analysis suggested that Scp3 functions independently from Ase1, a protein largely required for the bundling of the mitotic spindle. A strain lacking the ase1+ gene was more sensitive to CIPC, with the drug affecting the integrity of the mitotic spindle, indicating that CIPC has a mitotic target that has a role redundant with Ase1. These results suggested that multiple systems are independently involved to ensure microtubule orientation by bundling in fission yeast. PMID:25767875

  6. Modeling oscillatory microtubule polymerization Martin Hammele and Walter Zimmermann

    E-print Network

    Schmidt, Matthias

    dependence are presented. Both, a catastrophe rate that depends on the density of guanosine triphosphate guanosine triphosphate hy- drolysis is apparently the driving force of microtubule physi- ology. The rich observed either when GTP is regenerated enzymatically from endogenous GDP guanosine diphosphate 5

  7. Microtubules, MAPs and Mitosis: a holistic approach to

    E-print Network

    Mumby, Peter J.

    Microtubules, MAPs and Mitosis: a holistic approach to understanding cell division James Wake eld Zitzmann #12;Gene Ontology of MAPs uncharacterised 30% cell cycle/mitosis 9% cytoskeleton binding% cell cycle/mitosis 9% cytoskeleton binding 5% motors 3% vesicle-mediated transport 2% polarity 1

  8. A targeted multi-enzyme mechanism for selective microtubule

    E-print Network

    Paris-Sud XI, Université de

    1 A targeted multi-enzyme mechanism for selective microtubule polyglutamylation Juliette van Dijk: posttranslational modification, tubulin, motility, polyglutamylase, TTLL Running Title: The multi-enzyme mechanism of polyglutamylation Summary Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths

  9. Microtubule-organizing centers and nucleating sites in land plants.

    PubMed

    Vaughn, K C; Harper, J D

    1998-01-01

    Microtubule-organizing centers (MTOCs) are morphologically diverse cellular sites involved in the nucleation and organization of microtubules (MTs). These structures are synonymous with the centrosome in mammalian cells. In most land plant cells, however, no such structures are observed and some have argued that plant cells may not have MTOCs. This review summarizes a number of experimental approaches toward the elucidation of those subcellular sites involved in microtubule nucleation and organization. In lower land plants, structurally well-defined MTOCs are present, such as the blepharoplast, multilayered structure, and polar organizer. In higher plants, much of the nucleation and organization of MTs occurs on the nuclear envelope or other endomembranes, such as the plasmalemma and smooth (tubular) endoplasmic reticulum. In some instances, one endomembrane may serve as a site of nucleation whereas others serve as the site of organization. Structural and motor microtubule-associated proteins also appear to be involved in MT nucleation and organization. Immunochemical evidence indicates that at least several of the proteins found in mammalian centrosomes, gamma-tubulin, centrin, pericentrin, and polypeptides recognized by the monoclonal antibodies MPM-2, 6C6, and C9 also recognize putative lower land plant MTOCs, indicating shared mechanisms of nucleation/organization in plants and animals. The most recent data from tubulin incorporation in vivo, mutants with altered MT organization, and molecular studies indicate the potential of these research tools in investigation of MTOCs in plants. PMID:9522456

  10. Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat)

    NASA Technical Reports Server (NTRS)

    Lewis, M. L.; Reynolds, J. L.; Cubano, L. A.; Hatton, J. P.; Lawless, B. D.; Piepmeier, E. H.

    1998-01-01

    Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.

  11. BMP signaling and microtubule organization regulate synaptic strength.

    PubMed

    Ball, R W; Peled, E S; Guerrero, G; Isacoff, E Y

    2015-04-16

    The strength of synaptic transmission between a neuron and multiple postsynaptic partners can vary considerably. We have studied synaptic heterogeneity using the glutamatergic Drosophila neuromuscular junction (NMJ), which contains multiple synaptic connections of varying strengths between a motor axon and muscle fiber. In larval NMJs, there is a gradient of synaptic transmission from weak proximal to strong distal boutons. We imaged synaptic transmission with the postsynaptically targeted fluorescent calcium sensor SynapCam, to investigate the molecular pathways that determine synaptic strength and set up this gradient. We discovered that mutations in the Bone Morphogenetic Protein (BMP) signaling pathway disrupt production of strong distal boutons. We find that strong connections contain unbundled microtubules in the boutons, suggesting a role for microtubule organization in transmission strength. The spastin mutation, which disorganizes microtubules, disrupted the transmission gradient, supporting this interpretation. We propose that the BMP pathway, shown previously to function in the homeostatic regulation of synaptic growth, also boosts synaptic transmission in a spatially selective manner that depends on the microtubule system. PMID:25681521

  12. ORIGINAL PAPER The microtubule-associated protein, NUD-1, exhibits

    E-print Network

    Caldwell, Guy

    ORIGINAL PAPER The microtubule-associated protein, NUD-1, exhibits chaperone activity in vitro elegans NudC homolog, NUD-1, as a protein exhibiting molecular chaperone activity. All NudC/NUD-1 proteins for a member of this mitotically essential protein family as having chaperone activity and facilitates elucida

  13. Interaction of Actin Filaments with Microtubules Is Mediated by

    E-print Network

    unfractionated microtubule protein (containing protein kinases) with ATP and cyclic adenosine monophosphate (CAMP in the presence of adenosine triphosphate (ATP) and other nucleosidephosphates than in their absence.' Nishida et successivetreatments with kinases and protein phosphatases, we showedthat the effect of MAP phosphorylationon actin

  14. Force on spindle microtubule minus ends moves chromosomes.

    PubMed

    Elting, Mary Williard; Hueschen, Christina L; Udy, Dylan B; Dumont, Sophie

    2014-07-21

    The spindle is a dynamic self-assembling machine that coordinates mitosis. The spindle's function depends on its ability to organize microtubules into poles and maintain pole structure despite mechanical challenges and component turnover. Although we know that dynein and NuMA mediate pole formation, our understanding of the forces dynamically maintaining poles is limited: we do not know where and how quickly they act or their strength and structural impact. Using laser ablation to cut spindle microtubules, we identify a force that rapidly and robustly pulls severed microtubules and chromosomes poleward, overpowering opposing forces and repairing spindle architecture. Molecular imaging and biophysical analysis suggest that transport is powered by dynein pulling on minus ends of severed microtubules. NuMA and dynein/dynactin are specifically enriched at new minus ends within seconds, reanchoring minus ends to the spindle and delivering them to poles. This force on minus ends represents a newly uncovered chromosome transport mechanism that is independent of plus end forces at kinetochores and is well suited to robustly maintain spindle mechanical integrity. PMID:25023517

  15. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

    SciTech Connect

    Nieznanski, Krzysztof [Nencki Institute of Experimental Biology, Department of Muscle Biochemistry, Warsaw (Poland)]. E-mail: k.nieznanski@nencki.gov.pl; Podlubnaya, Zoya A. [Institute of Theoretical and Experimental Biophysics, Laboratory of Structure and Function of Muscle Proteins, Pushchino (Russian Federation); Pushchino State University, Pushchino (Russian Federation); Nieznanska, Hanna [Nencki Institute of Experimental Biology, Department of Muscle Biochemistry, Warsaw (Poland)

    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.

  16. Effects of kinesin-5 inhibition on dendritic architecture and microtubule organization

    PubMed Central

    Kahn, Olga I.; Sharma, Vandana; González-Billault, Christian; Baas, Peter W.

    2015-01-01

    Kinesin-5 is a slow homotetrameric motor protein best known for its essential role in the mitotic spindle, where it limits the rate at which faster motors can move microtubules. In neurons, experimental suppression of kinesin-5 causes the axon to grow faster by increasing the mobility of microtubules in the axonal shaft and the invasion of microtubules into the growth cone. Does kinesin-5 act differently in dendrites, given that they have a population of minus end–distal microtubules not present in axons? Using rodent primary neurons in culture, we found that inhibition of kinesin-5 during various windows of time produces changes in dendritic morphology and microtubule organization. Specifically, dendrites became shorter and thinner and contained a greater proportion of minus end–distal microtubules, suggesting that kinesin-5 acting normally restrains the number of minus end–distal microtubules that are transported into dendrites. Additional data indicate that, in neurons, CDK5 is the kinase responsible for phosphorylating kinesin-5 at Thr-926, which is important for kinesin-5 to associate with microtubules. We also found that kinesin-5 associates preferentially with microtubules rich in tyrosinated tubulin. This is consistent with an observed accumulation of kinesin-5 on dendritic microtubules, as they are known to be less detyrosinated than axonal microtubules. PMID:25355946

  17. 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 ?-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, ?TAT1, with microtubules and find that ?TAT1 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 ?TAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments. PMID:24227885

  18. Glycogen synthase kinase-3? activation mediates rotenone-induced cytotoxicity with the involvement of microtubule destabilization.

    PubMed

    Hongo, Haruyuki; Kihara, Takeshi; Kume, Toshiaki; Izumi, Yasuhiko; Niidome, Tetsuhiro; Sugimoto, Hachiro; Akaike, Akinori

    2012-09-14

    Rotenone, a mitochondrial complex I inhibitor, has been used to generate animal and cell culture models of Parkinson's disease. Recent studies suggest that microtubule destabilization causes selective dopaminergic neuronal loss. In this study, we investigated glycogen synthase kinase-3? (GSK3?) involvement in rotenone-induced microtubule destabilization. Rotenone-induced cytotoxicity in SH-SY5Y cells was attenuated by the GSK3? inhibitor SB216763. Tau, a microtubule-associated protein and substrate for GSK3?, has been implicated in the pathogenesis of tauopathies such as Alzheimer's disease. Rotenone induced an increase in phosphorylated tau, the effect of which was attenuated by concomitant treatment with SB216763. Rotenone treatment also decreased tau expression in the microtubule fraction and increased tau expression in the cytosol fraction. These effects were suppressed by SB216763, which suggests that rotenone reduces the capacity of tau to bind microtubules. Rotenone treatment increased the amount of free tubulin and reduced the amount of polymerized tubulin, indicating that rotenone destabilizes microtubules. Rotenone-induced microtubule destabilization was suppressed by SB216763 and taxol, a microtubule stabilizer. Taxol prevented rotenone-induced cytotoxicity and morphological changes. Taken together, these results suggest that rotenone-induced cytotoxicity is mediated by microtubule destabilization via GSK3? activation, and that microtubule destabilization is caused by reduction in the binding capacity of tau to microtubules, which is a result of tau phosphorylation via GSK3? activation. PMID:22922102

  19. Biophysical properties of stable microtubules in neurites revealed by optical techniques.

    PubMed

    Kurachi, M; Komiya, Y; Tashiro, T

    1999-10-01

    We tested the stability of microtubules in the neurites of cultured dorsal root ganglion cells by dissolving the cytoplasmic membrane with detergent and exposing them to defined extracellular medium under the microscope. Smooth cytoplasmic filaments visualized after membrane removal were suggested to be microtubules by the preservation of all of the filaments in the presence but not in the absence of taxol. They were further confirmed to be microtubules by immunostaining with anti-tubulin antibody. Significant number of microtubules in the established neurites remained longer than 1 hour after membrane removal. To investigate their stabilization mechanism, we transected the exposed microtubules by laser microbeam irradiation and observed their length changes with video-enhanced microscopy. Microtubule fragments started to shorten on both sides of the transection site, more rapidly from the newly generated plus ends than from the minus ends. The maximal rate as well as the pattern of shortening correlated with the time of transection; microtubules transected later than 30 min after membrane removal shortened at rates less than 20 microm/min and typically with intermittent pauses, while the more labile microtubules included in the earlier transections shortened continuously at higher rates. Microtubules in neurites were thus stabilized by (1) stopping disassembly at local sites including the plus ends, and (2) slowing disassembly along the length. Observations of the course of disassembly also suggested the presence of specialized points along microtubules which is involved in anchoring microtubules to the substratum or transiently stopping disassembly. PMID:15216898

  20. Cell Edges Accumulate Gamma Tubulin Complex Components and Nucleate Microtubules following Cytokinesis in Arabidopsis thaliana

    PubMed Central

    Ambrose, Chris; Wasteneys, Geoffrey O.

    2011-01-01

    Microtubules emanate from distinct organizing centers in fungal and animal cells. In plant cells, by contrast, microtubules initiate from dispersed sites in the cell cortex, where they then self-organize into parallel arrays. Previous ultrastructural evidence suggested that cell edges participate in microtubule nucleation but so far there has been no direct evidence for this. Here we use live imaging to show that components of the gamma tubulin nucleation complex (GCP2 and GCP3) localize at distinct sites along the outer periclinal edge of newly formed crosswalls, and that microtubules grow predominantly away from these edges. These data confirm a role for cell edges in microtubule nucleation, and suggest that an asymmetric distribution of microtubule nucleation factors contributes to cortical microtubule organization in plants, in a manner more similar to other kingdoms than previously thought. PMID:22110647

  1. Redistribution of microtubules and pericentriolar material during the development of polarity in mouse blastomeres

    PubMed Central

    1987-01-01

    The distribution of microtubules and microtubule organizing centers (MTOCs) during the development of cell polarity in eight-cell mouse blastomeres was studied by immunofluorescence and immunoelectron microscopy using monoclonal anti-tubulin antibodies and an anti- pericentriolar material (PCM) serum. In early eight-cell blastomeres microtubules were found mainly around the nucleus and in the cell cortex, whereas PCM foci were observed dispersed in the cytoplasm. During the eight-cell stage, microtubules disappeared from the area adjacent to the zone of intercellular contact and accumulated in the apical part of the cell while their number decreased in the basal domain. The PCM also relocalized to the apical domain of the cell, but this occurred after the redistribution of the microtubules by a mechanism that involved the microtubule network. The possible roles of both MTOCs and microtubules in establishing cell polarity are discussed. PMID:3571331

  2. Synergy between Multiple Microtubule-Generating Pathways Confers Robustness to Centrosome-Driven Mitotic Spindle Formation

    PubMed Central

    Hayward, Daniel; Metz, Jeremy; Pellacani, Claudia; Wakefield, James G.

    2014-01-01

    Summary The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. We have tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. Our work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules. PMID:24389063

  3. Dissecting the molecular mechanism underlying the intimate relationship between cellulose microfibrils and cortical microtubules

    PubMed Central

    Lei, Lei; Li, Shundai; Bashline, Logan; Gu, Ying

    2014-01-01

    A central question in plant cell development is how the cell wall determines directional cell expansion and therefore the final shape of the cell. As the major load-bearing component of the cell wall, cellulose microfibrils are laid down transversely to the axis of elongation, thus forming a spring-like structure that reinforces the cell laterally and while favoring longitudinal expansion in most growing cells. Mounting evidence suggests that cortical microtubules organize the deposition of cellulose microfibrils, but the precise molecular mechanisms linking microtubules to cellulose organization have remained unclear until the recent discovery of cellulose synthase interactive protein 1 , a linker protein between the cortical microtubules and the cellulose biosynthesizing machinery. In this review, we will focus on the intimate relationship between cellulose microfibrils and cortical microtubules, in particular, we will discuss microtubule arrangement and cell wall architecture, the linkage between cellulose synthase complexes and microtubules, and the feedback mechanisms between cell wall and microtubules. PMID:24659994

  4. Structural basis of motility in the microtubular axostyle: implications for cytoplasmic microtubule structure and function

    PubMed Central

    1980-01-01

    The gross morphology of the protozoan microtubule axostyle of Saccinobaculus ambloaxostylus can now be described in macromolecular detail. The left-handed coil of the axostyle is seen to be dependent upon the asymmetry inherent in the constituent microtubules as expressed by the specific array of linkages between microtubules and by a possible tendency for microtubules to coil into left-handed helices. The laminated sheets of microtubules are not aligned parallel to the long axis of the organelle, but become increasingly tilted off-axis as one descends through the sheets of microtubules from the convex to the concave surface of the axostyle. Fine-structural analysis of the axostyle indicates similarities of the linkages to dynein. The potential loci of the force-generating protein(s) are discussed as well as implications of the axostyle's structure on general microtubule function. PMID:6448863

  5. Tubulin bond energies and microtubule biomechanics determined from nanoindentation in silico

    E-print Network

    Kononova, Olga; Theisen, Kelly E; Marx, Kenneth A; Dima, Ruxandra I; Ataullakhanov, Fazly I; Grishchuk, Ekaterina L; Barsegov, Valeri

    2015-01-01

    Microtubules, the primary components of the chromosome segregation machinery, are stabilized by longitudinal and lateral non-covalent bonds between the tubulin subunits. However, the thermodynamics of these bonds and the microtubule physico-chemical properties are poorly understood. Here, we explore the biomechanics of microtubule polymers using multiscale computational modeling and nanoindentations in silico of a contiguous microtubule fragment. A close match between the simulated and experimental force-deformation spectra enabled us to correlate the microtubule biomechanics with dynamic structural transitions at the nanoscale. Our mechanical testing revealed that the compressed MT behaves as a system of rigid elements interconnected through a network of lateral and longitudinal elastic bonds. The initial regime of continuous elastic deformation of the microtubule is followed by the transition regime, during which the microtubule lattice undergoes discrete structural changes, which include first the reversib...

  6. Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos

    NASA Technical Reports Server (NTRS)

    Danilchik, Michael V.; Bedrick, Steven D.; Brown, Elizabeth E.; Ray, Kimberly

    2003-01-01

    In dividing Xenopus eggs, furrowing is accompanied by expansion of a new domain of plasma membrane in the cleavage plane. The source of the new membrane is known to include a store of oogenetically produced exocytotic vesicles, but the site where their exocytosis occurs has not been described. Previous work revealed a V-shaped array of microtubule bundles at the base of advancing furrows. Cold shock or exposure to nocodazole halted expansion of the new membrane domain, which suggests that these microtubules are involved in the localized exocytosis. In the present report, scanning electron microscopy revealed collections of pits or craters, up to approximately 1.5 micro m in diameter. These pits are evidently fusion pores at sites of recent exocytosis, clustered in the immediate vicinity of the deepening furrow base and therefore near the furrow microtubules. Confocal microscopy near the furrow base of live embryos labeled with the membrane dye FM1-43 captured time-lapse sequences of individual exocytotic events in which irregular patches of approximately 20 micro m(2) of unlabeled membrane abruptly displaced pre-existing FM1-43-labeled surface. In some cases, stable fusion pores, approximately 2 micro m in diameter, were seen at the surface for up to several minutes before suddenly delivering patches of unlabeled membrane. To test whether the presence of furrow microtubule bundles near the surface plays a role in directing or concentrating this localized exocytosis, membrane expansion was examined in embryos exposed to D(2)O to induce formation of microtubule monasters randomly under the surface. D(2)O treatment resulted in a rapid, uniform expansion of the egg surface via random, ectopic exocytosis of vesicles. This D(2)O-induced membrane expansion was completely blocked with nocodazole, indicating that the ectopic exocytosis was microtubule-dependent. Results indicate that exocytotic vesicles are present throughout the egg subcortex, and that the presence of microtubules near the surface is sufficient to mobilize them for exocytosis at the end of the cell cycle.

  7. A CLASP-modulated cell edge barrier mechanism drives cell-wide cortical microtubule organization in Arabidopsis

    PubMed Central

    Ambrose, Chris; Allard, Jun F.; Cytrynbaum, Eric N.; Wasteneys, Geoffrey O.

    2011-01-01

    It is well known that the parallel order of microtubules in the plant cell cortex defines the direction of cell expansion, yet it remains unclear how microtubule orientation is controlled, especially on a cell-wide basis. Here we show through 4D imaging and computational modelling that plant cell polyhedral geometry provides spatial input that determines array orientation and heterogeneity. Microtubules depolymerize when encountering sharp cell edges head-on, whereas those oriented parallel to those sharp edges remain. Edge-induced microtubule depolymerization, however, is overcome by the microtubule-associated protein CLASP, which accumulates at specific cell edges, enables microtubule growth around sharp edges and promotes formation of microtubule bundles that span adjacent cell faces. By computationally modelling dynamic 'microtubules on a cube' with edges differentially permissive to microtubule passage, we show that the CLASP-edge complex is a 'tuneable' microtubule organizer, with the inherent flexibility to generate the numerous cortical array patterns observed in nature. PMID:21847104

  8. Microtubule-Dependent Modulation of Adhesion Complex Composition

    PubMed Central

    Ng, Daniel H. J.; Humphries, Jonathan D.; Byron, Adam; Millon-Frémillon, Angélique; Humphries, Martin J.

    2014-01-01

    The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding ?5?1 integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, ?5-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, ?5-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183. PMID:25526367

  9. Carbendazim inhibits cancer cell proliferation by suppressing microtubule dynamics.

    PubMed

    Yenjerla, Mythili; Cox, Corey; Wilson, Leslie; Jordan, Mary Ann

    2009-02-01

    Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells, including drug- and multidrug-resistant and p53-deficient cell lines. Because of its promising preclinical anti-tumor activity, it has undergone phase I clinical trials and is under further clinical development. Although it weakly inhibits polymerization of brain microtubules and induces G(2)/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC(50), 10 microM) and half-maximally arrests mitosis at a similar concentration (8 microM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 microM, with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (K(d), 42.8 +/- 4.0 microM). Unlike some benzimidazoles that bind to the colchicine site in tubulin, carbendazim neither competes with colchicine nor competes with vinblastine for binding to brain tubulin. Thus, carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest. PMID:19001156

  10. Carbendazim Inhibits Cancer Cell Proliferation by Suppressing Microtubule Dynamics

    PubMed Central

    Yenjerla, Mythili; Cox, Corey; Wilson, Leslie; Jordan, Mary Ann

    2009-01-01

    Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells, including drug- and multidrug-resistant and p53-deficient cell lines. Because of its promising preclinical anti-tumor activity, it has undergone phase I clinical trials and is under further clinical development. Although it weakly inhibits polymerization of brain microtubules and induces G2/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC50, 10 ?M) and half-maximally arrests mitosis at a similar concentration (8 ?M), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 ?M, with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (Kd, 42.8 ± 4.0 ?M). Unlike some benzimidazoles that bind to the colchicine site in tubulin, carbendazim neither competes with colchicine nor competes with vinblastine for binding to brain tubulin. Thus, carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest. PMID:19001156

  11. Large-scale vortex lattice emerging from collectively moving microtubules.

    PubMed

    Sumino, Yutaka; Nagai, Ken H; Shitaka, Yuji; Tanaka, Dan; Yoshikawa, Kenichi; Chaté, Hugues; Oiwa, Kazuhiro

    2012-03-22

    Spontaneous collective motion, as in some flocks of bird and schools of fish, is an example of an emergent phenomenon. Such phenomena are at present of great interest and physicists have put forward a number of theoretical results that so far lack experimental verification. In animal behaviour studies, large-scale data collection is now technologically possible, but data are still scarce and arise from observations rather than controlled experiments. Multicellular biological systems, such as bacterial colonies or tissues, allow more control, but may have many hidden variables and interactions, hindering proper tests of theoretical ideas. However, in systems on the subcellular scale such tests may be possible, particularly in in vitro experiments with only few purified components. Motility assays, in which protein filaments are driven by molecular motors grafted to a substrate in the presence of ATP, can show collective motion for high densities of motors and attached filaments. This was demonstrated recently for the actomyosin system, but a complete understanding of the mechanisms at work is still lacking. Here we report experiments in which microtubules are propelled by surface-bound dyneins. In this system it is possible to study the local interaction: we find that colliding microtubules align with each other with high probability. At high densities, this alignment results in self-organization of the microtubules, which are on average 15?µm long, into vortices with diameters of around 400?µm. Inside the vortices, the microtubules circulate both clockwise and anticlockwise. On longer timescales, the vortices form a lattice structure. The emergence of these structures, as verified by a mathematical model, is the result of the smooth, reptation-like motion of single microtubules in combination with local interactions (the nematic alignment due to collisions)--there is no need for long-range interactions. Apart from its potential relevance to cortical arrays in plant cells and other biological situations, our study provides evidence for the existence of previously unsuspected universality classes of collective motion phenomena. PMID:22437613

  12. Functions of microtubules in the Saccharomyces cerevisiae cell cycle

    PubMed Central

    1988-01-01

    We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division. PMID:3049620

  13. Taxol, a microtubule stabilizer, prevents ischemic ventricular arrhythmias in rats

    PubMed Central

    Xiao, Junjie; Cao, Huaming; Liang, Dandan; Liu, Ying; Zhang, Hong; Zhao, Hong; Liu, Yi; Li, Jun; Yan, Biao; Peng, Luying; Zhou, Zhaonian; Chen, Yi-Han

    2011-01-01

    Abstract Microtubule integrity is important in cardio-protection, and microtubule disruption has been implicated in the response to ischemia in cardiac myocytes. However, the effects of Taxol, a common microtubule stabilizer, are still unknown in ischemic ventricular arrhythmias. The arrhythmia model was established in isolated rat hearts by regional ischemia, and myocardial infarction model by ischemia/reperfusion. Microtubule structure was immunohistochemically measured. The potential mechanisms were studied by measuring reactive oxygen species (ROS), activities of oxidative enzymes, intracellular calcium concentration ([Ca2+]i) and Ca2+ transients by using fluorometric determination, spectrophotometric assays and Fura-2-AM and Fluo-3-AM, respectively. The expression and activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) was also examined using real-time polymerase chain reaction, Western blot and pyruvate/Nicotinamide adenine dinucleotide-coupled reaction. Our data showed that Taxol (0.1, 0.3 and 1 ?M) effectively reduced the number of ventricular premature beats and the incidence and duration of ventricular tachycardia. The infarct size was also significantly reduced by Taxol (1 ?M). At the same time, Taxol preserved the microtubule structure, increased the activity of mitochondrial electron transport chain complexes I and III, reduced ROS levels, decreased the rise in [Ca2+]i and preserved the amplitude and decay times of Ca2+ transients during ischemia. In addition, SERCA2a activity was preserved by Taxol during ischemia. In summary, Taxol prevents ischemic ventricular arrhythmias likely through ameliorating abnormal calcium homeostasis and decreasing the level of ROS. This study presents evidence that Taxol may be a potential novel therapy for ischemic ventricular arrhythmias. PMID:20561109

  14. Search for multiple chiral doublets in rhodium isotopes

    SciTech Connect

    Peng, J. [Department of Physics, Beijing Normal University, Beijing 100875 (China); Center for Mathematical Sciences, University of Aizu, Aizu-Wakamatsu, 965-8580 Fukushima (Japan); School of Physics, and State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871 (China); Sagawa, H. [Center for Mathematical Sciences, University of Aizu, Aizu-Wakamatsu, 965-8580 Fukushima (Japan); Zhang, S. Q. [School of Physics, and State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871 (China); Institute of Theoretical Physics, Chinese Academy of Science, Beijing 100080 (China); Yao, J. M.; Zhang, Y. [School of Physics, and State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871 (China); Meng, J. [School of Physics, and State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871 (China); Institute of Theoretical Physics, Chinese Academy of Science, Beijing 100080 (China); Department of Physics, University of Stellenbosch, Stellenbosch (South Africa); Center of Theoretical Nuclear Physics, National Laboratory of Heavy Ion Accelerator, Lanzhou 730000 (China)

    2008-02-15

    The deformation in rhodium isotopes is investigated using adiabatic and configuration-fixed constrained triaxial relativistic mean field (RMF) approaches. The triaxial deformations are found in the ground states of {sup 102,104,106,108,110}Rh, which is consistent with triaxial Skyrme Hartree-Fock calculations. Several minima with triaxial deformation in {sup 104,106,108,110}Rh are obtained by the configuration-fixed constrained calculations. The corresponding configurations are characterized by the quantum numbers |nljm> obtained by transforming wave functions from a Cartesian basis to a spherical basis. The possible existence of multiple chiral doublets (M{chi}D) is demonstrated in {sup 104,106,108,110}Rh isotopes, based on different particle-hole configurations and triaxial deformations.

  15. Cold Electroweak Baryogenesis in the Two Higgs-Doublet Model

    E-print Network

    Anders Tranberg; Bin Wu

    2012-03-22

    We perform the first investigation of cold electroweak baryogenesis in the two Higgs-doublet model (2HDM). The electroweak symmetry breaking transition is assumed to occur through a spinodal instability from a super-cooled initial state. We consider the creation of net Chern-Simons number, which through the axial anomaly is equivalent to baryon number. CP-violation is explicit in the scalar potential, but only in combination with P-violation is it possible for an asymmetry to be generated. This is introduced through the leading C- and P-breaking, but CP-invariant, term expected to arise upon integrating out the fermions in the theory. We perform real-time lattice simulations of the transition, and find the coefficient of this term required for successful bayogenesis.

  16. Top-bottom doublet in the sphaleron background

    E-print Network

    J. M. Moreno; D. H. Oaknin; M. Quirós

    1995-08-30

    We consider the top-bottom doublet in the background of the sphaleron for the realistic case of large non-degeneracy of fermion masses, in particular $m_b=5$ GeV and $m_t=175$ GeV. We propose an axially symmetric $(r,\\theta)$-dependent ansatz for fermion fields and investigate the effects of the non-degeneracy on them. The exact solution is described, with an error less than 0.01\\%, by a set of ten radial functions. We also propose an approximate solution, in the $m_b/m_t\\rightarrow 0$ limit, with an error ${\\cal O}(m_b/m_t)$. We have found that the effects of non-degeneracy provide a $\\theta$-dependence typically $\\sim 10\\%$.

  17. Emulsion sheet doublets as interface trackers for the OPERA experiment

    E-print Network

    Anokhina, A.; Ariga, A.; Arrabito, L.; Autiero, D.; Badertscher, A.; Bay, F.; Greggio, F.Bersani; Bertolin, A.; Besnier, M.; Bick, D.; Bozza, C.; Brugiere, T.; Brugnera, R.; Brunetti, G.; Buontempo, S.; Carrara, E.; Cazes, A.; Chaussard, L.; Chernyavsky, M.; Chiarella, V.; Chon-Sen, N.; Chukanov, A.; Consiglio, L.; Cozzi, M.; Cuha, V.; Dal Corso, F.; D'Amato, G.; D'Ambrosio, N.; De Lellis, G.; Declais, Y.; De Serio, M.; Di Capua, F.; Di Ferdinando, D.; Di Giovanni, A.; Di Marco, N.; Di Troia, C.; Dmitrievski, S.; Dominjon, A.; Dracos, Marcos; Duchesneau, D.; Dusini, S.; Ebert, J.; Egorov, O.; Enikeev, R.; Ereditato, Antonio; Esposito, L.S.; Favier, J.; Felici, G.; Ferber, T.; Fini, R.; Frekers, D.; Fukuda, T.; Galkin, V.I.; Galkin, V.A.; Garfagnini, A.; Giacomelli, G.; Giorgini, M.; Goellnitz, C.; Goldberg, J.; Golubkov, D.; Gornushkin, Y.; Grella, G.; Grianti, F.; Guler, M.; Gusev, G.; Gustavino, C.; Hagner, Caren; Hara, T.; Hierholzer, M.; Hiramatsu, S.; Hoshino, Kaoru; Ieva, M.; Jakovcic, K.; Janicsko Csathy, J.; Janutta, B.; Jollet, C.; Juget, F.; Kawai, T.; Kazuyama, M.; Kim, S.H.; Knuesel, J.; Kodama, K.; Komatsu, M.; Kose, U.; Kreslo, I.; Laktineh, I.; Lazzaro, C.; Lenkeit, J.; Ljubicic, A.; Longhin, Andrea; Lutter, G.; Manai, K.; Mandrioli, G.; Marotta, A.; Marteau, J.; Matsuo, T.; Matsuoka, H.; Mauri, N.; Meisel, F.; Meregaglia, A.; Messina, M.; Migliozzi, P.; Mikado, S.; Miyamoto, S.; Monacelli, Piero; Morishima, Kunihiro; Moser, U.; Muciaccia, Maria Teresa; Naganawa, N.; Naka, T.; Nakamura, M.; Nakamura, T.; Nakano, T.; Nikitina, V.; Niwa, K.; Nonoyama, Y.; Ogawa, S.; Osedlo, V.; Ossetski, D.; Paoloni, A.; Park, B.D.; Park, I.G.; Pastore, A.; Patrizii, L.; Pennacchio, E.; Pessard, H.; Pilipenko, V.; Pistillo, C.; Polukhina, N.; Pozzato, M.; Pretzl, Klaus P.; Publichenko, P.; Pupilli, F.; Roganova, T.; Rosa, G.; Rostovtseva, I.; Rubbia, A.; Russo, A.; Ryazhskaya, O.; Ryzhikov, D.; Sato, O.; Sato, Y.; Saveliev, V.; Sazhina, G.; Schembri, A.; Scotto Lavina, L.; Shibuya, H.; Simone, S.; Sioli, Max; Sirignano, C.; Sirri, G.; Song, J.S.; Spinetti, M.; Stanco, L.; Starkov, N.; Stipcevic, M.; Strauss, T.; Strolin, Paolo Emilio; Sugonyaev, V.; Taira, Y.; Takahashi, S.; Tenti, M.; Terranova, F.; Tezuka, I.; Tioukov, V.; Tolun, P.; Tsarev, V.; Tufanli, S.; Ushida, N.; Vilain, P.; Vladimirov, M.; Votano, L.; Vuilleumier, J.L.; Wilquet, G.; Wonsak, B.; Wurtz, J.; Yoon, C.S.; Yoshida, J.; Zaitsev, Y.; Zemskova, S.; Zghiche, Amina; Zimmermann, R.

    2008-01-01

    New methods for efficient and unambiguous interconnection between electronic counters and target units based on nuclear photographic emulsion films have been developed. The application to the OPERA experiment, that aims at detecting oscillations between mu neutrino and tau neutrino in the CNGS neutrino beam, is reported in this paper. In order to reduce background due to latent tracks collected before installation in the detector, on-site large-scale treatments of the emulsions ("refreshing") have been applied. Changeable Sheet (CSd) packages, each made of a doublet of emulsion films, have been designed, assembled and coupled to the OPERA target units ("ECC bricks"). A device has been built to print X-ray spots for accurate interconnection both within the CSd and between the CSd and the related ECC brick. Sample emulsion films have been extensively scanned with state-of-the-art automated optical microscopes. Efficient track-matching and powerful background rejection have been achieved in tests with electronic...

  18. 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. PMID:17678013

  19. Warm Dark Matter in Two Higgs Doublet Models

    E-print Network

    Babu, K S; Mohapatra, Rabindra N

    2014-01-01

    We show that a neutral scalar field, \\sigma, of two Higgs doublet extensions of the Standard Model incorporating the seesaw mechanism for neutrino masses can be identified as a consistent {\\it warm} dark matter candidate with a mass of order keV. The relic density of $\\sigma$ is correctly reproduced by virtue of the late decay of a right-handed neutrino N participating in the seesaw mechanism. Constraints from cosmology determine the mass and lifetime of N to be M_N = 25 GeV - 20 TeV and \\tau_N = (10^{-4} - 1) sec. These models can also explain the 3.5 keV X-ray anomaly in the extra-galactic spectrum that has been recently reported in terms of the decay \\sigma \\to \\gamma \\gamma. Future tests of these models at colliders and in astrophysical settings are outlined.

  20. Leptophobic $Z'$ in Models with Multiple Higgs Doublet Fields

    E-print Network

    Cheng-Wei Chiang; Takaaki Nomura; Kei Yagyu

    2015-04-23

    We study the collider phenomenology of the leptophobic $Z'$ boson from an extra $U(1)'$ gauge symmetry in models with $N$-Higgs doublet fields. We assume that the $Z'$ boson at tree level has (i) no $Z$-$Z'$ mixing, (ii) no interaction with charged leptons, and (iii) no flavour-changing neutral current. Under such a setup, it is shown that in the $N=1$ case, all the $U(1)'$ charges of left-handed quark doublets and right-handed up- and down-type quarks are required to be the same, while in the $N \\ge 3$ case one can take different charges for the three types of quarks. The $N=2$ case is not well-defined under the above three requirements. We study the $pp\\to Z'V\\to b\\bar{b}V$ processes ($V=\\gamma,~Z$ and $W^\\pm$) with the leptonic decays of $Z$ and $W^\\pm$ at the LHC. The most promising discovery channel or the most stringent constraint on the $U(1)'$ gauge coupling constant comes from the $Z'\\gamma$ process below the $t\\bar{t}$ threshold and from the $t\\bar{t}$ process above the threshold. Assuming the collision energy of 8 TeV and the integrated luminosity of 19.6 fb$^{-1}$, we find that the constraint from the $Z'\\gamma$ search in the lower mass regime can be stronger than that from the UA2 experiment. In the $N \\ge 3$ case, we consider four benchmark points for the $Z'$ couplings with quarks. It is noted that if such a $Z'$ is discovered, a careful comparison between the $Z'\\gamma$ and $Z'W$ signals is crucial to reveal the nature of $Z'$ couplings with quarks. We also present the discovery reach of the $Z'$ boson at the 14-TeV LHC in both $N=1$ and $N\\geq 3$ cases.

  1. Leptophobic $Z'$ in Models with Multiple Higgs Doublet Fields

    E-print Network

    Cheng-Wei Chiang; Takaaki Nomura; Kei Yagyu

    2015-02-14

    We study the collider phenomenology of the leptophobic $Z'$ boson from an extra $U(1)'$ gauge symmetry in models with $N$-Higgs doublet fields. We assume that the $Z'$ boson at tree level has (i) no $Z$-$Z'$ mixing, (ii) no interaction with charged leptons, and (iii) no flavour-changing neutral current. Under such a setup, it is shown that in the $N=1$ case, all the $U(1)'$ charges of left-handed quark doublets and right-handed up- and down-type quarks are required to be the same, while in the $N \\ge 3$ case one can take different charges for the three types of quarks. The $N=2$ case is not well-defined under the above three requirements. We study the $pp\\to Z'V\\to b\\bar{b}V$ processes ($V=\\gamma,~Z$ and $W^\\pm$) with the leptonic decays of $Z$ and $W^\\pm$ at the LHC. The most promising discovery channel or the most stringent constraint on the $U(1)'$ gauge coupling constant comes from the $Z'\\gamma$ process below the $t\\bar{t}$ threshold and from the $t\\bar{t}$ process above the threshold. Assuming the collision energy of 8 TeV and the integrated luminosity of 19.6 fb$^{-1}$, we find that the constraint from the $Z'\\gamma$ search in the lower mass regime can be stronger than that from the UA2 experiment. In the $N \\ge 3$ case, we consider four benchmark points for the $Z'$ couplings with quarks. It is noted that if such a $Z'$ is discovered, a careful comparison between the $Z'\\gamma$ and $Z'W$ signals is crucial to reveal the nature of $Z'$ couplings with quarks. We also present the discovery reach of the $Z'$ boson at the 14-TeV LHC in both $N=1$ and $N\\geq 3$ cases.

  2. CLASPs Are CLIP115 and -170 Associating Proteins Involved in the Regional Regulation of Microtubule Dynamics in Motile Fibroblasts

    Microsoft Academic Search

    Anna Akhmanova; Casper C. Hoogenraad; Ksenija Drabek; Tatiana Stepanova; Bjorn Dortland; Ton Verkerk; Wim Vermeulen; Boudewijn M. Burgering; Chris I. De Zeeuw; Frank Grosveld; Niels Galjart

    2001-01-01

    CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit\\/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After

  3. Comparison of the effects of microtubule-associated protein 2 and tau on the packing density of in vitro assembled microtubules.

    PubMed Central

    Black, M M

    1987-01-01

    I have compared the effects of microtubule-associated protein 2 (MAP-2) and tau on the packing density of sedimented microtubules. Microtubules assembled in vitro in taxol were pelleted by centrifugation. The volumes of the resulting pellets were calculated from their weights assuming a specific gravity of 1 and then were normalized to the amount of protein in the pellet, yielding a value for pellet specific volume in microliter/mg of protein. The specific volume of the pellets reflects the intermicrotubule spacing within the pellet. Microtubules were assembled from tubulin alone or tubulin plus various amounts of MAP-2 or tau and collected by centrifugation, and the pellet specific volume was measured. The specific volume of microtubules composed of pure tubulin ranged from 6.4 to 7.7 microliter/mg of protein. Tau had no detectable effect on this value even at saturating levels on the microtubules. In contrast, MAP-2 increased pellet specific volume as the MAP-2/tubulin weight ratio increased; at the highest ratio examined, 0.43, the pellet specific volume was approximately 33. Even at the relatively low MAP-2/tubulin ratio of 0.09, pellet specific volume was approximately 2-fold greater than that of microtubules containing tubulin alone or tubulin plus tau. Electron microscopy confirmed that the observations on pellet specific volume reflected differences in the effects of MAP-2 and tau on the packing density of sedimented microtubules. These results are discussed in the context of observations showing that neighboring microtubules are more widely spaced in dendrites than in axons and that MAP-2 is enriched on microtubules in dendrites compared to microtubules in axons, whereas the converse is true for tau. Images PMID:3118376

  4. Augmin triggers microtubule-dependent microtubule nucleation in interphase plant cells.

    PubMed

    Liu, Ting; Tian, Juan; Wang, Guangda; Yu, Yanjun; Wang, Chaofeng; Ma, Yinping; Zhang, Xiaxia; Xia, Guixian; Liu, Bo; Kong, Zhaosheng

    2014-11-17

    Microtubule (MT)-dependent MT nucleation by ?-tubulin is required for interphase plant cells to establish a highly dynamic cortical MT network underneath the plasma membrane, which influences the deposition of cell wall materials and consequently governs patterns of directional cell expansion. Newly formed MTs either assume 40° angles or are parallel to the extant ones. To date, it has been enigmatic how the ?-tubulin complex is recruited to the sidewall of cortical MTs and initiates MT nucleation. Here, we discovered that the augmin complex was recruited to cortical MTs and initiated MT nucleation in both branching and parallel forms. The augmin complex overwhelmingly colocalized with the ?-tubulin complex. When the function of the augmin complex was compromised, MT nucleation frequency was drastically reduced, most obviously for the branching nucleation. Consequently, the augmin knockdown cells displayed highly parallel and bundled MTs, replacing the fine and mesh-like MT network in the wild-type cells. Our findings uncovered a mechanism by which the augmin complex functions in recruiting the ?-tubulin complex to cortical MTs and initiating MT nucleation, and they shifted the paradigm of the commonly perceived mitotic-specific function of augmin and established its crucial function in MT-dependent MT nucleation in interphase plant cells. PMID:25447999

  5. Asymmetric CLASP-dependent nucleation of non-centrosomal microtubules at the trans-Golgi network

    PubMed Central

    Efimov, Andrey; Kharitonov, Alexey; Efimova, Nadia; Loncarek, Jadranka; Miller, Paul M.; Andreyeva, Natalia; Gleeson, Paul; Galjart, Niels; Maia, Ana R. R.; McLeod, Ian X.; Yates, John R.; Maiato, Helder; Khodjakov, Alexey; Akhmanova, Anna; Kaverina, Irina

    2009-01-01

    Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need ?-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat non-centrosomal microtubule seeds. We show that CLASPs are recruited to trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi–emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front. PMID:17543864

  6. The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity

    PubMed Central

    Grode, Kyle D.; Rogers, Stephen L.

    2015-01-01

    Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules. PMID:25886649

  7. Tracking the Biogenesis and Inheritance of Subpellicular Microtubule in Trypanosoma brucei with Inducible YFP-?-Tubulin

    PubMed Central

    Sheriff, Omar; Lim, Li-Fern; He, Cynthia Y.

    2014-01-01

    The microtubule cytoskeleton forms the most prominent structural system in Trypanosoma brucei, undergoing extensive modifications during the cell cycle. Visualization of tyrosinated microtubules leads to a semiconservative mode of inheritance, whereas recent studies employing microtubule plus end tracking proteins have hinted at an asymmetric pattern of cytoskeletal inheritance. To further the knowledge of microtubule synthesis and inheritance during T. brucei cell cycle, the dynamics of the microtubule cytoskeleton was visualized by inducible YFP-?-tubulin expression. During new flagellum/flagellum attachment zone (FAZ) biogenesis and cell growth, YFP-?-tubulin was incorporated mainly between the old and new flagellum/FAZ complexes. Cytoskeletal modifications at the posterior end of the cells were observed with EB1, a microtubule plus end binding protein, particularly during mitosis. Additionally, the newly formed microtubules segregated asymmetrically, with the daughter cell inheriting the new flagellum/FAZ complex retaining most of the new microtubules. Together, our results suggest an intimate connection between new microtubule formation and new FAZ assembly, consequently leading to asymmetric microtubule inheritance and cell division. PMID:24800253

  8. 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.

  9. Ste20-related Protein Kinase LOSK (SLK) Controls Microtubule Radial Array in Interphase

    PubMed Central

    Burakov, Anton V.; Zhapparova, Olga N.; Kovalenko, Olga V.; Zinovkina, Liudmila A.; Potekhina, Ekaterina S.; Shanina, Nina A.; Weiss, Dieter G.; Kuznetsov, Sergei A.

    2008-01-01

    Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-?T or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-?T have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types. PMID:18287541

  10. Nezha/CAMSAP3 and CAMSAP2 cooperate in epithelial-specific organization of noncentrosomal microtubules

    PubMed Central

    Tanaka, Nobutoshi; Meng, Wenxiang; Nagae, Shigenori; Takeichi, Masatoshi

    2012-01-01

    Major microtubules in epithelial cells are not anchored to the centrosome, in contrast to the centrosomal radiation of microtubules in other cell types. It remains to be discovered how these epithelial microtubules are generated and stabilized at noncentrosomal sites. Here, we found that Nezha [also known as calmodulin-regulated spectrin-associated protein 3 (CAMSAP3)] and its related protein, CAMSAP2, cooperate in organization of noncentrosomal microtubules. These two CAMSAP molecules coclustered at the minus ends of noncentrosomal microtubules and thereby stabilized them. Depletion of CAMSAPs caused a marked reduction of microtubules with polymerizing plus ends, concomitantly inducing the growth of microtubules from the centrosome. In CAMSAP-depleted cells, early endosomes and the Golgi apparatus exhibited irregular distributions. These effects of CAMSAP depletion were maximized when both CAMSAPs were removed. These findings suggest that CAMSAP2 and -3 work together to maintain noncentrosomal microtubules, suppressing the microtubule-organizing ability of the centrosome, and that the network of CAMSAP-anchored microtubules is important for proper organelle assembly. PMID:23169647

  11. Microtubule behavior during guidance of pioneer neuron growth cones in situ

    PubMed Central

    1991-01-01

    The growth of an axon toward its target results from the reorganization of the cytoskeleton in response to environmental guidance cues. Recently developed imaging technology makes it possible to address the effect of such cues on the neural cytoskeleton directly. Although high resolution studies can be carried out on neurons in vitro, these circumstances do not recreate the complexity of the natural environment. We report here on the arrangement and dynamics of microtubules in live neurons pathfinding in response to natural guidance cues in situ using the embryonic grasshopper limb fillet preparation. A rich microtubule network was present within the body of the growth cone and normally extended into the distal growth cone margin. Complex microtubule loops often formed transiently within the growth cone. Branches both with and without microtubules were regularly observed. Microtubules did not extend into filopodia. During growth cone steering events in response to identified guidance cues, microtubule behaviour could be monitored. In turns towards guidepost cells, microtubules selectively invaded branches derived from filopodia that had contacted the guidepost cell. At limb segment boundaries, microtubules displayed a variety of behaviors, including selective branch invasion, and also invasion of multiple branches followed by selective retention in branches oriented in the correct direction. Microtubule invasion of multiple branches also was seen in growth cones migrating on intrasegmental epithelium. Both selective invasion and selective retention generate asymmetrical microtubule arrangements within the growth cone, and may play a key role in growth cone steering events. PMID:1918146

  12. Eribulin binds at microtubule ends to a single site on tubulin to suppress dynamic instability.

    PubMed

    Smith, Jennifer A; Wilson, Leslie; Azarenko, Olga; Zhu, Xiaojie; Lewis, Bryan M; Littlefield, Bruce A; Jordan, Mary Ann

    2010-02-16

    Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing dynamic instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086-1095]. Using [(3)H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall K(d) of 46 microM, but also showing a real or apparent very high affinity (K(d) = 0.4 microM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 +/- 1.3 molecules per microtubule (K(d) = 3.5 microM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that the binding of a single eribulin molecule at a microtubule end can potently inhibit its growth. Eribulin does not suppress dynamic instability at microtubule minus ends. Preincubation of microtubules with 2 or 4 microM vinblastine induced additional lower-affinity eribulin binding sites, most likely at splayed microtubule ends. Overall, our results indicate that eribulin binds with high affinity to microtubule plus ends and thereby suppresses dynamic instability. PMID:20030375

  13. Structure of dimension-six derivative interactions in pseudo Nambu-Goldstone N Higgs doublet models

    E-print Network

    Yohei Kikuta; Yasuhiro Okada; Yasuhiro Yamamoto

    2012-04-25

    We derive the general structure of dimension-six derivative interactions in the N Higgs doublet models, where Higgs fields arise as pseudo Nambu-Goldstone modes of a strongly interacting sector. We show that there are several relations among the dimension-six operators, and therefore the number of independent operators decreases compared with models on which only SU(2)_L x U(1)_Y invariance is imposed. As an explicit example, we derive scattering amplitudes of longitudinal gauge bosons and Higgs bosons at high energy on models involving two Higgs doublets, and compare them with the amplitudes in the case of one Higgs doublet.

  14. The octarepeat region of hamster PrP (PrP51-91) enhances the formation of microtubule and antagonize Cu(2+)-induced microtubule-disrupting activity.

    PubMed

    Li, Xiaoli; Dong, Chenfang; Shi, Song; Wang, Guirong; Li, Yuan; Wang, Xin; Shi, Qi; Tian, Chan; Zhou, Ruimin; Gao, Chen; Dong, Xiaoping

    2009-11-01

    Prion protein (PrP) is considered to associate with microtubule and its major component, tubulin. In the present study, octarepeat region of PrP (PrP51-91) was expressed in prokaryotic-expressing system. Using GST pull-down assay and co-immunoprecipitation, the molecular interaction between PrP51-91 and tubulin was observed. Our data also demonstrated that PrP51-91 could efficiently stimulate microtubule assembly in vitro, indicating a potential effect of PrP on microtubule dynamics. Moreover, PrP51-91 was confirmed to be able to antagonize Cu(2+)-induced microtubule-disrupting activity in vivo, partially protecting against Cu(2+) intoxication to culture cells and stabilize cellular microtubule structure. The association of the octarepeat region of PrP with tubulin may further provide insight into the biological function of PrP in the neurons. PMID:19902127

  15. 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 cannot rule out dynamic stress triggering due to the short time between the two main events. The 2008 May 29 earthquake doublet appears to be a continuation of the earthquake sequence that started in 2000 June, when two Mw6.5 events struck the eastern and central part of the South Iceland Seismic Zone, in the span of 81 hr. The 2000 June-2008 May sequence has released about half of the moment accumulated by plate motion since the previous earthquake sequence in 1896-1912. Therefore, continued earthquake activity with moderate size events rupturing N-S faults in the SISZ in the coming decades is likely.

  16. The thyroperoxidase doublet is not produced by alternative splicing.

    PubMed

    Cetani, F; Costagliola, S; Tonacchera, M; Panneels, V; Vassart, G; Ludgate, M

    1995-12-29

    Thyroperoxidase is a membrane-bound, heme-containing enzyme which catalyses iodination of thyroglobulin and coupling of resulting iodotyrosines to produce thyroid hormone. In addition to the full length molecule of 933 amino acids (TPO1), Northern blotting and sequencing have revealed several shorter transcripts. The most abundant is a species lacking 171 nucleotides in which the alternative splicing results in the deletion of codons 533-590 in exon 10 (TPO2). Evidence for TPO2 transcripts being translated into a protein is lacking, but in Western blots TPO invariably appears as a doublet of 110 and 105 kDa. In the present study we have produced two recombinant fusion proteins for: (i) the 57 amino acids which are spliced out in TPO2 and (ii) for the 20 amino acids which bridge the splice site (10 amino acids on both sides). Both recombinant fragments have been produced in the pMAL-cRI vector as a maltose-binding protein (MBP) fusion, permitting their purification from a bacterial lysate on an amylose column. Rabbits have been immunized by intradermal injection of 500 micrograms of fusion protein, initially in complete Freund's adjuvant followed by two boosts, at 2-week intervals, in incomplete Freund's adjuvant. The resulting high titre immune sera (IS) were reactive with the relevant immunising antigens, when tested by ELISA. Depletion of each serum by passage through an MBP-CNBr Sepharose column allowed purification of antibodies against the relevant peptides, as demonstrated by ELISA with the appropriate fusion protein and MBP. This demonstrates that we have produced specific polyclonal antibodies for the 57 amino acids unique to TPO1 and for the amino acid segment bridging the splice site, found in TPO2. These polyclonal antibodies were used in Western blotting experiments with normal and Graves' thyroid membranes, in reducing and non-reducing conditions. Monoclonal 47/C21 which recognises a linear epitope (amino acids residues 710-722) common to TPO1 and TPO2 was used as a control. In non-reducing conditions, we observed a broad signal at 105-110 kDa, which appeared to comprise two bands, with both polyclonal antibodies and the monoclonal. There was no difference in the image between the normal and the Graves' thyroid. In reducing conditions, the broad signal resolved clearly into two distinct bands, one at 105 and the other at 110 kDa. Once again we observed exactly the same pattern of reactivity with all three antibodies both in normal and Graves' glands. We conclude that the TPO doublet is not the consequence of translation of TPO2. PMID:8824887

  17. 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-shell vector bosons are obtained by numerical integration. Observables are computed (analytically or numerically) as function of the input parameters. Reasons for new version: Improved calculation of the oblique parameters. Summary of revisions: The computation of the oblique parameters has been improved to give reliable results in the case of degenerate masses for the Higgs bosons. Another issue in the oblique parameter calculation, affecting the numerical values of S, U, V, and X (independently of the Higgs boson masses), has been corrected. Restrictions: CP-violation is not treated. Running time: Less than 0.1 s on a standard PC.

  18. 2HDMC - two-Higgs-doublet model calculator

    NASA Astrophysics Data System (ADS)

    Eriksson, David; Rathsman, Johan; Stål, Oscar

    2010-01-01

    We describe 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. Program summaryProgram title:2HDMC Catalogue identifier: AEFI_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEFI_v1_0.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.: 12 032 No. of bytes in distributed program, including test data, etc.: 90 699 Distribution format: tar.gz Programming language: C++ Computer: Any computer running Linux Operating system: Linux RAM: 5 Mb Classification: 11.1 External routines: GNU Scientific Library ( http://www.gnu.org/software/gsl/) 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-shell vector bosons are obtained by numerical integration. Observables are computed (analytically or numerically) as function of the input parameters. Restrictions: CP-violation is not treated. Running time: Less than 0.1 s on a standard PC

  19. A Bio-Polymer Transistor: Electrical Amplification by Microtubules

    E-print Network

    Avner Priel; Arnolt J. Ramos; Jack A. Tuszynski; Horacio F. Cantiello

    2006-06-09

    Microtubules (MTs) are important cytoskeletal structures, engaged in a number of specific cellular activities, including vesicular traffic, cell cyto-architecture and motility, cell division, and information processing within neuronal processes. MTs have also been implicated in higher neuronal functions, including memory, and the emergence of "consciousness". How MTs handle and process electrical information, however, is heretofore unknown. Here we show new electrodynamic properties of MTs. Isolated, taxol-stabilized microtubules behave as bio-molecular transistors capable of amplifying electrical information. Electrical amplification by MTs can lead to the enhancement of dynamic information, and processivity in neurons can be conceptualized as an "ionic-based" transistor, which may impact among other known functions, neuronal computational capabilities.

  20. Regulation of microtubule motors by tubulin isotypes and posttranslational modifications

    PubMed Central

    Sirajuddin, Minhajuddin; Rice, Luke M.; Vale, Ronald D.

    2014-01-01

    The ‘tubulin-code’ hypothesis proposes that different tubulin genes or posttranslational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogenous tubulin species has hindered biochemical testing of this hypothesis. Here, we have engineered 25 ?/? tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single molecule assays. Our results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, we show that kinesin-1 motility on neuronal ?-tubulin (TUBB3) is increased by polyglutamylation and that robust kinesin-2 motility requires detyrosination of ?-tubulin. Our results also show that different molecular motors recognize distinctive tubulin “signatures”, which supports the premise of tubulin-code hypothesis. PMID:24633327

  1. Dynein, microtubule and cargo: a ménage à trois

    PubMed Central

    Pavin, Nenad; Toli?-Nørrelykke, Iva M.

    2013-01-01

    To exert forces, motor proteins bind with one end to cytoskeletal filaments, such as microtubules and actin, and with the other end to the cell cortex, a vesicle or another motor. A general question is how motors search for sites in the cell where both motor ends can bind to their respective binding partners. In the present review, we focus on cytoplasmic dynein, which is required for a myriad of cellular functions in interphase, mitosis and meiosis, ranging from transport of organelles and functioning of the mitotic spindle to chromosome movements in meiotic prophase. We discuss how dynein targets sites where it can exert a pulling force on the microtubule to transport cargo inside the cell. PMID:24256283

  2. The role of microtubules in contractile ring function

    NASA Technical Reports Server (NTRS)

    Conrad, A. H.; Paulsen, A. Q.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.

  3. INCREASED VISUALIZATION OF MICROTUBULES BY AN IMPROVED FIXATION PROCEDURE

    Microsoft Academic Search

    RONALD B. LUFTIG; PAUL N. McMILLAN; JAMES A. WEATHERBEED; ROBERT R. WEIHING

    1977-01-01

    tion mix, was used in the glutaraldehyde prefixation regimen instead of classical fixative buffers, i.e., isotonic cacodylate or phosphate buffer, the following features were observed in thin-sections of the cytoplasm of interphase HeLa cells: (a) a greater than 2-fold increase in total microtubule contour length, (b) a 2-fold increase in the number of microtubuleslz long, (c) an enhanced association of

  4. Antimitotic Action of Griseofulvin does not Involve Disruption of Microtubules

    Microsoft Academic Search

    Linda M. Grisham; Leslie Wilson; KLAUS G. BENSCH

    1973-01-01

    THE antimitotic action of griseofulvin in plant and mammalian cells has been attributed to a colchicine-like disruption of microtubules. Treatment of Vicia faba, Allium root tips or HeLa cells with griseofulvin, for example, resulted in the accumulation of cells at metaphase1-3. The griseo-fulvin-inhibited cells contained abnormal arrays of chromosomes, identical to those produced by treatment of dividing cells with colchicine.

  5. Microtubule orientation in globular leaflet cells of Chara inflata

    Microsoft Academic Search

    Kazuyoshi Iwata; Teruo Shimmen

    2007-01-01

    Chara inflata has globular leaflet cells and cylindrical internodal cells. The morphology of the leaflet cells is different from that of\\u000a other Characeae. The orientation of cortical microtubules (MTs) in young leaflet and internodal cells of this species was\\u000a analyzed by immunofluorescence microscopy. MTs with random orientation were observed in leaflet cells, while those relatively\\u000a transverse to the cell axis

  6. Characterization of microtubule-associated proteins in teleosts.

    PubMed

    Tomasiewicz, H G; Wood, J G

    1999-11-01

    Although microtubules are known to play an important role in many cellular processes, they have been virtually neglected in fish. In this report, microtubule-associated proteins (MAPs) in fish (teleost) were characterized using antibodies (Abs) directed against the mammalian MAPs tau, MAP1A and B, and MAP 2. Two different populations of tau-like proteins (TLPs) were found in fish brain using the anti-tau Abs Tau-1, Tau-2, tau5', and tau3'. The TLPs that were recognized by Tau-1, Tau-2, and tau5' were (1) heat-stable; (2) the same molecular weight as mammalian TLPs: 59-62 kDa; (3) not enriched in microtubules prepared from catfish brain; and (4) localized to the cell body of neurons in fish brains. While the TLPs recognized by tau3' Abs were (1) heat-stable; (2) lower molecular weight than mammalian TLPs: 32-55 vs. 50-65 kDa; (3) enriched in microtubule fractions prepared from catfish brain, and (4) localized to the axons of neurons. These results are consistent with two different populations of TLPs being present in fish brains. While MAP2 was found to be approximately the same molecular weight, 250 kDa, in zebrafish and goldfish as in mammals and to be distributed to dendrites in the fish brain, both MAP1A and MAP1B were found to be about 25% the mass of their mammalian homologs. These results suggest that MAPS in fish have some characteristics similar to their mammalian counterparts, but also possess some unique properties that require further study to elucidate their function. PMID:10542364

  7. Centrosome maturation requires YB-1 to regulate dynamic instability of microtubules for nucleus reassembly

    PubMed Central

    Kawaguchi, Atsushi; Asaka, Masamitsu N.; Matsumoto, Ken; Nagata, Kyosuke

    2015-01-01

    Microtubule formation from the centrosome increases dramatically at the onset of mitosis. This process is termed centrosome maturation. However, regulatory mechanisms of microtubule assembly from the centrosome in response to the centrosome maturation are largely unknown. Here we found that YB-1, a cellular cancer susceptibility protein, is required for the centrosome maturation. Phosphorylated YB-1 accumulated in the centrosome at mitotic phase. By YB-1 knockdown, microtubules were found detached from the centrosome at telophase and an abnormal nuclear shape called nuclear lobulation was found due to defective reassembly of nuclear envelope by mis-localization of non-centrosomal microtubules. In conclusion, we propose that YB-1 is important for the assembly of centrosomal microtubule array for temporal and spatial regulation of microtubules. PMID:25740062

  8. 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

  9. Shaping microtubules into diverse patterns: molecular connections for setting up both ends.

    PubMed

    Mimori-Kiyosue, Yuko

    2011-11-01

    Microtubules serve as rails for intracellular trafficking and their appropriate organization is critical for the generation of cell polarity, which is a foundation of cell differentiation, tissue morphogenesis, ontogenesis and the maintenance of homeostasis. The microtubule array is not just a static railway network; it undergoes repeated collapse and reassembly in diverse patterns during cell morphogenesis. In the last decade much progress has been made toward understanding the molecular mechanisms governing complex microtubule patterning. This review first revisits the basic principle of microtubule dynamics, and then provides an overview of how microtubules are arranged in highly shaped and functional patterns in cells changing their morphology by factors controlling the fate of microtubule ends. PMID:22021191

  10. Identification of MINUS, a small polypeptide that functions as a microtubule nucleation suppressor.

    PubMed Central

    Fanara, P; Oback, B; Ashman, K; Podtelejnikov, A; Brandt, R

    1999-01-01

    In eukaryotic cells, tubulin polymerization must be regulated precisely during cell division and differentiation. To identify new mechanisms involved in cellular microtubule formation, we isolated an activity that suppresses microtubule nucleation in vitro. The activity was due to a small acidic polypeptide of 4.7 kDa which we named MINUS (microtubule nucleation suppressor). MINUS inhibited tau- and taxol-mediated microtubule assembly in vitro and was inactivated by dephosphorylation. The protein was purified to homogeneity from cultured neural (PC12) cells and bovine brain. Microinjection of MINUS caused a transient loss of dynamic microtubules in Vero cells. The results suggest that MINUS acts with a novel mechanism on tubulin polymerization, thus regulating microtubule formation in living cells. PMID:9927416

  11. Centrosome maturation requires YB-1 to regulate dynamic instability of microtubules for nucleus reassembly.

    PubMed

    Kawaguchi, Atsushi; Asaka, Masamitsu N; Matsumoto, Ken; Nagata, Kyosuke

    2015-01-01

    Microtubule formation from the centrosome increases dramatically at the onset of mitosis. This process is termed centrosome maturation. However, regulatory mechanisms of microtubule assembly from the centrosome in response to the centrosome maturation are largely unknown. Here we found that YB-1, a cellular cancer susceptibility protein, is required for the centrosome maturation. Phosphorylated YB-1 accumulated in the centrosome at mitotic phase. By YB-1 knockdown, microtubules were found detached from the centrosome at telophase and an abnormal nuclear shape called nuclear lobulation was found due to defective reassembly of nuclear envelope by mis-localization of non-centrosomal microtubules. In conclusion, we propose that YB-1 is important for the assembly of centrosomal microtubule array for temporal and spatial regulation of microtubules. PMID:25740062

  12. NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

    PubMed Central

    Harkcom, William T.; Ghosh, Ananda K.; Sung, Matthew S.; Matov, Alexandre; Brown, Kevin D.; Giannakakou, Paraskevi; Jaffrey, Samie R.

    2014-01-01

    Nicotinamide adenine dinucleotide (NAD+) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD+. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD+ levels. We find that these effects of NAD+ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD+ on microtubule polymers. Taken together, these data demonstrate that NAD+ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606

  13. The relation of axonal transport of mitochondria with microtubules and other axoplasmic organelles.

    PubMed Central

    Friede, R L; Ho, K C

    1977-01-01

    Axonal transport of mitochondria was studied in frog sciatic nerves incubated in agents selected for their known or alleged effect on microtubules or axonal flow. Quantitative data on mitochondria, microtubules, neurofilaments, endoplasmic reticulum, and cross-sectional area of the axon indicate that axonal transport of mitochondria is dependent on microtubules. When more than half of the microtubules are destroyed, the axonal transport of mitochondria is diminished in proportion to the destruction of microtubules. Axonal transport of mitochondria is not related to neurofilaments and endoplasmic reticulum. Changes in the cross-sectional area of axons, even upon reduction to half the normal size, do not noticeably affect mitochondrial transport. Cyanide which blocks oxidative metabolism also blocks axonal transport of mitochondria, but analysis of fine structure indicates that cyanide is destructive to microtubules as well. Images Plate 2 Plate 3 Plate 4 Plate 5 Plate 1 PMID:66310

  14. NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents.

    PubMed

    Harkcom, William T; Ghosh, Ananda K; Sung, Matthew S; Matov, Alexandre; Brown, Kevin D; Giannakakou, Paraskevi; Jaffrey, Samie R

    2014-06-17

    Nicotinamide adenine dinucleotide (NAD(+)) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD(+)-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD(+). Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD(+) levels. We find that these effects of NAD(+) are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD(+) on microtubule polymers. Taken together, these data demonstrate that NAD(+) and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606

  15. Dual Detection of Chromosomes and Microtubules by the Chromosomal Passenger Complex Drives Spindle Assembly

    PubMed Central

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

    2010-01-01

    SUMMARY 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. PMID:20627073

  16. Dynamics of spindle microtubule organization: kinetochore fiber microtubules of plant endosperm

    PubMed Central

    1982-01-01

    Organization of kinetochore fiber microtubules (MTs) throughout mitosis in the endosperm of Haemanthus katherinae Bak. has been analysed using serial section reconstruction from electron micrographs. Accurate and complete studies have required careful analysis of individual MTs in precisely oriented serial sections through many (45) preselected cells. Kinetochore MTs (kMTs) and non-kinetochore MTs (nkMTs) intermingle within the fiber throughout division, undergoing characteristic, time- dependent, organizational changes. The number of kMTs increases progressively throughout the kinetochore during prometaphase-metaphase. Prometaphase chromosomes which were probably moving toward the pole at the time of fixation have unequally developed kinetochores associated with many nkMTs. The greatest numbers of kMTs (74-109/kinetochore), kinetochore cross-sectional area, and kMT central density all occur at metaphase. Throughout anaphase and telophase there is a decrease in the number of kMTs and, in the kinetochore cross-sectional area, an increased obliquity of kMTs and increased numbers of short MTs near the kinetochore. Delayed kinetochores possess more kMTs than do kinetochores near the poles, but fewer kMTs than chromosomes which have moved equivalent distances in other cells. The frequency of C-shaped proximal MT terminations within kinetochores is highest at early prometaphase and midtelophase, falling to zero at midanaphase. Therefore, in Haemanthus, MTs are probably lost from the periphery of the kinetochore during anaphase in a manner which is related to both time and position of the chromosome along the spindle axis. The complex, time-dependent organization of MTs in the kinetochore region strongly suggests that chromosome movement is accompanied by continual MT rearrangement and/or assembly/disassembly. PMID:7061596

  17. PTTG1/securin modulates microtubule nucleation and cell migration

    PubMed Central

    Moreno-Mateos, Miguel A.; Espina, Águeda G.; Torres, Belén; del Estal, María M. Gámez; Romero-Franco, Ana; Ríos, Rosa M.; Pintor-Toro, José A.

    2011-01-01

    Pituitary tumor transforming gene 1 (PTTG1), also known as securin, has been implicated in many biological functions, including inhibition of sister chromatid separation, DNA repair, organ development, and regulation of the expression and secretion of angiogenic and metastatic factors. Although most of these functions of securin seem to depend on the localization of PTTG1 in the nucleus of the cell, a fraction of the protein has been also detected in the cytoplasm. Here we demonstrate that, in different cell types, a portion of cytoplasmic PTTG1 is associated with the cis face of the Golgi apparatus and that this localization depends on PTTG1 phosphorylation status. In this organelle, PTTG1 forms a complex with proteins involved in microtubule nucleation, including GM130, AKAP450, and ?-tubulin. RNA interference–mediated depletion of PTTG1 produces a delay in centrosomal and noncentrosomal microtubule nucleation. Cells lacking PTTG1 show severe defects in both cell polarization and migration in wound-healing assays. To our knowledge, this is the first study reporting the role of PTTG1 in microtubule nucleation and cell polarization, two processes directly involved in cell migration. We believe that these findings will contribute to understanding the mechanisms underlying PTTG1-mediated biological functions. PMID:21937724

  18. Targeting Microtubules by Natural Agents for Cancer Therapy

    PubMed Central

    Mukhtar, Eiman; Adhami, Vaqar Mustafa; Mukhtar, Hasan

    2014-01-01

    Natural compounds that target microtubules and disrupt the normal function of the mitotic spindle have proven to be one of the best classes of cancer chemotherapeutic drugs available in clinics to date. There is increasing evidence showing that even minor alteration of microtubule dynamics can engage the spindle checkpoint, arresting cell cycle progression at mitosis and subsequently leading to cell death. Our improved understanding of tumor biology and our continued appreciation for what the microtubule target agents (MTAs) can do has helped pave the way for a new era in the treatment of cancer. The effectiveness of these agents for cancer therapy has been impaired, however, by various side effects and drug resistance. Several new MTAs have shown potent activity against the proliferation of various cancer cells, including resistance to the existing MTAs. Sustained investigation of the mechanisms of action of MTAs, development and discovery of new drugs, and exploring new treatment strategies that reduce side effects and circumvent drug resistance could provide more effective therapeutic options for cancer patients. This review focuses on the successful cancer chemotherapy from natural compounds in clinical settings and the challenges that may abort their usefulness. PMID:24435445

  19. Chromosome Movement in Mitosis Requires Microtubule Anchorage at Spindle Poles

    PubMed Central

    Gordon, Michael B.; Howard, Louisa; Compton, Duane A.

    2001-01-01

    Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis. PMID:11157972

  20. Microtubule-dependent transport in neurons: steps towards an understanding of regulation, function and dysfunction

    Microsoft Academic Search

    Brian W Guzik; Lawrence SB Goldstein

    2004-01-01

    Intracellular transport by microtubule-dependent motors is crucial for neuronal survival and function. Recent advances reveal novel strategies for the regulation of transport and the attachment of motors to cargoes. Current findings also illustrate the importance of directed transport in neuronal biology, including microtubule-motor-dependent transduction of neurotrophic signals and axonal damage signal complexes. Furthermore, recent data implicating the dysfunction of microtubule-dependent

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

    Microsoft Academic Search

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

    2007-01-01

    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

  2. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    SciTech Connect

    Iimori, Makoto; Ozaki, Kanako [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan)] [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Chikashige, Yuji [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan)] [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Habu, Toshiyuki [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan) [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan); Hiraoka, Yasushi [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan) [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, 565-0871 (Japan); Maki, Takahisa; Hayashi, Ikuko [Graduate School of Nanobioscience, Yokohama City University, Tsurumi, Yokohama, 230-0045 (Japan)] [Graduate School of Nanobioscience, Yokohama City University, Tsurumi, Yokohama, 230-0045 (Japan); Obuse, Chikashi [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan)] [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan); Matsumoto, Tomohiro, E-mail: tmatsumo@house.rbc.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan) [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan)

    2012-02-01

    Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

  3. Polo-like Kinase 1 Regulates Nlp, a Centrosome Protein Involved in Microtubule Nucleation

    Microsoft Academic Search

    Martina Casenghi; Patrick Meraldi; Ulrike Weinhart; Peter I. Duncan; Roman Körner; Erich A. Nigg

    2003-01-01

    In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization.

  4. Type II seesaw mechanism for Higgs doublets and the scale of new physics

    E-print Network

    W. Grimus; L. Lavoura; B. Radovcic

    2009-03-20

    We elaborate on an earlier proposal by Ernest Ma of a type II seesaw mechanism for suppressing the vacuum expectation values of some Higgs doublets. We emphasize that, by nesting this form of seesaw mechanism into various other seesaw mechanisms, one may obtain light neutrino masses in such a way that the new-physics scale present in the seesaw mechanism - the masses of scalar gauge-SU(2) triplets, scalar SU(2) doublets, or right-handed neutrinos - does not need to be higher than a few 10 TeV. We also investigate other usages of the type II seesaw mechanism for Higgs doublets. For instance, the suppression of the vacuum expectation values of Higgs doublets may realize Froggatt-Nielsen suppression factors in some entries of the fermion mass matrices.

  5. Gallagher-Moszkowski (GM) doublet bands in {sup 162}Ho

    SciTech Connect

    Liang Guodong; Wang Shouyu; Liu Yunzuo; Ma Yingjun; Lu Jingbin; Li Mingfei; Cui Xingzhu; Zhao Guangyi; Li Xianfeng; Wu Xiaoguang; Zhu Lihua; Li Guangshen; Wen Shuxian; Yang Chunxiang [Department of Physics, Jilin University, Changchun 130023 (China); China Institute of Atomic Energy, P.O. Box 275, Beijing 102413 (China)

    2005-12-15

    High-spin states in {sup 162}Ho have been populated in the reaction {sup 160}Gd({sup 7}Li, 5n) at a beam energy of 49 MeV. The K {sup {pi}}=1{sup -} band, the low-K Gallagher-Moszkowski (GM) partner band of known high-K(K {sup {pi}}=6{sup -}) band, based on the configuration {pi}7/2{sup -}[523]{center_dot}{nu}5/2{sup +}[642], and the K {sup {pi}}=6{sup +} band, the high-K GM partner band of known low-K(K{sup {pi}}=1{sup +}) band, based on the configuration {pi}7/2{sup -}[523]{center_dot}{nu}5/2{sup -}[523], have been identified. GM splitting energies, defined as {delta}E{sub GM}=E{sub int}{sup K-}E{sub int}{sup K,} 80 keV and -135 keV were extracted from these two sets of GM doublet bands, respectively. They are comparable with 65 keV and -145 keV, reported recently by Hojman et al. for the corresponding configurations {pi}7/2{sup -}[523]{center_dot}{nu}5/2{sup +}[642] and {pi}7/2{sup -}[523]{center_dot}{nu}5/2{sup -}[523] in {sup 164}Ho, respectively.

  6. Multiple chiral doublet candidate nucleus {sup 105}Rh in a relativistic mean-field approach

    SciTech Connect

    Li Jian; Zhang, S. Q.; Meng, J. [School of Physics, State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871 (China); School of Physics and Nuclear Energy Engineering, Beihang University, Beijing 100191 (China); School of Physics, State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871 (China); Department of Physics, University of Stellenbosch, Stellenbosch (South Africa)

    2011-03-15

    Following the reports of two pairs of chiral doublet bands observed in {sup 105}Rh, the adiabatic and configuration-fixed constrained triaxial relativistic mean-field calculations are performed to investigate their triaxial deformations with the corresponding configuration and the possible multiple chiral doublet (M{chi}D) phenomenon. The existence of the M{chi}D phenomenon in {sup 105}Rh is highly expected.

  7. Automatically R-Conserving SUSY SO(10) Models and Mixed Light Higgs Doublets

    E-print Network

    Dae-Gyu Lee; R. N. Mohapatra

    1994-06-24

    In automatic R-parity conserving SO(10) models, the simplest way to accomodate realistic fermion masses is to demand that that the light Higgs doublets be linear combinations of the {10} and {126}-bar grand unified Higgs representations. We study the realization of this mixed light Higgs property consistent with doublet-triplet splitting in a minimal model and discuss its predictions for neutrino masses and implications for proton decay.

  8. Altered microtubule organization in small-calibre axons of mice lacking tau protein

    Microsoft Academic Search

    A. Harada; K. Oguchi; S. Okabe; J. Kuno; S. Terada; T. Ohshima; R. Sato-Yoshitake; Y. Takei; T. Noda; N. Hirokawa

    1994-01-01

    THE tau gene encodes a protein (Tau) that is a major neuronal microtubule-associated protein localized mostly in axons1-4. It has microtubule-binding and tubulin-polymerizing activity in vitro 3,4 and is thought to make short crossbridges between axonal microtubules5,6. Further, tau-transfected non-neuronal cells extend long axon-like processes in which microtubule bundles resembling those in axons are formed6-8. In contrast, tau antisense oligo-nucleotides

  9. Estimating Microtubule Distributions from 2D Immunofluorescence Microscopy Images Reveals Differences among Human Cultured Cell Lines

    PubMed Central

    Wiking, Mikaela; Lundberg, Emma; Rohde, Gustavo K.; Murphy, Robert F.

    2012-01-01

    Microtubules are filamentous structures that are involved in several important cellular processes, including cell division, cellular structure and mechanics, and intracellular transportation. Little is known about potential differences in microtubule distributions within and across cell lines. Here we describe a method to estimate information pertaining to 3D microtubule distributions from 2D fluorescence images. Our method allows for quantitative comparisons of microtubule distribution parameters (number of microtubules, mean length) between different cell lines. Among eleven cell lines compared, some showed differences that could be accounted for by differences in the total amount of tubulin per cell while others showed statistically significant differences in the balance between number and length of microtubules. We also observed that some cell lines that visually appear different in their microtubule distributions are quite similar when the model parameters are considered. The method is expected to be generally useful for comparing microtubule distributions between cell lines and for a given cell line after various perturbations. The results are also expected to enable analysis of the differences in gene expression underlying the observed differences in microtubule distributions among cell types. PMID:23209697

  10. Notch signalling in adult neurons: a potential target for microtubule stabilization

    PubMed Central

    Bonini, Sara Anna; Ferrari-Toninelli, Giulia; Montinaro, Mery

    2013-01-01

    Cytoskeletal dysfunction has been proposed during the last decade as one of the main mechanisms involved in the aetiology of several neurodegenerative diseases. Microtubules are basic elements of the cytoskeleton and the dysregulation of microtubule stability has been demonstrated to be causative for axonal transport impairment, synaptic contact degeneration, impaired neuronal function leading finally to neuronal loss. Several pathways are implicated in the microtubule assembly/disassembly process. Emerging evidence is focusing on Notch as a microtubule dynamics regulator. We demonstrated that activation of Notch signalling results in increased microtubule stability and changes in axonal morphology and branching. By contrast, Notch inhibition leads to an increase in cytoskeleton plasticity with intense neurite remodelling. Until now, several microtubule-binding compounds have been tested and the results have provided proof of concept that microtubule-binding agents or compounds with the ability to stabilize microtubules may have therapeutic potential for the treatment of Alzheimer’s disease and other neurodegenerative diseases. In this review, based on its key role in cytoskeletal dynamics modulation, we propose Notch as a new potential target for microtubule stabilization. PMID:24228073

  11. Microtubules, the ER and Exu: New associations revealed by analysis of mini spindles mutations

    PubMed Central

    Pokrywka, Nancy Jo; Payne-Tobin, Anna; Raley-Susman, Kathleen M.; Swartzman, Sasha

    2009-01-01

    During Drosophila oogenesis, organized microtubule networks coordinate the localization of specific RNAs, the positioning of the oocyte nucleus, and ooplasmic streaming events. We used mutations in mini spindles (msps), a microtubule-associated protein, to disrupt microtubule function during mid- and late oogenesis, and show that msps is required for these microtubule-based events. Since endoplasmic reticulum (ER) organization is influenced by microtubules in other systems, we hypothesized that using msps to alter microtubule dynamics might affect the structure and organization of the ER in nurse cells and the oocyte. ER organization was monitored using GFP-tagged versions of Reticulon-like1 and protein disulfide isomerase. Analyses of living cells indicate microtubule associations mediate the movement of ER components within the oocyte. Surprisingly, the distribution and behavior of tubular ER in the oocyte differs from general ER, suggesting these two compartments of the ER interact differently with microtubules. We find that the morphology of Exu particles is msps-dependent, and that Exu is specifically associated with tubular ER in msps mutants. Our results extend previous descriptions of sponge bodies and the fusome, suggesting both are manifestations of a dynamic structure that interacts with microtubules and persists throughout oogenesis. PMID:19303437

  12. A phenotypic screen identifies microtubule plus end assembly regulators that can function in mitotic spindle orientation.

    PubMed

    Stolz, Ailine; Ertych, Norman; Bastians, Holger

    2015-03-19

    Proper regulation of microtubule dynamics during mitosis is essential for faithful chromosome segregation. In fact, recently we discovered increased microtubule plus end assembly rates that are frequently observed in human cancer cells as an important mechanism leading to whole chromosome missegregation and chromosomal instability (CIN). However, the genetic alterations responsible for increased microtubule polymerization rates in cancer cells remain largely unknown. The identification of such lesions is hampered by the fact that determining dynamic parameters of microtubules usually involves analyses of living cells, which is technically difficult to perform in large-scale screening settings. Therefore, we sought to identify alternative options to systematically identify regulators of microtubule plus end polymerization. Here, we introduce a simple and robust phenotypic screening assay that is based on the analyses of monopolar mitotic spindle structures that are induced upon inhibition of the mitotic kinesin Eg5/KIF11. We show that increased microtubule polymerization causes highly asymmetric monoasters in the presence of Eg5/KIF11 inhibition and this phenotype can be reliably assessed in living as well as in fixed cells. Using this assay we performed a siRNA screen, in which we identify several microtubule plus end binding proteins as well as centrosomal and cortex associated proteins as important regulators of microtubule plus end assembly. Interestingly, we demonstrate that a subgroup of these regulators function in the regulation of spindle orientation through their role in dampening microtubule plus end polymerization. PMID:25590964

  13. A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end

    PubMed Central

    Pecqueur, Ludovic; Duellberg, Christian; Dreier, Birgit; Jiang, Qiyang; Wang, Chunguang; Plückthun, Andreas; Surrey, Thomas; Gigant, Benoît; Knossow, Marcel

    2012-01-01

    Microtubules are cytoskeleton filaments consisting of ??-tubulin heterodimers. They switch between phases of growth and shrinkage. The underlying mechanism of this property, called dynamic instability, is not fully understood. Here, we identified a designed ankyrin repeat protein (DARPin) that interferes with microtubule assembly in a unique manner. The X-ray structure of its complex with GTP-tubulin shows that it binds to the ?-tubulin surface exposed at microtubule (+) ends. The details of the structure provide insight into the role of GTP in microtubule polymerization and the conformational state of tubulin at the very microtubule end. They show in particular that GTP facilitates the tubulin structural switch that accompanies microtubule assembly but does not trigger it in unpolymerized tubulin. Total internal reflection fluorescence microscopy revealed that the DARPin specifically blocks growth at the microtubule (+) end by a selective end-capping mechanism, ultimately favoring microtubule disassembly from that end. DARPins promise to become designable tools for the dissection of microtubule dynamic properties selective for either of their two different ends. PMID:22778434

  14. Microtubule-binding properties of dynactin p150 expedient for dynein motility.

    PubMed

    Kobayashi, Takuya; Shiroguchi, Katsuyuki; Edamatsu, Masaki; Toyoshima, Yoko Y

    2006-02-01

    Dynactin is a hetero-oligomeric protein complex that has an important role in dynein-based intracellular transport. The expressed N-terminal fragments of dynactin p150 bound to microtubules in the ratio of one to one tubulin dimer, independent from the binding of dynein stalk head. Single molecule observation revealed that these fragments moved around on microtubules by Brownian motion. When the dynein-dynactin complex moves on microtubules, p150 can support dynein to maintain contact with microtubules and does not interfere with the motility of dynein, and thus, the dynein-dynactin complex can efficiently achieve long-distance carriage of the cargo. PMID:16343429

  15. THE EFFECTS OF HIGH HYDROSTATIC PRESSURE ON THE MICROTUBULES OF TETRAHYMENA PYRIFORMIS

    PubMed Central

    Kennedy, John R.; Zimmerman, Arthur M.

    1970-01-01

    Exposure of Tetrahymena pyriformis to 7,500 or 10,000 psi of hydrostatic pressure for 2, 5, or 10 min intervals results in a change in cell shape and ciliary activity. Shape changes occur concurrently with a degradation of longitudinal microtubules in a posterior to anterior direction. High pressure also causes a disruption of ciliary activity. Fine structural analysis reveals a breakdown (presumably microtubule depolymerization) of the central ciliary microtubules. The depolymerization begins at the junction of the central ciliary microtubules with the axosome and progresses distally along the ciliary shaft for a distance of about 0.5 µ. PMID:5497538

  16. Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface

    E-print Network

    Welburn, Julie P. I.

    Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate ...

  17. Myomegalin is necessary for the formation of centrosomal and Golgi-derived microtubules

    PubMed Central

    Roubin, Régine; Acquaviva, Claire; Chevrier, Véronique; Sedjaï, Fatima; Zyss, Déborah; Birnbaum, Daniel; Rosnet, Olivier

    2013-01-01

    Summary The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in ?-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs), mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1), recently described as a nucleation activator, and is related to a family of ?-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits ?-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells. PMID:23430395

  18. [Relationship of microtubule and [Ca2+]cyt in wheat mesophyll protoplast].

    PubMed

    Liu, Gang; Wang, Dong Mei

    2005-08-01

    By using immunofluorescence labeling technique and incubating the Fluo-3AM of Ca2+ fluorescein, wheat (Triticum aestivum) cultivars Lovrin 10 was selected as mesophyll protoplast material in this paper for studying the relationship between the pattern of microtubule and [Ca2+]cyt. Laser scanning confocal microscopy(LSCM) was used and pharmacologic examination was set. The results indicated there was a connection between calcium signaling and microtubule organization in wheat mesophyll protoplast. When the cytosolic calcium concentration distinctly ascends, the pattern of microtubule showed depolymerization; Depolymerization of microtubule could enhance extracellar calcium afflux. PMID:16231700

  19. CLASPs function redundantly to regulate astral microtubules in the C. elegans embryo

    PubMed Central

    Espiritu, Eugenel B.; Krueger, Lori E.; Ye, Anna; Rose, Lesilee S.

    2012-01-01

    Microtubule dynamics are thought to play an important role in regulating microtubule interactions with cortical force generating motor proteins that position the spindle during asymmetric cell division. CLASPs are microtubule-associated proteins that have a conserved role in regulating microtubule dynamics in diverse cell types. Caenorhabditis elegans has three CLASP homologs in its genome. CLS-2 is known to localize to kinetochores and is needed for chromosome segregation at meiosis and mitosis; however CLS-1 and CLS-3 have not been reported to have any role in embryonic development. Here, we show that depletion of CLS-2 in combination with either CLS-1 or CLS-3 results in defects in nuclear rotation, maintenance of spindle length, and spindle displacement in the one-cell embryo. Polarity is normal in these embryos, but reduced numbers of astral microtubules reach all regions of the cortex at the time of spindle positioning. Analysis of the microtubule plus-end tracker EB1 also revealed a reduced number of growing microtubules reaching the cortex in CLASP depleted embryos, but the polymerization rate of astral microtubules was not slower than in wild type. These results indicate that C. elegans CLASPs act partially redundantly to regulate astral microtubules and position the spindle during asymmetric cell division. Further, we show that these spindle pole-positioning roles are independent of the CLS-2 binding proteins HCP-1 and HCP-2. PMID:22613359

  20. THE EFFECTS OF HIGH HYDROSTATIC PRESSURE ON THE MICROTUBULES OF TETR AHYMENA PYRIFORMIS

    E-print Network

    John R Kennedy; Arthur M Zimmerman

    Exposure of Tetrahymena pyriformis to 7,500 or 10,000 psi of hydrostatic pressure for 2, 5, or 10 min intervals results in a change in cell shape and ciliary activity. Shape changes occur concurrently with a degradation of longitudinal microtubules in a posterior to anterior direction. High pressure also causes a disruption of ciliary activity. Fine structural analysis reveals a breakdown (presumably microtubule depolymerization) of the central ciliary microtubules. The depolymerization begins at the junction of the central ciliary microtubules with the axosome and progresses distally along the ciliary shaft for a distance of about 0.5 µ.

  1. The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics

    PubMed Central

    Currie, Joshua D.; Stewman, Shannon; Schimizzi, Gregory; Slep, Kevin C.; Ma, Ao; Rogers, Stephen L.

    2011-01-01

    Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, we conducted an in vivo structure–function analysis of the Drosophila homologue Mini spindles (Msps). Msps exhibits EB1-dependent and spatially regulated MT localization, targeting to microtubule plus ends in the cell interior and decorating the lattice of growing and shrinking microtubules in the cell periphery. RNA interference rescue experiments revealed that the NH2-terminal four TOG domains of Msps function as paired units and were sufficient to promote microtubule dynamics and EB1 comet formation. We also identified TOG5 and novel inter-TOG linker motifs that are required for targeting Msps to the microtubule lattice. These novel microtubule contact sites are necessary for the interplay between the conserved TOG domains and inter-TOG MT binding that underlies the ability of Msps to promote MT dynamic instability. PMID:21965297

  2. Supracellular microtubule alignments in cell layers associated with the secretion of certain fish scales.

    PubMed

    Dane, P J; Tucker, J B

    1986-01-01

    Intercellularly aligned microtubule arrays are present in cell layers associated with the growth and secretion of scales in the zebra fish Brachydanio rerio and the neon tetra fish Hyphessobrycon innesi. The layers in question are: the osteoblast layer that covers the ossified outer surface of a scale, and the layer of fibroblasts that is situated immediately underneath the inner collagenous surface of a scale's fibrillary plate. In certain osteoblasts, the proximal portions of microtubules (with respect to centrosomes) run closely alongside the anterior margin of each cell where it flanks one of a scale's ridge-shaped circuli. These osteoblasts and microtubule portions are arranged in aligned rows that are parallel to circuli. However, the distal portions of the microtubules curve into an orientation that is approximately at right angles to circuli and they are aligned with each other and similar microtubule portions in adjacent osteoblasts. Such microtubule alignments only occur in osteoblasts that are associated with circuli. In Hyphessobrycon osteoblasts situated elsewhere on a scale's surface, microtubules radiate from cell centres but their distal portions curve into alignment with each other and are oriented alongside cell margins. The proximal portions of fibroblast microtubules radiate from centrally positioned centrosomes but the distal portions curve into alignment with each other and distal microtubule portions in neighbouring fibroblasts. The overall pattern of microtubule alignment is similar to that of collagen fibres, which these fibroblasts are secreting onto the fibrillary plate. The immunofluorescence protocol that was used to demonstrate the microtubule alignments described above did not reveal such alignments in the osteoblast and fibroblast layers associated with scales of the brown trout Salmo trutta fario. These findings are assessed in terms of intra-and inter-cellular control of microtubule alignment, and decentralized reorientation of microtubules at distances of several micrometres from centrosomal microtubule-organizing centres. The functional significance of the relationships between microtubule alignment and supracellular patterns of alignment that take place as collagen deposition and ossification proceed during scale formation is also considered. PMID:3308914

  3. 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.

  4. Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape

    PubMed Central

    1993-01-01

    Microtubules are long, proteinaceous filaments that perform structural functions in eukaryotic cells by defining cellular shape and serving as tracks for intracellular motor proteins. We report the first accurate measurements of the flexural rigidity of microtubules. By analyzing the thermally driven fluctuations in their shape, we estimated the mean flexural rigidity of taxol-stabilized microtubules to be 2.2 x 10(-23) Nm2 (with 6.4% uncertainty) for seven unlabeled microtubules and 2.1 x 10(-23) Nm2 (with 4.7% uncertainty) for eight rhodamine-labeled microtubules. These values are similar to earlier, less precise estimates of microtubule bending stiffness obtained by modeling flagellar motion. A similar analysis on seven rhodamine-phalloidin- labeled actin filaments gave a flexural rigidity of 7.3 x 10(-26) Nm2 (with 6% uncertainty), consistent with previously reported results. The flexural rigidity of these microtubules corresponds to a persistence length of 5,200 microns showing that a microtubule is rigid over cellular dimensions. By contrast, the persistence length of an actin filament is only approximately 17.7 microns, perhaps explaining why actin filaments within cells are usually cross-linked into bundles. The greater flexural rigidity of a microtubule compared to an actin filament mainly derives from the former's larger cross-section. If tubulin were homogeneous and isotropic, then the microtubule's Young's modulus would be approximately 1.2 GPa, similar to Plexiglas and rigid plastics. Microtubules are expected to be almost inextensible: the compliance of cells is due primarily to filament bending or sliding between filaments rather than the stretching of the filaments themselves. PMID:8432732

  5. Suppression of microtubule dynamic instability and turnover in MCF7 breast cancer cells by sulforaphane.

    PubMed

    Azarenko, Olga; Okouneva, Tatiana; Singletary, Keith W; Jordan, Mary Ann; Wilson, Leslie

    2008-12-01

    Sulforaphane (SFN), a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables. SFN arrests mitosis, possibly by affecting spindle microtubule function. A critical property of microtubules is their rapid and time-sensitive growth and shortening dynamics (dynamic instability), and suppression of dynamics by antimitotic anticancer drugs (e.g. taxanes and the vinca alkaloids) is central to the anticancer mechanisms of such drugs. We found that at concentrations that inhibited proliferation and mitosis of MCF7-green fluorescent protein-alpha-tubulin breast tumor cells by approximately 50% (~15 microM), SFN significantly modified microtubule organization in arrested spindles without modulating the spindle microtubule mass, in a manner similar to that of much more powerful antimitotic drugs. By using quantitative fluorescence video microscopy, we determined that at its mitotic concentration required to inhibit mitosis by 50%, SFN suppressed the dynamic instability of the interphase microtubules in these cells, strongly reducing the rate and extent of growth and shortening and decreasing microtubule turnover, without affecting the polymer mass. SFN suppressed the dynamics of purified microtubules in a similar fashion at concentrations well below those required to depolymerize microtubules, indicating that the suppression of dynamic instability by SFN in cells is due to a direct effect on the microtubules. The results indicate that SFN arrests proliferation and mitosis by stabilizing microtubules in a manner weaker than but similar to more powerful clinically used antimitotic anticancer drugs and strongly support the hypothesis that inhibition of mitosis by microtubule stabilization is important for SFN's chemopreventive activity. PMID:18952594

  6. Initiation of Hepatitis C Virus Infection Requires the Dynamic Microtubule Network

    PubMed Central

    Roohvand, Farzin; Maillard, Patrick; Lavergne, Jean-Pierre; Boulant, Steeve; Walic, Marine; Andréo, Ursula; Goueslain, Lucie; Helle, François; Mallet, Adeline; McLauchlan, John; Budkowska, Agata

    2009-01-01

    Early events leading to the establishment of hepatitis C virus (HCV) infection are not completely understood. We show that intact and dynamic microtubules play a key role in the initiation of productive HCV infection. Microtubules were required for virus entry into cells, as evidenced using virus pseudotypes presenting HCV envelope proteins on their surface. Studies carried out using the recent infectious HCV model revealed that microtubules also play an essential role in early, postfusion steps of the virus cycle. Moreover, low concentrations of vinblastin and nocodazol, microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules, inhibited infection, suggesting that microtubule dynamic instability and/or treadmilling mechanisms are involved in HCV internalization and early transport. By protein chip and direct core-dependent pull-down assays, followed by mass spectrometry, we identified ?- and ?-tubulin as cellular partners of the HCV core protein. Surface plasmon resonance analyses confirmed that core directly binds to tubulin with high affinity via amino acids 2-117. The interaction of core with tubulin in vitro promoted its polymerization and enhanced the formation of microtubules. Immune electron microscopy showed that HCV core associates, at least temporarily, with microtubules polymerized in its presence. Studies by confocal microscopy showed a juxtaposition of core with microtubules in HCV-infected cells. In summary, we report that intact and dynamic microtubules are required for virus entry into cells and for early postfusion steps of infection. HCV may exploit a direct interaction of core with tubulin, enhancing microtubule polymerization, to establish efficient infection and promote virus transport and/or assembly in infected cells. PMID:19269968

  7. She1-mediated inhibition of dynein motility along astral microtubules promotes polarized spindle movements

    PubMed Central

    Markus, Steven M.; Kalutkiewicz, Katelyn A.; Lee, Wei-Lih

    2012-01-01

    SUMMARY Background Cytoplasmic dynein motility along microtubules is critical for diverse cellular processes ranging from vesicular transport to nuclear envelope breakdown to mitotic spindle alignment. In yeast, we have proposed a regulated-offloading model to explain how dynein motility drives microtubule sliding along the cortex, powering transport of the nucleus into the mother-bud neck [1, 2]: the dynein regulator She1 limits dynein offloading by gating the recruitment of dynactin to the astral microtubule plus end, a prerequisite for offloading to the cortex. However, whether She1 subsequently affects cortically anchored dynein activity during microtubule sliding is unclear. Results Using single-molecule motility assays, we show that She1 strongly inhibits dynein movement along microtubules, acting directly on the motor domain in a manner independent of dynactin. She1 has no effect on the motility of either Kip2, a kinesin that utilizes the same microtubule track as dynein, or human kinesin-1, demonstrating the specificity of She1 for the dynein motor. At single-molecule resolution, She1 binds tightly to and exhibits diffusional behavior along microtubules. Diffusive She1 collides with and pauses motile dynein motors, prolonging their attachment to the microtubule. Furthermore, Aurora B/Ipl1 directly phosphorylates She1 and this modification appears to enhance the diffusive behavior of She1 along microtubules and its potency against dynein. In cells, She1 dampens productive microtubule-cortex interactions specifically in the mother compartment, polarizing spindle movements toward the bud cell. Conclusions Our data reveal how inhibitory microtubule-associated proteins selectively regulate motor activity to achieve unidirectional nuclear transport, and demonstrate a direct link between cell cycle machinery and dynein pathway activity. PMID:23142046

  8. beta-tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast

    E-print Network

    Bode, Claudia Janelle; Himes, Richard H.; Bloom, K. S.; Suprenant, Kathy A.; Pearson, C. G.; Thrower, D. A.; Gupta, M. L.

    2002-08-01

    Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast beta-tubulin C354, which is located near the ...

  9. Ciliary specializations in branchial stigmatal cells of protochordates.

    PubMed

    Martinucci, G B; Dallai, R; Burighel, P; Casagrande, L

    1992-01-01

    Tissues from the pharynx of five representative species of the protochordates (subphylum Tunicata, the three classes Ascidiacea, Thaliacea and Appendicularia, and subphylum Cephalochordata) were examined in both thin sections and freeze-fracture replicas. In all species, the stigmatal cilia of the branchial chamber are neatly arranged and move continuously to propel sea-water in a fixed direction for respiration and feeding of the organism. A number of specializations are found in the basal region of these cilia and are represented by: a) bridges connecting axonemal doublets numbers 5 and 6; b) dense fibrous material linking the doublet microtubules of the axoneme to the ciliary membrane, sometimes in the shape of longitudinal strands or as clusters of filaments; c) intramembrane particles (IMPs) associated with the P-face of the membrane, often arranged in clusters evenly aligned along the ciliary shaft in relation to the underlying axonemal doublets. Ciliary specializations are distributed along the plane of the effective stroke of the beat in both the ascidian Botryllus schlosseri and in the thaliacean Pyrosoma atlanticum and the amphioxus Branchiostoma lanceolatum, whereas in the thaliacean Doliolum nationalis and the appendicularian Oikopleura dioica a more uniform distribution of these specializations all around the basal portion of the cilia is observed. Whatever the disposition of the ciliary specializations in all the examined species, they are always present at the base of the water-propelling cilia. Some morphological evidence suggests that these specializations play a mechanical function in tethering the ciliary membrane to the axoneme. We propose that they help maintain the orientation of the cilia during beating, enhance their stiffness and improve their efficiency. PMID:18621208

  10. Evidence for initiation of microtubules in discrete regions of the cell cortex in Azolla root-tip cells, and an hypothesis on the development of cortical arrays of microtubules

    Microsoft Academic Search

    B. E. S. Gunning; A. R. Hardham; J. E. Hughes

    1978-01-01

    Complexes of microtubules, vesicles, and (to varying degrees) dense matrix material around the microtubules were seen along the edges of cells in root apices of Azolla pinnata R.Br. (viewing the cells as polyhedra with faces, vertices and edges). They are best developed after cytokinesis has been completed, when the daughter cells are reinstating their interphase arrays of microtubules. They are

  11. 2,5-Hexanedione and Carbendazim Coexposure Synergistically Disrupts Rat Spermatogenesis Despite Opposing Molecular Effects on Microtubules

    Microsoft Academic Search

    Robert J. Markelewicz; Susan J. Hall; Kim Boekelheide

    2004-01-01

    2,5-Hexanedione (2,5-HD), a taxol-like promoter of microtubule assembly, and carbendazim (CBZ), a colchicine-like inhibitor of microtubule assembly, are two environmental testicular toxicants that target and disrupt microtubule function in Sertoli cells. At the molecular level, these two toxicants have opposite effects on micro- tubule assembly, yet they share the common physiologic effect of inhibiting microtubule-dependent functions of Sertoli cells. By

  12. Katanin Localization Requires Triplet Microtubules in Chlamydomonas reinhardtii

    PubMed Central

    Esparza, Jessica M.; O’Toole, Eileen; Li, Linya; Giddings, Thomas H.; Kozak, Benjamin; Albee, Alison J.; Dutcher, Susan K.

    2013-01-01

    Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (?-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization. PMID:23320108

  13. Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

    PubMed Central

    Atherton, Joseph; Farabella, Irene; Yu, I-Mei; Rosenfeld, Steven S; Houdusse, Anne; Topf, Maya; Moores, Carolyn A

    2014-01-01

    Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ?7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface. DOI: http://dx.doi.org/10.7554/eLife.03680.001 PMID:25209998

  14. Force Transduction by the Microtubule-Bound Dam1 Ring Jonathan W. Armond

    E-print Network

    Turner, Matthew

    Force Transduction by the Microtubule-Bound Dam1 Ring Jonathan W. Armond and Matthew S. Turner of microtubules (MTs) and the motion of the Dam1 ring complex is now thought to play an important role protofilaments at the end of the depolymerizing MT physically prevent the Dam1 ring from falling off the end

  15. Microtubule-Induced Pins/Gai Cortical Polarity in Drosophila Neuroblasts

    E-print Network

    Doe, Chris

    Microtubule-Induced Pins/Gai Cortical Polarity in Drosophila Neuroblasts Sarah E. Siegrist1.09.043 SUMMARY Cortical polarity regulates cell division, mi- gration, and differentiation. Microtubules induce and Dlg/Pins co- immunoprecipitate, suggesting that micro- tubules induce Pins/Gai cortical polarity

  16. Aggregation Analysis of the Microtubule Binding Domain in Tau Protein by Spectroscopic Methods

    Microsoft Academic Search

    Tian-Ming Yao; Koji Tomoo; Toshimasa Ishida; Hiroshi Hasegawa; Masahiro Sasaki; Taizo Taniguchi

    2003-01-01

    The microtubule-associated protein tau is a highly soluble protein that shows hardly any tendency to assemble under physiological conditions. In the brains of Alzheimer's disease (AD) patients, however, tau dissociates from the axonal microtubule and abnormally aggregates to form paired helical filaments (PHFs). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. In recent

  17. Tau Isoform-Specific Modulation of Kinesin-Driven Microtubule Gliding Rates and Trajectories as

    E-print Network

    California at Santa Barbara, University of

    Tau Isoform-Specific Modulation of Kinesin-Driven Microtubule Gliding Rates and Trajectories as Determined with Tau-Stabilized Microtubules Austin Peck,1,2 M. Emre Sargin,3 Nichole E. LaPointe,1,2 Kenneth 1 October 2010; Accepted 1 October 2010 Monitoring Editor: George Bloom We have utilized tau

  18. Phosphorylation of Human Tau Protein by Microtubule-Associated Kinases: GSK3

    E-print Network

    Wood, John G.

    kinase 5; tau; Alzheimer's disease INTRODUCTION Microtubules (MTs) are the dynamic tracks on which cellsPhosphorylation of Human Tau Protein by Microtubule-Associated Kinases: GSK3 and cdk5 Are Key compartment is the tau protein. Tau, in the adult human brain, is a set of six protein isoforms, whose binding

  19. Pollen exine development precedes microtubule rearrangement in Vigna unguiculata (Fabaceae): A model for pollen wall patterning

    Microsoft Academic Search

    D. Southworth; J. A. Jernstedt

    1995-01-01

    Summary Although patterns on pollen exines are highly conserved, heritable traits, there is no prevailing explanation for control of pattern development. InVigna unguiculata (Fabaceae), the exine reticulum is unusually coarse so that development of the reticulum can be followed by light microscopy. Developing exine patterns were compared with the arrangement of microtubules in the microspore using immunofluorescence labeling of microtubules.

  20. Kinesin's Neck-Linker Determines its Ability to Navigate Obstacles on the Microtubule Surface

    E-print Network

    Hancock, William O.

    -linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence

  1. Microtubule organization and cell division in embryogenie protoplast cultures of white spruce ( Picea glauca )

    Microsoft Academic Search

    L. C. Fowke; S. M. Attree; H. Wang; D. I. Dunstan

    1990-01-01

    Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing

  2. Microtubule-targeting agents in oncology and therapeutic potential in hepatocellular carcinoma

    PubMed Central

    Loong, Herbert H; Yeo, Winnie

    2014-01-01

    In mammalian cells, microtubules are present both in interphase and dividing cells. In the latter, microtubules forming the mitotic spindle are highly dynamic and exquisitely sensitive to therapeutic inhibitors. Developed to alter microtubule function, microtubule-binding agents have been proven to be highly active as an anticancer treatment. Significant development of microtubule-binding agents has taken place in recent years, with newer anti-tubulin agents now showing novel properties of enhanced tumor specificity, reduced neurotoxicity, and insensitivity to chemoresistance mechanisms. Hepatocellular carcinoma remains one of the most difficult cancers to treat, with chemotherapies being relatively ineffective. There is now evidence to suggest that microtubule-binding agents may be effective in the treatment of hepatocellular carcinoma, especially when used in combination with mammalian target of rapamycin inhibitors. Preclinical models have suggested that the latter may be able to overcome resistance to microtubule binding agents. In this review article, recent developments of novel microtubule binding agents and their relevance to the treatment of hepatocellular carcinoma will be discussed. PMID:24790457

  3. Microtubules during germinal vesicle breakdown (GVBD) of Xenopus oocytes : effect of Ca2+ ionophore A-23187

    E-print Network

    Boyer, Edmond

    Microtubules during germinal vesicle breakdown (GVBD) of Xenopus oocytes : effect of Ca2+ ionophore in these experimental conditions (Germinal vesicle breakdown is induced). These results indicate that polymerization oocyte, Dumont, 1972), few microtubules have been observed near the germinal vesicle of the amphibian

  4. Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

    NASA Technical Reports Server (NTRS)

    Granger, C. L.; Cyr, R. J.

    2000-01-01

    Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

  5. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts

    Microsoft Academic Search

    Rebecca Heald; Régis Tournebize; Thiemo Blank; Raphael Sandaltzopoulos; Peter Becker; Anthony Hyman; Eric Karsenti

    1996-01-01

    Functional nuclei and mitotic spindles are shown to assemble around DNA-coated beads incubated in Xenopus egg extracts. Bipolar spindles assemble in the absence of centrosomes and kinetochores, indicating that bipolarity is an intrinsic property of microtubules assembling around chromatin in a mitotic cytoplasm. Microtubules nucleated at dispersed sites with random polarity rearrange into two arrays of uniform polarity. Spindle-pole formation

  6. Enhanced Mechanical Stability of Microtubules Polymerized with a Slowly Hydrolyzable Nucleotide Analogue

    E-print Network

    Smith, Marc L.

    of guanosine triphosphate. Using a combination of AFM imaging and local force spectroscopy, GMPCPP of guanosine triphosphate (GTP) bound between tubulin dimers in the microtubule lattice.3 Microtubules contain is greater in guanosine diphos- phate (GDP)-containing protofilaments than in GMPCPP- containing

  7. Genome stability is ensured by temporal control of kinetochore-microtubule dynamics.

    PubMed

    Bakhoum, Samuel F; Thompson, Sarah L; Manning, Amity L; Compton, Duane A

    2009-01-01

    Most solid tumours are aneuploid and many frequently mis-segregate chromosomes. This chromosomal instability is commonly caused by persistent mal-oriented attachment of chromosomes to spindle microtubules. Chromosome segregation requires stable microtubule attachment at kinetochores, yet those attachments must be sufficiently dynamic to permit correction of mal-orientations. How this balance is achieved is unknown, and the permissible boundaries of attachment stability versus dynamics essential for genome stability remain poorly understood. Here we show that two microtubule-depolymerizing kinesins, Kif2b and MCAK, stimulate kinetochore-microtubule dynamics during distinct phases of mitosis to correct mal-orientations. Few-fold reductions in kinetochore-microtubule turnover, particularly in early mitosis, induce severe chromosome segregation defects. In addition, we show that stimulation of microtubule dynamics at kinetochores restores stability to chromosomally unstable tumour cell lines, establishing a causal relationship between deregulation of kinetochore-microtubule dynamics and chromosomal instability. Thus, temporal control of microtubule attachment to chromosomes during mitosis is central to genome stability in human cells. PMID:19060894

  8. A R T I C L E S Drosophila katanin is a microtubule depolymerase that

    E-print Network

    Ross, Jennifer

    -mail: david.sharp@einstein.yu.edu) Received 16 November 2010; accepted 7 January 2011; published online 6-microtubule plus-end interactions and cell migration Dong Zhang1 , Kyle D. Grode2,5 , Shannon F. Stewman1,5 , Juan,5 and David J. Sharp1,6 Regulation of microtubule dynamics at the cell cortex is important for cell motility

  9. Identification of a Novel Protein Regulating Microtubule Stability through a Chemical Approach

    Microsoft Academic Search

    Sarah M Wignall; Nathanael S Gray; Young-Tae Chang; Lolita Juarez; Richard Jacob; Al Burlingame; Peter G Schultz; Rebecca Heald

    2004-01-01

    To identify novel proteins regulating the microtubule cytoskeleton, we screened a library of purine derivatives using mitotic spindle assembly in Xenopus egg extracts as an assay. Out of a collection of 1561 compounds, we identified 15 that destabilized microtubules without targeting tubulin directly, resulting in small spindles. Affinity chromatography with one compound, named diminutol, revealed a potential target as NQO1,

  10. Formation of a Dynamic Kinetochore Microtubule Interface through Assembly of the Dam1 Ring Complex

    Microsoft Academic Search

    Stefan Westermann; Agustin Avila-Sakar; Hong-Wei Wang; Hanspeter Niederstrasser; Jonathan Wong; David G. Drubin; Eva Nogales; Georjana Barnes

    2005-01-01

    How kinetochore proteins form a dynamic interface with microtubules is largely unknown. In budding yeast, the 10-protein Dam1 complex is an Aurora kinase target that plays essential roles maintaining the integrity of the mitotic spindle and regulating interactions with the kinetochore. Here, we investigated the biochemical properties of purified Dam1 complex. The complex oligomerized into rings around microtubules. Ring formation

  11. Mechanics of microtubule bundles in pillar cells from the inner ear.

    PubMed

    Tolomeo, J A; Holley, M C

    1997-10-01

    The mechanical properties of cross-linked microtubule bundles were measured from outer pillar cells isolated from the mammalian inner ear. Measurements were made using a three-point bending test and were incorporated into a mathematical model designed to distinguish between the stiffness contributions from microtubules and their cross-linking proteins. Outer pillar cells were composed of 1000-3000 parallel bundled microtubules in a square array that was interdigitated and cross-linked with actin filaments. The average midpoint bending stiffness of intact cells was 7 x 10(-4) N/m. After removal of both the actin filaments and cross-links with detergent in the presence of DNase I, the square array was disrupted and the stiffness decreased by a factor of 4, to 1.7 x 10(-4) N/m. The bending modulus for individual microtubules was calculated to be 7 x 10(-23) Nm2, and the Young's modulus for these 15 protofilament microtubules was 2 x 10(9) Pa. The shear modulus between microtubules in intact cells was calculated to be 10(3) Pa. It was concluded that cross-linking proteins provided shear resistance between microtubules, which resulted in a fourfold increase in stiffness. The model can be used to estimate the mechanical properties of cross-linked microtubule bundles in cells from which direct measurements are not available. PMID:9336220

  12. Formation of Membrane Networks In Vitro by Kinesin-driven Microtubule Movement

    E-print Network

    Vale, Ronald D.

    Formation of Membrane Networks In Vitro by Kinesin-driven Microtubule Movement Ronald D. Vale the translocating microtubule that can alter its direction of movement by dissociating or partially dissociating reform and tubules ofthe ER migrate towards the periphery in close proximity to the newly poly- merized

  13. A role of microtubules in the polarity of statocytes from roots of Lepidium sativum L

    Microsoft Academic Search

    W. Hensel

    1984-01-01

    When roots of Lepidium sativum L. are immersed in a colchicine solution (10-4 mol l-1), the cortical microtubules of statocytes are affected such that the dense network ofmicrotubules at the distal cell edges, between the endoplasmic reticulum and the plasma membrane, disappears almost completely, whereas the microtubules, lining the anticlinal cell walls are reduced only to a limited extent. Upon

  14. Microtubule acetylation amplifies p38 kinase signaling and anti-inflammatory IL-10 production

    PubMed Central

    Wang, Bin; Rao, Yan-Hua; Inoue, Makoto; Hao, Rui; Lai, Chun-Hsiang; Chen, David; McDonald, Stacey. L; Choi, Moon-Chang; Wang, Qiu; Shinohara, Mari; Yao, Tso-Pang

    2014-01-01

    Reversible acetylation of ?-tubulin is an evolutionarily conserved modification in microtubule networks. Despite its prevalence, the physiological function and regulation of microtubule acetylation remains poorly understood. Here we report that macrophages challenged by bacterial lipopolysaccharides (LPS) undergo extensive microtubule acetylation. Suppression of LPS-induced microtubule acetylation by inactivating the tubulin acetyltransferase, MEC17, profoundly inhibits the induction of anti-inflammatory interlukin-10 (IL-10), a phenotype effectively reversed by an acetylation-mimicking ?-tubulin mutant. Conversely, elevating microtubule acetylation by inhibiting the tubulin deacetylase, HDAC6, or stabilizing microtubules via Taxol, stimulates IL-10 hyper-induction. Supporting the anti-inflammatory function of microtubule acetylation, HDAC6 inhibition significantly protects mice from LPS toxicity. In HDAC6-deficient macrophages challenged by LPS, p38 kinase signaling becomes selectively amplified, leading to SP1-dependent IL-10 transcription. Remarkably, the augmented p38 signaling is suppressed by MEC17 inactivation. Our findings identify reversible microtubule acetylation as a kinase signaling modulator and a key component in the inflammatory response. PMID:24632940

  15. Steady-State Theory of the Interference of GTP Hydrolysis in the Mechanism of Microtubule Assembly

    Microsoft Academic Search

    Terrell L. Hill; Marie-France Carlier

    1983-01-01

    A model is presented for the interference of GTP hydrolysis in the mechanism of microtubule assembly. This model is suggested by previous results showing that both GTP and GDP are present at microtubule ends because of GTP hydrolysis and that tubulin does not bind to a GDP-bound end. The analytical theory developed here is aimed at calculation of the steady-state

  16. Small scale effects on the mechanical behaviors of protein microtubules based on the nonlocal elasticity theory

    SciTech Connect

    Gao, Yuanwen, E-mail: ywgao@lzu.edu.cn [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China)] [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China); Lei, Fang-Ming [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China)] [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China)

    2009-09-25

    Based on the nonlocal elastic theory, small scale effects are considered in the investigation of the mechanical properties of protein microtubules. A new prediction formula for the persistence lengths of microtubules with the consideration of the small scale effect is presented. Subsequently, the buckling of microtubules is studied based on a nonlocal elastic beam model. The predicted results of our model indicate that the length-dependence of persistence length is related not only to the shear terms, but also to the small scale effect. The Eular beam model, which is always considered unable to explain the length-dependence of microtubules, can capture the length-dependence of the persistence length of microtubules with the consideration of the small scale effect. The elastic buckling behaviors of microtubules in viscoelastic surrounding cytoplasm are also considered using the nonlocal Timoshenko beam model in this paper, and the results indicate that the small scale effect of microtubules also plays an important role in the buckling of microtubules.

  17. Still entangled: Assembly of the central spindle by multiple microtubule modulators

    Microsoft Academic Search

    Max E. Douglas; Masanori Mishima

    2010-01-01

    The central spindle is a microtubule-based structure that assembles during anaphase of mitosis in animal cells and is essential for multiple steps of cytokinesis. Central spindle assembly occurs by the cooperative action of multiple microtubule motors and modulators. Here, we review the mechanism by which the central spindle is formed, the role of several key proteins in this process and

  18. Microtubule stability affects the unique motility of F-actin in Marchantia polymorpha.

    PubMed

    Era, Atsuko; Kutsuna, Natsumaro; Higaki, Takumi; Hasezawa, Seiichiro; Nakano, Akihiko; Ueda, Takashi

    2013-01-01

    Actin microfilaments play crucial roles in diverse plant functions. Some specific cellular processes require interaction between F-actin and microtubules, and it is believed that there are direct or indirect connections between F-actin and microtubules. We previously reported that actin microfilaments exhibit unique dynamic motility in cells of the liverwort, Marchantia polymorpha; the relevance of this activity to microtubules has not been explored. To examine whether the dynamics of F-actin in M. polymorpha were somehow regulated by microtubules, we investigated the effects of stabilization or destabilization of microtubules on dynamics of actin bundles, which were visualized by Lifeact-Venus. To our surprise, both stabilization and destabilization of microtubules exerted similar effects on F-actin motility; apparent sliding movement of F-actin in M. polymorpha cells was accelerated by both oryzalin and paclitaxel, with the effect of paclitaxel more evident than that of oryzalin. Immunofluorescence staining revealed that some F-actin bundles were arrayed along with microtubules in M. polymorpha thallus cells. These results suggest that microtubules play regulatory roles in the unique F-actin dynamics in M. polymorpha. PMID:22678689

  19. A tethered delivery mechanism explains the catalytic action of a microtubule polymerase

    PubMed Central

    Ayaz, Pelin; Munyoki, Sarah; Geyer, Elisabeth A; Piedra, Felipe-Andrés; Vu, Emily S; Bromberg, Raquel; Otwinowski, Zbyszek; Grishin, Nick V; Brautigam, Chad A; Rice, Luke M

    2014-01-01

    Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple ??-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast ??-tubulin. Like TOG1, TOG2 binds selectively to a fully ‘curved’ conformation of ??-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two ??-tubulins. Preferential interactions between TOGs and fully curved ??-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized ??-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end. DOI: http://dx.doi.org/10.7554/eLife.03069.001 PMID:25097237

  20. Dynamic Instability of Individual Microtubules Analyzed byVideo Light Microscopy

    E-print Network

    Scholey, Jonathan

    Dynamic Instability of Individual Microtubules Analyzed byVideo Light Microscopy: Rate Constants-3280; and *DepartmentofAnatomy, DukeUniversity,Durham, North Carolina27710 Abstract. We have developed video microscopy, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concen- trations

  1. Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes

    PubMed Central

    1985-01-01

    Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti- fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies (Salmon, E. D., et al., 1984, J. Cell Biol., 99:2165-2174; Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186) which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules. PMID:3886672

  2. Topology of microtubules and actin in the life cycle of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

    Microsoft Academic Search

    Iva Slaninová; Judit Kucsera; Augustin Svoboda

    1999-01-01

    The morphology of budding and conjugating cells and associated changes in microtubules and actin distribution were studied in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by phase-contrast and fluorescence microscopy. The non-budding interphase cell showed a nucleus situated in the central position and bundles of cytoplasmic microtubules either stretching parallel to the longitudinal cell axis or randomly distributed in the cell;

  3. Feeding Cells Induced by Phytoparasitic Nematodes Require ?-Tubulin Ring Complex for Microtubule Reorganization

    PubMed Central

    Banora, Mohamed Youssef; Rodiuc, Natalia; Baldacci-Cresp, Fabien; Smertenko, Andrei; Bleve-Zacheo, Teresa; Mellilo, Maria Teresa; Karimi, Mansour; Hilson, Pierre; Evrard, Jean-Luc; Favery, Bruno; Engler, Gilbert; Abad, Pierre; de Almeida Engler, Janice

    2011-01-01

    Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound ?-tubulin ring complexes. Here we investigate the requirement of ?-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that ?-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis ?-tubulin (TUBG1 and TUBG2) and two ?-tubulin complex proteins genes (GCP3 and GCP4) are upregulated in galls. Electron microscopy demonstrates association of GCP3 and ?-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both ?-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the ?-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts. PMID:22144887

  4. Microtubule reorganization accompanying preprophase band formation in guard mother cells of Avena sativa L

    Microsoft Academic Search

    J. B. Mullinax; B. A. Palevitz

    1989-01-01

    Summary In order to study developmental changes in microtubule organization attending the formation of a longitudinally oriented preprophase band, the guard mother cells ofAvena were examined using a new procedure for anti-tubulin immunocytochemistry on large epidermal segments. We found that the interphase band (IMB) of transverse cortical microtubules present in these cells following asymmetric division is replaced after subsidiary cell

  5. The XMAP215-family protein DdCP224 is required for cortical interactions of microtubules

    PubMed Central

    Hestermann, Andrea; Gräf, Ralph

    2004-01-01

    Background Interactions of peripheral microtubule tips with the cell cortex are of crucial importance for nuclear migration, spindle orientation, centrosome positioning and directional cell movement. Microtubule plus end binding proteins are thought to mediate interactions of microtubule tips with cortical actin and membrane proteins in a dynein-dependent manner. XMAP215-family proteins are main regulators of microtubule plus end dynamics but so far they have not been implicated in the interactions of microtubule tips with the cell cortex. Results Here we show that overexpression of an N-terminal fragment of DdCP224, the Dictyostelium XMAP215 homologue, caused a collapse of the radial microtubule cytoskeleton, whereby microtubules lost contact with the cell cortex and were dragged behind like a comet tail of an unusually motile centrosome. This phenotype was indistinguishable from mutants overexpressing fragments of the dynein heavy chain or intermediate chain. Moreover, it was accompanied by dispersal of the Golgi apparatus and reduced cortical localization of the dynein heavy chain indicating a disrupted dynein/dynactin interaction. The interference of DdCP224 with cortical dynein function is strongly supported by the observations that DdCP224 and its N-terminal fragment colocalize with dynein and coimmunoprecipitate with dynein and dynactin. Conclusions Our data show that XMAP215-like proteins are required for the interaction of microtubule plus ends with the cell cortex in interphase cells and strongly suggest that this function is mediated by dynein. PMID:15186508

  6. Millimeter Scale Alignment of Magnetic Nanoparticle Functionalized Microtubules in Magnetic Fields

    E-print Network

    Hancock, William O.

    techniques,4 by viscous forces,5 or by strong magnetic or electric fields applied during tubule-modified microtubules, this cell was placed on top of a NdFeB permanent magnet for 3-10 min and visualized usingMillimeter Scale Alignment of Magnetic Nanoparticle Functionalized Microtubules in Magnetic Fields

  7. Interaction of microtubules with active principles of Xanthium strumarium.

    PubMed

    Menon, G S; Kuchroo, K; Dasgupta, D

    2001-01-01

    Indigenous variety of Xanthium strumarium (X. strumarium) was screened for its antimitotic activity using the microtubule-tubulin system isolated from mammalian tissue. A preliminary phytochemical screening of the whole extracts of the plant was carried out followed by partial purification of the whole extract of X.strumarium. The separated fractions obtained were identified and used for in vitro polymerization studies. The whole as well as partially separated chemical constituents of X. strumarium showed effective inhibition of tubulin polymerization. The results thus suggest that X. strumarium may possess antimitotic components. PMID:12002689

  8. Buckling of microtubules: An insight by molecular and continuum mechanics

    SciTech Connect

    Zhang, Jin; Meguid, S. A., E-mail: meguid@mie.utoronto.ca [Mechanics and Aerospace Design Laboratory, Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario M5S 3G8 (Canada)

    2014-10-27

    The molecular structural mechanics method has been extended to investigate the buckling of microtubules (MTs) with various configurations. The results indicate that for relative short MTs the shear deformation effect, rather than the nonlocal effect, is mainly responsible for the limitation of their widely used Euler beam description and the observed length-dependence of their bending stiffness. In addition, the configuration effect of MTs is also studied and considered as an explanation for the large scattering of the critical buckling force and bending stiffness observed in existing experiments. This configuration effect is also found to mainly originate from the geometry of the MTs and is mainly determined by the protofilament number.

  9. Actin-Dependent and -Independent Functions of Cortical Microtubules in the Differentiation of Arabidopsis Leaf Trichomes[W

    PubMed Central

    Sambade, Adrian; Findlay, Kim; Schäffner, Anton R.; Lloyd, Clive W.; Buschmann, Henrik

    2014-01-01

    Arabidopsis thaliana tortifolía2 carries a point mutation in ?-tubulin 4 and shows aberrant cortical microtubule dynamics. The microtubule defect of tortifolia2 leads to overbranching and right-handed helical growth in the single-celled leaf trichomes. Here, we use tortifolia2 to further our understanding of microtubules in plant cell differentiation. Trichomes at the branching stage show an apical ring of cortical microtubules, and our analyses support that this ring is involved in marking the prospective branch site. tortifolia2 showed ectopic microtubule bundles at this stage, consistent with a function for microtubules in selecting new branch sites. Overbranching of tortifolia2 required the C-terminal binding protein/brefeldin A-ADP ribosylated substrate protein ANGUSTIFOLIA1, and our results indicate that the angustifolia1 mutant is hypersensitive to alterations in microtubule dynamics. To analyze whether actin and microtubules cooperate in the trichome cell expansion process, we generated double mutants of tortifolia2 with distorted1, a mutant that is defective in the actin-related ARP2/3 complex. The double mutant trichomes showed a complete loss of growth anisotropy, suggesting a genetic interaction of actin and microtubules. Green fluorescent protein labeling of F-actin or microtubules in tortifolia2 distorted1 double mutants indicated that F-actin enhances microtubule dynamics and enables reorientation. Together, our results suggest actin-dependent and -independent functions of cortical microtubules in trichome differentiation. PMID:24714762

  10. A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity

    Microsoft Academic Search

    Claire E. Walczak; Isabelle Vernos; Timothy J. Mitchison; Eric Karsenti; Rebecca Heald

    1998-01-01

    Background: In eukaryotes, assembly of the mitotic spindle requires the interaction of chromosomes with microtubules. During this process, several motor proteins that move along microtubules promote formation of a bipolar microtubule array, but the precise mechanism is unclear. In order to examine the roles of different motor proteins in building a bipolar spindle, we have used a simplified system in

  11. The Initiation of Neurite Outgrowth by Sympathetic Neurons Grown In Vitro Does Not Depend on Assembly of Microtubules

    Microsoft Academic Search

    Carolyn L. Smith

    1994-01-01

    Neurite formation by dissociated chick sym- pathetic neurons in vitro begins when one of the many filopodia that emanate from the cell body of a neuron is invaded by cytoplasm containing microtubules and other components of axoplasm (Smith, 1994). This study was undertaken to determine whether this pro- cess depends on assembly of microtubules. To inhibit microtubule assembly, neurons were

  12. EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B

    PubMed Central

    Banerjee, Budhaditya; Kestner, Cortney A.

    2014-01-01

    The Aurora B kinase coordinates kinetochore–microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. We find that EB1, a microtubule plus end–tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). Our data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase. PMID:24616220

  13. High-resolution microtubule structures reveal the structural transitions in ??-tubulin upon GTP hydrolysis.

    PubMed

    Alushin, Gregory M; Lander, Gabriel C; Kellogg, Elizabeth H; Zhang, Rui; Baker, David; Nogales, Eva

    2014-05-22

    Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the ?-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both ?- and ?-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces. PMID:24855948

  14. Microtubule inhibitors: Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance.

    PubMed

    Perez, Edith A

    2009-08-01

    Microtubules are important cellular targets for anticancer therapy because of their key role in mitosis. Microtubule inhibitors (MTI) such as taxanes, vinca alkaloids, and epothilones stabilize or destabilize microtubules, thereby suppressing microtubule dynamics required for proper mitotic function, effectively blocking cell cycle progression and resulting in apoptosis. In spite of their antitumor activity, innate or acquired drug resistance to MTIs such as the taxanes is common, limiting their overall clinical efficacy. Further insight into the mechanisms of action of microtubule-targeting drugs has lead to the discovery of novel agents that may provide higher efficacy with limited toxicity and help overcome resistance to conventional MTIs. This review will focus on the different mechanisms of action of MTIs, potential factors related to resistance and tolerability, and will discuss the recent approval as well as the development of new antineoplastic agents. PMID:19671735

  15. Recent progress with microtubule stabilizers: new compounds, binding modes and cellular activities

    PubMed Central

    Rohena, Cristina C.

    2014-01-01

    Nature has yielded numerous classes of chemically distinct microtubule stabilizers. Several of these, including paclitaxel (Taxol) and docetaxel (Taxotere), are important drugs used in the treatment of cancer. New microtubule stabilizers and novel formulations of these agents continue to provide advances in cancer therapy. In this review we cover recent progress from late 2008 to August 2013 in the chemistry and biology of these diverse microtubule stabilizers focusing on the wide range of organisms that produce these compounds, their mechanisms of inhibiting microtubule-dependent processes, mechanisms of drug resistance, and their interactions with tubulin including their distinct binding sites and modes. A new potential role for microtubule stabilizers in neurodegenerative diseases is reviewed. PMID:24481420

  16. Functions of the Arabidopsis kinesin superfamily of microtubule-based motor proteins.

    PubMed

    Zhu, Chuanmei; Dixit, Ram

    2012-10-01

    Plants possess a large number of microtubule-based kinesin motor proteins. While the kinesin-2, 3, 9, and 11 families are absent from land plants, the kinesin-7 and 14 families are greatly expanded. In addition, some kinesins are specifically present only in land plants. The distinctive inventory of plant kinesins suggests that kinesins have evolved to perform specialized functions in plants. Plants assemble unique microtubule arrays during their cell cycle, including the interphase cortical microtubule array, preprophase band, anastral spindle and phragmoplast. In this review, we explore the functions of plant kinesins from a microtubule array viewpoint, focusing mainly on Arabidopsis kinesins. We emphasize the conserved and novel functions of plant kinesins in the organization and function of the different microtubule arrays. PMID:22038119

  17. Ring closure activates yeast ?TuRC for species-specific microtubule nucleation.

    PubMed

    Kollman, Justin M; Greenberg, Charles H; Li, Sam; Moritz, Michelle; Zelter, Alex; Fong, Kimberly K; Fernandez, Jose-Jesus; Sali, Andrej; Kilmartin, John; Davis, Trisha N; Agard, David A

    2015-02-01

    The ?-tubulin ring complex (?TuRC) is the primary microtubule nucleator in cells. ??TuRC is assembled from repeating ??-tubulin small complex (?TuSC) subunits and is thought to function as a template by presenting a ?-tubulin ring that mimics microtubule geometry. However, a previous yeast ?TuRC structure showed ?TuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that ?-tubulin complexes are in a closed conformation when attached to microtubules. To confirm the functional importance of the closed ?TuSC ring, we trapped the closed state and determined its structure, showing that the ?-tubulin ring precisely matches microtubule symmetry and providing detailed insight into ?TuRC architecture. Importantly, the closed state is a stronger nucleator, thus suggesting that this conformational switch may allosterically control ?TuRC activity. Finally, we demonstrate that ?TuRCs have a strong preference for tubulin from the same species. PMID:25599398

  18. Kinesin superfamily proteins and the regulation of microtubule dynamics in morphogenesis.

    PubMed

    Niwa, Shinsuke

    2015-01-01

    Kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motors that serve as sources of force for intracellular transport and cell division. Recent studies have revealed new roles of KIFs as microtubule stabilizers and depolymerizers, and these activities are fundamental to cellular morphogenesis and mammalian development. KIF2A and KIF19A have microtubule-depolymerizing activities and regulate the neuronal morphology and cilia length, respectively. KIF21A and KIF26A work as microtubule stabilizers that regulate axonal morphology. Morphological defects that are similar to human diseases are observed in mice in which these KIF genes have been deleted. Actually, KIF2A and KIF21A have been identified as causes of human neuronal diseases. In this review, the functions of these atypical KIFs that regulate microtubule dynamics are discussed. Moreover, some interesting unanswered questions and hypothetical answers to them are discussed. PMID:25347970

  19. EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B.

    PubMed

    Banerjee, Budhaditya; Kestner, Cortney A; Stukenberg, P Todd

    2014-03-17

    The Aurora B kinase coordinates kinetochore-microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. We find that EB1, a microtubule plus end-tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). Our data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase. PMID:24616220

  20. Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule- associated protein (cytoplasmic dynein)

    PubMed Central

    1988-01-01

    Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP- 1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule- activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3- nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties. PMID:2972729