Sample records for axonemal microtubule doublets

  1. The motor activity of mammalian axonemal dynein studied in situ on doublet microtubules

    Microsoft Academic Search

    David P. Lorch; Charles B. Lindemann; Alan J. Hunt

    2008-01-01

    Flagellar dynein generates forces that produce relative shearing between doublet microtubules in the axoneme; this drives propagated bending of flagella and cilia. To better understand dynein's role in coordinated flagellar and ciliary motion, we have developed an in situ assay in which polymerized single microtubules glide along doublet microtubules extruded from disintegrated bovine sperm flagella at a pH of 7.8.

  2. 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.

  3. Structural insights into microtubule doublet interactions inaxonemes

    SciTech Connect

    Downing, Kenneth H.; Sui, Haixin

    2007-06-06

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

  4. The axonemal axis and Ca2+-induced asymmetry of active microtubule sliding in sea urchin sperm tails

    PubMed Central

    1986-01-01

    Structural studies of stationary principal bends and of definitive patterns of spontaneous microtubule sliding disruption permitted description of the bending axis in sea urchin sperm tail axonemes. Lytechinus pictus sperm were demembranated in a buffer containing Triton X-100 and EGTA. Subsequent resuspension in a reactivation buffer containing 0.4 mM CaCl2 and 1.0 mM MgATP2- resulted in quiescent, rather than motile, cells and each sperm tail axoneme took on an extreme, basal principal bend of 5.2 rad. Thereafter, such flagellar axonemes began to disrupt spontaneously into two subsets of microtubules by active sliding requiring ATP. Darkfield light microscopy demonstrated that subset "1" is composed of microtubules from the inside edge of the principal bend. Subset "2" is composed of microtubules from the outside edge of the principal bend and always scatters less light in darkfield than subset 1. Subset 2, which always slides in the proximal direction, relative to subset 1, results in a basal loop of microtubules, and the subset 2 loop is restricted to the bend plane during sliding disruption. Electron microscopy revealed that doublets 8, 9, 1, 2, 3 and the central pair comprise subset 1, and doublets 4, 5, the bridge, 6, and 7 comprise subset 2. The microtubules of isolated subset 2 are maintained in a circular arc in the absence of spoke-central pair interaction. Longitudinal sections show that the bending plane bisects the central pair. We therefore conclude that the bend plane passes through doublet 1 and the 5-6 bridge and that doublet 1 is at the inside edge of the principal bend. Experimental definition of the axis permits explicit discussion of the location of active axonemal components which result in Ca2+-induced stationary basal bends and explicit description of components responsible for alternating basal principal and reverse bends. PMID:2940250

  5. 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

  6. Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules.

    PubMed Central

    Peterman, E J; Sosa, H; Goldstein, L S; Moerner, W E

    2001-01-01

    Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule. PMID:11606296

  7. 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

  8. Mechanochemical aspects of axonemal dynein activity studied by in vitro microtubule translocation.

    PubMed

    Hamasaki, T; Holwill, M E; Barkalow, K; Satir, P

    1995-12-01

    We have determined the relationship between microtubule length and translocation velocity from recordings of bovine brain microtubules translocating over a Paramecium 22S dynein substratum in an in vitro assay chamber. For comparison with untreated samples, the 22S dynein has been subjected to detergent and/or to pretreatments that induce phosphorylation of an associated 29 kDa light chain. Control and treated dyneins have been used at the same densities in the translocation assays. In any given condition, translocation velocity (v) shows an initial increase with microtubule length (L) and then reaches a plateau. This situation may be represented by a hyperbola of the general form v = aL/(L+b), which is formally analogous to the Briggs-Haldane relationship, which we have used to interpret our data. The results indicate that the maximum translocation velocity Vo(= a) is increased by pretreatment, whereas the length constant KL(= b), which corresponds to Km, does not change with pretreatment, implying that the mechanochemical properties of the pretreated dyneins differ from those of control dyneins. The conclusion that KL is constant for defined in vitro assays rules out the possibility that the velocity changes seen are caused by changes in geometry in the translocation assays or by the numbers of dyneins or dynein heads needed to produce maximal translocational velocity. From our analysis, we determine that f, the fraction of cycle time during which the dynein is in the force-generating state, is small--roughly 0.01, comparable to the f determined previously for heavy meromyosin. The practical limits of these mechanochemical changes imply that the maximum possible ciliary beat frequency is about 120 Hz, and that in the physiological range of 5-60 Hz, beat frequency could be controlled by varying the numbers of phosphorylated outer arm dyneins along an axonemal microtubule. PMID:8599664

  9. Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium

    PubMed Central

    1987-01-01

    The ciliary base is marked by a transition zone in which Y-shaped cross- linkers extend from doublet microtubules to the plasma membrane. Our goal was to investigate the hypothesis that the cross-linkers form a stable interaction between membrane or cell surface components and the underlying microtubule cytoskeleton. We have combined Triton X-100 extraction with lectin cytochemistry in the photoreceptor sensory cilium to investigate the relationship between cell surface glycoconjugates and the underlying cytoskeleton, and to identify the cell surface components involved. Wheat germ agglutinin (WGA) binds heavily to the cell surface in the region of the Y-shaped cross-linkers of the neonatal rat photoreceptor cilium. WGA binding is not removed by prior digestion with neuraminidase and succinyl-WGA also binds the proximal cilium, suggesting a predominance of N-acetylglucosamine containing glycoconjugates. Extraction of the photoreceptor plasma membrane with Triton X-100 removes the lipid bilayer, leaving the Y- shaped crosslinkers associated with the axoneme. WGA-binding sites are found at the distal ends of the crosslinkers after Triton X-100 extraction, indicating that the microtubule-membrane cross-linkers retain both a transmembrane and a cell surface component after removal of the lipid bilayer. To identify glycoconjugate components of the cross-linkers we used a subcellular fraction enriched in axonemes from adult bovine retinas. Isolated, detergent-extracted bovine axonemes show WGA binding at the distal ends of the cross-linkers similar to that seen in the neonatal rat. Proteins of the axoneme fraction were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. WGA labeling of the nitrocellulose transblots reveals three glycoconjugates, all of molecular mass greater than 400 kD. The major WGA-binding glycoconjugate has an apparent molecular mass of approximately 600 kD and is insensitive to prior digestion with neuraminidase. This glycoconjugate may correspond to the dominant WGA- binding component seen in cytochemical experiments. PMID:3693403

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

    PubMed Central

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

    2014-01-01

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

  11. Microtubule dynamics investigated by microinjection of Paramecium axonemal tubulin: lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase

    PubMed Central

    1989-01-01

    Microtubule (MT) dynamics in PtK2 cells have been investigated using in vivo injection of unmodified Paramecium ciliary tubulin and time-lapse fixation. The sites of incorporation of the axonemal tubulin were localized using a specific antibody which does not react with vertebrate cytoplasmic tubulin (Adoutte, A., M. Claisse, R. Maunoury, and J. Beisson. 1985. J. Mol. Evol. 22:220-229), followed by immunogold labeling, Nanovid microscopy, and ultrastructural observation of the same cells. We confirm data from microinjection of labeled tubulins in other cell types (Soltys, B. J., and G. G. Borisy. 1985. J. Cell Biol. 100:1682-1689; Mitchison, T., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527; Schulze, E., and M. Kirschner. 1986. J. Cell Biol. 102:1020-1031). In agreement with the dynamic instability model (Mitchison, T., and M. Kirschner. 1984. Nature (Lond.). 312:237-242), during interphase, fast (2.6 microns/min) distal growth of MTs occurs, together with new centrosomal nucleation. Most of the cytoplasmic MT complex is replaced within 15-30 min. During mitosis, astral MTs display the same pattern of renewal, but the turnover of the MT system is much faster (approximately 6 min). We have concentrated on the construction of the kinetochore fibers during prometaphase and observe that (a) incorporation of tubulin in the vicinity of the kinetochores is not seen during prophase and early prometaphase as long as the kinetochores are not yet connected to a pole by MTs; (b) proximal time- dependent incorporation occurs only into preexisting kinetochore MTs emanating from centrosomes. Consequently, in undisturbed prometaphase cells, the kinetochores probably do not act as independent nucleation sites. This confirms a model in which, at prometaphase, fast probing centrosomal MTs are grabbed by the kinetochores, where tubulin incorporation then takes place. PMID:2646309

  12. The axonemal axis and Ca2+-induced asymmetry of active microtubule sliding in sea urchin sperm tails

    Microsoft Academic Search

    W. S. Sale

    1986-01-01

    Abstract. Structural studies of stationary principal bends and of definitive patterns of spontaneous,mi- crotubule sliding disruption permitted,description of the bending,axis in sea urchin sperm,tail axonemes. Lytechinus pictus sperm,were demembranated,in a buffer containing,Triton X-100 and EGTA. Subse- quent resuspension,in a reactivation buffer containing 0.4 mM,CaC12 and 1.0 mM,MgATP 2- resulted in quiescent, rather than motile, cells and each sperm tail axoneme

  13. Motor regulation results in distal forces that bend partially disintegrated Chlamydomonas axonemes into circular arcs.

    PubMed

    Mukundan, V; Sartori, P; Geyer, V F; Jülicher, F; Howard, J

    2014-06-01

    The bending of cilia and flagella is driven by forces generated by dynein motor proteins. These forces slide adjacent microtubule doublets within the axoneme, the motile cytoskeletal structure. To create regular, oscillatory beating patterns, the activities of the axonemal dyneins must be coordinated both spatially and temporally. It is thought that coordination is mediated by stresses or strains, which build up within the moving axoneme, and somehow regulate dynein activity. During experimentation with axonemes subjected to mild proteolysis, we observed pairs of doublets associating with each other and forming bends with almost constant curvature. By modeling the statics of a pair of filaments, we show that the activity of the motors concentrates at the distal tips of the doublets. Furthermore, we show that this distribution of motor activity accords with models in which curvature, or curvature-induced normal forces, regulates the activity of the motors. These observations, together with our theoretical analysis, provide evidence that dynein activity can be regulated by curvature or normal forces, which may, therefore, play a role in coordinating the beating of cilia and flagella. PMID:24896122

  14. Motor Regulation Results in Distal Forces that Bend Partially Disintegrated Chlamydomonas Axonemes into Circular Arcs

    PubMed Central

    Mukundan, V.; Sartori, P.; Geyer, V.F.; Jülicher, F.; Howard, J.

    2014-01-01

    The bending of cilia and flagella is driven by forces generated by dynein motor proteins. These forces slide adjacent microtubule doublets within the axoneme, the motile cytoskeletal structure. To create regular, oscillatory beating patterns, the activities of the axonemal dyneins must be coordinated both spatially and temporally. It is thought that coordination is mediated by stresses or strains, which build up within the moving axoneme, and somehow regulate dynein activity. During experimentation with axonemes subjected to mild proteolysis, we observed pairs of doublets associating with each other and forming bends with almost constant curvature. By modeling the statics of a pair of filaments, we show that the activity of the motors concentrates at the distal tips of the doublets. Furthermore, we show that this distribution of motor activity accords with models in which curvature, or curvature-induced normal forces, regulates the activity of the motors. These observations, together with our theoretical analysis, provide evidence that dynein activity can be regulated by curvature or normal forces, which may, therefore, play a role in coordinating the beating of cilia and flagella. PMID:24896122

  15. Conformational change in the outer doublet microtubules from sea urchin sperm flagella

    Microsoft Academic Search

    T. Miki-Noumura; R. KAMIYA

    1979-01-01

    Dark-field microscopy with a high-powered light source revealed that the outer doublet microtubules (DMTs) from sea urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella assume helically coiled configurations (Miki-Noumura, T., and R. Kamiya. 1976. Exp. Cell Res. 97:451 .). We report here that the DMTs change shape when the pH or Ca-ion concentration is changed. The DMTs assumed a left-handed

  16. Is the curvature of the flagellum involved in the apparent cooperativity of the dynein arms along the "9+2" axoneme?

    PubMed

    Cibert, Christian; Ludu, Andrei

    2010-07-21

    In a recent study [Cibert, 2008. Journal of Theoretical Biology 253, 74-89], by assuming that walls of microtubules are involved in cyclic compression/dilation equilibriums as a consequence of cyclic curvature of the axoneme, it was proposed that local adjustments of spatial frequencies of both dynein arms and beta-tubulin monomers facing series create propagation of joint probability waves of interaction (JPI) between these two necessary partners. Modeling the occurrence of these probable interactions along the entire length of an axoneme between each outer doublet pair (without programming any cooperative dialog between molecular complexes) and the cyclic attachment of two facing partners, we show that such constituted active couples are clustered. Along a cluster the dynein arms exhibit a small phase shift with respect to the order according to which they began their cycle after being linked to a beta-tubulin monomer. The number of couples included in these clusters depends on the probability of interaction between the dynein arms and the beta-tubulin, on the location of the outer doublet pairs around the axonemal cylinder, and on the local bending of the axoneme; around the axonemal cylinder, the faster and the larger the sliding, the shorter the clusters. This mechanism could be involved in the apparent cooperativity of molecular motors and the beta-tubulin monomers, since it is partially controlled by local curvature, and the cluster length is inversely proportional to the sliding activity of the outer doublet pairs they link. PMID:20399794

  17. Dynein Binds to and Crossbridges Cytoplasmic Microtubules

    Microsoft Academic Search

    Leah T. Haimo; Bruce R. Telzer; Joel L. Rosenbaum

    1979-01-01

    Dynein isolated from Chlamydomonas flagellar axonemes binds to microtubules assembled in vitro from 6S brain tubulin dimers. The dynein arms bind periodically along the length of the microtubules with a center-to-center spacing of 24 nm, equal to the periodicity of dynein arms on intact axonemes. The arms project from the in vitro assembled microtubules at an angle of approximately 55

  18. 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

  19. The motility of axonemal dynein is regulated by the tubulin code.

    PubMed

    Alper, Joshua D; Decker, Franziska; Agana, Bernice; Howard, Jonathon

    2014-12-16

    Microtubule diversity, arising from the utilization of different tubulin genes and from posttranslational modifications, regulates many cellular processes including cell division, neuronal differentiation and growth, and centriole assembly. In the case of cilia and flagella, multiple cell biological studies show that microtubule diversity is important for axonemal assembly and motility. However, it is not known whether microtubule diversity directly influences the activity of the axonemal dyneins, the motors that drive the beating of the axoneme, nor whether the effects on motility are indirect, perhaps through regulatory pathways upstream of the motors, such as the central pair, radial spokes, or dynein regulatory complex. To test whether microtubule diversity can directly regulate the activity of axonemal dyneins, we asked whether in vitro acetylation or deacetylation of lysine 40 (K40), a major posttranslational modification of ?-tubulin, or whether proteolytic cleavage of the C-terminal tail (CTT) of ?- and ?-tubulin, the location of detyrosination, polyglutamylation, and polyglycylation modifications as well as most of the genetic diversity, can influence the activity of outer arm axonemal dynein in motility assays using purified proteins. By quantifying the motility with displacement-weighted velocity analysis and mathematically modeling the results, we found that K40 acetylation increases and CTTs decrease axonemal dynein motility. These results show that axonemal dynein directly deciphers the tubulin code, which has important implications for eukaryotic ciliary beat regulation. PMID:25658008

  20. 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

  1. Immunocytochemical localization of tubulin, actin, and myosin in axonemes of ciliated cells from quail oviduct.

    PubMed Central

    Sandoz, D; Gounon, P; Karsenti, E; Sauron, M E

    1982-01-01

    Tubulin, actin, and myosin have been localized in isolated demembranated ciliated cells from quail oviduct by immunocytochemistry in both light and electron microscopy by using purified antibodies. The peripheral doublets and the central tubules are stained by the antitubulin whereas the kinetosomes are poorly stained. Actin antibodies clearly stain the axonemes, but only on the proximal-half portion, whereas myosin antibodies stain a small area of the axonemes just above the ciliary neck region. Images PMID:7048302

  2. STUDIES ON THE MICROTUBULES IN HELIOZOA

    PubMed Central

    Tilney, Lewis G.; Porter, Keith R.

    1967-01-01

    When specimens of Actinosphaerium nucleofilum are placed at 4°C, the axopodia retract and the birefringent core (axoneme) of each axopodium disappears. In fixed specimens, it has been shown that this structure consists of a highly patterned bundle of microtubules, each 220 A in diameter; during cold treatment these microtubules disappear and do not reform until the organisms are removed to room temperature. Within a few minutes after returning the specimens to room temperature, the axonemes reappear and the axopodia begin to reform reaching normal length 30–45 min later. In thin sections of cells fixed during the early stages of this recovery period, microtubules, organized in the pattern of the untreated specimens, are found in each reforming axopodium. Reforming axopodia without birefringent axonemes (and thus without microtubules) are never encountered. From these observations we conclude that the microtubules may be instrumental not only in the maintenance of the axopodia but also in their growth. Thus, if the microtubules are destroyed, the axopodia should retract and not reform until these tubular units are reassembled. During the cold treatment short segments of a 340-A tubule appeared; when the organisms were removed from the cold, these tubular segments disappeared. It seems probable that they are one of the disintegration products of the microtubules. A model is presented of our interpretation of how a 220-A microtubule transforms into a 340-A tubule and what this means in terms of the substructure of the untreated microtubules. PMID:6033539

  3. Microtubule-associated proteins control the kinetics of microtubule nucleation.

    PubMed

    Wieczorek, Michal; Bechstedt, Susanne; Chaaban, Sami; Brouhard, Gary J

    2015-07-01

    Microtubules are born and reborn continuously, even during quiescence. These polymers are nucleated from templates, namely ?-tubulin ring complexes (?-TuRCs) and severed microtubule ends. Using single-molecule biophysics, we show that nucleation from ?-TuRCs, axonemes and seed microtubules requires tubulin concentrations that lie well above the critical concentration. We measured considerable time lags between the arrival of tubulin and the onset of steady-state elongation. Microtubule-associated proteins (MAPs) alter these time lags. Catastrophe factors (MCAK and EB1) inhibited nucleation, whereas a polymerase (XMAP215) and an anti-catastrophe factor (TPX2) promoted nucleation. We observed similar phenomena in cells. We conclude that GTP hydrolysis inhibits microtubule nucleation by destabilizing the nascent plus ends required for persistent elongation. Our results explain how MAPs establish the spatial and temporal profile of microtubule nucleation. PMID:26098575

  4. 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 methods to visualize the assembly and disas- sembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37°C

  5. The contribution of ??-tubulin curvature to microtubule dynamics.

    PubMed

    Brouhard, Gary J; Rice, Luke M

    2014-11-10

    Microtubules are dynamic polymers of ??-tubulin that form diverse cellular structures, such as the mitotic spindle for cell division, the backbone of neurons, and axonemes. To control the architecture of microtubule networks, microtubule-associated proteins (MAPs) and motor proteins regulate microtubule growth, shrinkage, and the transitions between these states. Recent evidence shows that many MAPs exert their effects by selectively binding to distinct conformations of polymerized or unpolymerized ??-tubulin. The ability of ??-tubulin to adopt distinct conformations contributes to the intrinsic polymerization dynamics of microtubules. ??-Tubulin conformation is a fundamental property that MAPs monitor and control to build proper microtubule networks. PMID:25385183

  6. Does axonemal dynein push, pull, or oscillate?

    PubMed

    Lindemann, Charles B; Hunt, Alan J

    2003-12-01

    Dynein is the molecular motor that provides motive force in cilia and flagella. Dynein is anchored to the A-subtubule of the outer doublets by a club-shaped extension called the stem, which supports the large globular head of the molecule. Dynein forms an attachment or cross-bridge to the B-subtubule of the adjacent outer doublet through a slender appendage extending from the head that is called the stalk or alternately the B-link. It is generally thought that the B-link mediates the interdoublet transfer of force that bends the flagellum. This requires that energy released at the site of ATP hydrolysis, located in the globular head, be transferred as mechanical work to the microtubule binding site at the tip of the B-link. It has been proposed that this is accomplished by a sideways or rotational translocation of the B-link caused by a rotation of the globular head. An estimate of the stiffness of the B-link and stem derived from the recently published data of Burgess et al. [2003: Nature 421:715-718] yields a maximum stiffness of 0.47 pN/nm for the B-link and 0.1 pN/nm for the stem. The B-link stiffness would allow transfer of 3.8 pN of force in response to an 8-nm displacement of the B-link tip. However, if as proposed the globular head of the dynein heavy chain is supported by the stem, the B-link and stem elasticity are in series. Thus, the flexibility of the stem would limit the force that can be transferred laterally by the entire dynein heavy chain to 0.6 pN at 8 nm displacement. This force is insufficient to support flagellar motility. So, if the stem were the only support for the globular head, then force would have to be transmitted linearly along the axis defined by the stem and B-link. Because this configuration is never observed, the hypothesis that dynein generates force by lateral displacement of the B-link is more attractive, but requires that the globular head of the dynein is stabilized by an additional means of support during the power stroke. We propose that the microtubule affinity of the tip of the B-link is independent of the ATP-dependent powerstroke, and that detachment from the B-subtubule is regulated by tension. A dynein cross-bridge cycle that incorporates an anchored head, together with a ratchet-like mechanism for microtubule translocation by the B-link, would have distinct advantages. This mechanism may reconcile dynein oscillation and interdoublet sliding within one cross-bridge mechanism. PMID:14584026

  7. Flow Cell Assays with Microtubules: Motility/Dynamics in Fluorescence and VE-DIC

    E-print Network

    Mitchison, Tim

    -microtubule interactions and action of severing/depolymerizing factors on microtubules. Described here are some general is essential and a glucose oxidase/catalase/glucose system works very well for this purpose. Axonemes (for motor proteins. This assay does not provide polarity information on the motility. Although

  8. Microtubule polarity and distribution in teleost photoreceptors.

    PubMed

    Troutt, L L; Burnside, B

    1988-07-01

    We have characterized the polarity orientation of microtubules in teleost retinal photoreceptors. The highly polarized rods and cones contain large numbers of paraxially aligned microtubules and exhibit dramatic cell shape changes. The myoid portion of the inner segments of both rods and cones undergoes contraction and elongation in response to light or circadian signals. Previous studies in our laboratory have demonstrated that in cones but not rods myoid elongation is microtubule-dependent. To determine polarity orientation, we decorated microtubules in photoreceptors of the green sunfish Lepomis cyanellus, with hooks formed from either exogenous or endogenous tubulin subunits. The direction of curvature of the attached hooks in cross section indicates microtubule polarity orientation by allowing one to determine the relative positions within the cell of the plus (fast-growing) and minus (slow-growing) ends of the microtubules. We found that virtually all cytoplasmic microtubules in photoreceptors are oriented with plus ends directed toward the synapse and minus ends toward the basal body at the base of the outer segment. Axonemal microtubules in photoreceptor outer segments are oriented with minus ends toward the basal body as in cilia and flagella. We have suggested previously that cone myoid elongation is mediated by mechanochemical sliding between microtubules. The polarity observations reported here indicate that if microtubules do slide in cones, sliding would necessarily occur between microtubules of parallel orientation as is observed in cilia and flagella. PMID:3249231

  9. REGULATION OF MICROTUBULES IN TETRAHYMENA

    PubMed Central

    Williams, Norman E.; Frankel, Joseph

    1973-01-01

    The coupled resorption and redifferentiation of oral structures which occurs in Tetrahymena pyriformis under conditions of amino acid deprivation has been studied by transmission electron microscopy. Two patterns of ciliary resorption have been found, (a) in situ, and (b) after withdrawal into the cytoplasm. No autophagic vacuoles containing cilia or ciliary axonemes have been seen. Stomatogenic field basal bodies arise by a process of rapid sequential nucleation, with new ones always appearing next to more mature ones, even though the latter may not be fully differentiated. Accessory radial ribbons of microtubules develop immediately adjacent to oral field basal bodies as a late step in their maturation. It can be seen that the formation of basal bodies and their orientation within the oral complex are separate processes. This is true for about 130 of the approximately 170 oral basal bodies; the remaining 40 or so form within the patterned groups of ciliary units as a later event. Clusters of randomly oriented thin-walled microtubules are found surrounding oral basal bodies at all times during stomatogenesis. They may either represent stores of microtubule subunit protein, or serve as effectors of basal body movement during their orientation into pattern. PMID:4345553

  10. Slow Axonemal Dynein e Facilitates the Motility of Faster Dynein c

    PubMed Central

    Shimizu, Youské; Sakakibara, Hitoshi; Kojima, Hiroaki; Oiwa, Kazuhiro

    2014-01-01

    We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (kcat = 0.27 s–1 and kcat,MT = 1.09 s–1, respectively) were lower than those of dynein c (kcat = 1.75 s–1 and kcat,MT = 2.03 s–1, respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (Vap = 1.2 ± 0.1 ?m/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (Vmax = 15.8 ± 1.5 ?m/s) and highly sensitive to decreases in the surface density (Vmin = 2.2 ± 0.7 ?m/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c’s power stroke. PMID:24853744

  11. Slow axonemal dynein e facilitates the motility of faster dynein c.

    PubMed

    Shimizu, Youské; Sakakibara, Hitoshi; Kojima, Hiroaki; Oiwa, Kazuhiro

    2014-05-20

    We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (kcat = 0.27 s(-1) and kcat,MT = 1.09 s(-1), respectively) were lower than those of dynein c (kcat = 1.75 s(-1) and kcat,MT = 2.03 s(-1), respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (Vap = 1.2 ± 0.1 ?m/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (Vmax = 15.8 ± 1.5 ?m/s) and highly sensitive to decreases in the surface density (Vmin = 2.2 ± 0.7 ?m/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c's power stroke. PMID:24853744

  12. Dynamic organization of microtubules and microtubule-organizing centers during the sexual phase of a parasitic protozoan, Lecudina tuzetae (Gregarine, Apicomplexa).

    PubMed

    Kuriyama, Ryoko; Besse, Colette; Gèze, Marc; Omoto, Charlotte K; Schrével, Joseph

    2005-12-01

    Lecudina tuzetae is a parasitic protozoan (Gregarine, Apicomplexa) living in the intestine of a marine polychaete annelid, Nereis diversicolor. Using electron and fluorescence microscopy, we have characterized the dynamic changes in microtubule organization during the sexual phase of the life cycle. The gametocyst excreted from the host worm into seawater consists of two (one male and one female) gamonts in which cortical microtubule arrays are discernible. Each gamont undergoes multiple nuclear divisions without cytokinesis, resulting in the formation of large multinucleate haploid cells. After cellularization, approximately 1000 individual gametes are produced from each gamont within 24 h. Female gametes are spherical and contain interphase cytoplasmic microtubule arrays emanating from a gamma-tubulin-containing site. In male gametes, both interphase microtubules and a flagellum with "6 + 0" axonemal microtubules extend from the same microtubule-organizing site. At the beginning of spore formation, each zygote secretes a wall to form a sporocyst. Following meiotic and mitotic divisions, each sporocyst gives rise to eight haploid cells that ultimately differentiate into sporozoites. The ovoid shaped sporocyst is asymmetric and forms at least two distinctive microtubule arrays: spindle microtubules and microtubule bundles originating from the protruding apical end corresponding to the dehiscence pole of the sporocyst. Because antibodies raised against mammalian centrosome components, such as gamma-tubulin, pericentrin, Cep135, and mitosis-specific phosphoproteins, react strongly with the microtubule-nucleating sites of Lecudina, this protozoan is likely to share common centrosomal antigens with higher eukaryotes. PMID:16240430

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

    PubMed Central

    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 the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell. PMID:511939

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

    PubMed

    Summers, K; Kirschner, M W

    1979-10-01

    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 the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell. PMID:511939

  15. 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

    2015-04-01

    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. PMID:25412847

  16. Micropatterning microtubules.

    PubMed

    Portran, Didier

    2014-01-01

    The following protocol describes a method to control the orientation and polarity of polymerizing microtubules (MTs). Reconstitution of specific geometries of dynamic MT networks is achieved using a ultraviolet (UV) micropatterning technique in combination with stabilized MT microseeds. The process is described in three main parts. First, the surface is passivated to avoid the non-specific absorption of proteins, using different polyethylene glycol (PEG)-based surface treatment. Second, specific adhesive surfaces (the micropatterns) are imprinted through a photomask using deep UVs. Lastly, MT microseeds are adhered to the micropatterns followed by MT polymerization. PMID:24484656

  17. X-Ray Fiber Diffraction Recordings from Oriented Demembranated Chlamydomonas Flagellar Axonemes.

    PubMed

    Toba, Shiori; Iwamoto, Hiroyuki; Kamimura, Shinji; Oiwa, Kazuhiro

    2015-06-16

    The high homology of its axonemal components with humans and a large repertoire of axonemal mutants make Chlamydomonas a useful model system for experiments on the structure and function of eukaryotic cilia and flagella. Using this organism, we explored the spatial arrangement of axonemal components under physiological conditions by small-angle x-ray fiber diffraction. Axonemes were oriented in physiological solution by continuous shear flow and exposed to intense and stable x rays generated in the synchrotron radiation facility SPring-8, BL45XU. We compared diffraction patterns from axonemes isolated from wild-type and mutant strains lacking the whole outer arm (oda1), radial spoke (pf14), central apparatus (pf18), or the ?-chain of the outer arm dynein (oda11). Diffraction of the axonemes showed a series of well-defined meridional/layer-line and equatorial reflections. Diffraction patterns from mutant axonemes exhibited a systematic loss/attenuation of meridional/layer-line reflections, making it possible to determine the origin of various reflections. The 1/24 and 1/12 nm(-1) meridional reflections of oda1 and oda11 were much weaker than those of the wild-type, suggesting that the outer dynein arms are the main contributor to these reflections. The weaker 1/32 and 1/13.7 nm(-1) meridional reflections from pf14 compared with the wild-type suggest that these reflections come mainly from the radial spokes. The limited contribution of the central pair apparatus to the diffraction patterns was confirmed by the similarity between the patterns of the wild-type and pf18. The equatorial reflections were complex, but a comparison with electron micrograph-based models allowed the density of each axonemal component to be estimated. Addition of ATP to rigor-state axonemes also resulted in subtle changes in equatorial intensity profiles, which could report nucleotide-dependent structural changes of the dynein arms. The first detailed description of axonemal reflections presented here serves as a landmark for further x-ray diffraction studies to monitor the action of constituent proteins in functional axonemes. PMID:26083924

  18. Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

    PubMed Central

    2014-01-01

    Toxoplasma gondii is an obligate intracellular parasite that causes serious opportunistic infections, birth defects, and blindness in humans. Microtubules are critically important components of diverse structures that are used throughout the Toxoplasma life cycle. As in other eukaryotes, spindle microtubules are required for chromosome segregation during replication. Additionally, a set of membrane-associated microtubules is essential for the elongated shape of invasive “zoites,” and motility follows a spiral trajectory that reflects the path of these microtubules. Toxoplasma zoites also construct an intricate, tubulin-based apical structure, termed the conoid, which is important for host cell invasion and associates with proteins typically found in the flagellar apparatus. Last, microgametes specifically construct a microtubule-containing flagellar axoneme in order to fertilize macrogametes, permitting genetic recombination. The specialized roles of these microtubule populations are mediated by distinct sets of associated proteins. This review summarizes our current understanding of the role of tubulin, microtubule populations, and associated proteins in Toxoplasma; these components are used for both novel and broadly conserved processes that are essential for parasite survival. PMID:25380753

  19. Ratchetlike Properties of In Vitro Microtubule Translocation by a Chlamydomonas Inner-Arm Dynein Species c in the Presence of Flow

    PubMed Central

    Kikushima, Kenji; Kamiya, Ritsu

    2009-01-01

    To investigate the force generation properties of Chlamydomonas axonemal inner-arm dyneins in response to external force, we analyzed microtubule gliding on dynein-coated surfaces under shear flow. When inner-arm dynein c was used, microtubule translocation in the downstream direction accelerated with increasing flow speed in a manner that depended on the dynein density and ATP concentration. In contrast, the microtubule translocation velocity in the upstream direction was unaffected by the flow speed. The number of microtubules on the glass surface was almost constant with and without flow, suggesting that gliding acceleration was not simply caused by weakened dynein-microtubule binding. With other inner-arm dynein species, the microtubule gliding velocity was unaffected by the flow regardless of the flow direction or nucleotide concentration. The flow-generated force acting on a single dynein was estimated to be as small as ?0.03 pN/dynein. These results indicate that dynein c possesses a ratchetlike property that allows acceleration only in one direction by a very small external force. This property should be important for slow- and fast-moving dyneins to function simultaneously within the axoneme. PMID:19751670

  20. Polymorphic Dynamics of Microtubules

    E-print Network

    Herve Mohrbach; Albert Johner; Igor M. Kulic

    2010-05-07

    Starting from the hypothesis that the tubulin dimer is a conformationally bistable molecule - fluctuating between a curved and a straight configuration at room temperature - we develop a model for polymorphic dynamics of the microtubule lattice. We show that tubulin bistability consistently explains unusual dynamic fluctuations, the apparent length-stiffness relation of grafted microtubules and the curved-helical appearance of microtubules in general. Analyzing experimental data we conclude that taxol stabilized microtubules exist in highly cooperative yet strongly fluctuating helical states. When clamped by the end the microtubule undergoes an unusual zero energy motion - in its effect reminiscent of a limited rotational hinge.

  1. Polymorphic Dynamics of Microtubules

    E-print Network

    Mohrbach, Herve; Kulic, Igor M

    2010-01-01

    Starting from the hypothesis that the tubulin dimer is a conformationally bistable molecule - fluctuating between a curved and a straight configuration at room temperature - we develop a model for polymorphic dynamics of the microtubule lattice. We show that tubulin bistability consistently explains unusual dynamic fluctuations, the apparent length-stiffness relation of grafted microtubules and the curved-helical appearance of microtubules in general. Analyzing experimental data we conclude that taxol stabilized microtubules exist in highly cooperative yet strongly fluctuating helical states. When clamped by the end the microtubule undergoes an unusual zero energy motion - in its effect reminiscent of a limited rotational hinge.

  2. Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs.

    PubMed

    Scholey, J M; Neighbors, B; McIntosh, J R; Salmon, E D

    1984-05-25

    Taxol was used to prepare microtubules from unfertilized eggs of sea urchins Lytechinus pictus, Strongylocentrotus droebachiensis , and Strongylocentrotus purpuratus. By electron microscopy, these microtubules possessed normal morphology and were decorated with projections. The polypeptides present were tubulin plus microtubule-associated proteins (MAPs) which included various high molecular weight polypeptides, and a Mr = 80,000 polypeptide. These MAPs were extracted from the microtubules by differential centrifugation in high ionic strength buffers, yielding a pellet of microtubules which were not decorated with projections. The Mr = 80,000 and high molecular weight MAPs were separated using Bio-Gel A-1.5 m chromatography, and shown to bind taxol-stabilized microtubules assembled from purified bovine brain tubulin. A dynein-like MgATPase activity is present in sea urchin egg extracts. 10-20% of this MgATPase co-pelleted with the taxol-assembled microtubules, under conditions where greater than 90% of the tubulin pelleted. During subsequent fractionation of the microtubules, by (i) high salt extraction followed by gel filtration or sucrose density gradient fractionation or (ii) ATP extraction, the MgATpase co-purified with high Mr MAPs. The MgATPase which remained in the microtubule-depleted egg extract was partially purified by (NH4)2SO4 fractionation, followed by Bio-Gel A-5 m and hydroxylapatite chromatography. The high Mr MAP MgATPase and the hydroxylapatite MgATPase both contained a prominent polypeptide (Mr approximately 350,000), which co-migrated on sodium dodecyl sulfate gels with the major heavy chain of dynein extracted from sperm axonemes. Our data suggest that this Mr approximately 350,000 polypeptide is cytoplasmic dynein. PMID:6144678

  3. Ca2+ and cAMP regulations of microtubule sliding in hyperactivated motility of bull spermatozoa

    PubMed Central

    ISHIJIMA, Sumio

    2015-01-01

    To reach and fertilize the egg, mammalian spermatozoa change their flagellar movement in the female reproductive tract, named hyperactivation. The biochemical analyses of the hyperactivated movement using demembranated spermatozoa defined the factors inducing this peculiar movement; namely, large asymmetrical flagellar movement observed in the early stage of the hyperactivation was induced with a high Ca2+ concentration while large symmetrical flagellar movement in the late stage of the hyperactivation was generated with low Ca2+ and high cAMP concentrations. Under these conditions, the microtubule sliding of bull sperm flagella was investigated by disintegrating the sperm flagella with MgATP2? after extracting their plasma membrane and mitochondria. The large asymmetrical flagellar movement was caused by a long sliding displacement of a fiber of the doublet microtubules. On the other hand, the large symmetrical flagellar movement was generated by a large amount of microtubule sliding by many doublet microtubules. PMID:25765012

  4. Microtubule-Associated Proteins and Microtubule-Interacting Proteins

    Microsoft Academic Search

    Maria Kavallaris; Sima Don; Nicole M. Verrills

    Microtubules are regulated by a range of proteins that interact with tubulin and regulate their stability. A large number\\u000a and variety of microtubule-associated proteins (MAPs) and microtubule-interacting proteins have been indentified and they\\u000a exhibit cell and tissue specific expression. MAPs and microtubule-interacting proteins carry out a wide range of functions\\u000a including regulation of microtubule stability, cross-linking microtubules and mediate interactions

  5. Dynamic instability of microtubule growth

    Microsoft Academic Search

    Tim Mitchison; Marc Kirschner

    1984-01-01

    We report here that microtubules in vitro coexist in growing and shrinking populations which interconvert rather infrequently. This dynamic instability is a general property of microtubules and may be fundamental in explaining cellular microtubule organization.

  6. Microtubules, Tubulins and Associated Proteins.

    ERIC Educational Resources Information Center

    Raxworthy, Michael J.

    1988-01-01

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

  7. Surfing on microtubule ends

    Microsoft Academic Search

    Pedro Carvalho; Jennifer S. Tirnauer; David Pellman

    2003-01-01

    A crowd of proteins seems to have gathered around the plus-ends of microtubules. A rapidly expanding group of proteins known as plus-end tracking proteins (+TIPs) have been identified that seem to be able to ‘surf’ the dynamic ends of microtubules. Microtubule plus-ends exist in multiple conformational and chemical states. In principle, altering this plus-end microenvironment is an appealing way for

  8. 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

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

    PubMed

    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, Peadar 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 M K; Shoemark, Amelia; Knowles, Michael R; Omran, Heymut; Mitchison, Hannah M

    2013-03-01

    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 disorganization 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 microtubular disorganisation defect," rather than "radial spoke defect." PMID:23255504

  10. Taccalonolide microtubule stabilizers.

    PubMed

    Li, Jing; Risinger, April L; Mooberry, Susan L

    2014-09-15

    This review focuses on a relatively new class of microtubule stabilizers, the taccalonolides. The taccalonolides are highly oxygenated pentacyclic steroids isolated from plants of the genus Tacca. Originally identified in a cell-based phenotypic screen, the taccalonolides have many properties similar to other microtubule stabilizers. They increase the density of interphase microtubules, causing microtubule bundling, and form abnormal multi-polar mitotic spindles leading to mitotic arrest and, ultimately, apoptosis. However, the taccalonolides differ from other microtubule stabilizers in that they retain efficacy in taxane resistant cell lines and in vivo models. Binding studies with the newly identified, potent taccalonolide AJ demonstrated covalent binding to ?-tubulin at or near the luminal and/or pore taxane binding site(s) which stabilizes microtubule protofilaments in a unique manner as compared to other microtubule stabilizers. The isolation and semi-synthesis of 21 taccalonolides helped to identify key structure activity relationships and the importance of multiple regions across the taccalonolide skeleton for optimal biological potency. PMID:24491636

  11. Microtubule composition: Cryptography of dynamic polymers

    E-print Network

    Microtubule composition: Cryptography of dynamic polymers Kerry Bloom* Department of Biology modification of microtubules reveals addi- tional levels of diversity within the cytoarchitecture. Modified forms of microtubules are differentially distributed in cells and harbor distinct sets of microtubule

  12. Lattice defects in microtubules: protofilament numbers vary within individual microtubules

    Microsoft Academic Search

    D. Chretien; E Metoz; E Verde; E. Karsenti; R. H. Wade

    1992-01-01

    We have used cryo-electron microscopy of vitrified specimens to study microtubules assembled both from three cycle purified tubulin (3 x-tubulin) and in cell free extracts of Xenopus eggs. In vitro assembled 3x-tubulin samples have a majority of microtubules with 14 protofilaments whereas in cell extracts most microtubules have 13 protofilaments. Microtubule poly- morphism was observed in both cases. The number

  13. Dark Matter with Two Inert Doublets plus One Higgs Doublet

    E-print Network

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

    2014-08-14

    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.

  14. 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

  15. Microtubule teardrop patterns.

    PubMed

    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

  16. Model for microtubule chemotaxis

    Microsoft Academic Search

    D. J. Oddel

    2002-01-01

    Dramatic reorganization of the microtubule (MT) cytoskeleton underlies a number of cellular processes including mitosis and axon initiation. MT assembly in vivo is regulated by MT assembly modulators, which are themselves regulated, often by antagonistic kinase\\/phosphatase pairs such that the phosphorylated form is active while the dephosphorylated form is inactive. If the antagonistic activities are spatially segregated in the cell,

  17. Fluorescent Taxoid Probes for Microtubule Research

    Microsoft Academic Search

    Isabel Barasoain; J. Fernando Díaz; José M. Andreu

    2010-01-01

    The use of the antitumor drug taxol as an experimental microtubule-stabilizing agent is widespread. Fluorescent taxol conjugates, although less employed, are very useful tools for several purposes in microtubule research. These include easily visualizing microtubule cytoskeletons in a variety of cells as well as in vitro assembled microtubules, studying the molecular recognition processes of taxoids by microtubules and investigating new

  18. Microtubule dynamics in fish melanophores

    PubMed Central

    1994-01-01

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

  19. 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

  20. Energy Consumption of Actively Beating Flagella

    NASA Astrophysics Data System (ADS)

    Chen, Daniel; Nicastro, Daniela; Dogic, Zvonimir

    2012-02-01

    Motile cilia and flagella are important for propelling cells or driving fluid over tissues. The microtubule-based core in these organelles, the axoneme, has a nearly universal ``9+2'' arrangement of 9 outer doublet microtubules assembled around two singlet microtubules in the center. Thousands of molecular motor proteins are attached to the doublets and walk on neighboring outer doublets. The motors convert the chemical energy of ATP hydrolysis into sliding motion between adjacent doublet microtubules, resulting in precisely regulated oscillatory beating. Using demembranated sea urchin sperm flagella as an experimental platform, we simultaneously monitor the axoneme's consumption of ATP and its beating dynamics while key parameters, such as solution viscosity and ATP concentration, are varied. Insights into motor cooperativity during beating and energetic consequences of hydrodynamic interactions will be presented.

  1. Chlamydomonas Axonemal Dynein Assembly Locus ODA8 Encodes a Conserved Flagellar Protein Needed for Cytoplasmic Maturation of Outer Dynein Arm Complexes

    PubMed Central

    Desai, Paurav B; Freshour, Judy R; Mitchell, David R

    2015-01-01

    The Chlamydomonas reinhardtii oda8 mutation blocks assembly of flagellar outer dynein arms (ODAs), and interacts genetically with ODA5 and ODA10, which encode axonemal proteins thought to aid dynein binding onto axonemal docking sites. We positionally cloned ODA8 and identified the gene product as the algal homolog of vertebrate LRRC56. Its flagellar localization depends on ODA5 and ODA10, consistent with genetic interaction studies, but phylogenomics suggests that LRRC56 homologs play a role in intraflagellar transport (IFT)-dependent assembly of outer row dynein arms, not axonemal docking. ODA8 distribution between cytoplasm and flagella is similar to that of IFT proteins and about half of flagellar ODA8 is in the soluble matrix fraction. Dynein extracted in vitro from wild type axonemes will rebind efficiently to oda8 mutant axonemes, without re-binding of ODA8, further supporting a role in dynein assembly or transport, not axonemal binding. Assays comparing preassembled ODA complexes from the cytoplasm of wild type and mutant strains show that dynein in oda8 mutant cytoplasm has not properly preassembled and cannot bind normally onto oda axonemes. We conclude that ODA8 plays an important role in formation and transport of mature dynein complexes during flagellar assembly. © 2014 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:25558044

  2. Lattice structure of cytoplasmic microtubules in a cultured Mammalian cell.

    PubMed

    McIntosh, J Richard; Morphew, Mary K; Grissom, Paula M; Gilbert, Susan P; Hoenger, Andreas

    2009-11-27

    Tubulin can polymerize in two distinct arrangements: "B-lattices," in which the alpha-tubulins of one protofilament lie next to alpha-tubulins in the neighboring protofilaments, or the "A" configuration, where alpha-tubulins lie beside beta-tubulins. Microtubules (MTs) in flagellar axonemes and those assembled from pure tubulin in vitro display only B-lattices, but recent work shows that A-lattices are found when tubulin co-polymerizes in vitro with an allele of end-binding protein 1 that lacks C-terminal sequences. This observation suggests that cytoplasmic MTs, which form in the presence of this "tip-associating protein," may have A-lattices. To test this hypothesis, we have decorated interphase MTs in 3T3 cells with monomeric motor domains from the kinesin-like protein Eg5. These MTs show only B-lattices, as confirmed by visual inspection of electron cryo-tomograms and power spectra of single projection views, imaged at higher electron dose. This result is significant because 13 protofilament MTs with B-lattices must include a "seam," one lateral domain where adjacent dimers are in the A-configuration. It follows that cytoplasmic MTs are not cylindrically symmetric; they have two distinct faces, which may influence the binding patterns of functionally significant MT-interacting proteins. PMID:19769986

  3. Noisy kink in microtubules

    E-print Network

    H. C. Rosu; J. A. Tuszy?ski; A. González

    1998-05-10

    We study the power spectrum of a class of noise effects generated by means of a digital-like disorder in the traveling variable of the conjectured Ginzburg-Landau-Montroll kink excitations moving along the walls of the microtubules. We have found a 1/f^{\\alpha} noise with \\alpha \\in (1.82-2.04) on all the time scales we have considered

  4. 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.

  5. Alcohol stimulates ciliary motility of isolated airway axonemes through a nitric oxide, cyclase and cyclic nucleotide-dependent kinase mechanism

    PubMed Central

    Sisson, Joseph H.; Pavlik, Jacqueline A.; Wyatt, Todd A.

    2009-01-01

    Background Lung mucociliary clearance provides the first line of defense from lung infections and is impaired in individuals who consume heavy amounts of alcohol. Previous studies have demonstrated that this alcohol-induced ciliary dysfunction (AICD) occurs through impairment of the nitric oxide (NO) and cyclic nucleotide-dependent kinase-signaling pathways in lung airway ciliated epithelial cells. Recent studies have established that all key elements of this alcohol-driven signaling pathway co-localize to the apical surface of the ciliated cells with the basal bodies. These findings led us to hypothesize that alcohol activates the cilia stimulation pathway at the organelle level. To test this hypothesis we performed experiments exposing isolated demembranated cilia (isolated axonemes) to alcohol and studied the effect of alcohol-stimulated ciliary motility on the pathways involved with isolated axoneme activation. Methods Isolated demembranated cilia were prepared from bovine trachea and activated with adenosine triphosphate (ATP). Ciliary beat frequency (CBF), NO production, adenylyl and guanylyl cyclase activities, cAMP- and cGMP-dependent kinase activities were measured following exposure to biologically relevant concentrations of alcohol. Results Alcohol rapidly stimulated axoneme beating 40% above baseline at very low concentrations of alcohol (1-10 mM). This activation was specific to ethanol, required the synthesis of NO, the activation of soluble adenylyl cyclase (sAC) and the activation of both cAMP- and cGMP-dependent kinases (PKA and PKG), all of which were present in the isolated organelle preparation. Conclusions Alcohol rapidly and sequentially activates the eNOS?NO?GC?cGMP?PKG and sAC?cAMP? PKA dual signaling pathways in isolated airway axonemes. These findings indicate a direct effect of alcohol on airway cilia organelle function and fully recapitulate the alcohol-driven activation of cilia known to exist in vivo and in intact lung ciliated cells in vitro following brief moderate alcohol exposure. Furthermore, these findings indicate that airway cilia are exquisitely sensitive to the effects of alcohol and substantiate a key role for alcohol in the alterations of mucociliary clearance associated with even low levels of alcohol intake. We speculate that this same axoneme-based alcohol activation pathway is down regulated following long term high alcohol exposure and that the isolated axoneme preparation provides an excellent model for studying the mechanism of alcohol-mediated cilia dysfunction. PMID:19183138

  6. 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

  7. Microtubule dynamics in interphase cells

    Microsoft Academic Search

    Eric Schulze; Marc Kirschner

    1986-01-01

    The sites of microtubule growth and the kinetics of elongation have been studied in vivo by microinjection of biotin-labeled tubulin and subse- quent visualization with immunocytochemic al probes. Immunofluorescen ce and immunoelectron micros- copy demonstrate that injected biotin-labeled subunits are incorporated into new segments of growth which are contiguous with unlabeled microtubules. Rapid in- corporation occurs by elongation of existing

  8. ?-Tubulin complexes and microtubule nucleation

    Microsoft Academic Search

    Michelle Moritz; David A Agard

    2001-01-01

    Microtubules are dynamic cytoskeletal polymers that assemble from ?\\/?-tubulin and are vital for the establishment of cell polarity, vesicle trafficking and formation of the mitotic\\/meiotic spindle. ?-Tubulin, a protein related to ?\\/?-tubulin, is required for initiating the polymerization of microtubules in vivo. ?-Tubulin has been found in two main protein complexes: the ?-tubulin ring complex and its subunit, the ?-tubulin

  9. Rf heating in Doublet IIA

    Microsoft Academic Search

    J. L. Luxon; R. L. Freeman; V. S. Chan; S. C. Chiu; J. C. DeBoo; R. W. Harvey; T. H. Jensen; R. J. La Haye; J. M. Lohr; C. P. Moeller; T. Ohkawa; J. C. Riordan; J. F. Tooker; D. F. Vaslow

    1980-01-01

    This report summarizes the results of the rf heating experiments in Doublet IIA. The experiments were designed to heat electrons using rf power at about twice the lower hybrid frequency. The parallel index of refraction was chosen in the range of 11 < n{sub â¥} < 28 for optimal deposition of power in the central regions of the plasma. Up

  10. Nonlinear ionic pulses along microtubules.

    PubMed

    Sekuli?, D L; Satari?, B M; Tuszynski, J A; Satari?, M V

    2011-05-01

    Microtubules are cylindrically shaped cytoskeletal biopolymers that are essential for cell motility, cell division and intracellular trafficking. Here, we investigate their polyelectrolyte character that plays a very important role in ionic transport throughout the intra-cellular environment. The model we propose demonstrates an essentially nonlinear behavior of ionic currents which are guided by microtubules. These features are primarily due to the dynamics of tubulin C-terminal tails which are extended out of the surface of the microtubule cylinder. We also demonstrate that the origin of nonlinearity stems from the nonlinear capacitance of each tubulin dimer. This brings about conditions required for the creation and propagation of solitonic ionic waves along the microtubule axis. We conclude that a microtubule plays the role of a biological nonlinear transmission line for ionic currents. These currents might be of particular significance in cell division and possibly also in cognitive processes taking place in nerve cells. PMID:21604102

  11. Running title: Binding of katanin to microtubules Arabidopsis katanin binds microtubules using a multimeric microtubule-binding

    E-print Network

    Boyer, Edmond

    1 Running title: Binding of katanin to microtubules Arabidopsis katanin binds microtubules using a multimeric microtubule-binding domain Virginie Stoppin-Melleta , Jérémie Gaillarda , Ton Timmersb-dependent destabilization of microtubules in animal cells. In plants, the catalytic subunit of Arabidopsis thaliana katanin

  12. THE “9 + 1” PATTERN OF MICROTUBULES IN SPERMATOZOA OF Mesostoma (PLATYHELMINTHES, TURBELLARIA)*

    PubMed Central

    Henley, Catherine; Costello, D. P.; Thomas, Mary Beth; Newton, W. D.

    1969-01-01

    The living spermatozoa of the flatworm Mesostoma georgianum have a sperm body about 100 ? long and 0.5 ? wide, and two motile free flagella, ca. 200 ? long. In sections examined with the electron microscope, these flagella have the usual nine pairs of peripheral doublet microtubules and have a single central core unit which is connected to the A members of the doublets by spokelike structures. There are also short connections between the doublets and the flagellar membrane. In material negatively stained with phosphotungstic acid, the doublet microtubules seem to have very different elastic properties than the core; they tend to fall on the copper grids in coils of rather uniform diameter (2-4 ?), while the core is much more rigid and is often found extending alone, along a relatively straight course, for very long distances (up to 73 ?). After negative staining, the core has a striking appearance with a dense center around which are wound two hollow structures in a double helix of 45° pitch. The center-to-center distance of each gyre is approximately 650 Å, and the hollow structures are ca. 180 Å in diameter. Images PMID:4190073

  13. Mini-Review Kinesin-II, a Membrane Traffic Motor in Axons, Axonemes, and Spindles

    E-print Network

    Scholey, Jonathan

    and kinesin-II to serve as ax- onal vesicle transport motors that can be deployed as membrane traffic motors, microtubule. Kinesin is a good candidate for being one of several fast anterograde axonal vesicle transport- channels in axons (12). These results suggest that kinesin transports vesicles containing Na

  14. Polymorphic Dynamics of Microtubules Herve Mohrbach1

    E-print Network

    Boyer, Edmond

    Polymorphic Dynamics of Microtubules Herv´e Mohrbach1 , Albert Johner2 and Igor M. Kuli´c2 1 Groupe configuration at room temperature - we develop a model for polymorphic dynamics of the microtubule lattice. We-stiffness relation of grafted microtubules and the curved-helical appearance of microtubules in general. Analyzing

  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. Microtubules containing detyrosinated tubulin are less dynamic.

    PubMed Central

    Kreis, T E

    1987-01-01

    Peptide antibodies specific for tyrosinated (tyr-tubulin) or detyrosinated alpha-tubulin (glu-tubulin) have been generated for studying the relative stability of microtubules enriched in either form of alpha-tubulin. Treatment of Vero cells with nocodazole has revealed that interphase microtubules rich in glu-tubulin (glu-microtubules) are resistant to higher concentrations of the microtubule-disrupting drug than the microtubules containing only tyr-tubulin (tyr-microtubules). Glu-tubulin is enriched in centrioles and mid-bodies, but absent from the first interphase microtubules that have repolymerized in late telophase. Tubulin (including both forms) has been labeled with rhodamine (rh-tubulin) and microinjected into Vero cells to study in vivo the dynamic properties and incorporation rates of tubulin into microtubules rich in either glu- or tyr-tubulin. Tyr-microtubules are significantly more rapidly labeled by the microinjected rh-tubulin than glu-microtubules. Ten minutes after injection, rh-tubulin is present in virtually all tyr-microtubules. The half-time of turnover of glu-microtubules is approximately 1 h. Even several hours after microinjection, some of the glu-microtubules have consistently not incorporated visible amounts of rh-tubulin. These results suggest that tyr- and glu-microtubules respectively represent relatively dynamic and stable subclasses of interphase microtubules. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:3315650

  17. Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins.

    PubMed Central

    Gibbons, B H; Asai, D J; Tang, W J; Hays, T S; Gibbons, I R

    1994-01-01

    Transcripts approximately 14.5 kilobases in length from 14 different genes that encode for dynein heavy chains have been identified in poly(A)+ RNA from sea urchin embryos. Analysis of the changes in level of these dynein transcripts in response to deciliation, together with their sequence relatedness, suggests that 11 or more of these genes encode dynein isoforms that participate in regeneration of external cilia on the embryo, whereas the single gene whose deduced sequence closely resembles that of cytoplasmic dynein in other organisms appears not to be involved in this regeneration. The four consensus motifs for phosphate binding found previously in the beta heavy chain of sea urchin dynein are present in all five additional isoforms for which extended sequences have been obtained, suggesting that these sites play a significant role in dynein function. Sequence analysis of a approximately 400 amino acid region encompassing the putative hydrolytic ATP-binding site shows that the dynein genes fall into at least six distinct classes. Most of these classes in sea urchin have a high degree of sequence identity with one of the dynein heavy chain genes identified in Drosophila, indicating that the radiation of the dynein gene family into the present classes occurred at an early stage in the evolution of eukaryotes. Evolutionary changes in cytoplasmic dynein have been more constrained than those in the axonemal dyneins. Images PMID:8186465

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

    PubMed

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

    2012-08-01

    Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ-ated 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

  20. Cellular/Molecular Acetylation of Microtubules Influences Their Sensitivity

    E-print Network

    Baas, Peter W.

    Cellular/Molecular Acetylation of Microtubules Influences Their Sensitivity to Severing by Katanin of microtubules to severing by katanin is regulated by acetylation of the microtubules. During interphase, fibroblasts display long microtubules with discrete regions rich in acetylated tubulin. Overexpression

  1. Microtubule asters as templates for nanomaterials assembly

    PubMed Central

    2012-01-01

    Self organization of the kinesin-microtubule system was implemented as a novel template to create percolated nanofiber networks. Asters of microtubule seeds were immobilized on glass surfaces and their growth was recorded over time. The individual aster islands became interconnected as microtubules grew and overlapped, resulting in a highly percolated network. Cellulose nanowhiskers were used to demonstrate the application of this system to nanomaterials organization. The size distribution of the cellulose nanowhiskers was comparable to that of microtubules. To link cellulose nanowhiskers to microtubules, the nanowhiskers were functionalized by biotin using cellulose binding domains. Fluorescence studies confirmed biotinylation of cellulose nanowhiskers and binding of cellulose nanowhiskers to biotinylated microtubules. PMID:23270559

  2. Microtubules in Plants

    PubMed Central

    Hashimoto, Takashi

    2015-01-01

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

  3. Doublecortin recognizes the 13-protofilament microtubule cooperatively and tracks microtubule ends

    PubMed Central

    Bechstedt, Susanne; Brouhard, Gary J.

    2014-01-01

    Neurons, like all cells, face the problem that tubulin forms microtubules with too many or too few protofilaments (pfs). Cells overcome this heterogeneity with the ?-tubulin ring complex, which provides a nucleation template for 13-pf microtubules. Doublecortin (DCX), a protein that stabilizes microtubules in developing neurons, also nucleates 13-pf microtubules in vitro. Using fluorescence microscopy assays, we show that the binding of DCX to microtubules is optimized for the lateral curvature of the 13-pf lattice. This sensitivity depends on a cooperative interaction wherein DCX molecules decrease the dissociation rate of their neighbors. Mutations in DCX found in patients with subcortical band heterotopia weaken these cooperative interactions. Using assays with dynamic microtubules, we discovered that DCX binds to polymerization intermediates at growing microtubule ends. These results support a mechanism for stabilizing 13-pf microtubules that allows DCX to template new 13-pf microtubules through associations with the sides of the microtubule lattice. PMID:22727374

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

    E-print Network

    Schnaufer, Achim

    and cilium is ancient and predates the radiation, over 800 million years ago, of the lineages that gave rise tetraurelia and Tetrahymena thermophila (FIG. 1a­f)) key biological questions can be addressed using either interference (RNAi) provides a powerful approach to study gene function. Thus, using protists as model systems

  5. Microtubule Severing Stymied by Free Tubulin

    NASA Astrophysics Data System (ADS)

    Ross, Jennifer; Bailey, Megan

    2015-03-01

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

  6. Electrophoresis of individual microtubules in microchannels

    E-print Network

    Dekker, Cees

    Electrophoresis of individual microtubules in microchannels M. G. L. van den Heuvel, M. P. de-sized fluidic channels to confine and measure electrophoresis of freely suspended individual microtubules. We measure orientation-dependent velocities of microtubules and the electro-osmotic flow mobility in our

  7. Length-dependent dynamics of microtubules

    E-print Network

    Vandana Yadav; Sutapa Mukherji

    2012-04-02

    Certain regulatory proteins influence the polymerization dynamics of microtubules by inducing catastrophe with a rate that depends on the microtubule length. Using a discrete formulation, here we show that, for a catastrophe rate proportional to the microtubule length, the steady-state probability distributions of length decay much faster with length than an exponential decay as seen in the absence of these proteins.

  8. Buckling and force propagation along intracellular microtubules

    E-print Network

    MacKintosh, F.C.

    OFFPRINT Buckling and force propagation along intracellular microtubules Moumita Das, Alex J propagation along intracellular microtubules Moumita Das1,2(a) , Alex J. Levine3 and F. C. MacKintosh1,2 1 September 2008 PACS 87.16.Ka ­ Filaments, microtubules, their networks, and supramolecular assemblies PACS

  9. Microtubule assembly nucleated by isolated centrosomes

    Microsoft Academic Search

    Tim Mitchison; Marc Kirschner

    1984-01-01

    Microtubules are involved in the morphogenesis of most cells and are the structural basis of the mitotic spindle. We report here that purified centrosomes nucleate the assembly of microtubules with unusual dynamic properties. This may have important implications for the mechanism by which microtubule arrays are organized and stabilized in cells.

  10. R760 Review Microtubules, membranes and cytokinesis

    E-print Network

    Straight, Aaron

    R760 Review Microtubules, membranes and cytokinesis Aaron F. Straight and Christine M. Field Proper. The microtubule midzone is able to stimulate the cortex of the cell to ensure proper ingression and completion of the cleavage furrow. Specialized microtubule structures are responsible for directing membrane vesicles

  11. High-Resolution Model of the Microtubule

    Microsoft Academic Search

    Eva Nogales; Michael Whittaker; Ronald A. Milligan; Kenneth H. Downing

    1999-01-01

    A high-resolution model of the microtubule has been obtained by docking the crystal structure of tubulin into a 20 Å map of the microtubule. The excellent fit indicates the similarity of the tubulin conformation in both polymers and defines the orientation of the tubulin structure within the microtubule. Long C-terminal helices form the crest on the outside of the protofilament,

  12. Length-dependent dynamics of microtubules

    E-print Network

    Yadav, Vandana

    2012-01-01

    Certain regulatory proteins influence the polymerization dynamics of microtubules by inducing catastrophe with a rate that depends on the microtubule length. Using a discrete formulation, here we show that, for a catastrophe rate proportional to the microtubule length, the steady-state probability distributions of length decay much faster with length than an exponential decay as seen in the absence of these proteins.

  13. Centrosome composition and microtubule anchoring mechanisms

    Microsoft Academic Search

    Michel Bornens

    2002-01-01

    Centrosomes of animal cells and spindle pole bodies of fungi are the major microtubule nucleating centers. Recent studies indicate that their capacity to organize microtubule arrays rests on elaborate control of the anchoring and release of the nucleated microtubules. Although common molecular mechanisms are likely to be involved in both cases, the centrosome from animal cells shows considerable complexity and

  14. 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

  15. Kinesin follows the microtubule's protofilament axis

    PubMed Central

    1993-01-01

    We tested the hypothesis that kinesin moves parallel to the microtubule's protofilament axis. We polymerized microtubules with protofilaments that ran either parallel to the microtubule's long axis or that ran along shallow helical paths around the cylindrical surface of the microtubule. When gliding across a kinesin-coated surface, the former microtubules did not rotate. The latter microtubules, those with supertwisted protofilaments, did rotate; the pitch and handedness of the rotation accorded with the supertwist measured by electron cryo- microscopy. The results show that kinesin follows a path parallel to the protofilaments with high fidelity. This implies that the distance between consecutive kinesin-binding sites along the microtubule must be an integral multiple of 4.1 nm, the tubulin monomer spacing along the protofilament, or a multiple of 8.2 nm, the dimer spacing. PMID:8099076

  16. Localized modulated waves in microtubules

    NASA Astrophysics Data System (ADS)

    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.

  17. Simulation of Microtubules: Mechanical properties

    NASA Astrophysics Data System (ADS)

    Stevens, Mark

    2015-03-01

    In order to understand microtubule assembly and the necessary monomeric properties to design artificial polymers that possess features similar to those of microtubules, we have developed a coarse-grained model of a monomer that self-assembles into tubules. In this model the monomer has a wedge shape which promotes tubule formation. There are attractive binding sites on the vertical and lateral sides of the monomer. We previously performed molecular dynamics simulations to calculate the set of structures that form upon self-assembly as we vary the lateral and vertical interaction strengths. In this talk, we will present the results of mechanical studies of the coarse-grained tubule system. The persistence length and various elastic moduli have been calculated. Microtubules have some of the largest persistence lengths of polymers. We have found that the persistence length is indeed very long for this coarse-grained model system. We calculate elastic moduli for varying the interaction strengths of the lateral and vertical interactions. We gain insight into the values that occur in microtubules, with respect to mechanical stability and stiffness.

  18. Higgs properties in the Stealth Doublet Model

    E-print Network

    Glenn Wouda

    2013-06-28

    I present a model with two scalar doublets and a softly broken $\\ztwo$ symmetry, where only one of the doublets gets a vacuum expectation value and couples to fermions at tree-level. The softly broken $\\ztwo$ symmetry leads to interesting phenomenology such as mixing between the two doublets and a charged scalar H^\\pm which can be light and dominantly decays into W \\gamma. The model can also naturally reproduce an enhanced \\gamma \\gamma signal of the newly observed Higgs boson at the LHC with mass 125 GeV.

  19. Higgs properties in the Stealth Doublet Model

    NASA Astrophysics Data System (ADS)

    Wouda, Glenn

    2013-11-01

    I present a model with two scalar doublets and a softly broken ?2 symmetry, where only one of the doublets gets a vacuum expectation value and couples to fermions at tree-level. The softly broken ?2 symmetry leads to interesting phenomenology such as mixing between the two doublets and a charged scalar H± which can be light and dominantly decays into H?. The model can also naturally reproduce an enhanced ?? signal of the newly observed Higgs boson at the LHC with mass 125 GeV.

  20. Higgs properties in the Stealth Doublet Model

    E-print Network

    Wouda, Glenn

    2013-01-01

    I present a model with two scalar doublets and a softly broken $\\ztwo$ symmetry, where only one of the doublets gets a vacuum expectation value and couples to fermions at tree-level. The softly broken $\\ztwo$ symmetry leads to interesting phenomenology such as mixing between the two doublets and a charged scalar H^\\pm which can be light and dominantly decays into W \\gamma. The model can also naturally reproduce an enhanced \\gamma \\gamma signal of the newly observed Higgs boson at the LHC with mass 125 GeV.

  1. Mutations in the DNAH11 (axonemal heavy chain dynein type 11) gene cause one form of situs inversus totalis and most likely primary ciliary dyskinesia

    Microsoft Academic Search

    Lucia Bartoloni; Jean-Louis Blouin; Yanzhen Pan; Corinne Gehrig; Amit K. Maiti; Nathalie Scamuffa; Colette Rossier; Mark Jorissen; Miguel Armengot; Maggie Meeks; Hannah M. Mitchison; Eddie M. K. Chung; Celia D. Delozier-Blanchet; William J. Craigen; Stylianos E. Antonarakis

    2002-01-01

    Primary ciliary dyskinesia (PCD; MIM 242650) is an autosomal recessive disorder of ciliary dysfunction with extensive genetic heterogeneity. PCD is characterized by bronchiectasis and upper respiratory tract infections, and half of the patients with PCD have situs inversus (Kartagener syndrome). We characterized the transcript and the genomic organization of the axonemal heavy chain dynein type 11 (DNAH11) gene, the human

  2. 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.

  3. Higgs phenomenology in the Stealth Doublet Model

    E-print Network

    Enberg, Rikard; Wouda, Glenn

    2013-01-01

    We analyze a model for the Higgs sector with two scalar doublets and a softly broken $Z_2$ symmetry. One of the doublets breaks the electroweak symmetry and has tree-level Yukawa couplings to fermions. The other doublet has no vacuum expectation value and no tree-level couplings to fermions. Because the $Z_2$ parity is broken the two doublets can mix, which leads to a distinct and novel phenomenology. This Stealth Doublet Model can be seen as a generalization of the Inert Doublet Model with a broken $Z_2$ symmetry. We outline the model and present constraints from theory, electroweak precision tests and collider searches, including the recent observation of a Higgs boson at the LHC. The CP-odd scalar $A$ and the charged scalar $H^\\pm$ couple to fermions at one-loop level. We compute the decays of $A$ and $H^\\pm$ and in particular the one-loop decays $A \\to f \\bar{f}$, $H^\\pm \\to f \\bar{f}^\\prime $, $H^\\pm \\to W^\\pm Z $ and $H^\\pm \\to W^\\pm \\gamma$. We also describe how to calculate and renormalize such proces...

  4. Disruption of microtubules uncouples budding and nuclear division in Toxoplasma gondii

    Microsoft Academic Search

    Naomi S. Morrissette; L. David Sibley

    The tachyzoite stage of the protozoan parasite Toxoplasma gondii has two populations of microtubules: spindle microtubules and subpellicular microtubules. To determine how these two microtubule populations are regulated, we investigated microtubule behavior during the cell cycle following treatment with microtubule-disrupting drugs. Previous work had established that the microtubule populations are individually nucleated by two distinct microtubule-organizing centers (MTOCs): the apical

  5. Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning and juvenile hydrocephalus

    PubMed Central

    Appelbe, Oliver K.; Bollman, Bryan; Attarwala, Ali; Triebes, Lindy A.; Muniz-Talavera, Hilmarie; Curry, Daniel J.; Schmidt, Jennifer V.

    2013-01-01

    SUMMARY Congenital hydrocephalus, the accumulation of excess cerebrospinal fluid (CSF) in the ventricles of the brain, affects one of every 1,000 children born today, making it one of the most common human developmental disorders. Genetic causes of hydrocephalus are poorly understood in humans, but animal models suggest a broad genetic program underlying the regulation of CSF balance. In this study, the random integration of a transgene into the mouse genome led to the development of an early onset and rapidly progressive hydrocephalus. Juvenile hydrocephalus transgenic mice (JhylacZ) inherit communicating hydrocephalus in an autosomal recessive fashion with dilation of the lateral ventricles observed as early as postnatal day 1.5. Ventricular dilation increases in severity over time, becoming fatal at 4-8 weeks of age. The ependymal cilia lining the lateral ventricles are morphologically abnormal and reduced in number in JhylacZ/lacZ brains, and ultrastructural analysis revealed disorganization of the expected 9+2 microtubule pattern. Rather, the majority of JhylacZ/lacZ cilia develop axonemes with 9+0 or 8+2 microtubule structures. Disruption of an unstudied gene, 4931429I11Rik (now named Jhy) appears to underlie the hydrocephalus of JhylacZ/lacZ mice, and the Jhy transcript and protein are decreased in JhylacZ/lacZ mice. Partial phenotypic rescue was achieved in JhylacZ/lacZ mice by the introduction of a bacterial artificial chromosome (BAC) carrying 60-70% of the JHY protein coding sequence. Jhy is evolutionarily conserved from humans to basal vertebrates, but the predicted JHY protein lacks identifiable functional domains. Ongoing studies are directed at uncovering the physiological function of JHY and its role in CSF homeostasis. PMID:23906841

  6. Mitosis. Microtubule detyrosination guides chromosomes during mitosis.

    PubMed

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

    2015-05-15

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

  7. The counterbend phenomenon in flagellar axonemes and cross-linked filament bundles.

    PubMed

    Gadêlha, Hermes; Gaffney, Eamonn A; Goriely, Alain

    2013-07-23

    Recent observations of flagellar counterbend in sea urchin sperm show that the mechanical induction of curvature in one part of a passive flagellum induces a compensatory countercurvature elsewhere. This apparent paradoxical effect cannot be explained using the standard elastic rod theory of Euler and Bernoulli, or even the more general Cosserat theory of rods. Here, we develop a geometrically exact mechanical model to describe the statics of microtubule bundles that is capable of predicting the curvature reversal events observed in eukaryotic flagella. This is achieved by allowing the interaction of deformations in different material directions, by accounting not only for structural bending, but also for the elastic forces originating from the internal cross-linking mechanics. Large-amplitude static configurations can be described analytically, and an excellent match between the model and the observed counterbend deformation was found. This allowed a simultaneous estimation of multiple sperm flagellum material parameters, namely the cross-linking sliding resistance, the bending stiffness, and the sperm head junction compliance ratio. We further show that small variations on the empirical conditions may induce discrepancies for the evaluation of the flagellar material quantities, so that caution is required when interpreting experiments. Finally, our analysis demonstrates that the counterbend emerges as a fundamental property of sliding resistance in cross-linked filamentous polymer bundles, which also suggests that cross-linking proteins may contribute to the regulation of the flagellar waveform in swimming sperm via counterbend mechanics. PMID:23824293

  8. Patterning Surface-bound Microtubules through Reversible DNA Hybridization

    E-print Network

    Hancock, William O.

    Patterning Surface-bound Microtubules through Reversible DNA Hybridization Gayatri Muthukrishnan and actuators in microscale devices. Existing efforts toward harnessing kinesin motors have involved microtubule hindered by the difficulty of immobilizing patterned and aligned microtubules on surfaces. Here we show

  9. Microtubules as Sensors for Abiotic Stimuli

    Microsoft Academic Search

    Peter Nick

    Microtubules are generally perceived as structural, static elements that basically function either\\u000a as supporting scaffolds or barriers. This view has been increasingly challenged during the last decade\\u000a of the last century, when in-vivo imaging of microtubules revealed that they are endowed with complex and\\u000a highly nonlinear dynamics. This indicates that, in addition to their traditional structural functions,\\u000a microtubules must play

  10. Microtubule-targeting-dependent reorganization of filopodia.

    PubMed

    Schober, Joseph M; Komarova, Yulia A; Chaga, Oleg Y; Akhmanova, Anna; Borisy, Gary G

    2007-04-01

    Interaction between the microtubule system and actin cytoskeleton has emerged as a fundamental process required for spatial regulation of cell protrusion and retraction activities. In our current studies, analysis of digital fluorescence images revealed targeting of microtubules to filopodia in B16F1 melanoma cells and fibroblasts. We investigated the functional consequence of targeting on filopodia reorganization and examined mechanisms by which microtubules may be guided to, or interact with, filopodia. Live cell imaging studies show that targeting events in lamellipodia wings temporally correlated with filopodia turning toward the lamellipodium midline and with filopodia merging. Rapid uncoupling of targeting with nocodazole decreased filopodia merging events and increased filopodia density. Total internal reflection fluorescence microscopy identified microtubules near the ventral surface and upward movement of targeted filopodia. The role of adhesion sites and microtubule plus-end proteins in targeting was investigated. Correlation of adhesion sites with microtubule targeting to filopodia was not observed and depletion of microtubule plus-end proteins did not significantly alter targeting frequency. We propose that microtubules target filopodia, independent of focal adhesions and plus-end proteins, causing filopodia movement and microtubules regulate filopodia density in lamellipodia wings through filopodia merging events. PMID:17356063

  11. Fluorescent taxoid probes for microtubule research.

    PubMed

    Barasoain, Isabel; Díaz, J Fernando; Andreu, José M

    2010-01-01

    The use of the antitumor drug taxol as an experimental microtubule-stabilizing agent is widespread. Fluorescent taxol conjugates, although less employed, are very useful tools for several purposes in microtubule research. These include easily visualizing microtubule cytoskeletons in a variety of cells as well as in vitro assembled microtubules, studying the molecular recognition processes of taxoids by microtubules and investigating new microtubule-stabilizing agents. This chapter describes both the methods for working with fluorescent taxol conjugates and several applications employing the active fluorescent taxoids Flutax-1, Flutax-2, Hexaflutax, Rotax, and FChitax-3. These methods include visualizing microtubules in native and mildly fixed cytoskeletons from cultured cells, ciliate and flagellate protozoans and in living tumor cells, purification of tubulin from tumor cell lines and measurement of its taxoid binding capacity. The applications discussed include a homogeneous assay to screen for compounds binding the taxol site, the determination of the pathway of taxol entry into microtubules and the design of high affinity microtubule-stabilizing agents. PMID:20466144

  12. Collision induced spatial organization of microtubules

    E-print Network

    Vladimir A. Baulin; Carlos M. Marques; Fabrice Thalmann

    2006-08-31

    The dynamic behavior of microtubules in solution can be strongly modified by interactions with walls or other structures. We examine here a microtubule growth model where the increase in size of the plus-end is perturbed by collisions with other microtubules. We show that such a simple mechanism of constrained growth can induce ordered structures and patterns from an initially isotropic and homogeneous suspension. First, microtubules self-organize locally in randomly oriented domains that grow and compete with each other. By imposing even a weak orientation bias, external forces like gravity or cellular boundaries may bias the domain distribution eventually leading to a macroscopic sample orientation.

  13. Higgs phenomenology in the stealth doublet model

    NASA Astrophysics Data System (ADS)

    Enberg, Rikard; Rathsman, Johan; Wouda, Glenn

    2015-05-01

    We analyze a model for the Higgs sector with two scalar doublets and a Z2 symmetry that is manifest in the Yukawa sector but broken in the potential. Thus, one of the doublets breaks the electroweak symmetry and has tree-level Yukawa couplings to fermions, whereas the other doublet has no vacuum expectation value and no tree-level couplings to fermions. Since the Z2 parity is broken the two doublets can mix, which leads to a distinct and novel phenomenology. This stealth doublet model can be seen as a generalization of the inert doublet model with a broken Z2 symmetry. We outline the model and present constraints from theory, electroweak precision tests, and collider searches, including the recent observation of a Higgs boson at the LHC. The charged scalar H± and the C P -odd scalar A couple to fermions at one-loop level. We compute the decays of H± and A and in particular the one-loop decays A ?f f ¯ , H±?f f¯ ' , H±?W±Z and H±?W±? . We also describe how to calculate and renormalize such processes in our model. We find that if one of H± or A is the lightest scalar, H±?W±? or A ?b b ¯ are typically their respective dominating decay channels. Otherwise, the dominating decays of H± and A are into a scalar and a vector. Due to the absence of tree-level fermion couplings for H± and A , we consider pair production and associated production with vector bosons and scalars at the LHC. If the parameter space of the model that favors H±?W±? is realized in Nature, we estimate that there could be a considerable amount of such events in the present LHC data.

  14. Microtubule-targeted anticancer agents and apoptosis

    Microsoft Academic Search

    Kapil N Bhalla

    2003-01-01

    Over the past decade, significant progress has been made in our understanding of the biology of microtubule (MT) assembly into the mitotic spindle during mitosis and the molecular signaling and execution of the various pathways to apoptosis. In the same period, the microtubule-targeted tubulin-polymerizing agents (MTPAs), notably paclitaxel and taxotere, have come to occupy a central role in the treatment

  15. 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.

  16. Brief Communications Microtubules in Dendritic Spine Development

    E-print Network

    Brief Communications Microtubules in Dendritic Spine Development Jiaping Gu,1 Bonnie L. Firestein,2 generallybelievedthatonlytheactincytoskeletonresidesindendriticspinesandcontrolsspinemorphologyandplasticity.Here,we report that microtubules (MTs) are present in spines and that shRNA knockdown of the MT plus-end-binding protein EB3 significantly reduces spine formation. Furthermore, stabilization

  17. A soluble adenylyl cyclase form targets to axonemes and rescues beat regulation in soluble adenylyl cyclase knockout mice.

    PubMed

    Chen, Xi; Baumlin, Nathalie; Buck, Jochen; Levin, Lonny R; Fregien, Nevis; Salathe, Matthias

    2014-12-01

    Ciliary beating is important for effective mucociliary clearance. Soluble adenylyl cyclase (sAC) regulates ciliary beating, and a roughly 50-kD sAC variant is expressed in axonemes. Normal human bronchial epithelial (NHBE) cells express multiple sAC splice variants: full-length sAC; variants with catalytic domain 1 (C1) deletions; and variants with partial C1. One variant, sACex5v2-ex12v2, contains two alternative splices creating new exons 5 (ex5v2) and 12 (ex12v2), encoding a roughly 45-kD protein. It is therefore similar in size to ciliary sAC. The variant increases in expression upon ciliogenesis during differentiation at the air-liquid interface. When expressed in NHBE cells, this variant was targeted to cilia. Exons 5v2-7 were important for ciliary targeting, whereas exons 2-4 prevented it. In vitro, cytoplasmic sACex2-ex12v2 (containing C1 and C2) was the only variant producing cAMP. Ciliary sACex5v2-ex12v2 was not catalytically active. Airway epithelial cells isolated from wild-type mice revealed sAC-dependent ciliary beat frequency (CBF) regulation, analogous to NHBE cells: CBF rescue from HCO3(-)/CO2-mediated intracellular acidification was sensitive to the sAC inhibitor, KH7. Compared with wild type, sAC C2 knockout (KO) mice revealed lower CBF baseline, and the HCO3(-)/CO2-mediated CBF decrease was not inhibited by KH7, confirming lack of functional sAC. Human sACex5v2-ex12v2 was targeted to cilia and sACex2-ex12v2 to the cytoplasm in these KO mice. Introduction of the ciliary sACex5v2-ex12v2 variant, but not the cytoplasmic sACex2-ex12v2, restored functional sAC activity in C2 KO mice. Thus, we show, for the first time, a mammalian axonemal targeting sequence that localizes a sAC variant to cilia to regulate CBF. PMID:24874272

  18. Nucleation and Transport Organize Microtubules in Metaphase Spindles

    E-print Network

    Needleman, Daniel

    Nucleation and Transport Organize Microtubules in Metaphase Spindles Jan Brugue´ s,1,2,* Valeria@fas.harvard.edu DOI 10.1016/j.cell.2012.03.027 SUMMARY Spindles are arrays of microtubules that segregate chromosomes microtubule polymeriza- tion dynamics are not spatially regulated, and the proper organization of microtubules

  19. 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.

  20. The Two-Higgs-Doublet Model: Past, Present and Future

    E-print Network

    California at Santa Cruz, University of

    The Two-Higgs-Doublet Model: Past, Present and Future Howard E. Haber 24 October 2008 Augusto Barroso Fest #12;Outline · A (biased) history of the two-Higgs-doublet model (2HDM) · The paradox of tan · The general Two-Higgs-Doublet Model ­ Basis-independent techniques ­ Basis-independent (invariant) form

  1. Model based dynamics analysis in live cell microtubule images

    PubMed Central

    Alt?nok, Alphan; Kiris, Erkan; Peck, Austin J; Feinstein, Stuart C; Wilson, Leslie; Manjunath, BS; Rose, Kenneth

    2007-01-01

    Background The dynamic growing and shortening behaviors of microtubules are central to the fundamental roles played by microtubules in essentially all eukaryotic cells. Traditionally, microtubule behavior is quantified by manually tracking individual microtubules in time-lapse images under various experimental conditions. Manual analysis is laborious, approximate, and often offers limited analytical capability in extracting potentially valuable information from the data. Results In this work, we present computer vision and machine-learning based methods for extracting novel dynamics information from time-lapse images. Using actual microtubule data, we estimate statistical models of microtubule behavior that are highly effective in identifying common and distinct characteristics of microtubule dynamic behavior. Conclusion Computational methods provide powerful analytical capabilities in addition to traditional analysis methods for studying microtubule dynamic behavior. Novel capabilities, such as building and querying microtubule image databases, are introduced to quantify and analyze microtubule dynamic behavior. PMID:17634094

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

    SciTech Connect

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

    2010-09-03

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

  3. Regulati on of Microtubule Dynamics by Protein: Interacti on Networks at Microtubule Tips

    Microsoft Academic Search

    Vaart van der B

    2011-01-01

    Microtubules are cytoskeletal fi laments, which play essenti al roles in cell division, morphology,\\u000amigrati on and organizati on of intracellular organelles. Many of these functi ons are regulated by\\u000athe associati on of microtubule plus ends with a group of structurally diverse and unrelated proteins\\u000a- the microtubule plus-end tracking proteins (+TIPs). This thesis describes how +TIPs infl uence

  4. The acetylation of alpha-tubulin and its relationship to the assembly and disassembly of microtubules

    Microsoft Academic Search

    Hiroshi Maruta; Karen Greet; Joel L. Rosenbaum

    1986-01-01

    A tight association between Chlamydo- monas alpha-tubulin acetyltransferase (TAT) and flagel- lar axonemes, and the cytoplasmic localization of both tubulin deacetylase (TDA) and an inhibitor of tubulin acetylation have been demonstrated by the use of calf brain tubulin as substrate for these enzymes. A major axonemal TAT of 130 kD has been solubilized by high salt treatment, purified, and characterized.

  5. Higgs phenomenology in the Stealth Doublet Model

    E-print Network

    Rikard Enberg; Johan Rathsman; Glenn Wouda

    2015-04-09

    We analyze a model for the Higgs sector with two scalar doublets and a $Z_2$ symmetry that is manifest in the Yukawa sector but broken in the potential. Thus, one of the doublets breaks the electroweak symmetry and has tree-level Yukawa couplings to fermions, whereas the other doublet has no vacuum expectation value and no tree-level couplings to fermions. Since the $Z_2$ parity is broken the two doublets can mix, which leads to a distinct and novel phenomenology. This Stealth Doublet Model can be seen as a generalization of the Inert Doublet Model with a broken $Z_2$ symmetry. We outline the model and present constraints from theory, electroweak precision tests and collider searches, including the recent observation of a Higgs boson at the LHC. The charged scalar $H^\\pm$ and the CP-odd scalar $A$ couple to fermions at one-loop level. We compute the decays of $H^\\pm$ and $A$ and in particular the one-loop decays $A \\to f \\bar{f}$, $H^\\pm \\to f \\bar{f}^\\prime $, $H^\\pm \\to W^\\pm Z $ and $H^\\pm \\to W^\\pm \\gamma$. We also describe how to calculate and renormalize such processes in our model. We find that if one of $H^\\pm$ or $A$ is the lightest scalar, $H^\\pm \\to W^\\pm \\gamma$ or $ A \\to b \\bar{b} $ are typically their respective dominating decay channels. Otherwise, the dominating decays of $H^\\pm$ and $A$ are into a scalar and a vector. Due to the absence of tree-level fermion couplings for $H^\\pm$ and $A$, we consider pair production and associated production with vector bosons and scalars at the LHC. If the parameter space of the model that favors $H^\\pm \\to W^\\pm \\gamma$ is realized in Nature, we estimate that there could be a considerable amount of such events in the present LHC data.

  6. Microtubules: Montroll's kink and Morse vibrations

    E-print Network

    H. C. Rosu

    1997-03-20

    Using a version of Witten's supersymmetric quantum mechanics proposed by Caticha, we relate Montroll's kink to a traveling, asymmetric Morse double-well potential suggesting in this way a connection between kink modes and vibrational degrees of freedom along microtubules

  7. Buckling and force propagation along intracellular microtubules

    E-print Network

    Moumita Das; Alex J. Levine; F. C. MacKintosh

    2008-07-22

    Motivated by recent experiments showing the buckling of microtubules in cells, we study theoretically the mechanical response of, and force propagation along elastic filaments embedded in a non-linear elastic medium. We find that, although embedded microtubules still buckle when their compressive load exceeds the critical value $f_c$ found earlier, the resulting deformation is restricted to a penetration depth that depends on both the non-linear material properties of the surrounding cytoskeleton, as well as the direct coupling of the microtubule to the cytoskeleton. The deformation amplitude depends on the applied load $f$ as $(f- f_c)^{1/2}$. This work shows how the range of compressive force transmission by microtubules can be as large as tens of microns and is governed by the mechanical coupling to the surrounding cytoskeleton.

  8. Microtubule dynamics in the peripheral nervous system

    PubMed Central

    Almeida-Souza, Leonardo

    2011-01-01

    The special architecture of neurons in the peripheral nervous system, with axons extending for long distances, represents a major challenge for the intracellular transport system. Two recent studies show that mutations in the small heat shock protein HSPB1, which cause an axonal type of Charcot-Marie-Tooth (CMT) neuropathy, affect microtubule dynamics and impede axonal transport. Intriguingly, while at presymptomatic age the neurons in the mutant HSPB1 mouse show a hyperstable microtubule network, at postsymptomatic age, the microtubule network completely lost its stability as reflected by a marked decrease in tubulin acetylation levels. We here propose a model explaining the role of microtubule stabilization and tubulin acetylation in the pathogenesis of HSPB1 mutations. PMID:22545178

  9. 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

  10. Emerging microtubule targets in glioma therapy.

    PubMed

    Katsetos, Christos D; Reginato, Mauricio J; Baas, Peter W; D'Agostino, Luca; Legido, Agustin; Tuszyn Ski, Jack A; Dráberová, Eduarda; Dráber, Pavel

    2015-03-01

    Major advances in the genomics and epigenomics of diffuse gliomas and glioblastoma to date have not been translated into effective therapy, necessitating pursuit of alternative treatment approaches for these therapeutically challenging tumors. Current knowledge of microtubules in cancer and the development of new microtubule-based treatment strategies for high-grade gliomas are the topic in this review article. Discussed are cellular, molecular, and pharmacologic aspects of the microtubule cytoskeleton underlying mitosis and interactions with other cellular partners involved in cell cycle progression, directional cell migration, and tumor invasion. Special focus is placed on (1) the aberrant overexpression of ?III-tubulin, a survival factor associated with hypoxic tumor microenvironment and dynamic instability of microtubules; (2) the ectopic overexpression of ?-tubulin, which in addition to its conventional role as a microtubule-nucleating protein has recently emerged as a transcription factor interacting with oncogenes and kinases; (3) the microtubule-severing ATPase spastin and its emerging role in cell motility of glioblastoma cells; and (4) the modulating role of posttranslational modifications of tubulin in the context of interaction of microtubules with motor proteins. Specific antineoplastic strategies discussed include downregulation of targeted molecules aimed at achieving a sensitization effect on currently used mainstay therapies. The potential role of new classes of tubulin-binding agents and ATPase inhibitors is also examined. Understanding the cellular and molecular mechanisms underpinning the distinct behaviors of microtubules in glioma tumorigenesis and drug resistance is key to the discovery of novel molecular targets that will fundamentally change the prognostic outlook of patients with diffuse high-grade gliomas. PMID:25976261

  11. Resistance to Microtubule-Targeting Drugs

    Microsoft Academic Search

    Paraskevi Giannakakou; James P. Snyder

    As essential components of cell shape, signaling, movement and division, microtubules (MTs) are crucial to normal cellular\\u000a functions and survival. Beginning with vincristine in the 1950’s numerous agents targeting the microtubules have been identified\\u000a and developed as anti-cancer agents. Their use in the clinic has at times led to complete regression of tumors, but unfortunately\\u000a in too many cases, regressions

  12. The role of dynamic instability in microtubule organization

    PubMed Central

    Horio, Tetsuya; Murata, Takashi

    2014-01-01

    Microtubules are one of the three major cytoskeletal components in eukaryotic cells. Heterodimers composed of GTP-bound ?- and ?-tubulin molecules polymerize to form microtubule protofilaments, which associate laterally to form a hollow microtubule. Tubulin has GTPase activity and the GTP molecules associated with ?-tubulin molecules are hydrolyzed shortly after being incorporated into the polymerizing microtubules. GTP hydrolysis alters the conformation of the tubulin molecules and drives the dynamic behavior of microtubules. Periods of rapid microtubule polymerization alternate with periods of shrinkage in a process known as dynamic instability. In plants, dynamic instability plays a key role in determining the organization of microtubules into arrays, and these arrays vary throughout the cell cycle. In this review, we describe the mechanisms that regulate microtubule dynamics and underlie dynamic instability, and discuss how dynamic instability may shape microtubule organization in plant cells. PMID:25339962

  13. Harnessing microtubule dynamic instability for nanostructure assembly.

    SciTech Connect

    Bouchard, Ann Marie; Osbourn, Gordon Cecil

    2004-06-01

    Intracellular molecular machines synthesize molecules, tear apart others, transport materials, transform energy into different forms, and carry out a host of other coordinated processes. Many molecular processes have been shown to work outside of cells, and the idea of harnessing these molecular machines to build nanostructures is attractive. Two examples are microtubules and motor proteins, which aid cell movement, help determine cell shape and internal structure, and transport vesicles and organelles within the cell. These molecular machines work in a stochastic, noisy fashion: microtubules switch randomly between growing and shrinking in a process known as dynamic instability; motor protein movement along microtubules is randomly interrupted by the motor proteins falling off. A common strategy in attempting to gain control over these highly dynamic, stochastic processes is to eliminate some processes (e.g., work with stabilized microtubules) in order to focus on others (interaction of microtubules with motor proteins). In this paper, we illustrate a different strategy for building nanostructures, which, rather than attempting to control or eliminate some dynamic processes, uses them to advantage in building nanostructures. Specifically, using stochastic agent-based simulations, we show how the natural dynamic instability of microtubules can be harnessed in building nanostructures, and discuss strategies for ensuring that 'unreliable' stochastic processes yield a robust outcome.

  14. 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.

  15. Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors.

    PubMed

    Fokin, A I; Brodsky, I B; Burakov, A V; Nadezhdina, E S

    2014-09-01

    This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles - transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting ?-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles. PMID:25385016

  16. Microtubule polarities indicate that nucleation and capture of microtubules occurs at cell surfaces in Drosophila

    PubMed Central

    1989-01-01

    Hook decoration with pig brain tubulin was used to assess the polarity of microtubules which mainly have 15 protofilaments in the transcellular bundles of late pupal Drosophila wing epidermal cells. The microtubules make end-on contact with cell surfaces. Most microtubules in each bundle exhibited a uniform polarity. They were oriented with their minus ends associated with their hemidesmosomal anchorage points at the apical cuticle-secreting surfaces of the cells. Plus ends were directed towards, and were sometimes connected to, basal attachment desmosomes at the opposite ends of the cells. The orientation of microtubules at cell apices, with minus ends directed towards the cell surface, is opposite to the polarity anticipated for microtubules which have elongated centrifugally from centrosomes. It is consistent, however, with evidence that microtubule assembly is nucleated by plasma membrane-associated sites at the apical surfaces of the cells (Mogensen, M. M., and J. B. Tucker. 1987. J. Cell Sci. 88:95- 107) after these cells have lost their centriole-containing, centrosomal, microtubule-organizing centers (Tucker, J. B., M. J. Milner, D. A. Currie, J. W. Muir, D. A. Forrest, and M.-J. Spencer. 1986. Eur. J. Cell Biol. 41:279-289). Our findings indicate that the plus ends of many of these apically nucleated microtubules are captured by the basal desmosomes. Hence, the situation may be analogous to the polar-nucleation/chromosomal-capture scheme for kinetochore microtubule assembly in mitotic and meiotic spindles. The cell surface-associated nucleation-elongation-capture mechanism proposed here may also apply during assembly of transcellular microtubule arrays in certain other animal tissue cell types. PMID:2925791

  17. 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

  18. [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

  19. 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

  20. Single file diffusion in microtubules

    NASA Astrophysics Data System (ADS)

    Rutenberg, Andrew; Farrell, Spencer; Brown, Aidan

    2015-03-01

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

  1. 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

  2. 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

  3. Reconstituting functional microtubule-barrier interactions.

    PubMed

    Taberner, Núria; Weber, Georges; You, Changjiang; Dries, Roland; Piehler, Jacob; Dogterom, Marileen

    2014-01-01

    Local interactions between the tips of microtubules and the cell cortex, or other cellular components such as kinetochores, play an important role in essential cellular processes like establishing cell polarity, distribution of organelles, and microtubule aster and chromosome positioning. Here we present two in vitro assays that specifically mimic microtubule-cortex interactions by employing selectively functionalized microfabricated barriers that allow for the immobilization of proteins with a range of affinities. We describe the microfabrication process to create gold or glass barriers and the subsequent functionalization of these barriers using self-assembled thiol monolayers or polylysine-poly(ethylene glycol), respectively. Near-permanent attachment of proteins is obtained using biotinylated surfaces combined with streptavidin and biotinylated proteins. Lower affinity interactions, further tunable with the addition of imidazole, are obtained using nickel-nitrilotriacetic acid (Ni(II)-NTA) functionalization combined with his-tagged proteins. Both mono-NTA and tris-NTA compounds are used. We show an assay to reconstitute the "end-on" interaction between dynamic microtubule tips and barrier-attached dynein, mimicking the cellular situation at the cortex and at kinetochores. In a second assay, we reconstitute microtubule-based delivery of end-tracking proteins to functionalized barriers, mimicking the transport of cell-end markers to the cell poles in interphase fission yeast cells. PMID:24484658

  4. 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.

  5. Dark energy via multi-Higgs doublet models: accelerated expansion of the Universe in inert doublet model scenario

    E-print Network

    Usman, Muhammad

    2015-01-01

    Scalar fields are among the possible candidates for dark energy. This paper is devoted to the scalar fields from the inert doublet model, where instead of one as in the standard model, two SU(2) Higgs doublets are used. The component fields of one SU(2) doublet ($\\phi_1$) act in an identical way to the standard model Higgs while the component fields of the second SU(2) doublet ($\\phi_2$) are taken to be the dark energy candidate (which is done by assuming that the phase transition in the field has not yet occurred). It is found that one can arrange for late time acceleration (dark energy) by using an SU(2) Higgs doublet in the inert Higgs doublet model, whose vacuum expectation value is zero, in the quintessential regime.

  6. CLIP170 Highlights Growing Microtubule Ends In Vivo

    Microsoft Academic Search

    Franck Perez; Georgios S. Diamantopoulos; Romaine Stalder; Thomas E. Kreis

    1999-01-01

    A chimera with the green fluorescent protein (GFP) has been constructed to visualize the dynamic properties of the endosome-microtubule linker protein CLIP-170 (GFP-CLIP170). GFP-CLIP170 binds in stretches along a subset of microtubule ends. These fluorescent stretches appear to move with the growing tips of microtubules at 0.15–0.4 ?m\\/s, comparable to microtubule elongation in vivo. Analysis of speckles along dynamic GFP-CLIP170

  7. Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium

    PubMed Central

    1985-01-01

    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 approximately 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 well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered. PMID:4055902

  8. Yukawa textures or dark doublets from Two Higgs Doublet Models with $Z_3$ symmetry

    E-print Network

    Aranda, Alfredo; Noriega-Papaqui, Roberto; Vaquera-Araujo, Carlos A

    2014-01-01

    The effect of $Z_3$ symmetry on the general Two Higgs Doublet Model is explored. Of particular interest is the question of what can a $Z_3$ symmetry do beyond the usual case with $Z_2$. There are two independent scenarios that give some interesting results: first, by giving non-trivial charges to the Standard Model fermions, it is possible to use the $Z_3$ symmetry of the scalar potential to generate potentially useful Yukawa textures. This is not possible with $Z_2$. A series of possibilities is presented where their viability is addressed and a specific example in the quark sector is given for concreteness. The second venue of interest is in the area of inert doublets. It is shown that by considering the Standard Model plus two additional inert doublet scalars, i.e. a Dark Two Higgs Doublet Model, together with $Z_3$, a scenario can be obtained that differs from the $Z_2$ case. Some general comments are presented on the potentially interesting phenomenology of such model.

  9. Méthode de calcul des antennes linéaires symétriques. applications au doublet, au doublet replié, a l’Antenne Yagi

    Microsoft Academic Search

    Louis Guillou; Jean-Pierre Daniel; Gérard Dubost

    1974-01-01

    \\u000a Résumé  Une étude théorique sur le doublet a amené les auteurs à élaborer une méthode numérique spécifique aux éléments rayonnants\\u000a linéaires symétriques. Des confrontations avec l’expérience sur les cas bien connus du doublet de longueur moyenne et du doublet\\u000a replié sont suivies d’une application aux antennes de type « Yagi ».

  10. Molecular Cell Structural Basis of Microtubule Plus End

    E-print Network

    Vale, Ronald D.

    Molecular Cell Article Structural Basis of Microtubule Plus End Tracking by XMAP215, CLIP-170: vale@cmp.ucsf.edu DOI 10.1016/j.molcel.2007.07.023 SUMMARY Microtubule plus end binding proteins (+TIPs) localize to the dynamic plus ends of microtu- bules, where they stimulate microtubule growth and recruit

  11. Extracting the Mechanical Properties of Microtubules from Thermal Fluctuation

    E-print Network

    Texas at Austin. University of

    CHAPTER 30 Extracting the Mechanical Properties of Microtubules from Thermal Fluctuation properties of microtubules have been the subject of intense study during recent decades because of their importance to the many cell functions that they are involved in. Observations of microtubule thermal

  12. Introduction Dynamic polymers known as microtubules (MTs) provide lines

    E-print Network

    Mogilner, Alex

    Introduction Dynamic polymers known as microtubules (MTs) provide lines of transport, communication ends of the other MTs 1381 Polar arrays of microtubules play many important roles in the cell. Normally, such arrays are organized by a centrosome anchoring the minus ends of the microtubules, while the plus ends

  13. Microtubule transport DOI: 10.1002/smll.200600410

    E-print Network

    Hancock, William O.

    Microtubule transport DOI: 10.1002/smll.200600410 Directing Transport of CoFe2O4-Functionalized Microtubules with Magnetic Fields** Benjamin M. Hutchins, Mark Platt, William O. Hancock,* and Mary Elizabeth microtubule tracks, which makes this motor an attrac- tive candidate for actuation and transport in hybrid

  14. Microtubules and vesicles under controlled tension D. Kuchnir Fygenson,1

    E-print Network

    Fygenson, Deborah Kuchnir

    Microtubules and vesicles under controlled tension D. Kuchnir Fygenson,1 M. Elbaum,2, * B. Shraiman Microtubules trapped inside cell-sized vesicles 10- m diameter define shapes reminiscent of living cells. In this work, three configurations are studied: an individual microtubule, a linear bundle, and a circular band

  15. Microtubules cut and run Peter W. Baas1

    E-print Network

    Baas, Peter W.

    Microtubules cut and run Peter W. Baas1 , Arzu Karabay1,2 and Liang Qiang1 1 Department that cells reconfigure their microtubules through rapid bouts of assembly and disassembly, as described by the mechanism known as dynamic instability. However, many cell types have complex patterns of microtubule

  16. Three fundamental cytoskeletal filaments Actin Microtubules (MT) Intermediate Filaments (IF)

    E-print Network

    Sniadecki, Nathan J.

    Session 4 #12;2 Three fundamental cytoskeletal filaments Actin Microtubules (MT) Intermediate;14 #12;15 Intermediate filaments (blue), plectin (green), Microtubules (red), anti-plectin labelled gold caps filament plus ends Latrunculin binds subunits and prevents their polymerization MICROTUBULE

  17. GDP-TUBULIN INCORPORATION INTO GROWING MICROTUBULES MODULATES POLYMER STABILITY*

    E-print Network

    Boyer, Edmond

    GDP-TUBULIN INCORPORATION INTO GROWING MICROTUBULES MODULATES POLYMER STABILITY* Odile VALIRON1, France and 3 From July 2010 1st, same adress as 1 Running head: GDP-tubulin incorporation in microtubules 05 37. Fax: (+33) 4 56 52 06 57. E-mail: odile.valiron@ujf-grenoble.fr Microtubule growth proceeds

  18. Critical Roles for Microtubules in Axonal Development and Disease

    E-print Network

    Baas, Peter W.

    Critical Roles for Microtubules in Axonal Development and Disease Aditi Falnikar and Peter W. Baas Abstract Axons are occupied by dense arrays of cytoskeletal elements called microtubules, which for the transport of organelles in both directions within the axon. Microtubules are organized and regulated

  19. A Mechanochemical Model Explains Interactions between Cortical Microtubules in Plants

    E-print Network

    Allard, Jun

    A Mechanochemical Model Explains Interactions between Cortical Microtubules in Plants Jun F. Allard, British Columbia, Canada ABSTRACT Microtubules anchored to the two-dimensional cortex of plant cells cortical microtubules arrays, which is required for proper cell wall growth. Although the cell-wide self

  20. Rles indirects des microtubules dans la morphogense nuclaire des spermatides

    E-print Network

    Paris-Sud XI, Université de

    Rôles indirects des microtubules dans la morphogenèse nucléaire des spermatides J.-L. COURTENS. Summary. The indirect roles of microtubules in the nuclear morphogenesis of spermatids. The depolymerization of the microtubules of the spermatid manchette was effective for 4 to 5 h in rat, starting 30 min

  1. TRACING MICROTUBULES IN LIVE CELL IMAGES M. E. Sargin1

    E-print Network

    California at Santa Barbara, University of

    TRACING MICROTUBULES IN LIVE CELL IMAGES M. E. Sargin1 , A. Altinok1 , E. Kiris2 , S. C. Feinstein2 93106 {msargin,alphan}@ece.ucsb.edu ABSTRACT Microtubule (MT) dynamics are traditionally analyzed from-- Biomedical image processing, Image line pattern analysis, Object detection 1. INTRODUCTION Microtubules (MTs

  2. Effects of Dynactin Disruption and Dynein Depletion on Axonal Microtubules

    E-print Network

    Baas, Peter W.

    Effects of Dynactin Disruption and Dynein Depletion on Axonal Microtubules Fridoon J. Ahmad1,2,a:PeterW.Baas,pbaas@drexelmed.edu We investigated potential roles of cytoplasmic dynein in organizing axonal microtubules either if the long microtubules in the axon also undergo dynein-dependent transport, we ascertained the rates of EGFP

  3. 2006 Nature Publishing Group Assembly dynamics of microtubules at

    E-print Network

    Perkins, Thomas

    © 2006 Nature Publishing Group Assembly dynamics of microtubules at molecular resolution Jacob W. J Dogterom1 Microtubules are highly dynamic protein polymers1 that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers

  4. Electrical Docking of Microtubules for Kinesin-Driven Motility in

    E-print Network

    Dekker, Cees

    Electrical Docking of Microtubules for Kinesin-Driven Motility in Nanostructures Martin G. L. van demonstrate localized electrical control of the docking of microtubules onto engineered kinesin-coated structures. After applying a voltage to a gold electrode, we observe an enhanced transport of microtubules

  5. Microtubule Reconfiguration during Axonal Retraction Induced by Nitric Oxide

    E-print Network

    Baas, Peter W.

    Microtubule Reconfiguration during Axonal Retraction Induced by Nitric Oxide Yan He, Wenqian Yu study, we sought to test this hypothesis with regard to microtubules. When a donor of nitric oxide that actually caused microtubule levels to increase. The retractions induced by nitric oxide were remarkably

  6. Optical Control of Microtubule Dynamics in Time and Space.

    PubMed

    Castle, Brian T; Odde, David J

    2015-07-16

    Small molecule inhibitors of microtubule dynamics are widely used as cell biology research tools and clinically as cancer chemotherapeutics. By slight modification to the chemical structure of a known microtubule inhibitor, combretastatin A-4, Borowiak et al. develop a photoswitchable derivative that can be turned "on" and "off" with low-intensity light to spatially and temporally control microtubule dynamics. PMID:26186185

  7. Microtubule-binding agents: a dynamic field of cancer therapeutics

    Microsoft Academic Search

    Mary Ann Jordan; Charles Dumontet

    2010-01-01

    Microtubules are dynamic filamentous cytoskeletal proteins composed of tubulin and are an important therapeutic target in tumour cells. Agents that bind to microtubules have been part of the pharmacopoeia of anticancer therapy for decades and until the advent of targeted therapy, microtubules were the only alternative to DNA as a therapeutic target in cancer. The screening of a range of

  8. Effect of microtubule polymerization on photoinduced hydrogen generation.

    PubMed

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

    2015-07-25

    Herein we report a novel reaction field for photoinduced H2 generation by using microtubules as a medium. By controlling the tubulin/microtubule hierarchical structure, synergistic effects by which the Ru(bpy)3(2+)-conjugated microtubule network causes suppression of energy loss by collision are clarified. PMID:26097911

  9. Molecular motor driven transportation on microtubule loops

    NASA Astrophysics Data System (ADS)

    Sikora, Aurelien; Federici, Filippo; Kim, Kyongwan; Nakazawa, Hikaru; Umetsu, Mitsuo; Hwang, Wonmuk; Teizer, Winfried

    2015-03-01

    Molecular motors such as kinesin are naturally fitted for the transport of cargo. By offering an unlimited path, microtubule loops allow the study of kinesin motility on distances exceeding that offered by a single microtubule. Moreover, the periodicity of the path allows the comparisons of trajectories between laps. Here we study the motility of quantum dot labeled kinesin on microtubule loops. Motility of kinesins over multiple laps is observed and their trajectories are extracted from kymograph using a custom algorithm. Distribution of velocities at given locations do not vary randomly but show a correlation with the presence of obstacles. Possible mechanisms responsible for the long range transport are discussed in the context of available theories.

  10. MARK, a Novel Family of Protein Kinases That Phosphorylate Microtubule-Associated Proteins and Trigger Microtubule Disruption

    Microsoft Academic Search

    Gerard Drewes; Andreas Ebneth; Ute Preuss; Eva-Maria Mandelkow; Eckhard Mandelkow

    1997-01-01

    MARK phosphorylates the microtubule-associated proteins tau, MAP2, and MAP4 on their microtubule-binding domain, causing their dissociation from microtubules and increased microtubule dynamics. We describe the molecular cloning, distribution, activation mechanism, and overexpression of two MARK proteins from rat that arise from distinct genes. They encode Ser\\/Thr kinases of 88 and 81 kDa, respectively, and show similarity to the yeast kin1+

  11. Interactions between Adaptor Protein-1 of the Clathrin Coat and Microtubules via Type 1a Microtubule-associated Proteins*

    E-print Network

    Lebendiker, Mario

    Interactions between Adaptor Protein-1 of the Clathrin Coat and Microtubules via Type 1a Microtubule-associated Proteins* Received for publication, February 4, 2001, and in revised form, May 1, 2001 1 (AP-1) adaptor of the trans-Golgi network clathrin inter- acts with microtubules. AP-1

  12. Binary Asteroids and the Formation of Doublet Craters

    Microsoft Academic Search

    William F. Bottke Jr.; H. Jay Melosh

    1996-01-01

    At least 10% (3 out of 28) of the largest known impact craters on Earth and a similar fraction of all impact structures on Venus are doublets (i.e., have a companion crater nearby), formed by the nearly simultaneous impact of objects of comparable size. Mars also has doublet craters, though the fraction found there is smaller (2%). These craters are

  13. Doublets in Arabic: Notes towards a Diachronic Phonological Study.

    ERIC Educational Resources Information Center

    Mahadin, Radwan S.

    1989-01-01

    Examines doublets in Arabic, discussing the alterations between the determinants in the doublets, and shows that the alterations are the result of phonological changes. It is concluded that the phonological changes are in agreement with changes that have occurred in other Semitic languages and in modern Arabic dialects. (30 references) (Author/VWL)

  14. Proto-Algic V: Doublets and Their Implications.

    ERIC Educational Resources Information Center

    Proulx, Paul

    1994-01-01

    This paper examines the reconstruction of doublets in the Proto-Algic Indian language. These doublets suggest dialect mixing before the breakup of Proto-Algic society, with frequent elements commonly manifesting the prestige-dialect innovations. An extensive Proto-Algic vocabulary is included. Two appendixes explain new or significantly revised…

  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. Integrin-Linked Kinase Regulates Interphase and Mitotic Microtubule Dynamics

    PubMed Central

    Lim, Simin; Kawamura, Eiko; Fielding, Andrew B.; Maydan, Mykola; Dedhar, Shoukat

    2013-01-01

    Integrin-linked kinase (ILK) localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells. PMID:23349730

  17. Three-dimensional structure of cytoplasmic dynein bound to microtubules

    PubMed Central

    Mizuno, Naoko; Narita, Akihiro; Kon, Takahide; Sutoh, Kazuo; Kikkawa, Masahide

    2007-01-01

    Cytoplasmic dynein is a large, microtubule-dependent molecular motor (1.2 MDa). Although the structure of dynein by itself has been characterized, its conformation in complex with microtubules is still unknown. Here, we used cryoelectron microscopy (cryo-EM) to visualize the interaction between dynein and microtubules. Most dynein molecules in the nucleotide-free state are bound to the microtubule in a defined conformation and orientation. A 3D image reconstruction revealed that dynein's head domain, formed by a ring-like arrangement of AAA+ domains, is located ?280 ? away from the center of the microtubule. The order of the AAA+ domains in the ring was determined by using recombinant markers. Furthermore, a 3D helical image reconstruction of microtubules with a dynein's microtubule binding domain [dynein stalk (DS)] revealed that the stalk extends perpendicular to the microtubule. By combining the 3D maps of the dynein-microtubule and DS-microtubule complexes, we present a model for how dynein in the nucleotide-free state binds to microtubules and discuss models for dynein's power stroke. PMID:18093913

  18. The role of microtubule movement in bidirectional organelle transport

    E-print Network

    Igor M. Kuli?; André E. X. Brown; Hwajin Kim; Comert Kural; Benjamin Blehm; Paul R. Selvin; Philip C. Nelson; Vladimir I. Gelfand

    2008-08-12

    We study the role of microtubule movement in bidirectional organelle transport in Drosophila S2 cells and show that EGFP-tagged peroxisomes in cells serve as sensitive probes of motor induced, noisy cytoskeletal motions. Multiple peroxisomes move in unison over large time windows and show correlations with microtubule tip positions, indicating rapid microtubule fluctuations in the longitudinal direction. We report the first high-resolution measurement of longitudinal microtubule fluctuations performed by tracing such pairs of co-moving peroxisomes. The resulting picture shows that motor-dependent longitudinal microtubule oscillations contribute significantly to cargo movement along microtubules. Thus, contrary to the conventional view, organelle transport cannot be described solely in terms of cargo movement along stationary microtubule tracks, but instead includes a strong contribution from the movement of the tracks.

  19. Isolation and analysis of microtubules and associated proteins.

    PubMed

    Sloboda, Roger D

    2015-02-01

    Microtubules, microtubule-associated proteins (MAPs), and motor proteins are essential components of all eukaryotic cells. They are all involved in mitosis and in the movement of organelles, proteins, and vesicles in cells. MAPs act as structural elements of the microtubule component of the cytoskeleton, whereas molecular motors propel cargo along microtubule tracks or translocate microtubules in the cytoplasm. This introduction provides an overview of procedures developed by many labs to isolate microtubules from cell homogenates, purify tubulin, MAPs, and motor proteins from microtubules preparations, and analyze kinesin and cytoplasmic dynein activity by video-enhanced differential interference contrast microscopy and fluorescence microscopy. These ingenious microscope-based assays, which were developed to determine the motility characteristics of kinesin and dynein, reveal, in clear and dramatic fashion, the activity of these amazing nanomachines in real time. PMID:25646506

  20. Cep70 regulates microtubule stability by interacting with HDAC6.

    PubMed

    Shi, Xingjuan; Yao, Yanjun; Wang, Yujue; Zhang, Yu; Huang, Qinghai; Zhou, Jun; Liu, Min; Li, Dengwen

    2015-07-01

    Microtubules, highly dynamic components of the cytoskeleton, are involved in mitosis, cell migration and intracellular trafficking. Our previous work has shown that the centrosomal protein Cep70 regulates microtubule organization and mitotic spindle orientation in mammalian cells. However, it remains elusive whether Cep70 is implicated in microtubule stability. Here we demonstrate that Cep70 enhances microtubule resistance to cold or nocodazole treatment. Our data further show that Cep70 promotes microtubule stability by regulating tubulin acetylation, and plays an important role in stabilizing microtubules. Mechanistic studies reveal that Cep70 interacts and colocalizes with histone deacetylase 6 (HDAC6) in the cytoplasm. These findings suggest that Cep70 promotes microtubule stability by interaction with HDAC6 and regulation of tubulin acetylation. PMID:26112604

  1. Kinetic theory for actively streaming microtubule suspensions

    NASA Astrophysics Data System (ADS)

    Gao, Tong; Blackwell, Robert; Glaser, Matt; Betterton, Meredith; Shelley, Michael

    2013-11-01

    Suspensions of polar biopolymers mixed with molecular motor proteins can exhibit surprising out-of-equilibrium phenomena. In a recent experiment by Sanchez et al., microtubules are driven into collective motion by plus-end walking motor complexes. In experiments where the suspension is confined to a fluid-fluid interface, they find the emergence of distinctive large-scale flows characterized by persistent time-dependence and formation/annihilation of disclination singularities in the nematic order. Here we develop a first-principles kinetic theory to investigate the nonlinear dynamics and pattern formation observed in active microtubule suspensions. We model the active stresses generated by motile microtubules by taking into account the extensile stresses due to both the antiparallel and the parallel microtubule pairs. In a concentrated system, the resultant particle-pair stresses can induce hydrodynamic instabilities, and lead to a large-scale flows. When the suspension is confined to a liquid-liquid interface, we recover much of the dynamics observed in the experiments.

  2. 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

  3. Microfilaments and microtubules regulate recycling from phagosomes

    Microsoft Academic Search

    Maria T Damiani; Maria I Colombo

    2003-01-01

    It is clear that the uptake of large particles is driven by a finely controlled rearrangement of the actin cytoskeleton. Here, we present evidence that myosin motors and microtubules also participate in the Fc?-mediated internalization process in macrophages. During phagocytosis, a substantial amount of plasma membrane is internalized without a net reduction in cell surface area, implying an active mechanism

  4. New one-dimensional conductors: Graphitic microtubules

    Microsoft Academic Search

    Noriaki Hamada; Shin-Ichi Sawada; Atsushi Oshiyama

    1992-01-01

    On the basis of realistic tight-binding band-structure calculations, we predict that carbon microtubules exhibit striking variations in electronic transport, from metallic to semiconducting with narrow and moderate band gaps, depending on the diameter of the tubule and on the degree of helical arrangement of the carbon hexagons. The origin of this drastic variation in the band structure is explained in

  5. MINI-REVIEW Engineering tubulin: microtubule functionalization

    E-print Network

    Hancock, William O.

    that contribute to cell shape and stiffness. Kinesin motor proteins bind to a variety of cargo, including vesicles for controlled actuation and transport at these length scales. The kinesin­microtubule system provides a highly and can be transported by kinesin motors, while adding utility such as the ability to reversibly bind

  6. Learning Generative Models of Microtubule Distributions

    E-print Network

    Matsuda, Noboru

    , Microtubules, Simulated Images, Computational Bi- ology, Bioimage Informatics #12;To my parents, my brother and my wife. #12;iv #12;Abstract The field of location proteomics seeks to characterize the distributions from cell images. Building on the work done by Zhao and Murphy [2007] where models of cell, nuclear

  7. Forces due to curving protofilaments in microtubules.

    PubMed

    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. PMID:24483487

  8. 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

  9. Cellular/Molecular Tau Protects Microtubules in the Axon from Severing

    E-print Network

    Baas, Peter W.

    Cellular/Molecular Tau Protects Microtubules in the Axon from Severing by Katanin Liang Qiang,1 is also afforded by microtubule-associated protein 2 (MAP2), which has a tau-like microtubule-binding domain, but not by MAP1b, which has a different microtubule-binding domain. The microtubule

  10. Higgs properties in a broken Inert Doublet Model

    E-print Network

    Rikard Enberg; Johan Rathsman; Glenn Wouda

    2014-12-22

    We consider a model for the Higgs sector with two scalar doublets and a broken $Z_2$ symmetry, the Stealth Doublet Model, where the $Z_2$ symmetry is manifest in the Yukawa sector but broken by the scalar potential. This model can be seen as a generalization of the Inert Doublet Model. One of the doublets is the Higgs doublet that participates in electroweak symmetry breaking and couples to fermions. The other doublet does not couple to fermions at tree level and does not acquire a vacuum expectation value. The broken $Z_2$ symmetry leads to interesting phenomenology such as mixing between the two doublets and charged and CP-odd scalars that can be light and have unusual decay channels. We present theoretical and experimental constraints on the model and consider the recent observation of a Higgs boson at the LHC. The data on the $H\\to\\gamma\\gamma$ channel can be naturally accommodated in the model, with either the lightest or the heaviest CP-even scalar playing the role of the observed particle.

  11. Making microtubules and mitotic spindles in cells without functional centrosomes.

    PubMed

    Mahoney, Nicole M; Goshima, Gohta; Douglass, Adam D; Vale, Ronald D

    2006-03-21

    Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes. PMID:16546079

  12. How do microtubule-targeted drugs work? An overview.

    PubMed

    Jordan, Mary Ann; Kamath, Kathy

    2007-12-01

    The importance of microtubules in mitosis makes them a superb target for a group of highly successful, chemically diverse anticancer drugs. Knowledge of the mechanistic differences among the many drugs of this class is vital to understanding their tissue and cell specificity, the development of resistance, the design of novel improved drugs, optimal scheduling of treatment, and potential synergistic combinations. This overview covers microtubule assembly dynamics, the exquisite regulation of microtubule dynamics in cells by endogenous regulators, the important role of microtubule dynamics in mitosis, the diversity and number of microtubule-targeted drugs undergoing clinical development, the antimitotic mechanisms of microtubule-targeted drugs with emphasis on suppression of microtubule dynamics by vinblastine and taxol, the role of drug uptake and retention in the efficacy of microtubule-targeted drugs, and the anti-angiogenic and vascular-disrupting mechanisms of microtubule targeted drugs. In view of the success of this class of drugs, it has been argued that microtubules represent the single best cancer target identified to date, and it seems likely that drugs in this class will continue to remain an important chemotherapeutic class of drugs even as more selective chemotherapeutic approaches are developed. PMID:18220533

  13. 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

  14. Arabidopsis AUGMIN Subunit8 Is a Microtubule Plus-End Binding Protein That Promotes Microtubule Reorientation in Hypocotyls[C][W

    PubMed Central

    Cao, Lingyan; Wang, Linhai; Zheng, Min; Cao, Hong; Ding, Lian; Zhang, Xiaolan; Fu, Ying

    2013-01-01

    In plant cells, cortical microtubules provide tracks for cellulose-synthesizing enzymes and regulate cell division, growth, and morphogenesis. The role of microtubules in these essential cellular processes depends on the spatial arrangement of the microtubules. Cortical microtubules are reoriented in response to changes in cell growth status and cell shape. Therefore, an understanding of the mechanism that underlies the change in microtubule orientation will provide insight into plant cell growth and morphogenesis. This study demonstrated that AUGMIN subunit8 (AUG8) in Arabidopsis thaliana is a novel microtubule plus-end binding protein that participates in the reorientation of microtubules in hypocotyls when cell elongation slows down. AUG8 bound to the plus ends of microtubules and promoted tubulin polymerization in vitro. In vivo, AUG8 was recruited to the microtubule branch site immediately before nascent microtubules branched out. It specifically associated with the plus ends of growing cortical microtubules and regulated microtubule dynamics, which facilitated microtubule reorientation when microtubules changed their growth trajectory or encountered obstacle microtubules during microtubule reorientation. This study thus reveals a novel mechanism underlying microtubule reorientation that is critical for modulating cell elongation in Arabidopsis. PMID:23735294

  15. The Y chromosomal fertility factor Threads in Drosophila hydei harbors a functional gene encoding an axonemal dynein beta heavy chain protein.

    PubMed Central

    Kurek, R; Reugels, A M; Glätzer, K H; Bünemann, H

    1998-01-01

    To understand the contradiction between megabase-sized lampbrush loops and putative protein encoding genes both associated with the loci of Y chromosomal fertility genes of Drosophila on the molecular level, we used PCR-mediated cloning to identify and isolate the cDNA sequence of the Y chromosomal Drosophila hydei gene DhDhc7(Y). Alignment of the sequences of the putative protein DhDhc7(Y) and the outer arm dynein beta heavy chain protein DYH2 of Tripneustes gratilla shows homology over the entire length of the protein chains. Therefore the proteins can be assumed to fulfill orthologous functions within the sperm tail axonemes of both species. Functional dynein beta heavy chain molecules, however, are necessary for the assembly and attachment of outer dynein arms within the sperm tail axoneme. Localization of DhDhc7(Y) to the fertility factor Threads, comprising at least 5.1 Mb of transcriptionally active repetitive DNA, results from an infertile Threads- mutant where large clusters of Threads specifically transcribed satellites and parts of DhDhc7(Y) encoding sequences are missing simultaneously. Consequently, the complete lack of the outer dynein arms in Threads- males most probably causes sperm immotility and hence infertility of the fly. Moreover, preliminary sequence analysis and several other features support the hypothesis that DhDhc7(Y) on the lampbrush loops Threads in D. hydei and Dhc-Yh3 on the lampbrush loops kl-5 in Drosophila melanogaster on the heterochromatic Y chromosome of both species might indeed code for orthologous dynein beta heavy chain proteins. PMID:9649526

  16. A novel microtubule-associated protein from mammalian nerve shows ATP- sensitive binding to microtubules

    PubMed Central

    1986-01-01

    We report the isolation of a protein from mammalian nerve which shows ATP-sensitive binding to microtubules and ATPase activity. This protein, which we have designated HMW4, was prepared from bovine spinal nerve roots by microtubule affinity and ATP-induced release, and was further purified by sucrose density gradient centrifugation. It is a high molecular weight protein with a denatured Mr of 315,000, a Stokes radius of 90 A, and a sedimentation value of approximately 19S. It can be resolved electrophoretically from the well-characterized bovine brain microtubule-associated proteins (MAPs) and also appears to be distinct from MAP 1C. HMW4 has a vanadate-sensitive and azide- insensitive ATPase activity which averages 20 nmol Pi/min per mg protein and is different from dynein and myosin ATPases. HMW4 prepared on sucrose gradients exhibits binding to MAP-free microtubules in the absence of ATP which is reduced by ATP addition. Assayed by darkfield microscopy, HMW4 causes bundling of MAP-free microtubules which is reversed by ATP addition. PMID:3639885

  17. A novel microtubule-associated protein from mammalian nerve shows ATP-sensitive binding to microtubules.

    PubMed

    Hollenbeck, P J; Chapman, K

    1986-10-01

    We report the isolation of a protein from mammalian nerve which shows ATP-sensitive binding to microtubules and ATPase activity. This protein, which we have designated HMW4, was prepared from bovine spinal nerve roots by microtubule affinity and ATP-induced release, and was further purified by sucrose density gradient centrifugation. It is a high molecular weight protein with a denatured Mr of 315,000, a Stokes radius of 90 A, and a sedimentation value of approximately 19S. It can be resolved electrophoretically from the well-characterized bovine brain microtubule-associated proteins (MAPs) and also appears to be distinct from MAP 1C. HMW4 has a vanadate-sensitive and azide-insensitive ATPase activity which averages 20 nmol Pi/min per mg protein and is different from dynein and myosin ATPases. HMW4 prepared on sucrose gradients exhibits binding to MAP-free microtubules in the absence of ATP which is reduced by ATP addition. Assayed by darkfield microscopy, HMW4 causes bundling of MAP-free microtubules which is reversed by ATP addition. PMID:3639885

  18. Fluorescent markers of the microtubule cytoskeleton in Zymoseptoria tritici.

    PubMed

    Schuster, M; Kilaru, S; Latz, M; Steinberg, G

    2015-06-01

    The microtubule cytoskeleton supports vital processes in fungal cells, including hyphal growth and mitosis. Consequently, it is a target for fungicides, such as benomyl. The use of fluorescent fusion proteins to illuminate microtubules and microtubule-associated proteins has led to a break-through in our understanding of their dynamics and function in fungal cells. Here, we introduce fluorescent markers to visualize microtubules and accessory proteins in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein to ?-tubulin (ZtTub2), to ZtPeb1, a homologue of the mammalian plus-end binding protein EB1, and to ZtGrc1, a component of the minus-end located ?-tubulin ring complex, involved in the nucleation of microtubules. In vivo observation confirms the localization and dynamic behaviour of all three markers. These marker proteins are useful tools for understanding the organization and importance of the microtubule cytoskeleton in Z. tritici. PMID:25857261

  19. The unusual microtubule polarity in teleost retinal pigment epithelial cells

    PubMed Central

    1988-01-01

    In cells of the teleost retinal pigment epithelium (RPE), melanin granules disperse into the RPE cell's long apical projections in response to light onset, and aggregate toward the base of the RPE cell in response to dark onset. The RPE cells possess numerous microtubules, which in the apical projections are aligned longitudinally. Nocodazole studies have shown that pigment granule aggregation is microtubule- dependent (Troutt, L. L., and B. Burnside, 1988b Exp. Eye Res. In press.). To investigate further the mechanism of microtubule participation in RPE pigment granule aggregation, we have used the tubulin hook method to assess the polarity of microtubules in the apical projections of teleost RPE cells. We report here that virtually all microtubules in the RPE apical projections are uniformly oriented with plus ends toward the cell body and minus ends toward the projection tips. This orientation is opposite that found for microtubules of dermal melanophores, neurons, and most other cell types. PMID:3170636

  20. A Mechanochemical Model Explains Interactions between Cortical Microtubules in Plants

    PubMed Central

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

    2010-01-01

    Microtubules anchored to the two-dimensional cortex of plant cells collide through plus-end polymerization. Collisions can result in rapid depolymerization, directional plus-end entrainment, or crossover. These interactions are believed to give rise to cellwide self-organization of plant cortical microtubules arrays, which is required for proper cell wall growth. Although the cell-wide self-organization has been well studied, less emphasis has been placed on explaining the interactions mechanistically from the molecular scale. Here we present a model for microtubule-cortex anchoring and collision-based interactions between microtubules, based on a competition between cross-linker bonding, microtubule bending, and microtubule polymerization. Our model predicts a higher probability of entrainment at smaller collision angles and at longer unanchored lengths of plus-ends. This model addresses observed differences between collision resolutions in various cell types, including Arabidopsis cells and Tobacco cells. PMID:20712991

  1. 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.

  2. Self-assembly of artificial microtubules

    E-print Network

    Cheng, Shengfeng; Stevens, Mark J

    2012-01-01

    Understanding the complex self-assembly of biomacromolecules is a major outstanding question. Microtubules are one example of a biopolymer that possesses characteristics quite distinct from standard synthetic polymers that are derived from its hierarchical structure. In order to understand how to design and build artificial polymers that possess features similar to those of microtubules, we have initially studied the self-assembly of model monomers into a tubule geometry. Our model monomer has a wedge shape with lateral and vertical binding sites that are designed to form tubules. We used molecular dynamics simulations to study the assembly process for a range of binding site interaction strengths. In addition to determining the optimal regime for obtaining tubules, we have calculated a diagram of the structures that form over a wide range of interaction strengths. Unexpectedly, we find that the helical tubules form, even though the monomer geometry is designed for nonhelical tubules. We present the detailed ...

  3. Prepared for submission to JHEP Higgs phenomenology in the Stealth Doublet Model

    E-print Network

    Lunds Universitet,

    Prepared for submission to JHEP Higgs phenomenology in the Stealth Doublet Model Rikard Enberg. This Stealth Doublet Model can be seen as a generalization of the Inert Doublet Model with a broken Z2 symmetry The Stealth Doublet Model 4 2.1 The scalar potential 4 2.2 Physical states and mass relations 5 2.3 Soft

  4. Simple model for lambda-doublet propensities in bimolecular reactions

    NASA Technical Reports Server (NTRS)

    Bronikowski, Michael J.; Zare, Richard N.

    1990-01-01

    A simple geometric model is presented to account for lambda-doublet propensities in bimolecular reactions A + BC - AB + C. It applies to reactions in which AB is formed in a pi state, and in which the unpaired molecular orbital responsible for lambda-doubling arises from breaking the B-C bond. The lambda-doublet population ratio is predicted to be 2:1 provided that: (1) the motion of A in the transition state determines the plane of rotation of AB; (2) the unpaired pi orbital lying initially along the B-C bond may be resolved into a projection onto the AB plane of rotation and a projection perpendicular to this plane; (3) there is no preferred geometry for dissociation of ABC. The 2:1 lambda-doublet ratio is the 'unconstrained dynamics prior' lambda-doublet distribution for such reactions.

  5. CVPR 2006 Submission #795. CONFIDENTIAL REVIEW COPY. DO NOT DISTRIBUTE. Spatiotemporal Contour Tracking of Microtubules

    E-print Network

    California at Santa Barbara, University of

    COPY. DO NOT DISTRIBUTE. Spatiotemporal Contour Tracking of Microtubules Anonymous CVPR submission Address Line2 Paper ID 795 Abstract Microtubules (MTs), one of three major cytoskeletal components, serve. Introduction Microtubules (MTs) are cylindrical, cytoskeletal protein polymers found in essentially all

  6. Host microtubules in the Hartig net region of ectomycorrhizas, ectendomycorrhizas, and

    E-print Network

    Massicotte, Hugues

    Host microtubules in the Hartig net region of ectomycorrhizas, ectendomycorrhizas, and monotropoid- labelling combined with confocal scanning laser microscopy, this study documents changes in microtubules in the three systems studied involve changes in the organization of microtubules. Key words: cytoskeleton

  7. Visualizing individual microtubules by bright field microscopy Braulio Gutirrez-Medinaa

    E-print Network

    Block, Steven

    Visualizing individual microtubules by bright field microscopy Braulio Gutiérrez-Medinaa Department; accepted 21 May 2010 Microtubules are slender 25 nm diameter , filamentous polymers involved in cellular structure and organization. Individual microtubules have been visualized via fluorescence imaging of dye

  8. Disorganization of Cortical Microtubules Stimulates Tangential Expansion and Reduces the Uniformity

    E-print Network

    Baskin, Tobias

    Disorganization of Cortical Microtubules Stimulates Tangential Expansion and Reduces the Uniformity, Columbia, Missouri 65211 To test the role of cortical microtubules in aligning cellulose microfibrils and controlling anisotropic expansion, we exposed Arabidopsis thaliana roots to moderate levels of the microtubule

  9. 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 ...

  10. Microtubule-Actin Cross-talk at Focal Adhesions

    NSDL National Science Digital Library

    Alexander F. Palazzo (Columbia University; Department of Anatomy and Cell Biology REV)

    2002-07-02

    Focal adhesions are dynamic structures in which traction forces are exerted against the substratum during cell migration and are sites for the organization of signaling complexes. Palazzo and Gundersen discuss how focal adhesions may also be the site of cross-talk between the actin-based and microtubule-based cytoskeletons. Microtubules appear to deliver factors that can regulate the formation and dissolution of focal adhesions, whereas focal adhesions contribute to microtubule localization and stability.

  11. Kin I Kinesins Are Microtubule-Destabilizing Enzymes

    Microsoft Academic Search

    Arshad Desai; Suzie Verma; Timothy J. Mitchison; Claire E. Walczak

    1999-01-01

    Using in vitro assays with purified proteins, we show that XKCM1 and XKIF2, two distinct members of the internal catalytic domain (Kin I) kinesin subfamily, catalytically destabilize microtubules using a novel mechanism. Both XKCM1 and XKIF2 influence microtubule stability by targeting directly to microtubule ends where they induce a destabilizing conformational change. ATP hydrolysis recycles XKCM1\\/XKIF2 for multiple rounds of

  12. 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

  13. 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.

  14. The role of molecular microtubule motors and the microtubule cytoskeleton in stress granule dynamics.

    PubMed

    Bartoli, Kristen M; Bishop, Darryl L; Saunders, William S

    2011-01-01

    Stress granules (SGs) are cytoplasmic foci that appear in cells exposed to stress-induced translational inhibition. SGs function as a triage center, where mRNAs are sorted for storage, degradation, and translation reinitiation. The underlying mechanisms of SGs dynamics are still being characterized, although many key players have been identified. The main components of SGs are stalled 48S preinitiation complexes. To date, many other proteins have also been found to localize in SGs and are hypothesized to function in SG dynamics. Most recently, the microtubule cytoskeleton and associated motor proteins have been demonstrated to function in SG dynamics. In this paper, we will discuss current literature examining the function of microtubules and the molecular microtubule motors in SG assembly, coalescence, movement, composition, organization, and disassembly. PMID:21760798

  15. Microtubule-binding agents: a dynamic field of cancer therapeutics

    PubMed Central

    Dumontet, Charles; Jordan, Mary Ann

    2010-01-01

    Preface Microtubules are dynamic filamentous cytoskeletal proteins that are an important therapeutic target in tumor cells. Microtubule binding agents have been part of the pharmacopoeia of cancer for decades, and until the advent of targeted therapy microtubules were the only alternative to DNA as a therapeutic target in cancer. The screening of a variety of botanical species and marine organisms has yielded promising new antitubulin agents with novel properties. Enhanced tumor specificity, reduced neurotoxicity, and insensitivity to chemoresistance mechanisms are the three main objectives in the current search for novel microtubule binding agents. PMID:20885410

  16. Toxoplasma gondii actively remodels the microtubule network in host cells

    PubMed Central

    Walker, Margaret E.; Hjort, Elizabeth E.; Smith, Sherri S.; Tripathi, Abhishek; Hornick, Jessica E.; Hinchcliffe, Edward H.; Archer, William; Hager, Kristin M.

    2009-01-01

    Toxoplasma gondii infection triggers host microtubule rearrangement and organelle recruitment around the parasite vacuole. Factors affecting initial stages of microtubule remodeling are unknown. To illuminate the mechanism, we tested the hypothesis that the parasite actively remodels host microtubules. Utilizing heat-killed parasites and time-lapse analysis, we determined microtubule rearrangement requires living parasites and is time dependent. We discovered a novel aster of microtubules (MTs) associates with the vacuole within 1 h of infection. This aster lacks the concentrated foci of gamma (?)-tubulin normally associated with MT nucleation sites. Unexpectedly, vacuole enlargement does not correlate with an increase in MT staining around the vacuole. We conclude microtubule remodeling does not result from steric constraints. Using nocodazole washout studies, we demonstrate the vacuole nucleates host microtubule growth in-vivo via ?-tubulin-associated sites. Moreover, superinfected host cells display multiple ?-tubulin foci. Microtubule dynamics are critical for cell cycle control in uninfected cells. Using non-confluent monolayers, we show host cells commonly fail to finish cytokinesis resulting in larger, multinucleated cells. Our data suggest intimate interactions between T. gondii and host microtubules result in suppression of cell division and/or cause a mitotic defect, thus providing a larger space for parasite duplication. PMID:18983931

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

    E-print Network

    Zelinski, Björn; Kierfeld, Jan

    2012-01-01

    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. 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.

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

    E-print Network

    Björn Zelinski; Nina Müller; Jan Kierfeld

    2012-12-14

    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. 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.

  19. ?-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

  20. 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).

  1. In vitro and in vivo analysis of microtubule destabilizing kinesins

    PubMed Central

    Stumpff, Jason; Cooper, Jeremy; Domnitz, Sarah; Moore, Ayana; Rankin, Kathleen; Wagenbach, Mike; Wordeman, Linda

    2010-01-01

    Cellular microtubules are rigid in comparison to other cytoskeletal elements [1, 2]. To facilitate cytoplasmic remodeling and timely responses to cell signaling events, microtubules depolymerize and repolymerize rapidly at their ends [3]. These dynamic properties are critically important for many cellular functions, such as spindle assembly, the capture and segregation of chromosomes during cell division and cell motility. Microtubule dynamics are spatially and temporally controlled in the cell by accessory proteins. Molecular motor proteins of the kinesin superfamily that act to destabilize microtubules play important roles in this regulation [4]. PMID:17951709

  2. The Kinesin-Related Protein MCAK Is a Microtubule Depolymerase that Forms an ATP-Hydrolyzing Complex at Microtubule Ends

    Microsoft Academic Search

    Andrew W. Hunter; Michael Caplow; David L. Coy; William O. Hancock; Stefan Diez; Linda Wordeman; Jonathon Howard

    2003-01-01

    MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has

  3. 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 ...

  4. Whole cell microtubule analysis by flow cytometry Karen C. Morrison, Paul J. Hergenrother

    E-print Network

    Hergenrother, Paul J.

    Whole cell microtubule analysis by flow cytometry Karen C. Morrison, Paul J. Hergenrother Roger and destabilize microtubule formation. This facile method is useful for conveniently, quantitatively, and cost

  5. Multi-mode electro-mechanical vibrations of a microtubule: In silico demonstration of electric pulse moving along a microtubule

    NASA Astrophysics Data System (ADS)

    Havelka, Daniel; Cifra, Michal; Ku?era, Ond?ej

    2014-06-01

    Microtubules are known to be involved in intracellular signaling. Here, we show in silico that electrically polar collective vibration modes of microtubules form electric oscillating potential which is quasi-periodic both in space and in time. While single mode microtubule vibration excites an electric field with spatially stationary local minima and maxima of the electric field, the multimode excitation causes the formation of an electric pulse and many transient local electric field minima. The biophysical mechanism we describe lends support to the view that microtubules may comprise a substrate for ultra-fast electrical signaling in neurons or other living cells.

  6. The microtubule plus-end tracking protein ARMADILLO-REPEAT KINESIN1 promotes microtubule catastrophe in Arabidopsis.

    PubMed

    Eng, Ryan Christopher; Wasteneys, Geoffrey O

    2014-08-01

    Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1's function is redundant in cells other than those forming root hairs. PMID:25159991

  7. The Microtubule Plus-End Tracking Protein ARMADILLO-REPEAT KINESIN1 Promotes Microtubule Catastrophe in Arabidopsis[W][OPEN

    PubMed Central

    Eng, Ryan Christopher; Wasteneys, Geoffrey O.

    2014-01-01

    Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1’s function is redundant in cells other than those forming root hairs. PMID:25159991

  8. Microtubule dynamics regulates Akt signaling via dynactin p150.

    PubMed

    Jo, Hakryul; Loison, Fabien; Luo, Hongbo R

    2014-08-01

    Following activation at the plasma membrane, Akt is subsequently deactivated in the cytoplasm. Although activation and deactivation of Akt must sometimes be separated in order to elicit and control cellular responses, the exact details of the spatiotemporal organization of Akt signaling are incompletely understood. Here we show that microtubule dynamics specifically modulate the deactivation phase of Akt signaling. Localization of Akt to microtubules sustains its activity, while disruption of microtubules attenuates Akt signaling independent of its initial activation. Conversely, stabilization of microtubules elevates Akt signaling both in vitro and in muscle tissues in vivo. Localization of Akt to microtubules is mediated by the microtubule binding protein dynactin p150, which is shown to be a direct target of Akt. Finally, microtubule disruption-induced Akt deactivation contributes to delayed cell cycle progression and accelerated cell death. Taken together, we revealed that, after initiation, the overall intensity and duration of oncogenic Akt signaling are determined by microtubule dynamics, a mechanism that could be exploited for therapeutic purposes. PMID:24726838

  9. Motion tracking of the outer tips of microtubules

    Microsoft Academic Search

    Stathis Hadjidemetriou; Derek Toomre; James S. Duncan

    2008-01-01

    ABSTRACT Microtubules are tubular biopolymers,of the cytoplasm. They play numerous,critical roles in a cell such as providing mechanical support and structural tracks for the anchoring and transport of chromosomes, organelles, and vesicles. They also form the microtubule assembly, which is critical for the coordination of cell division and migration. Abnormal function of the assembly is involved in cell pathology such

  10. Kinks and breathers in nonlinear dynamics of microtubules

    NASA Astrophysics Data System (ADS)

    Zdravkovi?, Slobodan

    2014-10-01

    Nonlinear dynamics of microtubules is studied. One radial degree of freedom per dimer is assumed and the used model is called ? - model. Four mathematical procedures yielding kink solitons are explained. In addition, semi discrete approximation is also explained and it is shown that this mathematical method brings about breathers moving along microtubules.

  11. Regulation of Cell Polarity by Microtubules in Fission Yeast

    Microsoft Academic Search

    Kenneth E. Sawin; Paul Nurse

    1998-01-01

    To investigate the role of microtubules in regulating cell polarity in Schizosaccharomyces pombe , we have developed a system in which normally cylindri- cal fission yeast synchronously form branched cells at high frequency upon treatment with the microtubule- depolymerizing drug thiabendazole (TBZ). Branching depends on both elevated temperature and cell cycle state and occurs at high frequency only when TBZ

  12. Microtubule distribution in gravitropic protonemata of the moss Ceratodon.

    PubMed

    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. PMID:11537091

  13. Microtubules, protoplasts and plant cell shape : An immunofluorescent study.

    PubMed

    Lloyd, C W; Slabas, A R; Powell, A J; Lowe, S B

    1980-02-01

    Indirect immunofluorescence has been used to study the function of cytoplasmic microtubules in controlling the shape of elongated carrot cells in culture. Using a purified wall-degrading preparation, the elongated cells are converted to spherical protoplasts and the transverse hoops of bundled microtubules are disorganised but not depolymerised in the process. Since microtubules remain attached to fragments of protoplast membrane adhering to coverslips and are still seen to be organised laterally in bundles, it would appear that re-orientation of the transverse bundles is due to loss of cell wall and not to the cleavage of microtubule bridges. After 24 h treatment in 10(-3) M colchicine, microtubules are depolymerised in elongated cells but, at this time, the cells retain their elongated shape. This suggests that wall which was organised in the presence of transverse microtubule bundles can retain asymmetric shape for short periods in the absence of those tubules. However, after longer periods of time the cells become spherical in colchicine. Neither wall nor tubules therefore exert individual control on continued cellular elongation and so we emphasize the fundamental nature of wall/microtubule interactions in shape control. It is concluded that the observations are best explained by a model in which hooped bundles of microtubules-which are directly or indirectly associated with molecules involved with cellulose biosynthesis at the cell surface-act as an essential template or scaffolding for the orientated deposition of cellulose. PMID:24311175

  14. 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

  15. Target Finding Mechanism of Microtubules in a Confined Geometry

    NASA Astrophysics Data System (ADS)

    Shojania Feizabadi, Mitra

    2007-03-01

    Discovery of a non-equilibrium dynamic of microtubules, called dynamic instability, raised this question: is stochastic polymerization dynamic of microtubules an advantage in the process of finding a chromosome as a target? Previous studies showed that compared to usual reversible polymerization, dynamic instability of microtubules with decreasing length distribution reduced the time required to find a target by several order of magnitude [1]. Dynamic Equations for growing and shrinking microtubules in a confined geometry is theoretically modeled by Govinden and Spillman [2]. This work calculates the target finding time for microtubules with exponentially increasing length distribution in a confined geometry. The efficiency of target finding mechanism based upon different dynamical parameters is discussed. [1] Holy TE, Leibler S. 1994, Proc. Natl. Acad. Sci. USA 91, 5682. [2] Govindan B, Spillman W. 2004, Phys. Rev. E 70, 032901.

  16. 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

  17. 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

  18. Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells.

    PubMed

    Akita, Kae; Higaki, Takumi; Kutsuna, Natsumaro; Hasezawa, Seiichiro

    2015-05-01

    Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulin? and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells. PMID:26039484

  19. Building the Microtubule Cytoskeleton Piece by Piece.

    PubMed

    Alfaro-Aco, Ray; Petry, Sabine

    2015-07-10

    The microtubule (MT) cytoskeleton gives cells their shape, organizes the cellular interior, and segregates chromosomes. These functions rely on the precise arrangement of MTs, which is achieved by the coordinated action of MT-associated proteins (MAPs). We highlight the first and most important examples of how different MAP activities are combined in vitro to create an ensemble function that exceeds the simple addition of their individual activities, and how the Xenopus laevis egg extract system has been utilized as a powerful intermediate between cellular and purified systems to uncover the design principles of self-organized MT networks in the cell. PMID:25957410

  20. Microtubule Length-Regulation by Molecular Motors

    E-print Network

    Melbinger, Anna; Frey, Erwin

    2012-01-01

    Length-regulation of microtubules (MTs) is essential for many cellular processes. Molecular motors like kinesin 8, which move along MTs and also act as depolymerases, are known as key players in MT dynamics. However, the regulatory mechanisms of length control remain elusive. Here, we investigate a stochastic model accounting for the interplay between polymerization kinetics and motor-induced depolymerization. We determine the dependence of MT length and variance on rate constants and motor concentration. Moreover, our analyses reveal how collective phenomena lead to a well-defined MT length.

  1. Self-organization of microtubules and motors.

    SciTech Connect

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

    2006-01-01

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

  2. Microtubule Length-Regulation by Molecular Motors

    E-print Network

    Anna Melbinger; Louis Reese; Erwin Frey

    2012-04-25

    Length-regulation of microtubules (MTs) is essential for many cellular processes. Molecular motors like kinesin 8, which move along MTs and also act as depolymerases, are known as key players in MT dynamics. However, the regulatory mechanisms of length control remain elusive. Here, we investigate a stochastic model accounting for the interplay between polymerization kinetics and motor-induced depolymerization. We determine the dependence of MT length and variance on rate constants and motor concentration. Moreover, our analyses reveal how collective phenomena lead to a well-defined MT length.

  3. Cryo-electron tomography of microtubule-kinesin motor complexes.

    PubMed

    Cope, Julia; Gilbert, Susan; Rayment, Ivan; Mastronarde, David; Hoenger, Andreas

    2010-05-01

    Microtubules complexed with molecular motors of the kinesin family or non-motor microtubule associated proteins (MAPs) such as tau or EB1 have been the subject of cryo-electron microcopy based 3-D studies for several years. Most of these studies that targeted complexes with intact microtubules have been carried out by helical 3-D reconstruction, while few were analyzed by single particle approaches or from 2-D crystalline arrays. Helical reconstruction of microtubule-MAP or motor complexes has been extremely successful but by definition, all helical 3-D reconstruction attempts require perfectly helical assemblies, which presents a serious limitation and confines the attempts to 15- or 16-protofilament microtubules, microtubule configurations that are very rare in nature. The rise of cryo-electron tomography within the last few years has now opened a new avenue towards solving 3-D structures of microtubule-MAP complexes that do not form helical assemblies, most importantly for the subject here, all microtubules that exhibit a lattice seam. In addition, not all motor domains or MAPs decorate the microtubule surface regularly enough to match the underlying microtubule lattice, or they adopt conformations that deviate from helical symmetry. Here we demonstrate the power and limitation of cryo-electron tomography using two kinesin motor domains, the monomeric Eg5 motor domain, and the heterodimeric Kar3Vik1 motor. We show here that tomography does not exclude the possibility of post-tomographic averaging when identical sub-volumes can be extracted from tomograms and in both cases we were able to reconstruct 3-D maps of conformations that are not possible to obtain using helical or other averaging-based methods. PMID:20025975

  4. Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3? phosphorylation

    Microsoft Academic Search

    Jürg Zumbrunn; Kazuhisa Kinoshita; Anthony A. Hyman; Inke S. Näthke

    2001-01-01

    Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating ?-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and

  5. Doublet-point method for supersonic unsteady lifting surfaces

    NASA Technical Reports Server (NTRS)

    Ueda, T.; Dowell, E. H.

    1984-01-01

    A method to predict unsteady aerodynamic forces on lifting surfaces in supersonic flow is presented. The wing is divided into small segments in which the lift force is expressed by a single-point doublet of the acceleration potential. This is the same concept as the doublet-point method developed by the authors for subsonic flows. In order to avoid sensitiveness to the Mach number, the upwash due to the point doublet is calculated by averaging over small areas. The integration is done analyticaly so that it requires no numerical quadrature. Pressure distributions are directly obtained as the unknowns of the algebraic equation. The results are compared with those obtained by other methods for various wing geometries, including the AGARD wing-tail configuration.

  6. Cytosolic Proteins From Tobacco Pollen Tubes That Crosslink Microtubules and Actin Filaments In Vitro

    E-print Network

    Baskin, Tobias

    Cytosolic Proteins From Tobacco Pollen Tubes That Crosslink Microtubules and Actin Filaments Monitoring Editor: Bruce Goode In plant cells, many processes require cooperative action of both microtubules the actin were incubated with microtubules, and finally those microtubule-binding proteins were pooled

  7. Estramustine phosphate inhibits germinal vesicle breakdown and induces depolymerization of microtubules

    E-print Network

    Paris-Sud XI, Université de

    of microtubules in mouse oocyte Hélène RIME Catherine JESSUS R. OZON Laboratoire de Physiologie de la Reproduction microtubules in ovo and dispersed non-tubulin antigens associated with microtubules of the metaphase spindle cytoplasmic asters. These results suggest that germinal vesicle breakdown and microtubule polymerization may

  8. Microtubules are tubular polymers that are composed of two proteins, -and -tubulin.

    E-print Network

    Stearns, Tim

    a b c Microtubules are tubular polymers that are composed of two proteins, - and -tubulin. The orientation of these proteins in the polymers gives microtubules an intrinsic polarity. Microtubules, microtubules form highly organ- ized, polarized arrays; in proliferating cells, these arrays undergo cell

  9. Segmentation and 3D Reconstruction of Microtubules in Total Internal Reflection

    E-print Network

    Hadjidemetriou, Efstathios "Stathis"

    Segmentation and 3D Reconstruction of Microtubules in Total Internal Reflection Fluorescence of the microtubules with the cell cortex plays numerous critical roles in a cell. For instance, it directs vesicle of the microtubules is involved in cancer. An effective method to observe microtubule function adjacent to the cortex

  10. Microtubule organization and morphogenesis in young spores of the moss Tetraphis pellucida Hedw

    Microsoft Academic Search

    R. C. Brown; B. E. Lemmon

    1983-01-01

    Summary Microtubule systems appear sequentially at the distal and proximal poles of tetrad members during mid-sporogenesis in the mossTetraphis pellucida Hedw. The distal microtubule system emanates from a microtubule organizing center (MTOC) located between the single plastid and the nucleus. The distal MTOC and associated microtubules, which appear immediately after cytokinesis, are ephemeral and do not appear to be associated

  11. Kinesin Is the Motor for Microtubule-mediated Golgi-to-ER Membrane Traffic

    Microsoft Academic Search

    Jennifer Lippincott-Sehwa; Nelson B. Cole; Alex Marotta; Patricia A. Conrad

    1995-01-01

    The distribution and dynamics of both the ER and Golgi complex in animal cells are known to be dependent on microtubules; in many cell types the ER extends toward the plus ends of microtubules at the cell periphery and the Golgi clusters at the minus ends of microtubules near the centrosome. In this study we provide evidence that the microtubule

  12. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility

    Microsoft Academic Search

    Ronald D. Vale; Thomas S. Reese; Michael P. Sheetz

    1985-01-01

    Summary Axoplasm from the squid giant axon contains a solu- ble protein translocator that induces movement of microtubules on glass, latex beads on microtubules, and axoplasmic organelles on microtubules. We now report the partial purification of a protein from squid giant axons and optic lobes that induces these microtubule-based movements and show that there is a homologous protein in bovine

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

    PubMed

    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 f(c) 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 f(c) 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. PMID:23214626

  14. Self-Reduction Rate of a Microtubule

    NASA Astrophysics Data System (ADS)

    Hiramatsu, Takashi; Matsui, Tetsuo; Sakakibara, Kazuhiko

    We formulate and study a quantum field theory of a microtubule, a basic element of living cells. Following the quantum theory of consciousness by Hameroff and Penrose, we let the system to reduce to one of the classical states without measurement if certain conditions are satisfied (self-reductions), and calculate the self-reduction time ?N (the mean interval between two successive self-reductions) of a cluster consisting of more than N neighboring tubulins (basic units composing a microtubule). ?N is interpreted there as an instance of the stream of consciousness. We analyze the dependence of ?N upon N and the initial conditions, etc. For relatively large electron hopping amplitude, ?N obeys a power law ?N ~ Nb, which can be explained by the percolation theory. For sufficiently small values of the electron hopping amplitude, ?N obeys an exponential law, ?N ~ exp (c'N). By using this law, we estimate the condition for ?N to take realistic values ?N ? 10-1 sec as N ? 1000.

  15. Self-assembly of artificial microtubules

    E-print Network

    Shengfeng Cheng; Ankush Aggarwal; Mark J. Stevens

    2012-01-10

    Understanding the complex self-assembly of biomacromolecules is a major outstanding question. Microtubules are one example of a biopolymer that possesses characteristics quite distinct from standard synthetic polymers that are derived from its hierarchical structure. In order to understand how to design and build artificial polymers that possess features similar to those of microtubules, we have initially studied the self-assembly of model monomers into a tubule geometry. Our model monomer has a wedge shape with lateral and vertical binding sites that are designed to form tubules. We used molecular dynamics simulations to study the assembly process for a range of binding site interaction strengths. In addition to determining the optimal regime for obtaining tubules, we have calculated a diagram of the structures that form over a wide range of interaction strengths. Unexpectedly, we find that the helical tubules form, even though the monomer geometry is designed for nonhelical tubules. We present the detailed dynamics of the tubule self-assembly process and show that the interaction strengths must be in a limited range to allow rearrangement within clusters. We extended previous theoretical methods to treat our system and to calculate the boundaries between different structures in the diagram.

  16. 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

  17. Towards systematic exploration of multi-Higgs-doublet models

    E-print Network

    I. P. Ivanov

    2015-04-24

    Conservative bSM models with rich scalar sector, such as multi-Higgs-doublet models, can easily accommodate the SM-like properties of the 125 GeV scalar observed at the LHC. Possessing a variety of bSM signals, they are worth investigating in fuller detail. Systematic study of these models is hampered by the highly multi-dimensional parameter space and by mathematical challenges. I outline some directions along which multi-Higgs-doublet models in the vicinity of a large discrete symmetry can be systematically explored.

  18. A Two Higgs Doublet Model for the Top Quark

    E-print Network

    Ashok Das; Chung Kao

    1995-11-20

    A two Higgs doublet model with special Yukawa interactions for the top quark and a softly broken discrete symmetry in the Higgs potential is proposed. In this model, the top quark is much heavier than the other quarks and leptons because it couples to a Higgs doublet with a much larger vacuum expectation value. The electric dipole moment (EDM) of the electron is evaluated with loop diagrams of the third generation fermions as well as the charm quark. The electron EDM is significantly enhanced for a naturally large $\\tan\\beta \\equiv |v_2|/|v_1|$.

  19. 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

  20. Modulation of Microtubule Interprotofilament Interactions by Modified Taxanes

    PubMed Central

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

    2011-01-01

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

  1. Endosperm Development in Barley: Microtubule Involvement in the Morphogenetic Pathway.

    PubMed Central

    Brown, R. C.; Lemmon, B. E.; Olsen, O. A.

    1994-01-01

    An immunofluorescence study of sectioned barley endosperm imaged by confocal laser scanning microscopy provided three-dimensional data on the relationship of microtubules to the cytoplasm, nuclei, and cell walls during development from 4 to 21 days after pollination (DAP). Microtubules play an important role throughout endosperm ontogeny. The syncytium is organized into units of nuclear-cytoplasmic domains by nuclear-based radial microtubule systems that appear to control the pattern of the first anticlinal walls at 5 to 6 DAP. After 7 DAP, phragmoplasts of two origins (interzonal and cytoplasmic) guide wall formation. Large compartments formed by the "free growing" walls in association with cytoplasmic phragmoplasts formed adventitiously at interfaces of opposing microtubule systems are subsequently subdivided by interzonal phragmoplast/cell plates to give rise to the starchy endosperm. During development of the aleurone layer from 8 to 21 DAP, the microtubule cycle is typical of plant histogenesis; cortical microtubules are hooplike, and preprophase bands of microtubules predict the division plane. PMID:12244271

  2. Microtubules in the Cerebral Cortex: Role in Memory and Consciousness

    NASA Astrophysics Data System (ADS)

    Woolf, Nancy J.

    This chapter raises the question whether synaptic connections in the cerebral cortex are adequate in accounting for higher cognition, especially cognition involving multimodal processing. A recent and novel approach to brain mechanics is outlined, one that involves microtubules and microtubule-associated protein-2 (MAP2). In addition to effects on the neuronal membrane, neurotransmitters exert actions on microtubules. These neurotransmitter effects alter the MAP2 phosphorylation state and rates of microtubule polymerization and transport. It is argued that these processes are important to the physical basis of memory and consciousness. In support of this argument, MAP2 is degraded with learning in discrete cortical modules. How this relates to synaptic change related to learning is unknown. The specific proposal is advanced that learning alters microtubules in the subsynaptic zone lying beneath the synapse, and that this forms the physical basis of long-term memory storage because microtubule networks determine the synapse strength by directing contacts with actin filaments and transport of synaptic proteins. It is argued that this is more probable than memory-related physical storage in the synapse itself. Comparisons to consciousness are made and it is concluded that there is a link between microtubules, memory and consciousness.

  3. 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

  4. Kinetics of microtubule catastrophe assessed by probabilistic analysis.

    PubMed Central

    Odde, D J; Cassimeris, L; Buettner, H M

    1995-01-01

    Microtubules are cytoskeletal filaments whose self-assembly occurs by abrupt switching between states of roughly constant growth and shrinkage, a process known as dynamic instability. Understanding the mechanism of dynamic instability offers potential for controlling microtubule-dependent cellular processes such as nerve growth and mitosis. The growth to shrinkage transitions (catastrophes) and the reverse transitions (rescues) that characterize microtubule dynamic instability have been assumed to be random events with first-order kinetics. By direct observation of individual microtubules in vitro and probabilistic analysis of their distribution of growth times, we found that while the slower growing and biologically inactive (minus) ends obeyed first-order catastrophe kinetics, the faster growing and biologically active (plus) ends did not. The non-first-order kinetics at plus ends imply that growing microtubule plus ends have an effective frequency of catastrophe that depends on how long the microtubules have been growing. This frequency is low initially but then rises asymptotically to a limiting value. Our results also suggest that an additional parameter, beyond the four parameters typically used to describe dynamic instability, is needed to account for the observed behavior and that changing this parameter can significantly affect the distribution of microtubule lengths at steady state. Images FIGURE 1 PMID:8519980

  5. 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.

  6. Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules

    E-print Network

    Hinow, Peter; Lopus, Manu; Jordan, Mary Ann; Tuszynski, Jack A

    2010-01-01

    We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady state microtubules assembled from MAP-free tubulin. Both experimentally and theoretically we study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. We find that to be an effective suppressor of microtubule dynamics a drug must primarily suppress the loss of GDP tubulin from the microtubule tip.

  7. 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. ...

  8. Elastic Behavior of Composite Actin and Microtubule Networks

    NASA Astrophysics Data System (ADS)

    Lin, Yi-Chia; Koenderink, Gijsje; Mackintosh, Frederick; Weitz, David

    2007-03-01

    We explore the non-linear shearing behavior of composite actin and microtubule networks. Large bending rigid microtubules are used as a probe of the deformation mode of cross-linked actin networks. For a sparsely cross-linked actin network that deforms non-affinely, adding microtubules can drive the system back to affine by suppressing local rearrangements of actin filaments. It applies to both permanently rigid cross-linker, such as scruin, and flexible cross-linker, such as filamin. This experiment also shows that filamin cross-linked actin networks are deforming in an affine manner.

  9. Fluorescent taxoids as probes of the microtubule cytoskeleton.

    PubMed

    Evangelio, J A; Abal, M; Barasoain, I; Souto, A A; Lillo, M P; Acuña, A U; Amat-Guerri, F; Andreu, J M

    1998-01-01

    Microtubules are specifically and efficiently visualized with the new fluorescent taxoids 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol (FLUTAX) and 7-O-[N-(4'-tetramethylrhodaminecarbonyl)-L-alanyl]taxol (ROTAX). Similarly to taxol, FLUTAX and ROTAX are able to drive inactive GDP-liganded tubulin into microtubule assembly. One molecule of FLUTAX binds per alphabeta-tubulin dimer assembled, competing with taxol for the same microtubule binding site with an eightfold smaller relative affinity. FLUTAX-induced microtubule elongation is markedly Mg2+-dependent, encompassing the binding of one Mg2+ ion more per tubulin dimer polymerized than in the case of taxol. A small perturbation of the absorption spectrum of bound FLUTAX is consistent with a cationic microenvironment relative to the solution. The fluorescence anisotropy of FLUTAX increases by an order of magnitude upon binding to microtubules and time-resolved measurements indicate that the fluorescein moiety remains considerably mobile on a protein surface. The rate of labeling suggests that this is the outer microtubule wall. Alternatively, the microtubule lumen would be functional. FLUTAX- and ROTAX-induced microtubules, radial structures, and organized microtubule bundles are readily observed under the fluorescence microscope. Rapid and accurate visualization of native (or very mildly fixed) cytoplasmic and spindle microtubules of a variety of permeabilized cells is simply obtained with micromolar FLUTAX, with an advantage over immunofluorescence. In addition, FLUTAX labels the centrosomes of PtK2 cells more intensely than antibodies to alpha- or beta-tubulin, and co-localizing with antibodies to gamma-tubulin. Two brightly fluorescent spots, probably separating or duplicating centrioles, can be resolved in the centrosomes of interphase cells. This finding indicates that centrosomes may well be additional targets of action of taxoids. FLUTAX strongly labels microtubules near the spindle poles, as well as microtubules at the telophase spindle equator and the central part of the midbody in cytokinesis (instead of the dark zone frequently observed with immunofluorescence), suggesting a predominant interaction of FLUTAX with sites at which tubulin is newly polymerized. Nanomolar concentrations of FLUTAX also permit specific imaging of centrosomes, half-spindles and midbodies in growing U937 cells. PMID:9453715

  10. The segmentation of microtubules in electron tomograms using Amira.

    PubMed

    Redemann, Stefanie; Weber, Britta; Möller, Marit; Verbavatz, Jean-Marc; Hyman, Anthony A; Baum, Daniel; Prohaska, Steffen; Müller-Reichert, Thomas

    2014-01-01

    The development of automatic tools for the three-dimensional reconstruction of the microtubule cytoskeleton is crucial for large-scale analysis of mitotic spindles. Recently, we have published a method for the semiautomatic tracing of microtubules based on 3D template matching (Weber et al., J Struct Biol 178:129-138, 2012). Here, we give step-by-step instructions for the automatic tracing of microtubules emanating from centrosomes in the early mitotic Caenorhabditis elegans embryo. This approach, integrated in the visualization and data analysis software Amira, is applicable to tomographic data sets from other model systems. PMID:24633801

  11. Microtubule attachment and spindle assembly checkpoint signaling at the kinetochore

    PubMed Central

    Foley, Emily A.; Kapoor, Tarun M.

    2013-01-01

    In eukaryotes, chromosome segregation during cell division is facilitated by the kinetochore, an assembly of proteins built on centromeric DNA. The kinetochore attaches chromosomes to spindle microtubules, modulates the stability of these attachments, and relays microtubule-binding status to the spindle assembly checkpoint, a cell cycle surveillance pathway that delays chromosome segregation in response to unattached kinetochores. Here, we discuss recent results that guide current thinking on how each of these kinetochore-centered processes is achieved, and how their integration ensures faithful chromosome segregation, focusing on the essential roles of kinase-phosphatase signaling and the microtubule-binding KMN protein network. PMID:23258294

  12. Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study

    Microsoft Academic Search

    Eva-Maria Mandelkow; Eckhard Mandelkow; Ronald A. Milligan

    1991-01-01

    Microtubulesdisplaytheuniquepropertyof dynamic instability characterizedby phase changesbe- tween growth and shrinkage,even inconstantenviron- mentalconditions.The phasescan be synchronized, leadingtobulkoscillations ofmicrotubules.To study thestructuralbasisofdynamic instability we have ex- amined growing,shrinking,and oscillating microtubules by time-resolvedcryo-EM .Inparticularwe have ad- dressedthreequestionswhich arecurrentlya matter ofdebate:(a)What istherelationship between micro- tubules,tubulinsubunits,and tubulinoligomersinmi- crotubuledynamics?;(b)How do microtubulesshrink? By releaseofsubunitsor viaoligomers?;and (c)Is

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

    We examine the production of dark matter by decaying topological defects in the high mass region mDM gg mW 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 ?-ray background and Big Bang Nucleosynthesis, together with constraints on the dark matter properties coming from direct and indirect detection limits. For the Inert Doublet Model high-mass region, an inert Higgs mass as low as ~ 200 GeV is permitted. There is also an upper limit on string mass per unit length, and hence the symmetry breaking scale, from the relic abundance in this scenario. Depending on assumptions made about the string decays, the limits are in the range 1012 GeV to 1013 GeV.

  14. 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.

  15. Effects of the KIF2C neck peptide on microtubules: lateral disintegration of microtubules and ?-structure formation.

    PubMed

    Shimizu, Youské; Shimizu, Takashi; Nara, Masayuki; Kikumoto, Mahito; Kojima, Hiroaki; Morii, Hisayuki

    2013-04-01

    Members of the kinesin-13 sub-family, including KIF2C, depolymerize microtubules. The positive charge-rich 'neck' region extending from the N-terminus of the catalytic head is considered to be important in the depolymerization activity. Chemically synthesized peptides, covering the basic region (A182-E200), induced a sigmoidal increase in the turbidity of a microtubule suspension. The increase was suppressed by salt addition or by reduction of basicity by amino acid substitutions. Electron microscopic observations revealed ring structures surrounding the microtubules at high peptide concentrations. Using the peptide A182-D218, we also detected free thin straight filaments, probably protofilaments disintegrated from microtubules. Therefore, the neck region, even without the catalytic head domain, may induce lateral disintegration of microtubules. With microtubules lacking anion-rich C-termini as a result of subtilisin treatment, addition of the peptide induced only a moderate increase in turbidity, and rings and protofilaments were rarely detected, while aggregations, also thought to be caused by lateral disintegration, were often observed in electron micrographs. Thus, the C-termini are not crucial for the action of the peptides in lateral disintegration but contribute to structural stabilization of the protofilaments. Previous structural studies indicated that the neck region of KIF2C is flexible, but our IR analysis suggests that the cation-rich region (K190-A204) forms ?-structure in the presence of microtubules, which may be of significance with regard to the action of the neck region. Therefore, the neck region of KIF2C is sufficient to cause disintegration of microtubules into protofilaments, and this may contribute to the ability of KIF2C to cause depolymerization of microtubules. PMID:23398918

  16. Nonlinear dynamics of microtubules - A new model

    E-print Network

    Zdravkovi?, Slobodan; Zekovi?, Slobodan

    2012-01-01

    In the present paper we describe a model of nonlinear dynamics of microtubules (MT) assuming a single longitudinal degree of freedom per tubulin dimer. This is a longitudinal displacement of a dimer at a certain position with respect to the neighbouring one. A nonlinear partial differential equation, describing dimer`s dynamics within MT, is solved both analytically and numerically. It is shown that such nonlinear model can lead to existence of kink solitons moving along the MTs. Internal electrical field strength is calculated using two procedures and a perfect agreement between the results is demonstrated. This enabled estimation of total energy, kink velocity and kink width. To simplify the calculation of the total energy we proved a useful theorem.

  17. 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

  18. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  20. INTRODUCTION Microtubules (MTs) are dynamic polymers in the sense that

    E-print Network

    Vorobjev, Ivan

    INTRODUCTION Microtubules (MTs) are dynamic polymers in the sense that they are continuously being in the cell. The recovery of bleaching (or loss of photoactivated mark) have been approximated by a single

  1. Kinetochores and disease: keeping microtubule dynamics in check!

    PubMed Central

    Bakhoum, Samuel F.; Compton, Duane A.

    2012-01-01

    The essential role of microtubules in cell division has long been known. Yet the mechanism by which microtubule attachment to chromosomes at kinetochores is regulated has only been recently revealed. Here, we review the role of kinetochore-microtubule (kMT) attachment dynamics in the cell cycle as well as emerging evidence linking deregulation of kMT attachments to diseases where chromosome mis-segregation and aneuploidy play a central role. Evidence indicates that the dynamic behavior of kMTs must fall within narrow permissible boundaries, which simultaneously allow a level of stability sufficient to establish and maintain chromosome-microtubule attachments and instability, which permits error correction required for accurate chromosome segregation. PMID:22196931

  2. Molecular analysis of kinetochore-microtubule attachment in budding yeast

    E-print Network

    Rines, Daniel R. (Daniel Roger), 1966-

    2003-01-01

    Kinetochores bind to microtubules and are responsible for chromosome segregation and the accurate transmission of genetic information during cell division. Kinetochores are DNA-protein complexes that assemble on centromeric ...

  3. 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.

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

    PubMed Central

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

    2015-01-01

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

  5. 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 ...

  6. Microtubule and calmodulin binding on Mn modules of MAP6 Structural Basis for the Association of MAP6 with Microtubules and its regulation by Calmodulin

    E-print Network

    Paris-Sud XI, Université de

    Microtubule and calmodulin binding on Mn modules of MAP6 1 Structural Basis for the Association of MAP6 with Microtubules and its regulation by Calmodulin Julien Lefèvre1# *, Philippe Savarin1 , Pierre Tronche, BP170, 38042 Grenoble Cedex 9, France *Running title: Microtubule and calmodulin binding on Mn

  7. 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

  8. Critical Roles for Microtubules in Axonal Development and Disease

    Microsoft Academic Search

    Aditi Falnikar; Peter W. Baas

    Axons are occupied by dense arrays of cytoskeletal elements called microtubules, which are critical for generating and maintaining\\u000a the architecture of the axon, and for acting as railways for the transport of organelles in both directions within the axon.\\u000a Microtubules are organized and regulated by molecules that affect their assembly and disassembly, their stabilization, their\\u000a association with other cytoskeletal elements,

  9. 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

  10. HDAC6 is a microtubule-associated deacetylase

    Microsoft Academic Search

    Charlotte Hubbert; Amaris Guardiola; Rong Shao; Yoshiharu Kawaguchi; Akihiro Ito; Andrew Nixon; Minoru Yoshida; Xiao-Fan Wang; Tso-Pang Yao

    2002-01-01

    Reversible acetylation of alpha-tubulin has been implicated in regulating microtubule stability and function. The distribution of acetylated alpha-tubulin is tightly controlled and stereotypic. Acetylated alpha-tubulin is most abundant in stable microtubules but is absent from dynamic cellular structures such as neuronal growth cones and the leading edges of fibroblasts. However, the enzymes responsible for regulating tubulin acetylation and deacetylation are

  11. Translocation and clustering of endosomes and lysosomes depends on microtubules

    Microsoft Academic Search

    Raffaele Matteoni; Thomas E. Kreis

    1987-01-01

    Indirect immunofluorescence labeling of normal rat kidney (NRK) cells with antibodies recog- nizing a lysosomal glycoprotein (LGP 120; Lewis, V., S. A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100:1839-1847) re- veals that lysosomes accumulate in the region around the microtubule-organizing center (MTOC). This clustering of lysosomes depends on microtubules. When the interphase

  12. Structure of cortical microtubule arrays in plant cells

    PubMed Central

    1978-01-01

    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 overlying developing xylem thickenings, and those in pre-prophase bands are oriented circumferentially but on average are only about one-eighth of the cell circumference in length, i.e., 2-4 micrometer. The arrays consist of overlapping component microtubules, interconnected by cross bridges where they are grouped and also connected to the plasma membrane. Microtubule lengths vary greatly in any given array, but the probability that any pass right around the cell is extremely low. The majority of the microtubule terminations lie in statistically random positions in the arrays, but nonrandomness in the form of groups of terminations and terminations in short lines parallel to the axis of cell elongation has been observed. Low temperature induces microtubule shortening and increases the frequency of C-shaped terminations over the 1.7% found under normal conditions; colchicine and high pressures produce abnormally large proportions of very short microtubules amongst those that survive the treatments. Deuterium oxide (D2O) treatment probably induces the formation of additional microtubules as distinct from increasing the length of those already present. The distribution of C-shaped terminations provides evidence for at least local polarity in the arrays. The validity of the findings is discussed, along with implications for the development, maintenance, and orientation of the arrays and their possible relationship to the orientation of cellulose deposition. PMID:350889

  13. Tau stabilizes microtubules by binding at the interface between tubulin heterodimers.

    PubMed

    Kadavath, Harindranath; Hofele, Romina V; Biernat, Jacek; Kumar, Satish; Tepper, Katharina; Urlaub, Henning; Mandelkow, Eckhard; Zweckstetter, Markus

    2015-06-16

    The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimer's disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubule-binding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tau-microtubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology. PMID:26034266

  14. Regulation of kinesin-transport by microtubule age and polymerization conditions

    NASA Astrophysics Data System (ADS)

    Xu, Jing; Liang, Winnie; King, Stephen; Faysal, K.

    2015-03-01

    Microtubules are fundamental biopolymers in cells, formed via self-assembly (``polymerization'') of tubulin dimers. Microtubule polymerization conditions have been shown to alter the presence of defects in microtubule lattices, including point defects (missing tubulin dimers) and line defects (protofilament disruption). Potential impact of these lattice defects on molecular motor-based transport is not yet understood. Here we investigate the impact of microtubule polymerization conditions on multiple-kinesin transport, using single-molecule-type optical trapping experiments. We find that kinesin-based cargoes pause preferentially at specific locations along individual microtubules, and that the pause frequency and duration is strongly dependent on microtubule age and polymerization condition. Within each polymerization condition and for fresh microtubules, we also observe significant variations in multiple-kinesin travel distances, depending on which microtubules the motors travel along. Taken together, our study suggests an important role of microtubule lattice defect in regulating intracellular transport.

  15. 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.

  16. LKB1 Destabilizes Microtubules in Myoblasts and Contributes to Myoblast Differentiation

    PubMed Central

    Dole, Neha; Gilberti, Renée M.; Dodge-Kafka, Kimberly; Tirnauer, Jennifer S.

    2012-01-01

    Background Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified. Findings We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMP-activated protein kinase (AMPK) and microtubule affinity regulating kinases (MARKs). LKB1 overexpression accelerated differentiation, whereas RNAi impaired it. Conclusions Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeletal changes required for differentiation. Transient destabilization of microtubules might be a useful strategy for enhancing and/or synchronizing myoblast differentiation. PMID:22348111

  17. Microtubules are essential for guard-cell function in Vicia and Arabidopsis.

    PubMed

    Eisinger, William; Ehrhardt, David; Briggs, Winslow

    2012-05-01

    Radially arranged cortical microtubules are a prominent feature of guard cells. Guard cells expressing GFP-tubulin showed consistent changes in the appearance of microtubules when stomata opened or closed. Guard cells showed fewer microtubule structures as stomata closed, whether induced by transfer to darkness, ABA, hydrogen peroxide, or sodium hydrogen carbonate. Guard cells kept in the dark (closed stomata) showed increases in microtubule structures and stomatal aperture on light treatment. GFP-EB1, marking microtubule growing plus ends, showed no change in number of plus ends or velocity of assembly on stomatal closure. Since the number of growing plus ends and the rate of plus-end growth did not change when microtubule structure numbers declined, microtubule instability and/or rearrangement must be responsible for the apparent loss of microtubules. Guard cells with closed stomata showed more cytosolic GFP-fluorescence than those with open stomata as cortical microtubules became disassembled, although with a large net loss in total fluorescence. Microtubule-targeted drugs blocked guard-cell function in Vicia and Arabidopsis. Oryzalin disrupted guard-cell microtubules and prevented stomatal opening and taxol stabilized guard-cell microtubules and delayed stomatal closure. Gas exchange measurements indicated that the transgenes for fluorescent-labeled proteins did not disrupt normal stomatal function. These dynamic changes in guard-cell microtubules combined with our inhibitor studies provide evidence for an active role of microtubules in guard-cell function. PMID:22402260

  18. Mammalian end binding proteins control persistent microtubule growth

    PubMed Central

    Komarova, Yulia; De Groot, Christian O.; Grigoriev, Ilya; Gouveia, Susana Montenegro; Munteanu, E. Laura; Schober, Joseph M.; Honnappa, Srinivas; Buey, Rubén M.; Hoogenraad, Casper C.; Dogterom, Marileen; Borisy, Gary G.; Steinmetz, Michel O.

    2009-01-01

    End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners. PMID:19255245

  19. Cooperative Lattice Dynamics and Anomalous Fluctuations of Microtubules

    E-print Network

    Hervé Mohrbach; Albert Johner; Igor M. Kuli?

    2011-11-19

    Microtubules have been in biophysical focus for several decades. Yet the confusing and mutually contradicting results regarding their elasticity and fluctuations have shed some doubts on their present understanding. In this paper we expose the empirical evidence for the existence of discrete GDP-tubulin fluctuations between a curved and a straight configuration at room temperature as well as for conformational tubulin cooperativity. Guided by a number of experimental findings, we build the case for a novel microtubule model, with the principal result that microtubules can spontaneously form micron size cooperative helical states with unique elastic and dynamic features. The polymorphic dynamics of the microtubule lattice resulting from the tubulin bistability quantitatively explains several experimental puzzles including anomalous scaling of dynamic fluctuations of grafted microtubules, their apparent length-stiffness relation and their remarkably curved-helical appearance in general. We point out that tubulin dimers's multistability and its cooperative switching could participate in important cellular processes, and could in particular lead to efficient mechanochemical signalling along single microtubules.

  20. 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.

  1. Quantum Computation in Brain Microtubules? Decoherence and Biological Feasibility

    E-print Network

    S. Hagan; S. R. Hameroff; J. A. Tuszy?ski

    2000-05-04

    The Penrose-Hameroff (`Orch OR') model of quantum computation in brain microtubules has been criticized as regards the issue of environmental decoherence. A recent report by Tegmark finds that microtubules can maintain quantum coherence for only $10^{-13}$ s, far too short to be neurophysiologically relevant. Here, we critically examine the assumptions behind Tegmark's calculation and find that: 1) Tegmark's commentary is not aimed at an existing model in the literature but rather at a hybrid that replaces the superposed protein conformations of the `Orch OR' theory with a soliton in superposition along the microtubule, 2) Tegmark predicts decreasing decoherence times at lower temperature, in direct contradiction of the observed behavior of quantum states, 3) recalculation after correcting Tegmark's equation for differences between his model and the `Orch OR' model (superposition separation, charge vs. dipole, dielectric constant) lengthens the decoherence time to $10^{-5} - 10^{-4}$ s and invalidates a critical assumption of Tegmark's derivation, 4) incoherent metabolic energy supplied to the collective dynamics ordering water in the vicinity of microtubules at a rate exceeding that of decoherence can counter decoherence effects (in the same way that lasers avoid decoherence at room temperature), and 5) phases of actin gelation may enhance the ordering of water around microtubule bundles, further increasing the decoherence-free zone by an order of magnitude and the decoherence time to $10^{-2} - 10^{-1}$ s. These revisions bring microtubule decoherence into a regime in which quantum gravity can interact with neurophysiology.

  2. Quantum computation in brain microtubules: Decoherence and biological feasibility

    NASA Astrophysics Data System (ADS)

    Hagan, S.; Hameroff, S. R.; Tuszy?ski, J. A.

    2002-06-01

    The Penrose-Hameroff orchestrated objective reduction (orch. OR) model assigns a cognitive role to quantum computations in microtubules within the neurons of the brain. Despite an apparently ``warm, wet, and noisy'' intracellular milieu, the proposal suggests that microtubules avoid environmental decoherence long enough to reach threshold for ``self-collapse'' (objective reduction) by a quantum gravity mechanism put forth by Penrose. The model has been criticized as regards the issue of environmental decoherence, and a recent report by Tegmark finds that microtubules can maintain quantum coherence for only 10-13 s, far too short to be neurophysiologically relevant. Here, we critically examine the decoherence mechanisms likely to dominate in a biological setting and find that (1) Tegmark's commentary is not aimed at an existing model in the literature but rather at a hybrid that replaces the superposed protein conformations of the orch. OR theory with a soliton in superposition along the microtubule; (2) recalculation after correcting for differences between the model on which Tegmark bases his calculations and the orch. OR model (superposition separation, charge vs dipole, dielectric constant) lengthens the decoherence time to 10-5-10-4 s (3) decoherence times on this order invalidate the assumptions of the derivation and determine the approximation regime considered by Tegmark to be inappropriate to the orch. OR superposition; (4) Tegmark's formulation yields decoherence times that increase with temperature contrary to well-established physical intuitions and the observed behavior of quantum coherent states; (5) incoherent metabolic energy supplied to the collective dynamics ordering water in the vicinity of microtubules at a rate exceeding that of decoherence can counter decoherence effects (in the same way that lasers avoid decoherence at room temperature); (6) microtubules are surrounded by a Debye layer of counterions, which can screen thermal fluctuations, and by an actin gel that might enhance the ordering of water in bundles of microtubules, further increasing the decoherence-free zone by an order of magnitude and, if the dependence on the distance between environmental ion and superposed state is accurately reflected in Tegmark's calculation, extending decoherence times by three orders of magnitude; (7) topological quantum computation in microtubules may be error correcting, resistant to decoherence; and (8) the decohering effect of radiative scatterers on microtubule quantum states is negligible. These considerations bring microtubule decoherence into a regime in which quantum gravity could interact with neurophysiology.

  3. SUR LE PROBLME PLAN DE PROPAGATION POUR LE DOUBLET LECTRIQUE HORIZONTAL Par G. BOUDOURIS et D. ILIAS.

    E-print Network

    Paris-Sud XI, Université de

    A par un doublet éléctrique vertical place au point B.et ayant la longueur 1 et le moment pcos qJ. On se compo- sante &1,. On remarquera maintenant que le doublet pcos q ne doit pas produire au point B la composante &r rayonn6e par le doublet pcos cp ou, ce qui revient au meme, par le doublet p

  4. Masses of physical scalars in two Higgs doublet models

    NASA Astrophysics Data System (ADS)

    Biswas, Ambalika; Lahiri, Amitabha

    2015-06-01

    We find bounds on scalar masses resulting from a criterion of naturalness, in a broad class of two Higgs doublet models. Specifically, we assume the cancellation of quadratic divergences in what are called the type I, type II, lepton-specific, and flipped two Higgs doublet models, with an additional U(1) symmetry. This results in a set of relations among masses of the physical scalars and coupling constants, a generalization of the Veltman conditions of the standard model. Assuming that the lighter C P -even neutral Higgs particle is the observed scalar particle of mass ˜125 GeV , and imposing further the constraints from the electroweak T-parameter, stability, and perturbative unitarity, we calculate the range of the mass of each of the remaining physical scalars.

  5. Fluctuations of doublet splittings using the annular billiard

    SciTech Connect

    Egydio de Carvalho, R.; Mijolaro, A.P. [Instituto de Geocie e CieExatas, Universidade Estadual Paulista-UNESP 13500-230 Rio Claro, SP (Brazil)

    2004-11-01

    We study the statistical distribution of quantum energy splittings due to a dynamical tunneling. The system, the annular billiard, has whispering quasimodes due to a discrete symmetry that exists even when chaos is present in the underlying classical dynamics. Symmetric and antisymmetric combinations of these quasimodes correspond to quantum doublet states whose degeneracies decrease as the circles become more eccentric. We construct numerical ensembles composed of splittings for two distinct regimes, one which we call semiclassical for high quantum numbers and high energies where the whispering regions are connected by chaos, and other which we call quantal for low quantum numbers, low energies, and near integrable where dynamical tunneling is not a dominant mechanism. In both cases we observe a variation on the fluctuation amplitudes, but their mean behaviors follow the formula of Leyvraz and Ullmo [J. Phys. A 29, 2529 (1996)]. A description of a three-level collision involving a doublet and a singlet is also provided through a numerical example.

  6. Power systems for the Doublet III 2-MW ECH system

    SciTech Connect

    Remsen, D.B. Jr.

    1981-10-01

    A system providing 5 second pulses at 60 GHz from ten 200 kW gyrotrons is being prepared for electron cyclotron heating experiments on Doublet III. The power supply for the gyrotron system is a power supply currently under construction by Universal Voltronics Corporation for the Doublet III neutral beam power supply, and is to have the option of reverse polarity (negative) to fill the needs of the system of ten gyrotons operating in parallel. The output of this power supply is 80 kV at 100A for 5 second pulses with good regulation. The output pulse rise and fall times and fault protection response time are all compatible with the gyrotron requirements.

  7. Inflation and dark matter in two Higgs doublet models

    Microsoft Academic Search

    Jinn-Ouk Gong; Sin Kyu Kang; Hyun Min Lee

    2012-01-01

    We consider the Higgs inflation in the extension of the Standard Model with two Higgs doublets coupled to gravity non-minimally. In the presence of an approximate global U(1) symmetry in the Higgs sector, both radial and angular modes of neutral Higgs bosons drive inflation where large non-Gaussianity is possible from appropriate initial conditions on the angular mode. We also discuss

  8. Open-shell doublet character in a hexaazatrinaphthylene trianion complex.

    PubMed

    Moilanen, Jani O; Day, Benjamin M; Pugh, Thomas; Layfield, Richard A

    2015-07-01

    Three-electron reduction of hexaazatrinaphthylene (HAN) with a magnesium(i) reagent leads to [(HAN){Mg(nacnac)}3] (), containing a [HAN](3-) ligand with a spin of S = 1/2. Ab initio calculations reveal that the [HAN](3-) ligand in has a ground-state wave function with multiconfigurational properties, and can be described as a triradicaloid species with a small amount of open-shell doublet character. PMID:26088395

  9. Pseudomonas aeruginosa exotoxin Y-mediated tau hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells.

    PubMed

    Balczon, Ron; Prasain, Nutan; Ochoa, Cristhiaan; Prater, Jason; Zhu, Bing; Alexeyev, Mikhail; Sayner, Sarah; Frank, Dara W; Stevens, Troy

    2013-01-01

    Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly. PMID:24023939

  10. Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly

    PubMed Central

    Nazarova, Elena; O'Toole, Eileen; Kaitna, Susi; Francois, Paul; Winey, Mark; Vogel, Jackie

    2013-01-01

    During spindle assembly, microtubules may attach to kinetochores or pair to form antiparallel pairs or interpolar microtubules, which span the two spindle poles and contribute to mitotic pole separation and chromosome segregation. Events in the specification of the interpolar microtubules are poorly understood. Using three-dimensional electron tomography and analysis of spindle dynamical behavior in living cells, we investigated the process of spindle assembly. Unexpectedly, we found that the phosphorylation state of an evolutionarily conserved Cdk1 site (S360) in ?-tubulin is correlated with the number and organization of interpolar microtubules. Mimicking S360 phosphorylation (S360D) results in bipolar spindles with a normal number of microtubules but lacking interpolar microtubules. Inhibiting S360 phosphorylation (S360A) results in spindles with interpolar microtubules and high-angle, antiparallel microtubule pairs. The latter are also detected in wild-type spindles <1 ?m in length, suggesting that high-angle microtubule pairing represents an intermediate step in interpolar microtubule formation. Correlation of spindle architecture with dynamical behavior suggests that microtubule pairing is sufficient to separate the spindle poles, whereas interpolar microtubules maintain the velocity of pole displacement during early spindle assembly. Our findings suggest that the number of interpolar microtubules formed during spindle assembly is controlled in part through activities at the spindle poles. PMID:23966467

  11. Pseudomonas aeruginosa Exotoxin Y-Mediated Tau Hyperphosphorylation Impairs Microtubule Assembly in Pulmonary Microvascular Endothelial Cells

    PubMed Central

    Balczon, Ron; Prasain, Nutan; Ochoa, Cristhiaan; Prater, Jason; Zhu, Bing; Alexeyev, Mikhail; Sayner, Sarah; Frank, Dara W.; Stevens, Troy

    2013-01-01

    Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly. PMID:24023939

  12. Automated stitching of microtubule centerlines across serial electron tomograms.

    PubMed

    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

  13. 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.

  14. Microtubule assembly and disassembly at alkaline pH

    PubMed Central

    Regula, CS; Pfeiffer; Berlin, RD

    1981-01-01

    Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of “treadmilling.” The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo. PMID:7228899

  15. Inflation and dark matter in two Higgs doublet models

    NASA Astrophysics Data System (ADS)

    Gong, Jinn-Ouk; Lee, Hyun Min; Kang, Sin Kyu

    2012-04-01

    We consider the Higgs inflation in the extension of the Standard Model with two Higgs doublets coupled to gravity non-minimally. In the presence of an approximate global U(1) symmetry in the Higgs sector, both radial and angular modes of neutral Higgs bosons drive inflation where large non-Gaussianity is possible from appropriate initial conditions on the angular mode. We also discuss the case with single-field inflation for which the U(1) symmetry is broken to a Z 2 subgroup. We show that inflationary constraints, perturbativity and stability conditions restrict the parameter space of the Higgs quartic couplings at low energy in both multi- and single-field cases. Focusing on the inert doublet models where Z 2 symmetry remains unbroken at low energy, we show that the extra neutral Higgs boson can be a dark matter candidate consistent with the inflationary constraints. The doublet dark matter is always heavy in multi-field inflation while it can be light due to the suppression of the co-annihilation in single-field inflation. The implication of the extra quartic couplings on the vacuum stability bound is also discussed in the light of the recent LHC limits on the Higgs mass.

  16. Asymmetric Inelastic Inert Doublet Dark Matter from Triplet Scalar Leptogenesis

    E-print Network

    Chiara Arina; Narendra Sahu

    2011-09-01

    The nature of dark matter (DM) particles and the mechanism that provides their measured relic abundance are currently unknown. In this paper we investigate inert scalar and vector like fermion doublet DM candidates with a charge asymmetry in the dark sector, which is generated by the same mechanism that provides the baryon asymmetry, namely baryogenesis-via-leptogenesis induced by decays of scalar triplets. At the same time the model gives rise to neutrino masses in the ballpark of oscillation experiments via type II seesaw. We discuss possible sources of depletion of asymmetry in the DM and visible sectors and solve the relevant Boltzmann equations for quasi-equilibrium decay of triplet scalars. A Monte-Carlo-Markov-Chain analysis is performed for the whole parameter space. The survival of the asymmetry in the dark sector leads to inelastic scattering off nuclei. We then apply bayesian statistic to infer the model parameters favoured by the current experimental data, in particular the DAMA annual modulation and Xenon100 exclusion limit. The latter strongly disfavours asymmetric scalar doublet DM of mass $\\mathcal{O}(\\TeV)$ as required by DM-$\\bar{\\rm DM}$ oscillations, while an asymmetric vector like fermion doublet DM with mass around 100 GeV is a good candidate for DAMA annual modulation yet satisfying the constraints from Xenon100 data.

  17. Two-Higgs-Doublet Type-II Seesaw Model

    E-print Network

    Chuan-Hung Chen; Takaaki Nomura

    2014-10-02

    Motivated by the new observed scalar boson of 126 GeV at ATLAS and CMS, various phenomena in two-Higgs-doublet model (THDM) are investigated broadly in the literature. For considering the model that possesses a solution to the massive neutrinos, we study the simplest extension of conventional type-II seesaw model to two Higgs doublets. We find that the new interactions in the scalar potential cause the sizable mixture of charged Higgses in triplet and doublet. As a result, we have a completely different decay pattern for doubly charged Higgs ($\\delta^{\\pm\\pm}$), even the vacuum expectation value (VEV) of Higgs triplet is at GeV level, which is limited by the precision measurement for $\\rho$-parameter. For illustrating the new characters of the model, we study the influence of new interactions on the new open channels $\\delta^{++}\\to ( H^+_1 W^{+^{(*)}}, H^+_1 H^+_1)$ with $H^+_1$ being the lightest charged Higgs. Additionally, due to the new mixing effect, the triplet charged Higgs could couple to quarks in the model; therefore, the search for $\\delta^{++}$ via $\\delta^{++}\\to tb W^+ \\to b \\bar b W^+ W^+$ by mediated $H^+_{1}$ becomes significant.

  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. 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. Microtubules: The neuronic system of the neurons?

    E-print Network

    N. E. Mavromatos; D. V. Nanopoulos

    1997-01-31

    In this talk we review recent work on integrable models for Microtubule (MT) networks, subneural paracrystalline cytosceletal structures, which seem to play a fundamental role in the neurons. We cast here the complicated MT dynamics in the form of a 1+1-dimensional non-critical string theory, which can be formulated in terms of (dual) Dirichlet branes, according to modern perspectives. We suggest that the MTs are the microsites in the brain, for the emergence of stable, macroscopic quantum coherent states, identifiable with the ``preconscious states''. Quantum space-time effects, as described by non-critical string theory, trigger then an ``organized collapse'' of the coherent states down to a specific or ``conscious state''. The whole process we estimate to take O(1 sec), in excellent agreement with a plethora of experimental/observational findings. The complete integrability of the stringy model for MT proves sufficient in providing a satisfactory solution to memory coding and capacity. Such features might turn out to be important for a model of the brain as a quantum computer.

  1. Tau induces cooperative Taxol binding to microtubules

    PubMed Central

    Ross, Jennifer L.; Santangelo, Christian D.; Makrides, Victoria; Fygenson, D. Kuchnir

    2004-01-01

    Taxol and tau are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds ? tubulin in the MT interior. Tau is a MT-associated protein that binds both ? and ? tubulin on the MT exterior. Both Taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts to bundle, stiffen, and space MTs. A structural study recently suggested that Taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces Taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau, yields the equilibrium dissociation constant of ?2 ?M, and determines the escape rate of Taxol through one pore to be 1.7 × 103 (M·s)–1. Extension of the model yields a measure of Taxol cooperativity with a Hill coefficient of at least 15 when tau is present at a 1:1 molar ratio with tubulin. PMID:15326286

  2. Self-organization of a radial microtubule array by dynein-dependent nucleation of microtubules

    PubMed Central

    Vorobjev, Ivan; Malikov, Viacheslav; Rodionov, Vladimir

    2001-01-01

    Polarized radial arrays of cytoplasmic microtubules (MTs) with minus ends clustered at the cell center define the organization of the cytoplasm through interaction with microtubule motors bound to membrane organelles or chromosomes. It is generally assumed that the radial organization results from nucleation of MTs at the centrosome. However, radial MT array can also be attained through self-organization that requires the activity of a minus-end-directed MT motor, cytoplasmic dynein. In this study we examine the role of cytoplasmic dynein in the self-organization of a radial MT array in cytoplasmic fragments of fish melanophores lacking the centrosome. After activation of dynein motors bound to membrane-bound organelles, pigment granules, the fragments rapidly form polarized radial arrays of MTs and position pigment aggregates at their centers. We show that rearrangement of MTs in the cytoplasm is achieved through dynein-dependent MT nucleation. The radial pattern is generated by continuous disassembly and reassembly of MTs and concurrent minus-end-directed transport of pigment granules bearing the nucleation sites. PMID:11504928

  3. 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

  4. Unusual ciliary abnormalities in three 9/11 response workers.

    PubMed

    McMahon, James T; Aslam, Rizwan; Schell, Stephen E

    2011-01-01

    After the 9/11 terrorist attacks on the World Trade Center in New York in 2001, thousands of response workers were exposed to complex mixtures of toxins, pollutants, and carcinogens. Many developed illnesses involving the respiratory tract. We report unusual ultrastructural ciliary abnormalities in 3 response workers that corresponded to their respiratory and ciliary functional abnormalities. Each patient had respiratory cilia biopsies that were evaluated for motility and ultrastructural changes. Impaired ciliary motility was seen in 2 of the 3 patients. Each of the patients showed monomorphic ultrastructural abnormalities. Two of the patients showed identical triangular disarray of axonemal microtubules with peripheral doublets 1,4, and 7 forming the corners of the triangle and doublet 9 always more medially displaced than doublets 2, 3, 5, 6, and 8. Two workers had cilia in which axonemes were replaced by homogeneously dense cores. One of these also had cilia with triangular axonemes as previously described. The other had cilia with a geometric triangular to pentagonal shape. The ciliary abnormalities described here may represent a new class of primary ciliary dyskinesia in which abnormalities may have a genetic basis and a phenotypic expression that is prompted at the cellular level by local environmental conditions. PMID:21370679

  5. Golgi-derived CLASP-dependent Microtubules Control Golgi Organization and Polarized Trafficking in Motile Cells

    PubMed Central

    Miller, Paul M.; Folkmann, Andrew W.; Maia, Ana R.R.; Efimova, Nadia; Efimov, Andrey; Kaverina, Irina

    2009-01-01

    Microtubules are indispensable for Golgi complex assembly and maintenance that is an integral part of cytoplasm organization in interphase mammalian cells. Here, we show that two discrete microtubule subsets drive two distinct, yet simultaneous, stages of Golgi assembly. In addition to the radial centrosomal microtubule array, which positions the Golgi in the cell center, we identify a role for microtubules that form at the Golgi membranes in a manner dependent on microtubule regulators CLASPs. These Golgi-derived microtubules draw Golgi mini-stacks together in tangential fashion and are critical for establishing continuity and proper morphology of the Golgi complex. We propose that specialized functions of these two microtubule arrays arise from their specific geometries. Further, we demonstrate that directional post-Golgi trafficking and cell migration depend on Golgi-associated CLASPs suggesting that correct organization of the Golgi complex by microtubules is essential for cell polarization and motility. PMID:19701196

  6. Neurotrophic Factors Stabilize Microtubules and Protect against Rotenone Toxicity on Dopaminergic Neurons*

    E-print Network

    Feng, Jian

    by the microtubule-stabilizing agent taxol. Here we show that nerve growth factor (NGF) significantly reduced rote of the mitochondria respiratory chain (7), rotenone is also a strong microtubule-depolymerizing agent (8

  7. Effects of anti-Alzheimer drugs on phosphorylation and assembly of microtubules from brain microtubular proteins.

    PubMed

    Shevtsov, P N; Shevtsova, E F; Burbaeva, G Sh; Bachurin, S O

    2014-04-01

    We studied the effects of anti-Alzheimer drugs (tacrine, amiridine, and memantine) on phosphorylation of tubulin and microtubule-associated proteins isolated from rat brain, evaluated the capacity of these proteins to polymerize into microtubules after addition of study pharmacological agents, and analyzed the structure of generated microtubules. It was shown that test substances impair assembly of microtubules to a different extent. Dose-dependent effects of these agents on phosphorylation of tubulin and microtubule-associated proteins were observed. Triazolam (not approved for clinical use as anti-Alzheimer drug) in the same concentrations was used as the reference substance in the same tests. It was observed that this substance even in minimal concentration induced the most pronounced changes in microtubule structure. A direct correlation between the capacity of the test substances to modulate tubulin phosphorylation and to impair microtubule structure was found: the more the substance inhibited tubulin phosphorylation, the more it disordered microtubule structure. PMID:24824692

  8. New tools reveal interaction determinants and post-mitotic function of crucial microtubule regulators 

    E-print Network

    Lesniewska, Karolina

    2014-11-27

    Microtubules are a major constituent of the cytoskeleton in all eukaryotic cells. They are essential for cell morphogenesis and motility. Specifically in the dividing cells, microtubules form the spindle which segregates ...

  9. Enhanced Mechanical Stability of Microtubules Polymerized with a Slowly Hydrolyzable Nucleotide Analogue

    E-print Network

    Smith, Marc L.

    Enhanced Mechanical Stability of Microtubules Polymerized with a Slowly Hydrolyzable Nucleotide of microtubules polymerized using guanylyl-R- -methylene diphosphonate (GMPCPP), a slowly hydrolyzable analogue- ing hydrolyzed GTP are intrinsically unstable, hindering direct in vitro studies of their mechanical

  10. Motor-induced sliding of microtubule and actin bundles Assaf Zemelab

    E-print Network

    Mogilner, Alex

    of the filament, i.e. dynein-toward microtubule minus end, kinesin-1-toward microtubule plus end, etc. The molecular motors are often deployed to transport vesicles and organelles, as well as to generate forces

  11. 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. PMID:25897332

  12. Noncentrosomal microtubules and type II myosins potentiate epidermal cell adhesion and barrier formation

    PubMed Central

    Sumigray, Kaelyn D.; Foote, Henry P.

    2012-01-01

    During differentiation, many cells reorganize their microtubule cytoskeleton into noncentrosomal arrays. Although these microtubules are likely organized to meet the physiological roles of their tissues, their functions in most cell types remain unexplored. In the epidermis, differentiation induces the reorganization of microtubules to cell–cell junctions in a desmosome-dependent manner. Here, we recapitulate the reorganization of microtubules in cultured epidermal cells. Using this reorganization assay, we show that cortical microtubules recruit myosin II to the cell cortex in order to engage adherens junctions, resulting in an increase in mechanical integrity of the cell sheets. Cortical microtubules and engaged adherens junctions, in turn, increase tight junction function. In vivo, disruption of microtubules or loss of myosin IIA and B resulted in loss of tight junction–mediated barrier activity. We propose that noncentrosomal microtubules act through myosin II recruitment to potentiate cell adhesion in the differentiating epidermis, thus forming a robust mechanical and chemical barrier against the external environment. PMID:23091070

  13. The feasibility of coherent energy transfer in microtubules.

    PubMed

    Craddock, Travis John Adrian; Friesen, Douglas; Mane, Jonathan; Hameroff, Stuart; Tuszynski, Jack A

    2014-11-01

    It was once purported that biological systems were far too 'warm and wet' to support quantum phenomena mainly owing to thermal effects disrupting quantum coherence. However, recent experimental results and theoretical analyses have shown that thermal energy may assist, rather than disrupt, quantum coherent transport, especially in the 'dry' hydrophobic interiors of biomolecules. Specifically, evidence has been accumulating for the necessary involvement of quantum coherent energy transfer between uniquely arranged chromophores in light harvesting photosynthetic complexes. The 'tubulin' subunit proteins, which comprise microtubules, also possess a distinct architecture of chromophores, namely aromatic amino acids, including tryptophan. The geometry and dipolar properties of these aromatics are similar to those found in photosynthetic units indicating that tubulin may support coherent energy transfer. Tubulin aggregated into microtubule geometric lattices may support such energy transfer, which could be important for biological signalling and communication essential to living processes. Here, we perform a computational investigation of energy transfer between chromophoric amino acids in tubulin via dipole excitations coupled to the surrounding thermal environment. We present the spatial structure and energetic properties of the tryptophan residues in the microtubule constituent protein tubulin. Plausibility arguments for the conditions favouring a quantum mechanism of signal propagation along a microtubule are provided. Overall, we find that coherent energy transfer in tubulin and microtubules is biologically feasible. PMID:25232047

  14. Single molecule studies reveal new mechanisms for microtubule severing

    NASA Astrophysics Data System (ADS)

    Ross, Jennifer; Diaz-Valencia, Juan Daniel; Morelli, Margaret; Zhang, Dong; Sharp, David

    2011-03-01

    Microtubule-severing enzymes are hexameric complexes made from monomeric enzyme subunits that remove tubulin dimers from the microtubule lattice. Severing proteins are known to remodel the cytoskeleton during interphase and mitosis, and are required in proper axon morphology and mammalian bone and cartilage development. We have performed the first single molecule imaging to determine where and how severing enzymes act to cut microtubules. We have focused on the original member of the group, katanin, and the newest member, fidgetin to compare their biophysical activities in vitro. We find that, as expected, severing proteins localize to areas of activity. Interestingly, the association is very brief: they do not stay bound nor do they bind cooperatively at active sites. The association duration changes with the nucleotide content, implying that the state in the catalytic cycle dictates binding affinity with the microtubule. We also discovered that, at lower concentrations, both katanin and fidgetin can depolymerize taxol-stabilized microtubules by removing terminal dimers. These studies reveal the physical regulation schemes to control severing activity in cells, and ultimately regulate cytoskeletal architecture. This work is supported by the March of Dimes Grant #5-FY09-46.

  15. 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

  16. Length control of microtubules by depolymerizing motor proteins

    E-print Network

    Bindu S. Govindan; Manoj Gopalakrishnan; Debashish Chowdhury

    2008-07-08

    In many intracellular processes, the length distribution of microtubules is controlled by depolymerizing motor proteins. Experiments have shown that, following non-specific binding to the surface of a microtubule, depolymerizers are transported to the microtubule tip(s) by diffusion or directed walk and, then, depolymerize the microtubule from the tip(s) after accumulating there. We develop a quantitative model to study the depolymerizing action of such a generic motor protein, and its possible effects on the length distribution of microtubules. We show that, when the motor protein concentration in solution exceeds a critical value, a steady state is reached where the length distribution is, in general, non-monotonic with a single peak. However, for highly processive motors and large motor densities, this distribution effectively becomes an exponential decay. Our findings suggest that such motor proteins may be selectively used by the cell to ensure precise control of MT lengths. The model is also used to analyze experimental observations of motor-induced depolymerization.

  17. Changes in Neurofilament and Microtubule Distribution following Focal Axon Compression

    PubMed Central

    Fournier, Adam J.; Hogan, James D.; Rajbhandari, Labchan; Shrestha, Shiva; Venkatesan, Arun; Ramesh, K. T.

    2015-01-01

    Although a number of cytoskeletal derangements have been described in the setting of traumatic axonal injury (TAI), little is known of early structural changes that may serve to initiate a cascade of further axonal degeneration. Recent work by the authors has examined conformational changes in cytoskeletal constituents of neuronal axons undergoing traumatic axonal injury (TAI) following focal compression through confocal imaging data taken in vitro and in situ. The present study uses electron microscopy to understand and quantify in vitro alterations in the ultrastructural composition of microtubules and neurofilaments within neuronal axons of rats following focal compression. Standard transmission electron microscopy processing methods are used to identify microtubules, while neurofilament identification is performed using antibody labeling through gold nanoparticles. The number, density, and spacing of microtubules and neurofilaments are quantified for specimens in sham Control and Crushed groups with fixation at <1min following load. Our results indicate that the axon caliber dependency known to exist for microtubule and neurofilament metrics extends to axons undergoing TAI, with the exception of neurofilament spacing, which appears to remain constant across all Crushed axon diameters. Confidence interval comparisons between Control and Crushed cytoskeletal measures suggests early changes in the neurofilament spatial distributions within axons undergoing TAI may precede microtubule changes in response to applied loads. This may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level, and suggests the rapid removal of neurofilament sidearms as one possible mechanism. PMID:26111004

  18. 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

  19. Microtubule guiding in a multi-walled carbon nanotube circuit.

    PubMed

    Sikora, Aurélien; Ramón-Azcón, Javier; Sen, Mustafa; Kim, Kyongwan; Nakazawa, Hikaru; Umetsu, Mitsuo; Kumagai, Izumi; Shiku, Hitoshi; Matsue, Tomokazu; Teizer, Winfried

    2015-08-01

    In nanotechnological devices, mass transport can be initiated by pressure driven flow, diffusion or by employing molecular motors. As the scale decreases, molecular motors can be helpful as they are not limited by increased viscous resistance. Moreover, molecular motors can move against diffusion gradients and are naturally fitted for nanoscale transportation. Among motor proteins, kinesin has particular potential for lab-on-a-chip applications. It can be used for sorting, concentrating or as a mechanical sensor. When bound to a surface, kinesin motors propel microtubules in random directions, depending on their landing orientation. In order to circumvent this complication, the microtubule motion should be confined or guided. To this end, dielectrophoretically aligned multi-walled-carbon nanotubes (MWCNT) can be employed as nanotracks. In order to control more precisely the spatial repartition of the MWCNTs, a screening method has been implemented and tested. Polygonal patterns have been fabricated with the aim of studying the guiding and the microtubule displacement between MWCNT segments. Microtubules are observed to transfer between MWCNT segments, a prerequisite for the guiding of microtubules in MWCNT circuit-based biodevices. The effect of the MWCNT organization (crenellated or hexagonal) on the MT travel distance has been investigated as well. PMID:26162482

  20. 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

  1. 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

  2. ASAP, a human microtubule-associated protein required for bipolar spindle assembly and cytokinesis

    Microsoft Academic Search

    Jean-Michel Saffin; Magali Venoux; Claude Prigent; Julien Espeut; Francis Poulat; Dominique Giorgi; Ariane Abrieu; Sylvie Rouquier

    2005-01-01

    We have identified a unique human microtubule-associated protein (MAP) named ASAP for ASter-Associated Protein. ASAP localizes to microtubules in interphase, associates with the mitotic spindle during mitosis, localizes to the central body during cytokinesis and directly binds to purified microtubules by its COOH-terminal domain. Overexpression of ASAP induces profound bundling of cytoplasmic microtubules in interphase cells and aberrant monopolar spindles

  3. The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis

    Microsoft Academic Search

    José A. Sánchez-Alcázar; Ángeles Rodríguez-Hernández; Mario D. Cordero; Daniel J. M. Fernández-Ayala; Gloria Brea-Calvo; Katherina Garcia; Plácido Navas

    2007-01-01

    It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate\\u000a that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic\\u000a microtubule network possesses a novel organization, whose nucleation appears independent of conventional ?-tubulin ring complex\\u000a containing structures. Our analysis suggests that microtubules

  4. 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

  5. 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

  6. Tracking the ends: a dynamic protein network controls the fate of microtubule tips

    Microsoft Academic Search

    Anna Akhmanova; Michel O. Steinmetz

    2008-01-01

    Microtubule plus-end tracking proteins (+TIPs) are a diverse group of evolutionarily conserved cellular factors that accumulate at the ends of growing microtubules. They form dynamic networks through the interaction of a limited set of protein modules, repeat sequences and linear motifs that bind to each other with moderate affinities. +TIPs regulate different aspects of cell architecture by controlling microtubule dynamics,

  7. Nuclear envelope radiating microtubules in plant cells during interphase mitosis transition

    Microsoft Academic Search

    R. Bakhuizen; P. C. van Spronsen; F. A. J. Sluiman-den Hertog; C. J. Venverloo; L. Goosen-de Roo

    1985-01-01

    Summary The microtubule distribution during the transition from interphase to the mitotic phase was examined at ultrastructural level in large highly vacuolated cells ofNautilocalyx lynchii and in small non-vacuolated cells ofPisum sativum. Both cell types contain, besides preprophase bands and perinuclear microtubules, also microtubules radiating from the nucleus into the transvacuolar cytoplasmic strands and cytoplasm respectively.

  8. Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination Involved?

    E-print Network

    Boyer, Edmond

    and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies called plus- end tracking proteins (+TIPs) associate specifically with growing microtubule+ends whereCap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination Involved? Anouk Bosson, Jean

  9. PHYSICAL REVIEW E 86, 041918 (2012) Dynamics and length distribution of microtubules under force and confinement

    E-print Network

    Kierfeld, Jan

    2012-01-01

    PHYSICAL REVIEW E 86, 041918 (2012) Dynamics and length distribution of microtubules under force Dortmund, Germany (Received 10 July 2012; published 31 October 2012) We investigate the microtubule typical cellular environments: (i) The microtubule is confined by rigid and fixed walls, (ii) it grows

  10. Effect of tubulin diffusion on polymerization of microtubules P. A. Deymier,1

    E-print Network

    Deymier, Pierre

    Effect of tubulin diffusion on polymerization of microtubules P. A. Deymier,1 Y. Yang,1 and J of microtubules MT's growing from a nucleation center is simulated with a kinetic Monte Carlo model that includes is synchronous and bounded. The microtubules form an aster with a monotonously decreasing long-time distribution

  11. J Biol Chem . Author manuscript GDP-tubulin incorporation into growing microtubules modulates polymer

    E-print Network

    Boyer, Edmond

    J Biol Chem . Author manuscript Page /1 10 GDP-tubulin incorporation into growing microtubules.valiron@ujf-grenoble.fr > Abstract Microtubule growth proceeds through endwise addition of nucleotide bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits which are thought to come exclusively from

  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 direct evidence supporting this hypothesis is lacking. Previous studies have shown that the microtubule

  13. Kinesin and Dynein-Dynactin at Intersecting Microtubules: Motor Density Affects Dynein Function

    E-print Network

    Ross, Jennifer

    Kinesin and Dynein-Dynactin at Intersecting Microtubules: Motor Density Affects Dynein Function and cytoplasmic dynein are microtubule-based motor proteins that actively transport material throughout the cell. Microtubules can intersect at a variety of angles both near the nucleus and at the cell periphery

  14. EXTRACTION OF 3D MICROTUBULES AXES FROM CELLULAR ELECTRON TOMOGRAPHY IMAGES

    E-print Network

    EXTRACTION OF 3D MICROTUBULES AXES FROM CELLULAR ELECTRON TOMOGRAPHY IMAGES Lichen Liang Dept. Health Albany, NY 12201-0509 Abstract Microtubules are structural and motile elements. They are essential structure, which requires extracting microtubules from the Cellular Electron Tomography Images. Manual seg

  15. Models of spatial and orientational self-organization of microtubules under the influence of gravitational fields

    E-print Network

    Portet, Stéphanie

    Models of spatial and orientational self-organization of microtubules under the influence-organization of microtubules from purified tubulin solutions is sensitive to gravi- tational conditions. In this paper, we propose two models of spatial and orientational self-organization of microtubules in a gravitational field

  16. Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules

    E-print Network

    Hinow, Peter

    Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules Peter Hinow (Petr G and Natural Sciences, Albena, Bulgaria, June 20­25, 2011 Peter Hinow et al. Microtubule Dynamics #12 Ann Jordan (University of California, Santa Barbara, CA, USA) Peter Hinow et al. Microtubule Dynamics

  17. Mitochondrial positioning in fission yeast is driven by association with dynamic microtubules and mitotic

    E-print Network

    Vale, Ronald D.

    Mitochondrial positioning in fission yeast is driven by association with dynamic microtubules, University of California, San Francisco, CA 94107 Contributed by Ronald D. Vale, July 24, 2003 Microtubules. In higher eukaryotes, kinesin motor proteins have been shown to transport mitochondria along microtubules

  18. Collision induced spatial organization of microtubules Vladimir A. Baulin, Carlos M. Marques, Fabrice Thalmann

    E-print Network

    Paris-Sud XI, Université de

    Collision induced spatial organization of microtubules Vladimir A. Baulin, Carlos M. Marques, 2006) Abstract The dynamic behavior of microtubules in solution can be strongly modified by interactions with walls or other structures. We examine here a microtubule growth model where the increase

  19. ENHANCEMENT OF MICROTUBULES IN EM TOMOGRAPHY Ming Jiang and Qiang Ji

    E-print Network

    ENHANCEMENT OF MICROTUBULES IN EM TOMOGRAPHY Ming Jiang and Qiang Ji Dept of ECSE Rensselaer, NY 12201-0509 ABSTRACT The interpretation of the EM tomography of microtubules is challenging due of microtubules, it is particu- larly challenging to interpret the acquired data due to its low SNR and the fact

  20. Theory of self-assembly of microtubules and motors Igor S. Aranson1

    E-print Network

    Hasty, Jeff

    Theory of self-assembly of microtubules and motors Igor S. Aranson1 and Lev S. Tsimring2 1 Argonne describing spatiotemporal organization of an array of microtubules interacting via molecular motors. Starting functions of molecular motors is to organize a network of long filaments microtubules during cell division

  1. Abstract Electron micrographs of tips of growing and shrinking microtubules are analyzed and interpreted. The

    E-print Network

    Jánosi, Imre M.

    Abstract Electron micrographs of tips of growing and shrinking microtubules are analyzed with competing intrinsic curvatures. Observations are also consistent with growing and shrinking microtubules shrinking microtubules. If this is so, the lateral bonds between protofilaments are responsible for the dif

  2. L E T T E R S Pivoting of microtubules around the spindle pole

    E-print Network

    Pavin, Nenad

    L E T T E R S Pivoting of microtubules around the spindle pole accelerates kinetochore capture Iana,5 During cell division, spindle microtubules attach to chromosomes through kinetochores, protein complexes on the chromosome1 . The central question is how microtubules find kinetochores. According to the pioneering idea

  3. Axonal Transport of Microtubules: the Long and Short Peter W. Baas*, C. Vidya Nadar and Kenneth

    E-print Network

    Baas, Peter W.

    Review Axonal Transport of Microtubules: the Long and Short of It Peter W. Baas*, C. Vidya Nadar studies on cultured neurons have demonstrated that microtubules are transported down the axon in the form, and occurs at the fast rate of known motors. The majority of the microtubule mass in the axon exists

  4. Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

    E-print Network

    Texas at Austin. University of

    Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence (received for review June 25, 2005) Microtubules are hollow cylindrical structures that constitute one and the architecture of the microtubule. Here, we use single-particle tracking methods com- bined with a fluctuation

  5. Pattern formation of microtubules and motors: Inelastic interaction of polar rods Igor S. Aranson1

    E-print Network

    Hasty, Jeff

    Pattern formation of microtubules and motors: Inelastic interaction of polar rods Igor S. Aranson1 organization of an array of microtubules interacting via molecular motors. Starting from a stochastic model or vortices depending on the type and concentration of MM. After MM binds to a microtubule at a random

  6. Model-Based Automated Extraction of Microtubules from Electron Tomography Volume

    E-print Network

    1 Model-Based Automated Extraction of Microtubules from Electron Tomography Volume Ming Jiang, Qiang Ji*, Senior Member, IEEE, and Bruce F. McEwen I. INTRODUCTION Microtubules form the skeletons, the microtubules attach to chromosomes via kinetochore to drive the movement of chromosomes during mitosis. Re

  7. DOI: 10.1002/adma.200700608 Morphology of Microtubules Grown in Agarose Gels: Effect

    E-print Network

    Deymier, Pierre

    DOI: 10.1002/adma.200700608 Morphology of Microtubules Grown in Agarose Gels: Effect of Diffusion that microtubules (MTs) might serve as active biomolecular nanostructures that can assemble into hierarchi- cal polymerization of microtubules.[14] We have recently reported the controlled nucleation and growth

  8. Transport and Detection of Unlabeled Nucleotide Targets by Microtubules Functionalized With

    E-print Network

    Discher, Dennis

    ARTICLE Transport and Detection of Unlabeled Nucleotide Targets by Microtubules Functionalized of the present study is to create mobile microscale biosensors by attaching molecular beacons to microtubules and using kinesin molecular motors to trans- port these functionalized microtubules across two-dimen- sional

  9. Metallization of nanobiostructures: a theoretical study of copper nanowires growth in microtubules

    E-print Network

    Deymier, Pierre

    Metallization of nanobiostructures: a theoretical study of copper nanowires growth in microtubules-surface copper metallization of microtubules over outer-surface metallization. The inner-surface metallization be important elements for the manufacturing of nanoscale electronic circuits and devices. Microtubules (MTs

  10. Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules

    E-print Network

    Hinow, Peter

    Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules Peter Hinow1 for the growth, catastrophe and rescue processes of steady state microtubules assembled from MAP-free tubulin in the possible presence of a microtubule associated drug. As an example for the latter, we both experimentally

  11. Force-Velocity Relation for Growing Microtubules Anatoly B. Kolomeisky* and Michael E. Fisher

    E-print Network

    Force-Velocity Relation for Growing Microtubules Anatoly B. Kolomeisky* and Michael E. Fisher and Technology, University of Maryland, College Park, Maryland 20742 USA ABSTRACT Forces generated by microtubule:856­860) for single microtubules growing in vitro, but their analysis of the data suggested that V decreased more

  12. Theoretical Analysis of Microtubules Dynamics Using a Physical-Chemical Description of Hydrolysis

    E-print Network

    Theoretical Analysis of Microtubules Dynamics Using a Physical- Chemical Description of Hydrolysis, United States *S Supporting Information ABSTRACT: Microtubules are cytoskeleton multifilament pro- teins of microtubule dynamics based on discrete-state stochastic models that explicitly takes into account all relevant

  13. MAL3 MASKS CATASTROPHE EVENTS IN SCHIZOSACCHAROMYCES POMBE MICROTUBULES BY INHIBITING SHRINKAGE AND PROMOTING RESCUE*

    E-print Network

    Cross, Robert

    MAL3 MASKS CATASTROPHE EVENTS IN SCHIZOSACCHAROMYCES POMBE MICROTUBULES BY INHIBITING SHRINKAGE Microtubule Dynamics Address correspondence to: Robert A. Cross, The Chart, Oxted, Surrey, RH8 0TL,UK, Tel in a tip- tracking network that regulates microtubule dynamics in cells. How Mal3 itself influences

  14. Microtubule-severing enzymes at the cutting edge David J. Sharp1,

    E-print Network

    Ross, Jennifer

    Microtubule-severing enzymes at the cutting edge David J. Sharp1, * and Jennifer L. Ross2 1-dependent severing of microtubules was first reported in Xenopus laevis egg extracts in 1991. Two years later this observation led to the purification of the first known microtubule-severing enzyme, katanin. Katanin homologs

  15. Controlled Microtubules Transport on Patterned Non-fouling Surfaces R. C. Lipscomb1

    E-print Network

    Controlled Microtubules Transport on Patterned Non-fouling Surfaces R. C. Lipscomb1 , J. Clemmens2 adhere exclusively to the fouling areas, thus forming kinesin tracks suited for microtubule guidance. Keywords - Protein patterns, microfluidics, kinesin, microtubules, non-fouling coatings, plasma deposition

  16. The FASEB Journal Research Communication Mechanical breaking of microtubules in axons during

    E-print Network

    Baas, Peter W.

    The FASEB Journal · Research Communication Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration Min D to degeneration of almost all axons by 24 h. Stabilizing the microtubules with taxol maintained the undulating

  17. 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

  18. The role of kinesin and other soluble factors in organelle movement along microtubules

    Microsoft Academic Search

    Trina A. Schroer; Bruce J. Schnapp; Thomas S. Reese; Michael P. Sheetz

    1988-01-01

    Kinesin is a force-generating ATPase that drives the sliding movement of microtubules on glass coverslips and the movement of plastic beads along microtubules. Although kinesin is suspected to partici- pate in microtubule-based organelle transport, the ex- act role it plays in this process is unclear. To address this question, we have developed a quantitative assay that allows us to determine

  19. Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

    SciTech Connect

    Scaife, R.M. (Fred Hutchinson Cancer Research Center, Seattle, WA (United States)); Wilson, L. (Univ. of California, Santa Barbara (United States)); Purich, D.L. (Univ. of Florida, Gainesville (United States))

    1992-01-14

    Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

  20. Calcineurin ensures a link between the DNA replication checkpoint and microtubule-dependent polarized growth

    Microsoft Academic Search

    Kazunori Kume; Takayuki Koyano; Muneyoshi Kanai; Takashi Toda; Dai Hirata

    2011-01-01

    Microtubules are central to eukaryotic cell morphogenesis. Microtubule plus-end tracking proteins (+TIPs) transport polarity factors to the cell cortex, thereby playing a key role in both microtubule dynamics and cell polarity. However, the signalling pathway linking +TIPs to cell polarity control remains elusive. Here we show that the fission yeast checkpoint kinase Cds1 (Chk2 homologue) delays the transition of growth

  1. Methods Mol Med. Author manuscript Purification and mass spectrometry identification of microtubule-binding

    E-print Network

    Paris-Sud XI, Université de

    egg extracts. Isolated proteins are analysed using SDS-gel electrophoresis and identified by various of microtubule-binding proteins from Xenopus egg extracts Gache Vincent 1 , Waridel Patrice 2 , Luche Sylvie 3 ; microtubule ; microtubule-associated protein ; MAP ; motor ; Xenopus ; egg extracts ; mass

  2. 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

  3. 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

  4. 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

  5. Interactive Domains in the Molecular Chaperone Human aB Crystallin Modulate Microtubule Assembly and

    E-print Network

    Clark, John

    Interactive Domains in the Molecular Chaperone Human aB Crystallin Modulate Microtubule Assembly. Interactive sequences on the surface of human aB crystallin collectively modulate microtubule assembly through Chaperone Human aB Crystallin Modulate Microtubule Assembly and Disassembly. PLoS ONE 2(6): e498. doi:10

  6. 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

  7. 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

  8. 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.

  9. Cell prestress. II. Contribution of microtubules

    NASA Technical Reports Server (NTRS)

    Stamenovic, Dimitrije; Mijailovich, Srboljub M.; Tolic-Norrelykke, Iva Marija; Chen, Jianxin; Wang, Ning; Ingber, D. E. (Principal Investigator)

    2002-01-01

    The tensegrity model hypothesizes that cytoskeleton-based microtubules (MTs) carry compression as they balance a portion of cell contractile stress. To test this hypothesis, we used traction force microscopy to measure traction at the interface of adhering human airway smooth muscle cells and a flexible polyacrylamide gel substrate. The prediction is that if MTs balance a portion of contractile stress, then, upon their disruption, the portion of stress balanced by MTs would shift to the substrate, thereby causing an increase in traction. Measurements were done first in maximally activated cells (10 microM histamine) and then again after MTs had been disrupted (1 microM colchicine). We found that after disruption of MTs, traction increased on average by approximately 13%. Because in activated cells colchicine induced neither an increase in intracellular Ca(2+) nor an increase in myosin light chain phosphorylation as shown previously, we concluded that the observed increase in traction was a result of load shift from MTs to the substrate. In addition, energy stored in the flexible substrate was calculated as work done by traction on the deformation of the substrate. This result was then utilized in an energetic analysis. We assumed that cytoskeleton-based MTs are slender elastic rods supported laterally by intermediate filaments and that MTs buckle as the cell contracts. Using the post-buckling equilibrium theory of Euler struts, we found that energy stored during buckling of MTs was quantitatively consistent with the measured increase in substrate energy after disruption of MTs. This is further evidence supporting the idea that MTs are intracellular compression-bearing elements.

  10. 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

  11. Non-equilibrium microtubule fluctuations in a model cytoskeleton

    E-print Network

    C. P. Brangwynne; G. H. Koenderink; F. C. MacKintosh; D. A. Weitz

    2007-09-19

    Biological activity gives rise to non-equilibrium fluctuations in the cytoplasm of cells; however, there are few methods to directly measure these fluctuations. Using a reconstituted actin cytoskeleton, we show that the bending dynamics of embedded microtubules can be used to probe local stress fluctuations. We add myosin motors that drive the network out of equilibrium, resulting in an increased amplitude and modified time-dependence of microtubule bending fluctuations. We show that this behavior results from step-like forces on the order of 10 pN driven by collective motor dynamics.

  12. Shape coexistence and parity doublet in Zr isotopes

    NASA Astrophysics Data System (ADS)

    Kumar, Bharat; Singh, S. K.; Patra, S. K.

    2015-03-01

    The ground and excited states properties of Zr isotopes are studied from proton to neutron drip lines using the relativistic (RMF) and nonrelativistic (SHF) mean-field formalisms with Bardeen-Cooper-Schrieffer (BCS) and Bogoliubov pairing, respectively. The well-known NL3* and SLy4 parameter sets are used in the calculations. We have found spherical ground and low-lying large deformed excited states in most of the isotopes. Several couples of ?? = 1/2± parity doublets configurations are noticed, while analyzing the single-particle energy levels of the large deformed configurations.

  13. Delta wing flutter based on doublet lattice method in NASTRAN

    NASA Technical Reports Server (NTRS)

    Jew, H.

    1975-01-01

    The subsonic doublet-lattice method (DLM) aeroelastic analysis in NASTRAN was successfully applied to produce subsonic flutter boundary data in parameter space for a large delta wing configuration. Computed flow velocity and flutter frequency values as functions of air density ratio, flow Mach number, and reduced frequency are tabulated. The relevance and the meaning of the calculated results are discussed. Several input-deck problems encountered and overcome are cited with the hope that they may be helpful to NASTRAN Rigid Format 45 users.

  14. Shape co-existence and parity doublet in Zr isotopes

    E-print Network

    Bharat Kumar

    2014-12-05

    We studied the ground and excited states properties for Zr isotopes starting^M from proton to neutron drip-lines using the relativistic and non-relativistic mean field formalisms with BCS and Bogolyubov pairing. The celebrity NL3 and SLy4 parameter sets are used in the calculations. We find spherical ground and low-lying^M superdeformed excited states in most of the isotopes. Several couples of^M $\\Omega^{\\pi}=1/2^{\\pm}$ parity doublets configurations are found,^M while analyzing the single-particle energy levels of the superdeformed ^M configurations.

  15. Anaphase A Chromosome Movement and Poleward Spindle Microtubule Flux Occur At Similar Rates in Xenopus Extract Spindles

    Microsoft Academic Search

    Arshad Desai; Paul S. Maddox; Timothy J. Mitchison; E. D. Salmon

    1998-01-01

    We have used local fluorescence photoacti- vation to mark the lattice of spindle microtubules dur- ing anaphase A in Xenopus extract spindles. We find that both poleward spindle microtubule flux and anaphase A chromosome movement occur at similar rates ( z 2 m m\\/min). This result suggests that poleward microtubule flux, coupled to microtubule depolymer- ization near the spindle poles,

  16. MAP6-F is a temperature sensor that directly binds to and protects microtubules from cold-induced depolymerization*

    E-print Network

    Paris-Sud XI, Université de

    MAP6-F is a temperature sensor that directly binds to and protects microtubules from cold, France *Running title: Microtubule stabilization by MAP6 § To whom correspondence should be addressed-Mail: christian.delphin@ujf-grenoble.fr Keywords: Microtubules; MAP6; hypothermia Background: Microtubules

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

    E-print Network

    Paris-Sud XI, Université de

    Krit 1 interactions with microtubules and membranes are regulated by Rap1 and integrin cytoplasmic in vitro to microtubules through its N and C-termini and that Rap1 and ICAP-1 inhibit Krit1 binding to microtubules. Consistently, YFP-Krit1 localizes on CFP-labelled microtubules in BHK cells and is delocalized

  18. Synchrotron X-ray Diffraction Study of Microtubules Buckling and Bundling under Osmotic Stress: A Probe of Interprotofilament Interactions

    E-print Network

    Weeks, Eric R.

    Synchrotron X-ray Diffraction Study of Microtubules Buckling and Bundling under Osmotic Stress 93106, USA (Received 21 April 2004; published 4 November 2004) Microtubules are hollow cylinders the microtubule wall. Synchrotron x-ray diffraction of microtubules under increasing osmotic stress shows

  19. Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain

    Microsoft Academic Search

    V. I. Rodionov; Fatima K. Gyoeva; Elly Tanaka; Alexander D. Bershadsky; Juri M. Vasiliev; Vladimlr I. Gelfand

    1993-01-01

    One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a criti- cal role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may

  20. Three-Dimensional Microtubule Behavior in Xenopus Egg Extracts Reveals Four Dynamic States and State-Dependent Elastic Properties

    Microsoft Academic Search

    Philipp J. Keller; Francesco Pampaloni; Gianluca Lattanzi; Ernst H. K. Stelzer

    2008-01-01

    Although microtubules are key players in many cellular processes, very little is known about their dynamic and mechanical properties in physiological three-dimensional environments. The conventional model of microtubule dynamic instability postulates two dynamic microtubule states, growth and shrinkage. However, several studies have indicated that such a model does not provide a comprehensive quantitative and qualitative description of microtubule behavior. Using

  1. Survival of the aligned: ordering of the plant cortical microtubule array

    E-print Network

    Tindemans, Simon H; Mulder, Bela M

    2009-01-01

    The microtubule cortical array is a structure consisting of highly aligned microtubules, observed in all growing plant cells, which plays a crucial role in the characteristic plant cell growth by uniaxial expansion along the axis perpendicular to the microtubules. To investigate the orientational ordering of microtubules in this system, we present both a coarse-grained theoretical model and stochastic particle-based simulations, and compare the results from these complementary approaches. Our results indicate that collisions that induce depolymerization are the main driving factor in the alignment of microtubules in the cortical array.

  2. A nonmotor microtubule binding site in kinesin-5 is required for filament crosslinking and sliding.

    PubMed

    Weinger, Joshua S; Qiu, Minhua; Yang, Ge; Kapoor, Tarun M

    2011-01-25

    Kinesin-5, a widely conserved motor protein required for assembly of the bipolar mitotic spindle in eukaryotes, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell-shaped molecule [1-3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5's ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a nonmotor microtubule binding site in its C terminus [6]. However, it is not known how the nonmotor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four nonmotor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the nonmotor C terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5's microtubule binding without substantially reducing motor activity. Our results suggest that tetramerization of kinesin-5's low-processivity motor domains is not sufficient for microtubule sliding because the motor domains alone are unlikely to maintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes nonmotor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function. PMID:21236672

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

    PubMed Central

    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

  4. 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

  5. Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules

    PubMed Central

    Hinow, Peter; Rezania, Vahid; Lopus, Manu; Jordan, Mary Ann; Tuszy?ski, Jack A.

    2011-01-01

    We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady state microtubules assembled from MAP-free tubulin in the possible presence of a microtubule associated drug. As an example for the latter, we both experimentally and theoretically study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. Our model predicts that among drugs that act locally at the microtubule tip, primary inhibition of the loss of GDP tubulin results in stronger damping of microtubule dynamics than inhibition of GTP tubulin addition. On the other hand, drugs whose action occurs in the interior of the microtubule need to be present in much higher concentrations to have visible effects. PMID:21836336

  6. 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

  7. Metallic Glass Wire Based Localization of Kinesin/Microtubule Bio-molecular Motility System

    NASA Astrophysics Data System (ADS)

    Kim, K.; Sikora, A.; Yaginuma, S.; Nakayama, K. S.; Nakazawa, H.; Umetsu, M.; Hwang, W.; Teizer, W.

    2014-03-01

    We report electrophoretic accumulation of microtubules along metallic glass (Pd42.5Cu30Ni7.5P20) wires free-standing in solution. Microtubules are dynamic cytoskeletal filaments. Kinesin is a cytoskeletal motor protein. Functions of these bio-molecules are central to various dynamic cellular processes. Functional artificial organization of bio-molecules is a prerequisite for transferring their native functions into device applications. Fluorescence microscopy at the individual-microtubule level reveals microtubules aligning along the wire axis during the electrophoretic migration. Casein-treated electrodes are effective for releasing trapped microtubules upon removal of the external field. Furthermore, we demonstrate gliding motion of microtubules on kinesin-treated metallic glass wires. The reversible manner in the local adsorption of microtubules, the flexibility of wire electrodes, and the compatibility between the wire electrode and the bio-molecules are beneficial for spatio-temporal manipulation of the motility machinery in 3 dimensions.

  8. 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

  9. 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...

  10. Spinning Janus doublets driven in uniform ac electric fields.

    PubMed

    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. PMID:24580163

  11. Fitting the Two-Higgs-Doublet model of type II

    E-print Network

    Otto Eberhardt

    2014-06-16

    We present the current status of the Two-Higgs-Doublet model of type II. Taking into account all available relevant information, we exclude at $95$% CL sizeable deviations of the so-called alignment limit, in which all couplings of the light CP-even Higgs boson $h$ are Standard-Model-like. While we can set a lower limit of $240$ GeV on the mass of the pseudoscalar Higgs boson at $95$% CL, the mass of the heavy CP-even Higgs boson $H$ can be even lighter than $200$ GeV. The strong constraints on the model parameters also set limits on the triple Higgs couplings: the $hhh$ coupling in the Two-Higgs-Doublet model of type II cannot be larger than in the Standard Model, while the $hhH$ coupling can maximally be $2.5$ times the size of the Standard Model $hhh$ coupling, assuming an $H$ mass below $1$ TeV. The selection of benchmark scenarios which maximize specific effects within the allowed regions for further collider studies is illustrated for the $H$ branching fraction to fermions and gauge bosons. As an example, we calculate the cross section of $gg\\to hh$ for four benchmark points and show that a resonant $H$ could enhance it by almost a factor of $70$ at a centre-of-mass energy of $14$ TeV.

  12. 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.

  13. 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.

  14. Next-to-Minimal Two Higgs Doublet Model

    DOE PAGESBeta

    Chen, Chien-Yi; Freid, Michael; Sher, Marc

    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 discoveredmore »in this mass range, then the upper limit on the mixing will be sufficiently strong such that there will be no significant effects on the couplings of the h for most of parameter space. The reverse is true in the type-II model, the limits from measurements of the couplings of the h will dominate over the limits from non-observation of the heavy Higgs.« less

  15. Doublet III neutral beam injector test tank cryopanel design

    SciTech Connect

    Doll, D.W.; Kamperschroer, J.H.; Arend, P.V.

    1980-03-01

    A simple condensing cryopanel has been designed for the Doublet III neutral beam test tank with a 320,000 liters per second pumping capacity for hydrogen. This maintains a vacuum in the test tank which simulates the Doublet III vessel, 1.3 x 10/sup -3/ Pa (approx.10/sup -5/ torr). The hydrogen gas load comes from the beam striking the test tank calorimeter and amounts to about 7.2 torr liters per second. The cryopanel is cylindrical shaped with a liquid helium (LHe) surface that pumps through liquid nitrogen (LN) cooled aluminum chevrons located in squirrel-cage fashion around the inside surface of the cylinder. The LHe cooled surface is a smooth cylinder 2.09m in diameter by .69m long with LHe flowing in a approx. 1mm annular space between concentric cylinders. The chevrons which are not blackened are cooled from each end with LN flowing in ring manifolds that serve as the primary cryopanel structure. The LHe is force fed at 55.2 kPa remaining in the liquid phase through the panel. External heat exchanger capability permits use of helium at 3.8 to 4.2/sup 0/K. Normal operating flow rate is 1.4 g/sec for a heat load expected to be 12.2 W total.

  16. 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

  17. 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

  18. 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.

  19. Microtubule depolymerization induces traction force increase through two distinct pathways

    PubMed Central

    Rape, Andrew; Guo, Wei-hui; Wang, Yu-li

    2011-01-01

    Traction forces increase after microtubule depolymerization; however, the signaling mechanisms underlying this, in particular the dependence upon myosin II, remain unclear. We investigated the mechanism of traction force increase after nocodazole-induced microtubule depolymerization by applying traction force microscopy to cells cultured on micropatterned polyacrylamide hydrogels to obtain samples of homogeneous shape and size. Control cells and cells treated with a focal adhesion kinase (FAK) inhibitor showed similar increases in traction forces, indicating that the response is independent of FAK. Surprisingly, pharmacological inhibition of myosin II did not prevent the increase of residual traction forces upon nocodazole treatment. This increase was abolished upon pharmacological inhibition of FAK. These results suggest two distinct pathways for the regulation of traction forces. First, microtubule depolymerization activates a myosin-II-dependent mechanism through a FAK-independent pathway. Second, microtubule depolymerization also enhances traction forces through a myosin-II-independent, FAK-regulated pathway. Traction forces are therefore regulated by a complex network of complementary signals and force-generating mechanisms. PMID:22193960

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

    Microsoft Academic Search

    MARIAN L. LEWIS; JULIE L. REYNOLDS; LUIS A. CUBANO; JASON P. HATTON; B. DESALES LAWLESS; EDWARD H. PIEPMEIER

    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 apopto- sis. Cell growth was stimulated in microgravity by in- creasing serum concentration. After 4 and 48 h, cells filtered

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

    E-print Network

    Holly Freedman; Vahid Rezania; Avner Priel; Eric Carpenter; Sergei Y. Noskovd; Jack A. Tuszynski

    2009-12-09

    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 when an external potential is 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 a net current to be pumped across the microtubule wall and propagate down the microtubule through the lumen. This effect is demonstrated to enhance and add directly to the longitudinal current through the lumen resulting from an external voltage source, and could be significant in amplifying low-intensity endogenous currents within the cellular environment or as a nano-bioelectronic device.

  2. Microtubule Binding to Smads May Regulate TGF? Activity

    Microsoft Academic Search

    Chunming Dong; Zhiru Li; Rene Alvarez; Xin-Hua Feng; Pascal J. Goldschmidt-Clermont

    2000-01-01

    Smad proteins are intracellular signaling effectors of the TGF? superfamily. We show that endogenous Smad2, 3, and 4 bind microtubules (MTs) in several cell lines. Binding of Smads to MTs does not require TGF? stimulation. TGF? triggers dissociation from MTs, phosphorylation, and nuclear translocation of Smad2 and 3, with consequent activation of transcription in CCL64 cells. Destabilization of the MT

  3. Nuclear centering in Spirogyra: force integration by microfilaments along microtubules.

    PubMed

    Grolig, F

    1998-01-01

    The contribution of microtubules and microfilaments to the cytomechanics of transverse nuclear centering were investigated in the charophycean green alga Spirogyra crassa (Zygnematales). Cytoplasmic strands of enhanced rigidity and fasciate appearance radiate from the rim of the lenticular nucleus through the vacuole, frequently split once or twice and are attached to the helical chloroplast bands in the peripheral cytoplasm. The nucleus is encased in tubulin and a web of F-actin. Bundles of microtubules, emerging from the nuclear rim, are organized into dividing fascicles within the strands and reach to the inner surface of the chloroplast envelope. Organelles are translocated in both directions along similarly arranged fascicles of microfilament bundles which extend from the nucleus to the peripheral actin cytoskeleton. Application of microtubule- and/or microfilament-depolymerizing drugs affected the position of the nucleus only slowly, but in distinct ways. The differential effects suggest that nuclear centering depends on the tensional integrity of the perinuclear scaffold, with microfilaments conveying tension along stabilized microtubules and the actin cytoskeleton integrating the translocation forces generated within the scaffold. PMID:9443384

  4. Fission yeast Scp3 potentially maintains microtubule orientation through bundling.

    PubMed

    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

  5. Light Chain-dependent Regulation of Kinesin's Interaction with Microtubules

    Microsoft Academic Search

    Kristen J. Verhey; Donna L. Lizotte; Tatiana Abramson; Linda Barenboim; Bruce J. Schnapp; Tom A. Rapoport

    1998-01-01

    We have investigated the mechanism by which conventional kinesin is prevented from binding to microtubules (MTs) when not transporting cargo. Kinesin heavy chain (HC) was expressed in COS cells either alone or with kinesin light chain (LC). Immuno- fluorescence microscopy and MT cosedimentation ex- periments demonstrate that the binding of HC to MTs is inhibited by coexpression of LC. Association

  6. Microtubule and Motor-dependent Endocytic Vesicle Sorting In Vitro

    Microsoft Academic Search

    Eustratios Bananis; John W. Murray; Richard J. Stockert; Peter Satir; Allan W. Wolkoff

    2000-01-01

    Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluo- rescence and video imaging, we provide the first in vitro reconstitution of receptor-ligand sorting in early en- docytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate

  7. MODEL BASED DYNAMICS ANALYSIS IN MICROTUBULE VIDEOS A. Altinok1

    E-print Network

    California at Santa Barbara, University of

    and machine learning based methods for ex- tracting novel dynamics information from microtubule video in videos, and introducing machine learn- ing tools that provide novel analysis capabilities. Recently, we. Rose2 Dept. of Computer Science1 , Electrical and Computer Engineering2 , Molecular Cellular

  8. 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.

  9. Central microtubular agenesis causing primary ciliary dyskinesia.

    PubMed

    Stannard, Wendy; Rutman, Andrew; Wallis, Colin; O'Callaghan, Chris

    2004-03-01

    Primary ciliary dyskinesia is an autosomal recessive disorder characterized by chronic upper and lower respiratory tract symptoms. We report the diagnosis of primary ciliary dyskinesia associated with a circular ciliary beat pattern in three siblings. This beat pattern is consistent with a ciliary transposition defect, where a peripheral microtubule doublet is transposed to the center of the ciliary axoneme to replace the absent central microtubule pair. However, in these siblings, ultrastructural analysis of the cilia revealed an absence of the central microtubule pair only. This variant of transposition with a circular ciliary beat pattern has not been described previously. In addition, this defect, together with the transposition defect, may help explain the mechanism of the circular beat pattern and also the absence of situs inversus in these patients. PMID:14982824

  10. Structural Basis for the Association of MAP6 Protein with Microtubules and Its Regulation by Calmodulin

    PubMed Central

    Lefèvre, Julien; Savarin, Philippe; Gans, Pierre; Hamon, Loïc; Clément, Marie-Jeanne; David, Marie-Odile; Bosc, Christophe; Andrieux, Annie; Curmi, Patrick A.

    2013-01-01

    Microtubules are highly dynamic ??-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90–177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90–177) to microtubules with a 1:1 MAP6(90–177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90–177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca2+-calmodulin competes with microtubules for MAP6(90–177) binding and that the binding mode of MAP6(90–177) to microtubules and Ca2+-calmodulin involves a common stretch of amino acid residues on the MAP6(90–177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca2+-calmodulin. PMID:23831686

  11. 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.

  12. 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

  13. 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

  14. The effect of solution composition on microtubule dynamic instability.

    PubMed Central

    Schilstra, M J; Bayley, P M; Martin, S R

    1991-01-01

    The exchange of tubulin dimer into steady-state microtubules was studied over a range of solution conditions, in order to assess the effects of various common buffer components on the dynamic instability of microtubules. In comparison with standard buffer conditions (100 mM-Pipes buffer, pH 6.5, containing 0.1 mM-EGTA, 1.8 mM-MgC12 and 1 M-glycerol), the rate and extent of exchange, and thus of dynamic instability, are suppressed by increasing the concentration of glycerol above 2 M. Exchange is enhanced by the addition of further Mg2+ (up to 17 mM) or by the addition of Ca2+ (up to 0.4 mM). Phosphate ion (150 mM) has relatively little effect on the dynamic behaviour of microtubules, as judged by the exchange method. The findings are interpreted within the framework of the Lateral Cap model for microtubule dynamic instability, in terms of the effects of these changes on the intrinsic rate constants of the system. By contrast, the extent of tubulin exchange depends selectively on the value of the dissociation rate constant for tubulin-GDP. A decrease in the extent of exchange, and hence in dynamic activity, is associated with a decreased value for this rate constant, and vice versa. The results also show good agreement of predictions of the model in treating the observed variations in the dynamic properties of individual microtubules, induced by different solution conditions. PMID:1678598

  15. 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…

  16. Asymmetric microtubule arrays organize the endoplasmic reticulum during polarity establishment in the brown alga Silvetia compressa.

    PubMed

    Peters, Nick T; Kropf, Darryl L

    2010-02-01

    Polarity is a fundamental characteristic of most cell types, and is crucial to early development of the brown alga Silvetia compressa. In eukaryotes the cytoskeleton plays an important role in generating cellular asymmetries. While it is known that F-actin is required for polarization and growth in most tip-growing cells, the roles of microtubules are less clear. We examined the distribution and function of microtubules in S. compressa zygotes as they polarized and initiated tip growth. Microtubules formed asymmetric arrays oriented toward the rhizoid hemisphere early in the polarization process. These arrays were spatially coupled with polar adhesive deposition, a marker of the rhizoid pole. Reorientation of the light vector during polarization led to sequential redistribution of polar axis components, with the microtubules and the polar axis reorienting nearly simultaneously, followed by cell wall loosening and then deposition of new polar adhesive. These findings suggested that microtubules may organize and target endomembrane arrays. We therefore examined the distribution of the endoplasmic reticulum during polarization and found it colocalized with microtubules and became targeted toward the rhizoid pole as microtubule asymmetry was generated. Endoplasmic reticulum association with microtubules remained fully intact following pharmacological disruption of F-actin, whereas microtubule disruption led to aggregation of the endoplasmic reticulum around the nucleus. We propose that brown algae utilize microtubules for organization of the endoplasmic reticulum and migration of exocytotic components to the rhizoid cortex, and present a model for polarity establishment to account for these new findings. PMID:20169534

  17. Cyclin G-associated kinase promotes microtubule outgrowth from chromosomes during spindle assembly.

    PubMed

    Tanenbaum, Marvin E; Vallenius, Tea; Geers, Erica F; Greene, Lois; Mäkelä, Tomi P; Medema, Rene H

    2010-08-01

    During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation. Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the mitotic spindle. Here, we have performed a large scale RNAi screen and identify cyclin G-associated kinase (GAK) as a novel regulator of microtubule generation at kinetochores/chromatin. This function of GAK requires its C-terminal J-domain, which is essential for clathrin recycling from endocytic vesicles. Consistently, cells lacking GAK show strongly reduced levels of clathrin on the mitotic spindle, and reduction of clathrin levels also inhibits microtubule generation at kinetochores/chromosomes. Finally, we present evidence that association of clathrin with the spindle is promoted by a signal coming from the chromosomes. These results identify a role for GAK and clathrin in microtubule outgrowth from kinetochores/chromosomes and suggest that GAK acts through clathrin to control microtubule outgrowth around chromosomes. PMID:20237935

  18. 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.

  19. Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein280-deficient Melanoma Cell Line

    Microsoft Academic Search

    C. Casey Cunningham; Nicole Leclerc; Lisa A. Flanagan; Mei Lu; Paul A. Janmey; Kenneth S. Kosik

    1997-01-01

    The emergence of processes from cells often involves interactions between microtubules and mi- crofilaments. Interactions between these two cytoskele- tal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal mi- crotubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates inter- actions between microfilaments and microtubules. To approach this problem in

  20. CPV Phenomenology of Flavor Conserving Two Higgs Doublet Models

    E-print Network

    Satoru Inoue; Michael J. Ramsey-Musolf; Yue Zhang

    2014-07-31

    We analyze the constraints on CP-violating, flavor conserving Two Higgs Doublet Models (2HDMs) implied by measurements of Higgs boson properties at the Large Hadron Collider (LHC) and by the non-observation of permanent electric dipole moments (EDMs) of molecules, atoms and the neutron. We find that the LHC and EDM constraints are largely complementary, with the LHC studies constraining the mixing between the neutral CP-even states and EDMs probing the effect of mixing between the CP-even and CP-odd scalars. The presently most stringent constraints are implied by the non-observation of the ThO molecule EDM signal. Future improvements in the sensitivity of neutron and diamagnetic atom EDM searches could yield competitive or even more severe constraints. We analyze the quantitative impact of hadronic and nuclear theory uncertainties on the interpretation of the latter systems and conclude that these uncertainties cloud the impact of projected improvements in the corresponding experimental sensitivities.

  1. Compatible abelian symmetries in N-Higgs-doublet models

    NASA Astrophysics Data System (ADS)

    Nishi, C. C.

    2015-03-01

    We analyze the compatibility between abelian symmetries acting in two different sectors of a theory using the Smith Normal Form method. We focus on N-Higgs-doublet models (NHDMs) and on the compatibility between symmetries in the Higgs potential and in the Yukawa interactions, which were separately analyzed in previous works. It is shown that two equal (isomorphic) symmetry groups that act in two separate sectors are not necessarily compatible in the whole theory and an upper bound is found for the size of the group that can be implemented in the entire NHDM. We also develop useful techniques to analyze compatibility and extend a symmetry from one sector to another. Consequences to the supersymmetric case are briefly discussed.

  2. The Electroweak Phase Transition in the Inert Doublet Model

    E-print Network

    Blinov, Nikita; Stefaniak, Tim

    2015-01-01

    We study the strength of a first-order electroweak phase transition in the Inert Doublet Model (IDM), where particle dark matter (DM) is comprised of the lightest neutral inert Higgs boson. We improve over previous studies in the description and treatment of the finite-temperature effective potential and of the electroweak phase transition. We focus on a set of benchmark models inspired by the key mechanisms in the IDM leading to a viable dark matter particle candidate, and illustrate how to enhance the strength of the electroweak phase transition by adjusting the masses of the yet undiscovered IDM Higgs states. We argue that across a variety of DM masses, obtaining a strong enough first-order phase transition is a generic possibility in the IDM. We find that due to direct dark matter searches and collider constraints, a sufficiently strong transition and a thermal relic density matching the universal DM abundance is possible only in the Higgs funnel regime.

  3. 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

  4. Microtubule Alterations Occur Early in Experimental Parkinsonism and The Microtubule Stabilizer Epothilone D Is Neuroprotective

    PubMed Central

    Cartelli, Daniele; Casagrande, Francesca; Busceti, Carla Letizia; Bucci, Domenico; Molinaro, Gemma; Traficante, Anna; Passarella, Daniele; Giavini, Erminio; Pezzoli, Gianni; Battaglia, Giuseppe; Cappelletti, Graziella

    2013-01-01

    The role of microtubule (MT) dysfunction in Parkinson's disease is emerging. It is still unknown whether it is a cause or a consequence of neurodegeneration. Our objective was to assess whether alterations of MT stability precede or follow axonal transport impairment and neurite degeneration in experimental parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57Bl mice. MPTP induced a time- and dose-dependent increase in fibres with altered mitochondria distribution, and early changes in cytoskeletal proteins and MT stability. Indeed, we observed significant increases in neuron-specific ?III tubulin and enrichment of deTyr tubulin in dopaminergic neurons. Finally, we showed that repeated daily administrations of the MT stabilizer Epothilone D rescued MT defects and attenuated nigrostriatal degeneration induced by MPTP. These data suggest that alteration of ??s is an early event specifically associated with dopaminergic neuron degeneration. Pharmacological stabilization of MTs may be a viable strategy for the management of parkinsonism. PMID:23670541

  5. Tau binding to microtubules does not directly affect microtubule-based vesicle motility.

    PubMed

    Morfini, Gerardo; Pigino, Gustavo; Mizuno, Naoko; Kikkawa, Masahide; Brady, Scott T

    2007-09-01

    Tau protein is a major microtubule (MT)-associated brain protein enriched in axons. Multiple functional roles are proposed for tau protein, including MT stabilization, generation of cell processes, and targeting of phosphotransferases to MTs. Recently, experiments involving exogenous tau expression in cultured cells suggested a role for tau as a regulator of kinesin-1-based motility. Tau was proposed to inhibit attachment of kinesin-1 to MTs by competing for the kinesin-1 binding site. In this work, we evaluated effects of tau on fast axonal transport (FAT) by using vesicle motility assays in isolated squid axoplasm. Effects of recombinant tau constructs on both kinesin-1 and cytoplasmic dynein-dependent FAT rates were evaluated by video microscopy. Exogenous tau binding to endogenous squid MTs was evidenced by a dramatic change in individual MT morphologies. However, perfusion of tau at concentrations approximately 20-fold higher than physiological levels showed no effect on FAT. In contrast, perfusion of a cytoplasmic dynein-derived peptide that competes with kinesin-1 and cytoplasmic dynein binding to MTs in vitro rapidly inhibited FAT in both directions. Taken together, our results indicate that binding of tau to MTs does not directly affect kinesin-1- or cytoplasmic dynein-based motilities. In contrast, our results provide further evidence indicating that the functional binding sites for kinesin-1 and cytoplasmic dynein on MTs overlap. PMID:17265463

  6. Charge exchange measurements on the Doublet III tokamak

    SciTech Connect

    Lohr, J.; Armentrout, C.J.

    1985-08-01

    Two passive charge exchange analyzers were installed on the Doublet III tokamak. Both were of the E parallel B type, permitting H-D discrimination by mass. Deuterons with energies up to about 90 keV could be observed at the highest spectrometer magnetic fields available. Beam injection energy on Doublet III was typically 75 keV. One of the analyzers could scan across the beam injection angle of approximately 27/sup 0/ at the magnetic axis, while the other analyzer observed parallel neutral flux across nearly the entire cross section from about 10 cm inside the limiter to tangency radii of about 85 cm, intersecting the centerpost. Beam injection was angled toward the direction of positive plasma current and co-going particles were generally observed by both analyzers. When neutral beam power was increased in steps, generally the observed fast neutral flux did not increase proportionally at higher power levels. In addition, the parallel analyzer in a few cases showed evidence for a fast particle loss at a single energy, with the distribution function being filled in from higher and lower energies. Flux bursts were observed in synchronism with limiter H/sub ..cap alpha../ spikes at the low energy range of the parallel analyzer. The perpendicular analyzer, observing fast particles near their injected pitch angle, detected bursts at all energies, with especially pronounced correlation with H/sub ..cap alpha../ activity at high energies. When fishbone activity was seen magnetically, simultaneous bursts were often, but not always, observed on the perpendicular analyzer, but were never seen on the parallel instrument.

  7. Oxaliplatin-Based Doublets Versus Cisplatin or Carboplatin-Based Doublets in the First-Line Treatment of Advanced Nonsmall Cell Lung Cancer.

    PubMed

    Yu, Jing; Xiao, Jing; Yang, Yifan; Cao, Bangwei

    2015-07-01

    The efficacy and toxicity of oxaliplatin-based versus carboplatin/cisplatin-based doublets in patients with previously untreated nonsmall cell lung cancer (NSCLC) have been compared.We searched published randomized controlled trials of oxaliplatin-based or carboplatin/cisplatin-based medications for NSCLC. A fixed effect model was used to analyze outcomes which were expressed as the hazard ratio for overall survival (OS) and time-to-progression (TTP), relative risk, overall response rate (ORR), disease control rate (DCR), 1-year survival, and the odds ratios for toxicity were pooled.Eight studies involving 1047 patients were included. ORR tended to favor carboplatin/cisplatin but the effect was not significantly different compared with oxaliplatin doublets (P?=?0.05). The effects of OS, TTP, DCR, and 1-year survival between the 2 regimens were comparable. Oxaliplatin doublets caused less grade 3/4 leukocytopenia and neutropenia. Grades 3 to 4 nonhematological toxicities and grades 3 to 4 hematological toxicities showed little difference between oxaliplatin doublets and carboplatin/cisplatin doublets.Meta-analysis shows that the efficacy of oxaliplatin doublets is similar to that of other currently used platinum doublets. The lack of significant differences in the statistic analysis does not preclude genuine differences in clinical efficacy, because higher diversities between the studies covered differences between the 2 groups in each study. Oxaliplatin combined with a third-generation agent should be considered for use as alternative chemotherapy in patients who cannot tolerate conventional platinum-based regimens because the toxicity profile is much more favorable. PMID:26166081

  8. 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

  9. Relative velocity of sliding of microtubules by the action of Kinesin-5

    E-print Network

    Sthitadhi Roy

    2011-03-10

    Kinesin-5, also known as Eg5 in vertebrates is a processive motor with 4 heads, which moves on filamentous tracks called microtubules. The basic function of Kinesin-5 is to slide apart two anti-parallel microtubules by simultaneously walking on both the microtubules. We develop an analytical expression for the steady-state relative velocity of this sliding in terms of the rates of attachments and detachments of motor heads with the ATPase sites on the microtubules. We first analyse the motion of one pair of motor heads on one microtubule and then couple it to the motion of the other pair of motor heads of the same motor on the second microtubule to get the relative velocity of sliding.

  10. Relative velocity of sliding of microtubules by the action of Kinesin-5

    E-print Network

    Roy, Sthitadhi

    2011-01-01

    Kinesin-5, also known as Eg5 in vertebrates is a processive motor with 4 heads, which moves on filamentous tracks called microtubules. The basic function of Kinesin-5 is to slide apart two anti-parallel microtubules by simultaneously walking on both the microtubules. We develop an analytical expression for the steady-state relative velocity of this sliding in terms of the rates of attachments and detachments of motor heads with the ATPase sites on the microtubules. We first analyse the motion of one pair of motor heads on one microtubule and then couple it to the motion of the other pair of motor heads of the same motor on the second microtubule to get the relative velocity of sliding.

  11. 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

  12. Regulation of microtubule stability and organization by mammalian Par3 in specifying neuronal polarity

    PubMed Central

    Chen, She; Chen, Jia; Shi, Hang; Wei, Michelle; Castaneda-Castellanos, David R.; Bultje, Ronald S.; Pei, Xin; Kriegstein, Arnold R.; Zhang, Mingjie; Shi, Song-Hai

    2012-01-01

    SUMMARY Polarization of mammalian neurons with a specified axon requires precise regulation of microtubule and actin dynamics in the developing neurites. Here we show that mammalian partition defective 3 (mPar3), a key component of the Par polarity complex that regulates the polarization of many cell types including neurons, directly regulates microtubule stability and organization. The N-terminal portion of mPar3 exhibits strong microtubule binding, bundling and stabilization activity, which can be suppressed by its C-terminal portion via an intra-molecular interaction. Interestingly, the inter-molecular oligomerization of mPar3 is able to relieve the intra-molecular interaction and thereby promote microtubule bundling and stabilization. Furthermore, disruption of this microtubule regulatory activity of mPar3 impairs its function in axon specification. Together, these results demonstrate a role for mPar3 in directly regulating microtubule organization that is crucial for neuronal polarization. PMID:23273878

  13. Contributions of microtubule rotation and dynamic instability to kinetochore capture

    NASA Astrophysics Data System (ADS)

    Sweezy-Schindler, Oliver; Edelmaier, Christopher; Blackwell, Robert; Glaser, Matt; Betterton, Meredith

    2014-03-01

    The capture of lost kinetochores (KCs) by microtubules (MTs) is a crucial part of prometaphase during mitosis. Microtubule dynamic instability has been considered the primary mechanism of KC capture, but recent work discovered that lateral KC attachment to pivoting MTs enabled rapid capture even with significantly reduced MT dynamics. We aim to understand the relative contributions of MT rotational diffusion and dynamic instability to KC capture, as well as KC capture through end-on and/or lateral attachment. Our model consists of rigid MTs and a spherical KC, which are allowed to diffuse inside a spherical nuclear envelope consistent with the geometry of fission yeast. For simplicity, we include a single spindle pole body, which is anchored to the nuclear membrane, and its associated polar MTs. Brownian dynamics treats the diffusion of the MTs and KC and kinetic Monte Carlo models stochastic processes such as dynamic instability. NSF 1546021.

  14. 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

  15. The Feasibility of Coherent Energy Transfer in Microtubules

    E-print Network

    Craddock, Travis John Adrian; Mane, Jonathan; Hameroff, Stuart; Tuszynski, Jack A

    2014-01-01

    It was once purported that biological systems were far too warm and wet to support quantum phenomena mainly due to thermal effects disrupting quantum coherence. However recent experimental results and theoretical analyses have shown that thermal energy may assist, rather than disrupt, quantum coherence, especially in the dry hydrophobic interiors of biomolecules. Specifically, evidence has been accumulating for the necessary involvement of quantum coherence and entanglement between uniquely arranged chromophores in light harvesting photosynthetic complexes. Amazingly, the tubulin subunit proteins, which comprise microtubules, also possess a distinct architecture of chromophores, namely aromatic amino acids including tryptophan. The geometry and dipolar properties of these aromatics are similar to those found in photosynthetic units indicating that tubulin may support coherent energy transfer. Tubulin aggregated into microtubule geometric lattices may support such energy transfer, which could be of import for ...

  16. Microtubule dynamic instability: the role of cracks between protofilaments

    E-print Network

    Li, Chunlei; Goodson, Holly V; Alber, Mark S

    2013-01-01

    Microtubules (MTs) are cytoplasmic protein polymers that are essential for fundamental cellular processes including the maintenance of cell shape, organelle transport and formation of the mitotic spindle. Microtubule dynamic instability is critical for these processes, but it remains poorly understood, in part because the relationship between the structure of the MT tip and the growth/depolymerization transitions is enigmatic. What are the functionally significant aspects of a tip structure that is capable of promoting MT growth, and how do changes in these characteristics cause the transition to depolymerization (catastrophe)? Here we use computational models to investigate the connection between cracks (laterally unbonded regions) between protofilaments and dynamic instability. Our work indicates that it is not the depth of the cracks per se that governs MT dynamic instability. Instead it is whether the cracks terminate in GTP-rich or GDP-rich areas of the MT that governs whether a particular MT tip structu...

  17. Engineering tubulin: microtubule functionalization approaches for nanoscale device applications

    Microsoft Academic Search

    Jennelle L. Malcos; William O. Hancock

    2011-01-01

    With the emergences of engineered devices at microscale and nanoscale dimensions, there is a growing need for controlled actuation\\u000a and transport at these length scales. The kinesin–microtubule system provides a highly evolved biological transport system\\u000a well suited for these tasks. Accordingly, there is an ongoing effort to create hybrid nanodevices that integrate biological\\u000a components with engineered materials for applications such

  18. Nuclear centering in Spirogyra : force integration by microfilaments along microtubules

    Microsoft Academic Search

    Franz Grolig

    1997-01-01

    .   The contribution of microtubules and microfilaments to the cytomechanics of transverse nuclear centering were investigated\\u000a in the charophycean green alga Spirogyracrassa (Zygnematales). Cytoplasmic strands of enhanced rigidity and fasciate appearance radiate from the rim of the lenticular nucleus\\u000a through the vacuole, frequently split once or twice and are attached to the helical chloroplast bands in the peripheral cytoplasm.\\u000a The

  19. Involvement of microtubules in rhizoid differentiation of Spirogyra species

    Microsoft Academic Search

    K. Yoshida; N. Inoue; S. Sonobe; T. Shimmen

    2003-01-01

    Summary.?Some species of Spirogyra form rosette-shaped or rod-shaped rhizoids in the terminal cell of the filaments. In the present study, we analyzed an involvement of microtubules (MTs) in rhizoid differentiation. Before rhizoid differentiation, cortical MTs were arranged transversely to the long axis of cylindrical cells, reflecting the diffuse growth. At the beginning of rhizoid differentiation, MTs were absent from the

  20. Regulation of the orientation of cortical microtubules in Spirogyra cells

    Microsoft Academic Search

    Kazuyoshi Iwata

    1995-01-01

    The orientation of cortical microtubules (MTs) was synchronously regulated inSpirogyra cells. While the reorganized MTs in distilled water for 1.5 hr, after 1 hr treatment with amiprophos-methyl (APM) and complete\\u000a depolymerization of the MTs, were all transverse, those reorganized in 0.30 M mannitol were all oblique or longitudinal. After\\u000a the MTs had reorganized in 0.30 M mannitol, these cells were

  1. Single Fungal Kinesin Motor Molecules Move Processively along Microtubules

    Microsoft Academic Search

    Stefan Lakämper; Athina Kallipolitou; Günther Woehlke; Manfred Schliwa; Edgar Meyhöfer

    2003-01-01

    Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin)

  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. 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

  4. Visualization of the dynamic instability of individual microtubules by dark-field microscopy

    Microsoft Academic Search

    Tetsuya Horio; Hirokazu Hotani

    1986-01-01

    It has previously been shown that two populations of microtubules coexist in a dynamically unstable manner in vitro: those in one population elongate while those in the other shorten and finally disappear1,2. This conclusion was based on changes in the number and length distribution of microtubules after dilution of the micro-tubule solution. Here, we demonstrate directly that growing and shortening

  5. Cytoplasmic organization and quantitation of microtubules in bovine mammary epithelial cells during lactation and involution.

    PubMed

    Nickerson, S C; Akers, R M; Weinland, B T

    1982-01-01

    Ultrastructural examination of milk secretory cells from lactating bovine mammary gland revealed presence of numerous microtubules in the apical and paranuclear cytoplasm, particularly in the vicinity of Golgi components. Most microtubules were oriented perpendicular to the apical plasma membrane and appeared to form a framework around Golgi dictyosomal elements and secretory vesicles. In comparison, non-secretory cells obtained from involuting glands displayed few microtubules and these were randomly located throughout the cytoplasm with no particular orientation. PMID:7199973

  6. Krit1 interactions with microtubules and membranes are regulated by Rap1 and ICAP-1.

    E-print Network

    Paris-Sud XI, Université de

    1 Krit1 interactions with microtubules and membranes are regulated by Rap1 and ICAP-1. Sophie-33-476-54-94-74; Fax: 00-33-476-54-94-25; E-Mail:faurobert@ipmc.cnrs.fr Short title: Rap1 regulates Krit1 microtubule in vitro to microtubules through its N and C-termini and that Rap1 and ICAP-1 inhibit Krit1 binding

  7. Two Phases of Astral Microtubule Activity during Cytokinesis in C. elegans Embryos

    Microsoft Academic Search

    Fumio Motegi; Nathalie V. Velarde; Fabio Piano; Asako Sugimoto

    2006-01-01

    Summary Microtubules of the mitotic spindle are believed to provide positional cues for the assembly of the actin- based contractile ring and the formation of the sub- sequent cleavagefurrow during cytokinesis.InCaeno- rhabditis elegans, astral microtubules have been thought to inhibit cortical contraction outside the cleavage furrow.Here,wedemonstrate by liveimaging and RNA interference (RNAi) that astral microtubules play two distinct roles in

  8. Short-circuiting microtubule plus and minus end proteins in spindle positioning

    PubMed Central

    Rogers, Stephen L

    2014-01-01

    Proteins residing at the plus and minus ends of microtubules have been thought not to communicate with each other, but recent findings on bona fide nucleation factors also regulating microtubule dynamics have challenged this notion. New work by Bouissou et al (2014) in The EMBO Journal now reveals that interplay between the nucleation factor ?-TuRC and the plus-end tracking protein EB1 controls mitotic spindle positioning by affecting the stability and dynamics of astral microtubules. PMID:24421323

  9. Insights into microtubule nucleation from the crystal structure of human g-tubulin

    Microsoft Academic Search

    Hector Aldaz; Luke M. Rice; Tim Stearns; David A. Agard

    2005-01-01

    Microtubules are hollow polymers of ab-tubulin that show GTP- dependent assembly dynamics and comprise a critical part of the eukaryotic cytoskeleton. Initiation of new microtubules in vivo requires g-tubulin, organized as an oligomer within the 2.2-MDa g-tubulin ring complex (g-TuRC) of higher eukaryotes1-3. Struc- tural insight is lacking regarding g-tubulin, its oligomerization and how it promotes microtubule assembly. Here we

  10. Kinesins Are Indispensable for Interdigitation of Phragmoplast Microtubules in the Moss Physcomitrella patens

    Microsoft Academic Search

    Yuji Hiwatashi; Mari Obara; Yoshikatsu Sato; Tomomichi Fujita; T. Murata; M. Hasebe

    2008-01-01

    Microtubules form arrays with parallel and antiparallel bundles and function in various cellular processes, including subcellular transport and cell division. The antiparallel bundles in phragmoplasts, plant-unique microtubule arrays, are mostly unexplored and potentially offer new cellular insights. Here, we report that the Physcomitrella patens kinesins KINID1a and KINID1b (for kinesin for interdigitated microtubules 1a and 1b), which are specific to

  11. HIV-1 Rev Depolymerizes Microtubules to Form Stable Bilayered Rings

    PubMed Central

    Watts, Norman R.; Sackett, Dan L.; Ward, Rita D.; Miller, Mill W.; Wingfield, Paul T.; Stahl, Stephen S.; Steven, Alasdair C.

    2000-01-01

    We describe a novel interaction between HIV-1 Rev and microtubules (MTs) that results in the formation of bilayered rings that are 44–49 nm in external diameter, 3.4–4.2 MD (megadaltons) in mass, and have 28-, 30-, or 32-fold symmetry. Ring formation is not sensitive to taxol, colchicine, or microtubule-associated proteins, but requires Mg2+ and is inhibited by maytansine. The interaction involves the NH2-terminal domain of Rev and the face of tubulin exposed on the exterior of the MTs. The NH2-terminal half of Rev has unexpected sequence similarity to the tubulin-binding portion of the catalytic/motor domains of the microtubule-destabilizing Kin I kinesins. We propose a model wherein binding of Rev dimers to MTs at their ends causes segments of two neighboring protofilaments to peel off and close into rings, circumferentially containing 14, 15, or 16 tubulin heterodimers, with Rev bound on the inside. Rev has a strong inhibitory effect on aster formation in Xenopus egg extracts, demonstrating that it can interact with tubulin in the presence of normal levels of cellular constituents. These results suggest that Rev may interact with MTs to induce their destabilization, a proposition consistent with the previously described disruption of MTs after HIV-1 infection. PMID:10908577

  12. Rearrangement of microtubule network under biochemical and mechanical stimulations.

    PubMed

    Celik, Emrah; Abdulreda, Midhat H; Maiguel, Dony; Li, Jie; Moy, Vincent T

    2013-04-01

    Cells are constantly under the influence of various external forces in their physiological environment. These forces are countered by the viscoelastic properties of the cytoskeleton. To understand the response of the cytoskeleton to biochemical and mechanical stimuli, GFP-tubulin expressing CHO cells were investigated using scanning laser confocal microscopy. Cells treated with nocodazole revealed disruption in the microtubule network within minutes of treatment while keeping the cell shape intact. By contrast, trypsin, a proteolytic agent, altered the shape of CHO cells by breaking the peptide bonds at adhesion sites. CHO cells were also stimulated mechanically by applying an indentation force with an atomic force microscope (AFM) and by shear stress in a parallel plate flow chamber. Mechanical stimulation applied using AFM showed two distinct cytoskeletal responses to the applied force: an immediate response that resulted in the depolymerization and displacement of the microtubules out of the contact zone, and a slower response characterized by tubulin polymerization at the periphery of the indented area. Flow chamber experiments revealed that shear force did not induce formation of new microtubules in CHO cells and that detachment of adherent cells from the substrate occurred independent from the flow direction. Overall, the experimental system described here allows real-time characterization of dynamic changes in cell cytoskeleton in response to the mechano-chemical stimuli and, therefore, provides better understanding of the biophysical and functional properties of cells. PMID:23466787

  13. 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

  14. Microtubules and pancreatic amylase release by mouse pancreas in vitro

    PubMed Central

    1976-01-01

    The effects of vinblastine and colchicine on pancreatic acinar cells were studied by use of in vitro mouse pancreatic fragments. Vinblastine inhibited the release of amylase stimulated by bethanechol, caerulein, or ionophore A23187. Inhibition required preincubation with vinblastine,and maximum inhibition was observed after 90 min. Inhibition was relatively irreversible and could not be overcome by a high concentration of stimulant. Inhibition could also be produced by colchicine although longer preincubation was required and inhibition was only partial. Uptake of [3H]vinblastine and [3H]colchicine by pancreatic fragments was measured and found not to be responsible for the slow onset of inhibition by these drugs. In incubated pancreas, microtubules were present primarily in the apical pole of the cell and in association with the Golgi region. Vinblastine, under time and dose conditions that inhibited the release of stimulated amylase, also reduced the number of microtubules. The only other consistent structural effects of vinblastine were the presence of vinblastine- induced crystals and an increased incidence of autophagy. The remainder of cell structure was not affected nor were overall tissue ATP and electrolyte contents or the stimulant-induced increase in 45Ca++ efflux. It is concluded that the antisecretory effects of vinblastine and colchicine are consistent with a microtubular action, but that acinar cell microtubules are more resistant to the drugs than many other cell types. PMID:791957

  15. CHLAMYDOMONAS FLAGELLA

    PubMed Central

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

    1972-01-01

    Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of ? 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively PMID:4558009

  16. Dynamics of microtubules visualized by darkfield microscopy: treadmilling and dynamic instability.

    PubMed

    Hotani, H; Horio, T

    1988-01-01

    Individual microtubules undergoing treadmilling in vitro were visualized by darkfield light microscopy, and the relationship between treadmilling and dynamic instability was studied as a function of microtubule-associated proteins (MAPs). In order to demonstrate treadmilling directly by real-time observation, we constructed three-block microtubules, the center-block of which was decorated with Tetrahymena dynein. The decorated block can easily be distinguished from undecorated blocks in the darkfield microscope because the decorated one appears much thicker. At steady-state conditions, the length of an undecorated block at one end increased and that at another end decreased, while the decorated center-block did not change in its length. The results from these direct observations show that calf brain 3X-microtubules exhibit a treadmilling flux of 0.9 micron/h. Using a similar microscopy technique, we previously demonstrated that phosphocellulose PC-microtubules existed in either the growing or the shortening phase and alternated quite frequently at steady-state conditions (dynamic instability). How does treadmilling relate to dynamic instability? An image recording of individual 3X-microtubules containing MAPs revealed that the microtubules undergo treadmilling and do not exhibit any dynamic instability. This evidence shows that MAPs suppress the dynamic instability of microtubules. That is, treadmilling can take place in the steady state only after microtubules have been stabilized by MAPs. PMID:2972399

  17. Attachment of the cap to the central microtubules of tetrahymena cilia

    E-print Network

    Dentler, William L., Jr

    1984-03-01

    J. Cell Sci. 66, 167-173 (1984) 167 Printed in Great Britain ? 'Hie Company of Biologists Limited 1984 ATTACHMENT OF THE CAP TO THE CENTRAL MICROTUBULES OF TETRAHYMENA CILIA WILLIAM L. DENTLER Department of Physiology and Cell Biology, Center... in appearance to the distal filament plugs attached to the ends of the A-microtubules. The caps have been separated from the microtubules and are composed of a bead, two plates, and two peg-like plugs to which the microtubules are attached. The structure...

  18. 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

  19. Prophase Microtubule Arrays Undergo Flux-like Behavior in Mammalian Cells

    PubMed Central

    Ferenz, Nick P.

    2007-01-01

    In higher eukaryotic cells, microtubules within metaphase and anaphase spindles undergo poleward flux, the slow, poleward movement of tubulin subunits through the spindle microtubule lattice. Although a number of studies have documented this phenomenon across a wide range of model systems, the possibility of poleward flux before nuclear envelope breakdown (NEB) has not been examined. Using a mammalian cell line expressing photoactivatable green fluorescent protein (GFP)-tubulin, we observe microtubule motion, both toward and away from centrosomes, at a wide range of rates (0.5–4.5 ?m/min) in prophase cells. Rapid microtubule motion in both directions is dynein dependent. In contrast, slow microtubule motion, which occurs at rates consistent with metaphase flux, is insensitive to inhibition of dynein but sensitive to perturbation of Eg5 and Kif2a, two proteins with previously documented roles in flux. Our results demonstrate that microtubules in prophase cells are unexpectedly dynamic and that a subpopulation of these microtubules shows motion that is consistent with flux. We propose that the marked reduction in rate and directionality of microtubule motion from prophase to metaphase results from changes in microtubule organization during spindle formation. PMID:17671163

  20. The Kinetochore-Bound Ska1 Complex Tracks Depolymerizing Microtubules and Binds to Curved Protofilaments

    E-print Network

    Arthanari, Haribabu

    To ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive chromosome movements. How kinetochores associate with depolymerizing ...

  1. An ELMO2-RhoG-ILK network modulates microtubule dynamics.

    PubMed

    Jackson, Bradley C; Ivanova, Iordanka A; Dagnino, Lina

    2015-07-15

    ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting ?1 integrin-dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3?, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca(2+)-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes. PMID:25995380

  2. 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

  3. Microtubules are a target for self-incompatibility signaling in Papaver pollen.

    PubMed

    Poulter, Natalie S; Vatovec, Sabina; Franklin-Tong, Vernonica E

    2008-03-01

    Perception and integration of signals into responses is of crucial importance to cells. Both the actin and microtubule cytoskeleton are known to play a role in mediating diverse stimulus responses. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization. SI in Papaver rhoeas triggers a Ca(2+)-dependent signaling network to trigger programmed cell death (PCD), providing a neat way to inhibit and destroy incompatible pollen. We previously established that SI stimulates F-actin depolymerization and that altering actin dynamics can push pollen tubes into PCD. Very little is known about the role of microtubules in pollen tubes. Here, we investigated whether the pollen tube microtubule cytoskeleton is a target for the SI signals. We show that SI triggers very rapid apparent depolymerization of cortical microtubules, which, unlike actin, does not reorganize later. Actin depolymerization can trigger microtubule depolymerization but not vice versa. Moreover, although disruption of microtubule dynamics alone does not trigger PCD, alleviation of SI-induced PCD by taxol implicates a role for microtubule depolymerization in mediating PCD. Together, our data provide good evidence that SI signals target the microtubule cytoskeleton and suggest that signal integration between microfilaments and microtubules is required for triggering of PCD. PMID:18192439

  4. 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 ...

  5. Astral Microtubule Pivoting Promotes Their Search for Cortical Anchor Sites during Mitosis in Budding Yeast

    PubMed Central

    Baumgärtner, Stephan; Toli?, Iva M.

    2014-01-01

    Positioning of the mitotic spindle is crucial for proper cell division. In the budding yeast Saccharomyces cerevisiae, two mechanisms contribute to spindle positioning. In the Kar9 pathway, astral microtubules emanating from the daughter-bound spindle pole body interact via the linker protein Kar9 with the myosin Myo2, which moves the microtubule along the actin cables towards the neck. In the dynein pathway, astral microtubules off-load dynein onto the cortical anchor protein Num1, which is followed by dynein pulling on the spindle. Yet, the mechanism by which microtubules target cortical anchor sites is unknown. Here we quantify the pivoting motion of astral microtubules around the spindle pole bodies, which occurs during spindle translocation towards the neck and through the neck. We show that this pivoting is largely driven by the Kar9 pathway. The microtubules emanating from the daughter-bound spindle pole body pivot faster than those at the mother-bound spindle pole body. The Kar9 pathway reduces the time needed for an astral microtubule inside the daughter cell to start pulling on the spindle. Thus, we propose a new role for microtubule pivoting: By pivoting around the spindle pole body, microtubules explore the space laterally, which helps them search for cortical anchor sites in the context of spindle positioning in budding yeast. PMID:24721997

  6. The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

    PubMed Central

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

  7. 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.

  8. Radiative neutrino masses in the singlet-doublet fermion dark matter model with scalar singlets

    NASA Astrophysics Data System (ADS)

    Restrepo, Diego; Rivera, Andrés; Sánchez-Peláez, Marta; Zapata, Oscar; Tangarife, Walter

    2015-07-01

    When the singlet-doublet fermion dark matter model is extended with additional Z2-odd real singlet scalars, neutrino masses and mixings can be generated at the one-loop level. In this work, we discuss the salient features arising from the combination of the two resulting simplified dark matter models. When the lightest Z2-odd particle is a scalar singlet, Br (? ?e ? ) could be measurable provided that the singlet-doublet fermion mixing is small enough. In this scenario, the new decay channels of vector-like fermions into scalars can also generate interesting leptonic plus missing transverse energy signals at the LHC. On the other hand, in the case of doublet-like fermion dark matter, scalar coannihilations lead to an increase in the relic density which allows one to lower the bound of doublet-like fermion dark matter.

  9. 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

  10. 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

  11. Spinning Janus doublets driven in uniform AC electric fields

    E-print Network

    Alicia Boymelgreen; Gilad Yossifon; Sinwook Park; Touvia Miloh

    2013-10-06

    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 electroosmosis. 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 non-linear electrokinetics. A simple kinematic rigid body model is used to predict the paths and double velocities (angular and linear) based on their relative orientations with good agreement.

  12. Asymmetric Inelastic Inert Doublet Dark Matter from Triplet Scalar Leptogenesis

    E-print Network

    Arina, Chiara

    2011-01-01

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

  13. Regulation of microtubule nucleation in Schizosaccharomyces pombe: recruitment of Mto1 to the site of the prospective eMTOC 

    E-print Network

    Miller, Victoria Jane

    2010-01-01

    Mto1 recruits ?-tubulin to the sites of cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe. The regulation of Mto1 localisation is central to re-modelling of the microtubule cytoskeleton ...

  14. Identification of a homozygous splice site mutation in the dynein axonemal light chain 4 gene on 22q13.1 in a large consanguineous family from Pakistan with congenital mirror movement disorder.

    PubMed

    Ahmed, Iltaf; Mittal, Kirti; Sheikh, Taimoor I; Vasli, Nasim; Rafiq, Muhammad Arshad; Mikhailov, Anna; Ohadi, Mehrnaz; Mahmood, Huda; Rouleau, Guy A; Bhatti, Attya; Ayub, Muhammad; Srour, Myriam; John, Peter; Vincent, John B

    2014-11-01

    Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976-39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV. PMID:25098561

  15. 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

  16. Growth Cone Turning Induced by Direct Local Modification of Microtubule Dynamics

    E-print Network

    Growth Cone Turning Induced by Direct Local Modification of Microtubule Dynamics Kenneth B. Buck Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 Pathfinding by nerve growth present direct evidence to demonstrate an essential and instructive role for microtubules (MTs) in growth

  17. 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

  18. Rearrangement of microtubule polarity orientation during conversion of dendrites to axons in cultured pyramidal neurons

    Microsoft Academic Search

    Daisuke Takahashi; Wenqian Yu; Peter W. Baas; Rika Kawai-Hirai; Kensuke Hayashi

    2007-01-01

    Axons and dendrites of neurons differ in the polarity orientation of their microtu- bules. Whereas the polarity orientation of microtubules in axons is uniform, with all plus ends distal, that in dendrites is nonuniform. The mechanisms responsible for establishment and maintenance of microtubule polarity orientation in neuronal proc- esses remain unclear, however. We previously described a culture system in which

  19. A Bending Mode Analysis for Growing Microtubules: Evidence for a Velocity-Dependent Rigidity

    PubMed Central

    Janson, Marcel E.; Dogterom, Marileen

    2004-01-01

    Microtubules are dynamic protein polymers that continuously switch between elongation and rapid shrinkage. They have an exceptional bending stiffness that contributes significantly to the mechanical properties of eukaryotic cells. Measurements of the persistence length of microtubules have been published since 10 years but the reported values vary over an order of magnitude without an available explanation. To precisely measure the rigidity of microtubules in their native growing state, we adapted a previously developed bending mode analysis of thermally driven shape fluctuations to the case of an elongating filament that is clamped at one end. Microtubule shapes were quantified using automated image processing, allowing for the characterization of up to five bending modes. When taken together with three other less precise measurements, our rigidity data suggest that fast-growing microtubules are less stiff than slow-growing microtubules. This would imply that care should be taken in interpreting rigidity measurements on stabilized microtubules whose growth history is not known. In addition, time analysis of bending modes showed that higher order modes relax more slowly than expected from simple hydrodynamics, possibly by the effects of internal friction within the microtubule. PMID:15454464

  20. 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