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Sample records for axonemal microtubule doublets

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

    SciTech Connect

    Sui, Haixin; Downing, Kenneth H.

    2006-05-22

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

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

    PubMed

    Seetharam, Raviraja N; Satir, Peter

    2008-07-01

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

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

    PubMed Central

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

    2013-01-01

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

  4. 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.

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

    PubMed

    Stepanek, Ludek; Pigino, Gaia

    2016-05-06

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

  6. The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme.

    PubMed

    Alford, Lea M; Stoddard, Daniel; Li, Jennifer H; Hunter, Emily L; Tritschler, Douglas; Bower, Raqual; Nicastro, Daniela; Porter, Mary E; Sale, Winfield S

    2016-06-01

    We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of ∼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Si, Y; Okuno, M

    1995-11-01

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

  9. High speed sliding of axonemal microtubules produced by outer arm dynein.

    PubMed

    Seetharam, Raviraja N; Satir, Peter

    2005-02-01

    To study dynein arm activity at high temporal resolution, axonemal sliding was measured field by field for wild type and dynein arm mutants of Tetrahymena thermophila. For wt SB255 cells, when the rate of data acquisition was 60 fps, about 5x greater than previously published observations, sliding was observed to be discontinuous with very high velocity sliding (average 196 microm/sec) for a few msec (1 or 2 fields) followed by a pause of several fields. The sliding velocities measured were an order of magnitude greater than rates previously measured by video analysis. However, when the data were analyzed at 12 fps for the same axonemes, consistent with previous observations, sliding was linear as the axonemes extended several times their original length with an average velocity of approximately 10 microm/sec. The pauses or stops occurred at approximately 200 and 300% of the initial length, suggesting that dynein arms on one axonemal doublet were initially active to the limit of extension, and then the arms on the next doublet became activated. In contrast, in a mutant where OADs are missing, sliding observed at 60 fps was continuous and slow (5 microm/sec), as opposed to the discontinuous high-velocity sliding of SB255 and of the mutant at the permissive temperature where OADs are present. High-velocity step-wise sliding was also present in axonemes from an inner arm dynein mutant (KO6). These results indicate that the high-speed discontinuous pattern of sliding is produced by the mechanochemical activity of outer arm dynein. The rate of sliding is consistent with a low duty ratio of the outer arm dynein and with the operation of each arm along a doublet once per beat.

  10. Axonemal Dynein Arms.

    PubMed

    King, Stephen M

    2016-11-01

    Axonemal dyneins form the inner and outer rows of arms associated with the doublet microtubules of motile cilia. These enzymes convert the chemical energy released from adenosine triphosphate (ATP) hydrolysis into mechanical work by causing the doublets to slide with respect to each other. Dyneins form two major groups based on the number of heavy-chain motors within each complex. In addition, these enzymes contain other components that are required for assembly of the complete particles and/or for the regulation of motor function in response to phosphorylations status, ligands such as Ca(2+), changes in cellular redox state and which also apparently monitor and respond to the mechanical state or curvature in which any given motor finds itself. It is this latter property, which is thought to result in waves of motor function propagating along the axoneme length. Here, I briefly describe our current understanding of axonemal dynein structure, assembly, and organization.

  11. Axoneme Structure from Motile Cilia.

    PubMed

    Ishikawa, Takashi

    2017-01-03

    The axoneme is the main extracellular part of cilia and flagella in eukaryotes. It consists of a microtubule cytoskeleton, which normally comprises nine doublets. In motile cilia, dynein ATPase motor proteins generate sliding motions between adjacent microtubules, which are integrated into a well-orchestrated beating or rotational motion. In primary cilia, there are a number of sensory proteins functioning on membranes surrounding the axoneme. In both cases, as the study of proteomics has elucidated, hundreds of proteins exist in this compartmentalized biomolecular system. In this article, we review the recent progress of structural studies of the axoneme and its components using electron microscopy and X-ray crystallography, mainly focusing on motile cilia. Structural biology presents snapshots (but not live imaging) of dynamic structural change and gives insights into the force generation mechanism of dynein, ciliary bending mechanism, ciliogenesis, and evolution of the axoneme.

  12. The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

    PubMed Central

    Kuhns, Stefanie; Schmidt, Kerstin N.; Reymann, Jürgen; Gilbert, Daniel F.; Neuner, Annett; Hub, Birgit; Carvalho, Ricardo; Wiedemann, Philipp; Zentgraf, Hanswalter; Erfle, Holger; Klingmüller, Ursula; Boutros, Michael

    2013-01-01

    Despite the critical contributions of cilia to embryonic development and human health, key regulators of cilia formation await identification. In this paper, a functional RNA interference–based screen linked 30 novel protein kinases with ciliogenesis. Of them, we have studied the role of the microtubule (MT)-associated protein/MT affinity regulating kinase 4 (MARK4) in depth. MARK4 associated with the basal body and ciliary axoneme in human and murine cell lines. Ultrastructural and functional analyses established that MARK4 kinase activity was required for initiation of axoneme extension. We identified the mother centriolar protein ODF2 as an interaction partner of MARK4 and showed that ODF2 localization to the centriole partially depended on MARK4. Our data indicated that, upon MARK4 or ODF2 knockdown, the ciliary program arrested before the complete removal of the CP110–Cep97 inhibitory complex from the mother centriole, suggesting that these proteins act at this level of axonemal extension. We propose that MARK4 is a critical positive regulator of early steps in ciliogenesis. PMID:23400999

  13. 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.

  14. Structural biology of cytoplasmic and axonemal dyneins.

    PubMed

    Ishikawa, Takashi

    2012-08-01

    Dyneins are microtubule-based, ATP-driven motor proteins with six tandemly linked AAA+ domains, a long N-terminal tail and a coiled-coil stalk. Cytoplasmic dyneins function as individual homodimers and are responsible for minus-end-oriented transport along microtubules. Axonemal dyneins of flagella/cilia are anchored in arrays to peripheral microtubule doublets by their N-terminal tails, and generate sliding motions of adjacent microtubule doublets toward the plus end. The coiled-coil stalk is responsible for communication between the AAA+ domains and the microtubule binding domain. A number of isoforms of axonemal dyneins are integrated to generate bending motion. In this article I will review recent structural studies and address the question as to how dyneins generate force and cause bending in flagella/cilia.

  15. Heterogeneity of dynein structure implies coordinated suppression of dynein motor activity in the axoneme.

    PubMed

    Maheshwari, Aditi; Ishikawa, Takashi

    2012-08-01

    Axonemal dyneins provide the driving force for flagellar/ciliary bending. Nucleotide-induced conformational changes of flagellar dynein have been found both in vitro and in situ by electron microscopy, and in situ studies demonstrated the coexistence of at least two conformations in axonemes in the presence of nucleotides (the apo and the nucleotide-bound forms). The distribution of the two forms suggested cooperativity between adjacent dyneins on axonemal microtubule doublets. Although the mechanism of such cooperativity is unknown it might be related to the mechanism of bending. To explore the mechanism by which structural heterogeneity of axonemal dyneins is induced by nucleotides, we used cilia from Tetrahymena thermophila to examine the structure of dyneins in a) the intact axoneme and b) microtubule doublets separated from the axoneme, both with and without additional pure microtubules. We also employed an ATPase assay on these specimens to investigate dynein activity functionally. Dyneins on separated doublets show more activation by nucleotides than those in the intact axoneme, both structurally and in the ATPase assay, and this is especially pronounced when the doublets are coupled with added microtubules, as expected. Paralleling the reduced ATPase activity in the intact axonemes, a lower proportion of these dyneins are in the nucleotide-bound form. This indicates a coordinated suppression of dynein activity in the axoneme, which could be the key for understanding the bending mechanism.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Linck, R W; Stephens, R E

    1987-04-01

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

  18. Biochemical characterization of tektins from sperm flagellar doublet microtubules

    PubMed Central

    1987-01-01

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

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

    PubMed

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

    2014-06-20

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

  20. Integrated Control of Axonemal Dynein AAA+ Motors

    PubMed Central

    King, Stephen M.

    2012-01-01

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

  1. Curvature regulation of the ciliary beat through axonemal twist.

    PubMed

    Sartori, Pablo; Geyer, Veikko F; Howard, Jonathon; Jülicher, Frank

    2016-10-01

    Cilia and flagella are hairlike organelles that propel cells through fluid. The active motion of the axoneme, the motile structure inside cilia and flagella, is powered by molecular motors of the axonemal dynein family. These motors generate forces and torques that slide and bend the microtubule doublets within the axoneme. To create regular waveforms, the activities of the dyneins must be coordinated. It is thought that coordination is mediated by stresses due to radial, transverse, or sliding deformations, and which build up within the moving axoneme and feed back on dynein activity. However, which particular components of the stress regulate the motors to produce the observed waveforms of the many different types of flagella remains an open question. To address this question, we describe the axoneme as a three-dimensional bundle of filaments and characterize its mechanics. We show that regulation of the motors by radial and transverse stresses can lead to a coordinated flagellar motion only in the presence of twist. We show that twist, which could arise from torque produced by the dyneins, couples curvature to transverse and radial stresses. We calculate emergent beating patterns in twisted axonemes resulting from regulation by transverse stresses. The resulting waveforms are similar to those observed in flagella of Chlamydomonas and sperm. Due to the twist, the waveform has nonplanar components, which result in swimming trajectories such as twisted ribbons and helices, which agree with observations.

  2. Curvature regulation of the ciliary beat through axonemal twist

    NASA Astrophysics Data System (ADS)

    Sartori, Pablo; Geyer, Veikko F.; Howard, Jonathon; Jülicher, Frank

    2016-10-01

    Cilia and flagella are hairlike organelles that propel cells through fluid. The active motion of the axoneme, the motile structure inside cilia and flagella, is powered by molecular motors of the axonemal dynein family. These motors generate forces and torques that slide and bend the microtubule doublets within the axoneme. To create regular waveforms, the activities of the dyneins must be coordinated. It is thought that coordination is mediated by stresses due to radial, transverse, or sliding deformations, and which build up within the moving axoneme and feed back on dynein activity. However, which particular components of the stress regulate the motors to produce the observed waveforms of the many different types of flagella remains an open question. To address this question, we describe the axoneme as a three-dimensional bundle of filaments and characterize its mechanics. We show that regulation of the motors by radial and transverse stresses can lead to a coordinated flagellar motion only in the presence of twist. We show that twist, which could arise from torque produced by the dyneins, couples curvature to transverse and radial stresses. We calculate emergent beating patterns in twisted axonemes resulting from regulation by transverse stresses. The resulting waveforms are similar to those observed in flagella of Chlamydomonas and sperm. Due to the twist, the waveform has nonplanar components, which result in swimming trajectories such as twisted ribbons and helices, which agree with observations.

  3. Are the local adjustments of the relative spatial frequencies of the dynein arms and the beta-tubulin monomers involved in the regulation of the "9+2" axoneme?

    PubMed

    Cibert, Christian

    2008-07-07

    The "9+2" axoneme is a highly specific cylindrical machine whose periodic bending is due to the cumulative shear of its 9 outer doublets of microtubules. Because of the discrete architecture of the tubulin monomers and the active appendices that the outer doublets carry (dynein arms, nexin links and radial spokes), this movement corresponds to the relative shear of these topological verniers, whose characteristics depend on the geometry of the wave train. When an axonemal segment bends, this induces the compressed and dilated conformations of the tubulin monomers and, consequently, the modification of the spatial frequencies of the appendages that the outer doublets carry. From a dynamic point of view, the adjustments of the spatial frequencies of the elements of the two facing verniers that must interact create different longitudinal periodic patterns of distribution of the joint probability of the molecular interaction as a function of the location of the doublet pairs around the axonemal cylinder and their spatial orientation within the axonemal cylinder. During the shear, these patterns move along the outer doublet intervals at a speed that ranges from one to more than a thousand times that of sliding, in two opposite directions along the two opposite halves of the axoneme separated by the bending plane, respecting the polarity of the dynein arms within the axoneme. Consequently, these waves might be involved in the regulation of the alternating activity of the dynein arms along the flagellum, because they induce the necessary intermolecular dialog along the axoneme since they could be an element of the local dynamic stability/instability equilibrium of the axoneme. This complements the geometric clutch model [Lindemann, C., 1994. A "geometric clutch" hypothesis to explain oscillations of the axoneme of cilia and flagella. J. Theor. Biol. 168, 175-189].

  4. Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension.

    PubMed

    Kasahara, Kousuke; Kawakami, Yoshitaka; Kiyono, Tohru; Yonemura, Shigenobu; Kawamura, Yoshifumi; Era, Saho; Matsuzaki, Fumio; Goshima, Naoki; Inagaki, Masaki

    2014-10-01

    Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis.

  5. Bending, twisting and beating trunk robot bioinspired from the '3 + 0' axoneme.

    PubMed

    Cibert, Christian

    2013-06-01

    The axoneme is the skeleton and motor axis of flagella and cilia in eukaryotic organisms. Basically it consists of a series of longitudinal fibers (outer doublets of microtubules) that design a cylinder and whose sliding, due to the coordinated activities of dedicated molecular motors (the dynein arms), is converted into a bending because outer doublets pairs are stabilized by elastic links (the nexine molecules). In spite of these interesting mechanical properties, mechanical and robotics engineers have never considered this amazing molecular machinery as a model. The aim of this paper is to propose the robotic design and the kinematic modeling of the '3 + 0' axoneme that makes motile the flagellum of Diplauxis hatti, the simplest that exists. The model that we propose bends and twists and combines the two movements. It is able to propagate wave trains that could be involved in the development of biomimetic actuators of various mechanisms such as (sub)aquatic robotic propellers as well as robotic trunks.

  6. 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

  7. Sensing the Mechanical State of the Axoneme and Integration of Ca2+ Signaling by Outer Arm Dynein

    PubMed Central

    King, Stephen M.

    2010-01-01

    Axonemal dyneins have been demonstrated to monitor the mechanical state of the axoneme and must also alter activity in response to various signaling pathways. The central pair/radial spoke systems are clearly involved in controlling inner dynein arm function; however, the mechanisms by which the outer dynein arm transduces regulatory signals appear quite distinct at the molecular level. In Chlamydomonas, these regulatory components include thioredoxins involved in response to redox changes, molecules that tether the γ heavy chain motor unit to the A-tubule of the outer doublet and a Ca2+-binding protein that controls the structure of the γ heavy chain N-terminal domain. Together, these studies now suggest that the γ heavy chain acts as a key regulatory node for controlling outer arm function in response to alterations in curvature and ligand binding. Furthermore, they allow us to propose a testable molecular mechanism by which altered Ca2+ levels might lead to a change in ciliary waveform by controlling whether one heavy chain of outer arm dynein acts as a microtubule translocase or as an ATP-dependent brake that limits the amount of inter-doublet sliding. PMID:20186692

  8. X-ray diffraction recording from single axonemes of eukaryotic flagella.

    PubMed

    Nishiura, Masaya; Toba, Shiori; Takao, Daisuke; Miyashiro, Daisuke; Sakakibara, Hitoshi; Matsuo, Tatsuhito; Kamimura, Shinji; Oiwa, Kazuhiro; Yagi, Naoto; Iwamoto, Hiroyuki

    2012-06-01

    We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 μm, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-μm segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, ~2 μm) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20°, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general.

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

    PubMed Central

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

    1995-01-01

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

  10. Cloning, localization, and axonemal function of Tetrahymena centrin.

    PubMed

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

    2003-01-01

    Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.

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

    PubMed Central

    1985-01-01

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

  12. Mammalian Axoneme Central Pair Complex Proteins: Broader Roles Revealed by Gene Knockout Phenotypes

    PubMed Central

    Teves, Maria E.; Nagarkatti-Gude, David R.; Zhang, Zhibing; Strauss, Jerome F.

    2016-01-01

    The axoneme genes, their encoded proteins, their functions and the structures they form are largely conserved across species. Much of our knowledge of the function and structure of axoneme proteins in cilia and flagella is derived from studies on model organisms like the green algae, Chlamydomonas reinhardtii. The core structure of cilia and flagella is the axoneme, which in most motile cilia and flagella contains a 9 + 2 configuration of microtubules. The two central microtubules are the scaffold of the central pair complex (CPC). Mutations that disrupt CPC genes in Chlamydomonas and other model organisms result in defects in assembly, stability and function of the axoneme, leading to flagellar motility defects. However, targeted mutations generated in mice in the orthologous CPC genes have revealed significant differences in phenotypes of mutants compared to Chlamydomonas. Here we review observations that support the concept of cell-type specific roles for the CPC genes in mice, and an expanded repertoire of functions for the products of these genes in cilia, including non-motile cilia, and other microtubule-associated cellular functions. PMID:26785425

  13. ida4-1, ida4-2, and ida4-3 are intron splicing mutations affecting the locus encoding p28, a light chain of Chlamydomonas axonemal inner dynein arms.

    PubMed Central

    LeDizet, M; Piperno, G

    1995-01-01

    We recently determined the nucleotide sequence of the gene encoding p28, a light chain of inner dynein arms of Chlamydomonas axonemes. Here, we show that p28 is the protein encoded by the IDA4 locus. p28, and the dynein heavy chains normally associated with it, are completely absent from the flagella and cell bodies of three allelic strains of ida4, named ida4-1, ida4-2, and ida4-3. We determined the nucleotide sequence of the three alleles of the p28 gene and found in each case a single nucleotide change, affecting the splice sites of the first, second, and fourth introns, respectively. Reverse transcriptase-polymerase chain reaction amplification of RNAs prepared from ida4 cells confirmed that these mutations prevent the correct splicing of the affected introns, thereby blocking the synthesis of full-length p28. These are the first intron splicing mutations described in Chlamydomonas and the first inner dynein arm mutations characterized at the molecular level. The absence in ida4 axonemes of the dynein heavy chains normally found in association with p28 suggests that p28 is necessary for stable assembly of a subset of inner dynein arms or for the binding of these arms to the microtubule doublets. Images PMID:7579690

  14. Active sliding between cytoplasmic microtubules.

    PubMed

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

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

  15. Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing

    PubMed Central

    Karak, Somdatta; Jacobs, Julie S.; Kittelmann, Maike; Spalthoff, Christian; Katana, Radoslaw; Sivan-Loukianova, Elena; Schon, Michael A.; Kernan, Maurice J.; Eberl, Daniel F.; Göpfert, Martin C.

    2015-01-01

    Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly’s ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility. PMID:26608786

  16. Torque generation by axonemal outer-arm dynein.

    PubMed

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

    2015-02-17

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

  17. A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

    PubMed Central

    Gagnon, C; White, D; Huitorel, P; Cosson, J

    1994-01-01

    To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes. Images PMID:7841521

  18. An integrative model of internal axoneme mechanics and external fluid dynamics in ciliary beating.

    PubMed

    Dillon, R H; Fauci, L J

    2000-12-07

    We present a fluid-mechanical model of an individual cilium which incorporates discrete representations of the dynein arms, the passive elastic structure of the axoneme including the microtubules and nexin links. This model, based upon the immersed boundary method (Peskin, 1977), couples the internal force generation of the molecular motors through the passive elastic structure with the external fluid mechanics governed by the Navier-Stokes equations. Detailed geometric information is available, such as the spacing and shear between the microtubules, the local curvature of individual microtubules and the stretching of the nexin links. In addition, the explicit representation of the dynein motors allows us the flexibility to incorporate a variety of activation theories. In this article, we choose a simple activation theory so that the ciliary beat is not present, but is an emergent property of the interacting components of the coupled fluid-axoneme system. We present numerical results from computer simulations of sliding disintegration and ciliary beating with several different viscosities.

  19. Effects of iodide on the coupling between ATP hydrolysis and motile activity in axonemal dynein.

    PubMed

    Nakano, Izumi; Fujiwara, Rin; Wada, Mikiyo; Shingyoji, Chikako

    2011-05-01

    Dynein transduces the chemical energy of ATP hydrolysis into mechanical work through conformational changes. To identify the factors governing the coupling between the ATPase activity and the motile activity of the dynein molecule, we examined the effects of potassium iodide, which can unfold protein tertiary structures, on dynein activity in reactivated sea urchin sperm flagella. The presence of low concentrations of KI (0.05-0.1 M) in the reactivating solution did not influence the stable beating of demembranated flagella at 0.02-1 mM ATP, when the total concentration of potassium was kept at 0.15 M by adding K-acetate. However, double-reciprocal plots of ATP concentration and beat frequency showed a mixed type of inhibition by KI, indicating the possibility that KI inhibits the ATP hydrolysis and decreases the maximum sliding velocity. The ATPase activity of 21S dynein with or without microtubules did not decrease with the KI concentration. In the elastase-treated axonemes, KI decreased the velocity of sliding disintegration, while it increased the frequency of occurrence of axonemes showing no sliding. This may be related to some defect in the coordination of dynein activities. On 21S dynein adsorbed on a glass surface, however, the velocity of microtubule sliding was increased by KI, while KI lowered the dynein-microtubule affinity. The velocity further increased under lower salt conditions enhancing the dynein-microtubule interactions. The results suggest the importance of organized regulation of the dynamic states of dynein-microtubule interactions through the stalk for the coupling between the ATPase activity and the motile activity of dynein in beating flagella.

  20. Cellular Samurai: katanin and the severing of microtubules.

    PubMed

    Quarmby, L

    2000-08-01

    Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

  1. Doublet craters on Venus

    NASA Astrophysics Data System (ADS)

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

    2003-09-01

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

  2. Microtubules in protozoan cells. III. Ultrastructural changes during disintegration and reformation of heliozoan microtubules.

    PubMed

    Toyohara, A; Shigenaka, Y; Mohri, H

    1978-08-01

    In the heliozoan, Echinosphaerium nucleofilum strain MA, cold temperature (2 degrees C) induced axopodial retraction to about 36% of the initial length after 3 h. By electron microscopy, it was found that such axopodial shortening is accompanied by degradation of axonemal microtubules (25 nm in diameter), followed by the appearance of macrotubles (37 nm in diameter) and filamentous structures (14 nm in diameter) of a tubular and twisted appearance. All of these structures (microtubules, macrotubules and filamentous structures) were found to be depolymerized completely by applying 10 mM colchicine for 1-2 h, and to be replaced by regions with low electron density. Axopodial re-extension was induced rapidly by returning the cold-treated organisms to room temperature (20 degrees C). At a very early stage of axopodial re-extension, the filamentous structures were often observed to be continuous with the macrotubules. At a late stage of axopodial re-extension, the reforming axoneme was composed only of normal microtubules, while the macrotubules and filamentous structures had disappeared. On the basis of these results, the processes of disintegration and reformation of microtubules are discussed.

  3. Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: spatial arrangement and properties

    PubMed Central

    1986-01-01

    A monoclonal antibody, 6-11B-1, specific for acetylated alpha-tubulin (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) was used to study the distribution of this molecule in interphase cells of Chlamydomonas reinhardtii. Double-label immunofluorescence was performed using 6-11B-1, and 3A5, an antibody specific for all alpha- tubulin isoforms. It was found that acetylated alpha-tubulin is not restricted to the axonemes, but is also present in basal bodies and in a subset of cytoplasmic microtubules that radiate from the basal bodies just beneath the plasma membrane. Immunoblotting experiments of basal body polypeptide components using 6-11B-1 as a probe confirmed that basal bodies contain acetylated alpha-tubulin. In the cell body, 6-11B- 1 stained an average of 2.2 microtubules/cell, while 3A5 stained an average of 6.5 microtubules. Although exposure to 0 degrees C depolymerized both types of cytoplasmic microtubules, exposure to various concentrations of colchicine or nocodazole showed that the acetylated microtubules are much more resistant to drug-induced depolymerization than nonacetylated microtubules. Axonemes and basal bodies are already known to be colchicine-resistant. All acetylated microtubules appear, therefore, to be more drug-resistant than nonacetylated microtubules. The acetylation of alpha-tubulin may be part of a mechanism that stabilizes microtubules. PMID:3722261

  4. Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro

    SciTech Connect

    Walker, R.A.; Inoue, S.; Salmon, E.D.

    1989-03-01

    The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a GTP cap. A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of stabilizing cap, possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

  5. CYTOPLASMIC MICROTUBULES

    PubMed Central

    Slautterback, David B.

    1963-01-01

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

  6. Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

    PubMed

    Mitchell, V; Sigala, J; Ballot, C; Jumeau, F; Barbotin, A L; Duhamel, A; Rives, N; Rigot, J M; Escalier, D; Peers, M C

    2015-03-01

    Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk.

  7. Basal body multipotency and axonemal remodelling are two pathways to a 9+0 flagellum

    PubMed Central

    Wheeler, R. J.; Gluenz, E.; Gull, K.

    2015-01-01

    Eukaryotic cilia/flagella exhibit two characteristic ultrastructures reflecting two main functions; a 9+2 axoneme for motility and a 9+0 axoneme for sensation and signalling. Whether, and if so how, they interconvert is unclear. Here we analyse flagellum length, structure and molecular composition changes in the unicellular eukaryotic parasite Leishmania during the transformation of a life cycle stage with a 9+2 axoneme (the promastigote) to one with a 9+0 axoneme (the amastigote). We show 9+0 axonemes can be generated by two pathways: by de novo formation and by restructuring of existing 9+2 axonemes associated with decreased intraflagellar transport. Furthermore, pro-basal bodies formed under conditions conducive for 9+2 axoneme formation can form a 9+0 axoneme de novo. We conclude that pro-centrioles/pro-basal bodies are multipotent and not committed to form either a 9+2 or 9+0 axoneme. In an alternative pathway structures can also be removed from existing 9+2 axonemes to convert them to 9+0. PMID:26667778

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

    PubMed

    Bibring, T; Baxandall, J

    1971-02-01

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

  9. BLD10/CEP135 is a microtubule-associated protein that controls the formation of the flagellum central microtubule pair.

    PubMed

    Carvalho-Santos, Zita; Machado, Pedro; Alvarez-Martins, Inês; Gouveia, Susana M; Jana, Swadhin C; Duarte, Paulo; Amado, Tiago; Branco, Pedro; Freitas, Micael C; Silva, Sara T N; Antony, Claude; Bandeiras, Tiago M; Bettencourt-Dias, Mónica

    2012-08-14

    Cilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled by a central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. We characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. We show that assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and that the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135's role in assembling highly stable MT-based structures, such as motile axonemes and centrioles.

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

    PubMed

    Sloboda, Roger D

    2015-02-02

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

  11. Ciliary microtubule capping structures contain a mammalian kinetochore antigen

    PubMed Central

    1990-01-01

    Structures that cap the plus ends of microtubules may be involved in the regulation of their assembly and disassembly. Growing and disassembling microtubules in the mitotic apparatus are capped by kinetochores and ciliary and flagellar microtubules are capped by the central microtubule cap and distal filaments. To compare the ciliary caps with kinetochores, isolated Tetrahymena cilia were stained with CREST (Calcinosis/phenomenon esophageal dysmotility, sclerodactyly, telangiectasia) antisera known to stain kinetochores. Immunofluorescence microscopy revealed that a CREST antiserum stained the distal tips of cilia that contained capping structures but did not stain axonemes that lacked capping structures. Both Coomassie blue- stained gels and Western blots probed with CREST antiserum revealed that a 97-kD antigen copurifies with the capping structures. Affinity- purified antibodies to the 97-kD ciliary protein stained the tips of cap-containing Tetrahymena cilia and the kinetochores in HeLa, Chinese hamster ovary, and Indian muntjak cells. These results suggest that at least one polypeptide found in the kinetochore is present in ciliary microtubule capping structures and that there may be a structural and/or functional homology between these structures that cap the plus ends of microtubules. PMID:2106524

  12. Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs

    PubMed Central

    1987-01-01

    We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s- 1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus. PMID:3680377

  13. Automation design of cemented doublet

    NASA Astrophysics Data System (ADS)

    Romanova, Galina; Ivanova, Tatiana; Korotkova, Natalia

    2015-09-01

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

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

    PubMed Central

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

    1971-01-01

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

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

    PubMed Central

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

    2015-01-01

    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 α-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

  16. 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 α-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

  17. Compressing the Inert Doublet Model

    SciTech Connect

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

    2016-02-16

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

  18. SEROLOGICAL SIMILARITY OF FLAGELLAR AND MITOTIC MICROTUBULES

    PubMed Central

    Fulton, Chandler; Kane, R. E.; Stephens, R. E.

    1971-01-01

    An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins. PMID:4106543

  19. Microtubule dynamics and organization

    NASA Astrophysics Data System (ADS)

    Dogterom, Marileen

    2000-03-01

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

  20. Entropy and information in flagellar axoneme cybernetics: a radial spokes integrative function.

    PubMed

    Cibert, Christian

    2003-04-01

    Radial spokes and the consequences of their relationships with the central apparatus seem to play a very important role in the regulation of axonemal activity. We modeled their behavior and observed that it appears to differ in the cilium and the flagellum with respect to the development of bending as a function of time. Specifically, our calculation raises the question of the real function of the radial spokes in the regulation of the axoneme, because a given curvature of the flagellar axoneme may correspond to two opposite of their tilts. The stable nil/low amplitude shear points that we had characterized along the flagellum allowed us to describe their axoneme as a series of modules [Cibert, 2002: Cell Motil. Cytoskeleton 51:89-111]. We observed that a nil/low shearing point moves along each module during beating when a new bend is created at the base of the flagellum [Cibert, 2001: Cell Motil. Cytoskeleton 49:161-175]. We propose that the structural gradients of isoforms of tubulin could be basic verniers that act as structural references for the axonemal machinery during the beating. This allowed us to interpret the axonemal organization as a segmented structure, that could be analyzed according to the complexion(1) theory and Shannon's information theory, which associate entropy and probability in the creation of information. The important consequence of this interpretation is that regulation of the axonemal machinery appears to be due to the upstream and downstream cross-talk between the axonemal segments that do not involve any dedicated integrative structure but depend on the energy level of the entire length of each module.

  1. Modified axonemes and ciliary membranes in three polychaete species.

    PubMed

    Pfannenstiel, H D

    1982-01-01

    In living Ophryotrocha puerilis, Polyophthalmus pictus and Dinophilus gyrociliatus no modified cilia are present. Treatment with hyper- and hypotonic magnesium chloride solutions leads to the formation of either cilia with dilated tips or discocilia (paddle cilia). Discocilia show axoneme loops within distal swellings of the ciliary membranes. Both types of modified cilia regain their normal appearance if they are allowed to recover in seawater. The total number of discocilia and the diameter of the loops are inversely related to the osmolarity of the magnesium chloride solution used. Even isotonic solutions of magnesium chloride, which are usually used to anaesthetize marine worms, readily induce modified cilia. This indicates that the effect is not merely due to osmotic conditions. Glutaraldehyde and osmium tetroxide may act in the same way to induce modified cilia, a fact which may account for the numerous TEM and SEM documentations of modified cilia in various marine invertebrates. Which cilia in a particular species are modified varies from one specimen to another.

  2. Fluorescent ATP analog mant-ATP reports dynein activity in the isolated Chlamydomonas axoneme

    NASA Astrophysics Data System (ADS)

    Feofilova, Maria; Howard, Jonathon

    Eukaryotic flagella are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins located all along the length of the axoneme. How the force production of motors is controlled spatially and temporally is still an open question. Therefore, monitoring dynein activity in the axonemal structure is expected to provide novel insights in regulation of the beat. We use high sensitivity fluorescence microscopy to monitor the binding and hydrolysis kinetics of the fluorescently labeled ATP analogue mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate), which is known to support dynein activity. By studying the kinetics of mant-ATP fluorescence, we identified distinct mant-ATP binding sites in the axoneme. The application of this method to axonemes with reduced amounts of dynein, showed evidence that one of the sites is associated with binding to dynein. In the future, we would like to use this method to find the spatial distribution of dynein activity in the axoneme.

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

    PubMed Central

    1981-01-01

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

  4. 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

  5. Targeting Toxoplasma tubules: tubulin, microtubules, and associated proteins in a human pathogen.

    PubMed

    Morrissette, Naomi

    2015-01-01

    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.

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

    PubMed Central

    1984-01-01

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

  7. Phosphorylation in isolated Chlamydomonas axonemes: a phosphoprotein may mediate the Ca2+-dependent photophobic response

    PubMed Central

    1985-01-01

    An in vitro system was devised for studying phosphorylation of Chlamydomonas reinhardtii axonemal proteins. Many of the polypeptides phosphorylated in this system could be identified as previously described axonemal components that are phosphorylated in vivo. The in vitro system apparently preserved the activities of diverse axonemal kinases without greatly altering the substrate specificity of the enzymes. The in vitro system was used to study the effect of calcium concentration on axonemal protein phosphorylation. Calcium has previously been demonstrated to initiate the axonemal reversal reaction of the photophobic response; the in vitro system made it possible to investigate the possibility that this calcium effect is mediated by protein phosphorylation. Calcium specifically altered the phosphorylation of only two axonemal proteins; the phosphorylation of an otherwise unidentified 85,000 Mr protein was repressed by calcium concentrations greater than or equal to 10(-6) M, while the phosphorylation of the previously identified 95,000 Mr protein b4 was stimulated by calcium at concentrations greater than 10(-6) M. Protein b4 is one of six polypeptides that are deficient in the mbo mutants, strains that do not exhibit a photophobic reversal reaction. Therefore, this calcium-stimulated phosphorylation may be involved in initiating the photophobic response. Neither calmodulin nor the C-kinase could be implicated in b4 phosphorylation. The calcium-dependent activation of the b4 kinase was not affected by several drugs that bind to and inhibit calmodulin, or by the addition of exogenous calmodulin. Activators and inhibitors of the calcium-phospholipid-dependent C kinase also had no effect on b4 phosphorylation. PMID:4055893

  8. 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)

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

    PubMed

    Vater, W; Böhm, K J; Unger, E

    1993-02-01

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

  10. Singlet-Doublet Dark Matter

    SciTech Connect

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

    2012-02-15

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

  11. Bug22 influences cilium morphology and the post-translational modification of ciliary microtubules

    PubMed Central

    Mendes Maia, Teresa; Gogendeau, Delphine; Pennetier, Carole; Janke, Carsten; Basto, Renata

    2014-01-01

    Summary Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs) such as polyglycylation, polyglutamylation or acetylation, are determinants of microtubule (MT) functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme. PMID:24414207

  12. Achromatic doublets using group indices of refraction

    NASA Astrophysics Data System (ADS)

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

    2008-03-01

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

  13. Inert doublet model and LEP II limits

    SciTech Connect

    Lundstroem, Erik; Gustafsson, Michael; Edsjoe, Joakim

    2009-02-01

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

  14. Microtubule dissassembly in vivo: intercalary destabilization and breakdown of microtubules in the heliozoan Actinocoryne contractilis

    PubMed Central

    1992-01-01

    In the marine heliozoan Actinocoryne contractilis, uninterrupted rods of microtubules stiffen the axopodia and the stalk. Stimulation in sea water elicits an extremely fast contraction (millisecond range) accompanied by almost complete Mt dissociation. Using high-speed cinematography and light transmittance measurements, we have studied the process of Mt disassembly in real time. In sea water, Mt disassembly follows an exponential decrease (mean half time of 4 ms) or proceeds by short steps. Cell contraction and Mt disassembly have been inhibited or slowed down through the use of artificial media. Although kinetics are slower (mean half time of 3 s), the curves of the length change against time look similar. The rapid as well as the slower process are accompanied by the formation of breakpoints on the stalk, from which disassembly proceeds. In specimens fixed during the slowed contraction, the presence across the Mt rods, of a single or multiple destabilization band that may consist of granular material and polymorphic forms of tubulin supports the hypothesis of "intercalary destabilization and breakdown" of axonemal Mts. PMID:1639845

  15. Do prokaryotes contain microtubules?

    PubMed Central

    Bermudes, D; Hinkle, G; Margulis, L

    1994-01-01

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

  16. Do prokaryotes contain microtubules?

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

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

    SciTech Connect

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

    2009-04-23

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

  18. Estrogen related receptor is required for the testicular development and for the normal sperm axoneme/mitochondrial derivatives in Drosophila males

    PubMed Central

    Misra, Snigdha; Pandey, Anuj Kumar; Gupta, Snigdha; Kumar, Ajay; Khanna, Priyanka; shankar, Jai; Ravi Ram, Kristipati

    2017-01-01

    Estrogen related receptors (ERRs), categorized as orphan nuclear receptors, are critical for energy homeostasis and somatic development. However, significance of ERRs in the development of reproductive organs/organelles/cells remain poorly understood, albeit their homology to estrogen receptors. In this context, here, we show that knockdown of ERR in the testes leads to improperly developed testes with mis-regulation of genes (aly, mia, bruce, bam, bgcn, fzo and eya) involved in spermatogenesis, resulting in reduced male fertility. The observed testicular deformity is consistent with the down-regulation of SOX-E group of gene (SOX100B) in Drosophila. We also show dispersion/disintegration of fusomes (microtubule based structures associated with endoplasmic reticulum derived vesicle, interconnecting spermatocytes) in ERR knockdown testes. A few ERR knockdown testes go through spermatogenesis but have significantly fewer sperm. Moreover, flagella of these sperm are defective with abnormal axoneme and severely reduced mitochondrial derivatives, suggesting a possible role for ERR in mitochondrial biogenesis, analogous to mammalian ERRα. Interestingly, similar knockdown of remaining seventeen nuclear receptors did not yield a detectable reproductive or developmental defect in Drosophila. These findings add newer dimensions to the functions envisaged for ERR and provide the foundation for deciphering the relevance of orphan nuclear receptors in ciliopathies and testicular dysgenesis. PMID:28094344

  19. BRIDGES BETWEEN MICROTUBULES

    PubMed Central

    McIntosh, J. R.

    1974-01-01

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

  20. EYS Is a Protein Associated with the Ciliary Axoneme in Rods and Cones

    PubMed Central

    Alfano, Giovanna; Kruczek, Przemyslaw M.; Shah, Amna Z.; Kramarz, Barbara; Jeffery, Glen; Zelhof, Andrew C.; Bhattacharya, Shomi S.

    2016-01-01

    Purpose Mutations in the EYS gene are a common cause of autosomal recessive retinitis pigmentosa (arRP), yet the role of the EYS protein in humans is presently unclear. The aim of this study was to investigate the isoform structure, expression and potential function of EYS in the mammalian retina in order to better understand its involvement in the pathogenesis of arRP. Methods To achieve the objective, we examined the expression of mRNA transcripts of EYS isoforms in human tissues and cell lines by RT-PCR. We also investigated the localisation of EYS in cultured cells and retinal cryo-sections by confocal fluorescence microscopy and Western blot analysis. Results RT-PCR analysis confirmed that EYS has at least four isoforms. In addition to the previously reported EYS isoforms 1 and 4, we present the experimental validation of two smaller variants referred to as EYS isoforms 2 and 3. All four isoforms are expressed in the human retina and Y79 cells and the short variants were additionally detected in the testis. Immunofluorescent confocal microscopy and Western blot analysis revealed that all EYS isoforms preferentially localise to the cytoplasm of Y79 and HeLa cells. Moreover, an enrichment of the endogenous protein was observed near the centrosomes in Y79 cells. Interestingly, EYS was observed at the ciliary axoneme in Y79 ciliated cells. In macaque retinal cryosections, EYS was found to localise in the region of the photoreceptor ciliary axoneme in both rods and cones as well as in the cytoplasm of the ganglion cells. Conclusion The results obtained in this study lead us to speculate that, in photoreceptor cells, EYS could be a protein involved in maintaining the stability of the ciliary axoneme in both rods and cones. The variability of its isoform structure suggests that other roles are also possible and yet to be established. PMID:27846257

  1. Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

    PubMed Central

    Vasquez, R J; Howell, B; Yvon, A M; Wadsworth, P; Cassimeris, L

    1997-01-01

    Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of

  2. Doublet Tracer Testing in Klamath Falls, Oregon

    SciTech Connect

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

    1983-12-15

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

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

    NASA Astrophysics Data System (ADS)

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

    1987-11-01

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

  4. 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

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

    PubMed

    Gardiner, John; Overall, Robyn; Marc, Jan

    2012-01-01

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

  6. Chlamydomonas shortens its flagella by activating axonemal disassembly, stimulating IFT particle trafficking, and blocking anterograde cargo loading.

    PubMed

    Pan, Junmin; Snell, William J

    2005-09-01

    Almost all eukaryotic cells form cilia/flagella, maintain them at their genetically specified lengths, and shorten them. Here, we define the cellular mechanisms that bring about shortening of flagella prior to meiotic cell division and in response to environmental cues in the biflagellated green alga Chlamydomonas. We show that the flagellar shortening pathway is distinct from the one that enforces transient shortening essential for length control. During flagellar shortening, disassembly of the axoneme is stimulated over the basal rate, and the rate of entry into flagella of intraflagellar transport (IFT) particles is increased. Moreover, the particles entering the disassembling flagella lack cargo. Thus, flagellar shortening depends on the interplay between dynamic properties of the axoneme and the IFT machinery; a cell triggered to shorten its flagellum activates disassembly of the axoneme and stimulates entry into the flagellum of IFT particles possessing empty cargo binding sites available to retrieve the disassembled components.

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

    PubMed

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

    2014-10-01

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

  8. Localization of EB1, IFT polypeptides, and kinesin-2 in Chlamydomonas flagellar axonemes via immunogold scanning electron microscopy.

    PubMed

    Sloboda, Roger D; Howard, Louisa

    2007-06-01

    Intraflagellar transport (IFT) refers to the bi-directional movement of particles and associated cargo along the axonemes of eukaryotic flagella and cilia. To provide a new perspective on the morphology of IFT particles, their association with the axoneme, and their composition, we have used immunogold localization coupled to detection via scanning electron microscopy. Here we co-localize in the Chlamydomonas flagellar axoneme polypeptides labeled with specific antibodies. Chlamydomonas EB1 localizes to the distal tip of the axoneme, as expected from previous immunofluorescent data (Pedersen et al. Curr Biol2003;13(22):1969-1974), thus demonstrating the utility of this approach. Using antibodies to IFT-related polypeptides, particles can be identified associated with the axoneme that fall into one of two classes: The first class is composed of IFT particles labeled with polyclonal antibodies to kinesin-2 and monoclonal antibodies to either IFT139 (an IFT complex A polypeptide) or IFT172 (a complex B polypeptide). The second class is comprised of particles that label with antibodies to IFT139 alone; thus, discrete particles are present associated with the axoneme that are composed only of complex A polypeptides. When IFT particles were purified by sucrose gradient ultracentrifugation, they appeared as more or less spherical aggregates of varying dimensions labeled with antibodies to IFT139 and to the motor protein kinesin-2. By contrast, isolated IFT particles that were labeled with IFT172 antibodies were not labeled with kinesin-2 antibodies. The data are discussed in terms of the total polypeptide composition of an IFT particle and the interaction of the particles with the motors that power IFT.

  9. Three doublet lepton-specific model

    NASA Astrophysics Data System (ADS)

    Merchand, Marco; Sher, Marc

    2017-03-01

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

  10. Dynein axonemal heavy chain 8 promotes androgen receptor activity and associates with prostate cancer progression

    PubMed Central

    Wang, Yu; Ledet, Russell J.; Imberg-Kazdan, Keren; Logan, Susan K.; Garabedian, Michael J.

    2016-01-01

    To gain insight into cellular factors regulating AR action that could promote castration resistant prostate cancer (CRPC), we performed a genome-wide RNAi screen for factors that promote ligand-independent AR transcriptional activity and integrated clinical databases for candidate genes that are positively associated with prostate cancer metastasis and recurrence. From this analysis, we identified Dynein Axonemal Heavy Chain 8 (DNAH8) as an AR regulator that displayed higher mRNA expression in metastatic than in primary tumors, and showed high expression in patients with poor prognosis. Axonemal dyneins function in cellular motility, but the function of DNAH8 in prostate cancer or other cell types has not been reported. DNAH8 is on chromosome 6q21.2, a cancer-associated amplicon, and is primarily expressed in prostate and testis. Its expression is higher in primary tumors compared to normal prostate, and is further increased in metastatic prostate cancers. Patients expressing high levels of DNAH8 have a greater risk of relapse and a poor prognosis after prostatectomy. Depletion of DNAH8 in prostate cancer cells suppressed AR transcriptional activity and proliferation. Androgen treatment increased DNAH8 mRNA expression, and AR bound the DNAH8 promoter sequence indicating DNAH8 is an AR target gene. Thus, DNAH8 is a new regulator of AR associated with metastatic tumors and poor prognosis. PMID:27363033

  11. Cell Biology: Microtubule Collisions to the Rescue.

    PubMed

    Gardner, Melissa K

    2016-12-19

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

  12. The ciliary transition zone functions in cell adhesion but is dispensable for axoneme assembly in C. elegans.

    PubMed

    Schouteden, Clementine; Serwas, Daniel; Palfy, Mate; Dammermann, Alexander

    2015-07-06

    Cilia are cellular projections that perform sensory and motile functions. A key ciliary subdomain is the transition zone, which lies between basal body and axoneme. Previous work in Caenorhabditis elegans identified two ciliopathy-associated protein complexes or modules that direct assembly of transition zone Y-links. Here, we identify C. elegans CEP290 as a component of a third module required to form an inner scaffolding structure called the central cylinder. Co-inhibition of all three modules completely disrupted transition zone structure. Surprisingly, axoneme assembly was only mildly perturbed. However, dendrite extension by retrograde migration was strongly impaired, revealing an unexpected role for the transition zone in cell adhesion.

  13. 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.

  14. 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.

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

    PubMed Central

    1986-01-01

    A tight association between Chlamydomonas alpha-tubulin acetyltransferase (TAT) and flagellar 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. Using the Chlamydomonas TAT with brain tubulin as substrate, we have studied the effects of acetylation on the assembly and disassembly of microtubules in vitro. We also determined the relative rates of acetylation of tubulin dimers and polymers. The acetylation does not significantly affect the temperature-dependent polymerization or depolymerization of tubulin in vitro. Furthermore, polymerization of tubulin is not a prerequisite for the acetylation, although the polymer is a better substrate for TAT than the dimer. The acetylation is sensitive to calcium ions which completely inhibit the acetylation of both dimers and polymers of tubulin. Acetylation of the dimer is not inhibited by colchicine; the effect of colchicine on acetylation of the polymer can be explained by its depolymerizing effect on the polymer. PMID:3733880

  16. Effective theory for electroweak doublet dark matter

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  17. Doublet III: status and future plans

    SciTech Connect

    Rawls, J.M.

    1980-04-01

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

  18. Anomalous Flexural Behaviors of Microtubules

    PubMed Central

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

    2012-01-01

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

  19. Microtubule dynamics in plant cells.

    PubMed

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

    2010-01-01

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

  20. A "MICROTUBULE" IN A BACTERIUM

    PubMed Central

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

    1967-01-01

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

  1. Equimolar heterodimers in microtubules

    PubMed Central

    1982-01-01

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

  2. Evolution of microtubule organizing centers across the tree of eukaryotes.

    PubMed

    Yubuki, Naoji; Leander, Brian S

    2013-07-01

    The architecture of eukaryotic cells is underpinned by complex arrrays of microtubules that stem from an organizing center, referred to as the MTOC. With few exceptions, MTOCs consist of two basal bodies that anchor flagellar axonemes and different configurations of microtubular roots. Variations in the structure of this cytoskeletal system, also referred to as the 'flagellar apparatus', reflect phylogenetic relationships and provide compelling evidence for inferring the overall tree of eukaryotes. However, reconstructions and subsequent comparisons of the flagellar apparatus are challenging, because these studies require sophisticated microscopy, spatial reasoning and detailed terminology. In an attempt to understand the unifying features of MTOCs and broad patterns of cytoskeletal homology across the tree of eukaryotes, we present a comprehensive overview of the eukaryotic flagellar apparatus within a modern molecular phylogenetic context. Specifically, we used the known cytoskeletal diversity within major groups of eukaryotes to infer the unifying features (ancestral states) for the flagellar apparatus in the Plantae, Opisthokonta, Amoebozoa, Stramenopiles, Alveolata, Rhizaria, Excavata, Cryptophyta, Haptophyta, Apusozoa, Breviata and Collodictyonidae. We then mapped these data onto the tree of eukaryotes in order to trace broad patterns of trait changes during the evolutionary history of the flagellar apparatus. This synthesis suggests that: (i) the most recent ancestor of all eukaryotes already had a complex flagellar apparatus, (ii) homologous traits associated with the flagellar apparatus have a punctate distribution across the tree of eukaryotes, and (iii) streamlining (trait losses) of the ancestral flagellar apparatus occurred several times independently in eukaryotes.

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

    SciTech Connect

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

    2008-12-01

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

  4. Doublet-singlet model and unitarity

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

  5. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2011-08-20

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

  6. Microtubule organization during human parthenogenesis.

    PubMed

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

    2009-04-01

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

  7. What Organizes the Molecular Ballet that Promotes the Movement of the Axoneme in Such a Way that its Molecular Machinery Seems to be a Whole?

    NASA Astrophysics Data System (ADS)

    Cibert, Christian

    2005-03-01

    The axonemal machinery constitutes a highly organized structure whose mechanisms seem to be very simple but whose regulation remains unknown. This apparent simplicity is reinforced by the fact that many models are able to perfectly mimic the axonemal wave trains that propagate along cilia and flagella. However nobody knows what are the actual mechanisms that coordinate the molecular ballet that exist during the beat. Here we present some theoretical elements that show that if the radial spokes are one of the main elements that promote axonemal regulation, they must be involved in a complex mechanism that makes the axoneme a discrete structure whose regulation could depend on local entropy that promotes the emergence of new molecular properties.

  8. X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.

    PubMed

    Olcese, Chiara; Patel, Mitali P; Shoemark, Amelia; Kiviluoto, Santeri; Legendre, Marie; Williams, Hywel J; Vaughan, Cara K; Hayward, Jane; Goldenberg, Alice; Emes, Richard D; Munye, Mustafa M; Dyer, Laura; Cahill, Thomas; Bevillard, Jeremy; Gehrig, Corinne; Guipponi, Michel; Chantot, Sandra; Duquesnoy, Philippe; Thomas, Lucie; Jeanson, Ludovic; Copin, Bruno; Tamalet, Aline; Thauvin-Robinet, Christel; Papon, Jean-François; Garin, Antoine; Pin, Isabelle; Vera, Gabriella; Aurora, Paul; Fassad, Mahmoud R; Jenkins, Lucy; Boustred, Christopher; Cullup, Thomas; Dixon, Mellisa; Onoufriadis, Alexandros; Bush, Andrew; Chung, Eddie M K; Antonarakis, Stylianos E; Loebinger, Michael R; Wilson, Robert; Armengot, Miguel; Escudier, Estelle; Hogg, Claire; Amselem, Serge; Sun, Zhaoxia; Bartoloni, Lucia; Blouin, Jean-Louis; Mitchison, Hannah M

    2017-02-08

    By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

  9. X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3

    PubMed Central

    Olcese, Chiara; Patel, Mitali P.; Shoemark, Amelia; Kiviluoto, Santeri; Legendre, Marie; Williams, Hywel J.; Vaughan, Cara K.; Hayward, Jane; Goldenberg, Alice; Emes, Richard D.; Munye, Mustafa M.; Dyer, Laura; Cahill, Thomas; Bevillard, Jeremy; Gehrig, Corinne; Guipponi, Michel; Chantot, Sandra; Duquesnoy, Philippe; Thomas, Lucie; Jeanson, Ludovic; Copin, Bruno; Tamalet, Aline; Thauvin-Robinet, Christel; Papon, Jean- François; Garin, Antoine; Pin, Isabelle; Vera, Gabriella; Aurora, Paul; Fassad, Mahmoud R.; Jenkins, Lucy; Boustred, Christopher; Cullup, Thomas; Dixon, Mellisa; Onoufriadis, Alexandros; Bush, Andrew; Chung, Eddie M. K.; Antonarakis, Stylianos E.; Loebinger, Michael R.; Wilson, Robert; Armengot, Miguel; Escudier, Estelle; Hogg, Claire; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Beales, Philip L.; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Allan, Daly; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Foley, A. Reghan; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; McCarthy, Shane; Muddyman, Dawn; Muntoni, Francesco; Parker, Victoria; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter J.; Schmidts, Miriam; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Amselem, Serge; Sun, Zhaoxia; Bartoloni, Lucia; Blouin, Jean-Louis; Mitchison, Hannah M.

    2017-01-01

    By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins. PMID:28176794

  10. Microtubules in Plants

    PubMed Central

    Hashimoto, Takashi

    2015-01-01

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

  11. Cortical microtubule rearrangements and cell wall patterning

    PubMed Central

    Oda, Yoshihisa

    2015-01-01

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

  12. Self-repair promotes microtubule rescue

    PubMed Central

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

    2016-01-01

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

  13. GAR22β regulates cell migration, sperm motility, and axoneme structure

    PubMed Central

    Gamper, Ivonne; Fleck, David; Barlin, Meltem; Spehr, Marc; Sayad, Sara El; Kleine, Henning; Maxeiner, Sebastian; Schalla, Carmen; Aydin, Gülcan; Hoss, Mareike; Litchfield, David W.; Lüscher, Bernhard; Zenke, Martin; Sechi, Antonio

    2016-01-01

    Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β−/− Sertoli cells moved faster than wild-type cells. In addition, GAR22β−/− cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β−/− cells reduced cell motility and focal adhesion turnover. GAR22β–actin interaction was stronger than GAR22β–microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β–EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes. PMID:26564797

  14. Possible multiple chiral doublet bands in 107Ag

    NASA Astrophysics Data System (ADS)

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

    2013-08-01

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

  15. Partially natural two Higgs doublet models

    SciTech Connect

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

    2016-06-21

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

  16. Electromagnetic transitions in multiple chiral doublet bands

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

  17. Microtubule defects & Neurodegeneration.

    PubMed

    Baird, Fiona J; Bennett, Craig L

    2013-12-06

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

  18. Identification of a Novel Microtubule-Binding Protein in Giardia lamblia

    PubMed Central

    Kim, Juri; Park, Soon-Jung

    2016-01-01

    Giardia lamblia is a protozoan that causes diarrheal diseases in humans. Cytoskeletal structures of Giardia trophozoites must be finely reorganized during cell division. To identify Giardia proteins which interact with microtubules (MTs), Giardia lysates were incubated with in vitro-polymerized MTs and then precipitated by ultracentifugation. A hypothetical protein (GL50803_8405) was identified in the precipitated fraction with polymerized MTs and was named GlMBP1 (G. lamblia microtubule-binding protein 1). Interaction of GlMBP1 with MTs was confirmed by MT binding assays using recombinant GlMBP1 (rGlMBP1). In vivo expression of GlMBP1 was shown by a real-time PCR and western blot analysis using anti-rGlMBP1 antibodies. Transgenic G. lamblia trophozoites were constructed by integrating a chimeric gene encoding hemagglutinin (HA)-tagged GlMBP1 into a Giardia chromosome. Immunofluorescence assays of this transgenic G. lamblia, using anti-HA antibodies, revealed that GlMBP1 mainly localized at the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. This result indicates that GlMBP1 is a component of the G. lamblia cytoskeleton. PMID:27658598

  19. Identification of a Novel Microtubule-Binding Protein in Giardia lamblia.

    PubMed

    Kim, Juri; Park, Soon-Jung

    2016-08-01

    Giardia lamblia is a protozoan that causes diarrheal diseases in humans. Cytoskeletal structures of Giardia trophozoites must be finely reorganized during cell division. To identify Giardia proteins which interact with microtubules (MTs), Giardia lysates were incubated with in vitro-polymerized MTs and then precipitated by ultracentifugation. A hypothetical protein (GL50803_8405) was identified in the precipitated fraction with polymerized MTs and was named GlMBP1 (G. lamblia microtubule-binding protein 1). Interaction of GlMBP1 with MTs was confirmed by MT binding assays using recombinant GlMBP1 (rGlMBP1). In vivo expression of GlMBP1 was shown by a real-time PCR and western blot analysis using anti-rGlMBP1 antibodies. Transgenic G. lamblia trophozoites were constructed by integrating a chimeric gene encoding hemagglutinin (HA)-tagged GlMBP1 into a Giardia chromosome. Immunofluorescence assays of this transgenic G. lamblia, using anti-HA antibodies, revealed that GlMBP1 mainly localized at the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. This result indicates that GlMBP1 is a component of the G. lamblia cytoskeleton.

  20. Optimization of isopolar microtubule arrays.

    PubMed

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

    2013-02-19

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

  1. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2014-03-05

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

  2. How dynein moves along microtubules

    PubMed Central

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

    2015-01-01

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

  3. 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.

  4. On and around microtubules: an overview.

    PubMed

    Wade, Richard H

    2009-10-01

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

  5. Doublet craters and the tidal disruption of binary asteroids

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

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

  6. Dear microtubule, I see you

    PubMed Central

    Nogales, Eva

    2016-01-01

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

  7. Partially natural two Higgs doublet models

    DOE PAGES

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

    2016-06-21

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

  8. Microtubule catastrophe from protofilament dynamics.

    PubMed

    Jemseena, V; Gopalakrishnan, Manoj

    2013-09-01

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

  9. Microtubule alignment and manipulation using AC electrokinetics.

    PubMed

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

    2008-09-01

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

  10. Low Scale Thermal Leptogenesis in Neutrinophilic Higgs Doublet Models

    NASA Astrophysics Data System (ADS)

    Haba, N.; Seto, O.

    2011-06-01

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

  11. Evidence for Octupole Correlations in Multiple Chiral Doublet Bands

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

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

    PubMed

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

    2007-03-01

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

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

    PubMed

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

    2016-08-23

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

  14. Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate

    PubMed Central

    1991-01-01

    Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build- up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP

  15. Doublets and other allied well patterns

    SciTech Connect

    Brigham, W.E.

    1997-06-01

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

  16. Microtubule-associated proteins from Antarctic fishes.

    PubMed

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

    1990-01-01

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

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

    PubMed

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

    2013-05-28

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

  18. Microtubule detyrosination guides chromosomes during mitosis

    PubMed Central

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

    2015-01-01

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

  19. Biological Information Processing in Single Microtubules

    DTIC Science & Technology

    2012-02-15

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

  20. Proteomic Analysis of Bovine Axonemes Exposed to Acute Alcohol: Role of eNOS and HSP90 in Cilia Stimulation

    PubMed Central

    Simet, Samantha M.; Pavlik, Jacqueline A.; Sisson, Joseph H.

    2012-01-01

    Background Cilia are fingerlike motor-driven organelles, which propel inhaled particles and mucus from the lung and airways. We have previously shown that brief alcohol exposure stimulates ciliary motility through an endothelial nitric oxide (eNOS)-dependent pathway localized in the ciliary metabolon. However, the signaling molecules of the ciliary metabolon involved in alcohol-triggered cilia beat frequency (CBF) stimulation upstream of eNOS activation are unknown. Methods and Results We hypothesized that brief alcohol exposure alters threonine and serine phosphorylation of proteins involved in stimulating ciliary beat frequency. Two-dimensional electrophoresis indicated both increases and deceases in the serine and threonine phosphorylation states of several proteins. One of the proteins identified was heat shock protein 90 (HSP90), which undergoes increased threonine phosphorylation after brief alcohol exposure. Because HSP90 has been shown to associate with eNOS in lung tissue, we hypothesized that HSP90 is a key component in alcohol-triggered eNOS activation and that these two proteins co-localize within the ciliary metabolon. Immunofluorescence experiments demonstrate that eNOS and HSP90 co-localize within basal bodies of the ciliary metabolon and partially translocate to the axoneme upon brief alcohol exposure. Pretreatment with geldanamycin, which disrupts HSP90 chaperone functions, prevented eNOS-HSP90 association and prevented the translocation of eNOS from the ciliary metabolon to the axoneme. Functional cilia motility studies revealed that geldanamycin blocked alcohol-stimulated ciliary motility in bovine bronchial epithelial cells and mouse tracheal rings. Conclusions Based on the HSP90 localization with eNOS, alcohol activation of HSP90 phosphorylation, and geldanamycin’s ability inhibit HSP90-eNOS association, prevent eNOS translocation to the axoneme, and block alcohol-stimulated ciliary motility, we conclude that alcohol-induced cilia stimulation

  1. Ectopic A-lattice seams destabilize microtubules.

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2016-11-01

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

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

    SciTech Connect

    Carlucci, Maria Valentina

    2010-12-22

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

  4. Candidates for chiral doublet bands in 136Nd

    NASA Astrophysics Data System (ADS)

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

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

  5. Two Higgs doublet models with an S3 symmetry

    NASA Astrophysics Data System (ADS)

    Cogollo, D.; Silva, João P.

    2016-05-01

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

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

    PubMed

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

    2016-08-08

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

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

    PubMed

    Feizabadi, Mitra Shojania; Rosario, Brandon

    2017-03-04

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

  8. Microtubule motors: moving forward on many fronts.

    PubMed

    Allan, Viki

    2009-07-08

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

  9. Microtubule Control of Metabolism in Prostate Cancer

    DTIC Science & Technology

    2013-06-01

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

  10. Mechanical Properties of Doubly Stabilized Microtubule Filaments

    PubMed Central

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

    2013-01-01

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

  11. Movement of chromosomes with severed kinetochore microtubules.

    PubMed

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

    2015-05-01

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

  12. Mechanical properties of doubly stabilized microtubule filaments.

    PubMed

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

    2013-04-02

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

  13. On complex, curved trajectories in microtubule gliding

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  14. Tubulin bistability and polymorphic dynamics of microtubules.

    PubMed

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

    2010-12-31

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

  15. FLUCTUATING MOTOR FORCES BEND GROWING MICROTUBULES

    PubMed Central

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

    2013-01-01

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

  16. Visualizing individual microtubules by bright field microscopy

    NASA Astrophysics Data System (ADS)

    Gutiérrez-Medina, Braulio; Block, Steven M.

    2010-11-01

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

  17. Targeting Microtubules for Wound Repair

    PubMed Central

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

    2016-01-01

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

  18. Active contraction of microtubule networks

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2015-05-26

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

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

    SciTech Connect

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

    2015-05-01

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

  1. Effects of fluid propagation on occurrence of doublet earthquakes

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

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

    PubMed Central

    Laan, Liedewij; Roth, Sophie; Dogterom, Marileen

    2012-01-01

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

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

    PubMed

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

    1989-05-01

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

  4. Reovirus Cell Entry Requires Functional Microtubules

    PubMed Central

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

    2013-01-01

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

  5. Microtubules regulate disassembly of epithelial apical junctions

    PubMed Central

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

    2006-01-01

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

  6. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Weber, K; Pollack, R; Bibring, T

    1975-01-01

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

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

    PubMed

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

    2013-12-06

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2010-09-03

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

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

    PubMed

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

    2016-05-23

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

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

    PubMed

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

    2012-01-01

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

  13. Rigidity of microtubules is increased by stabilizing agents

    PubMed Central

    1995-01-01

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

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

    SciTech Connect

    Pich, Antonio; Tuzon, Paula

    2009-11-01

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

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

    NASA Astrophysics Data System (ADS)

    Hussein, O. A.; Sise, O.

    2017-02-01

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

  16. A search for close-mass lepton doublet

    SciTech Connect

    Riles, J.K.

    1989-04-01

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

  17. Microtubules, polarity and vertebrate neural tube morphogenesis

    PubMed Central

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

    2016-01-01

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

  18. Axonemal dynein intermediate-chain gene (DNAI1) mutations result in situs inversus and primary ciliary dyskinesia (Kartagener syndrome).

    PubMed

    Guichard, C; Harricane, M C; Lafitte, J J; Godard, P; Zaegel, M; Tack, V; Lalau, G; Bouvagnet, P

    2001-04-01

    Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).

  19. Kinetochore-microtubule interactions during cell division.

    PubMed

    Maiato, Helder; Sunkel, Claudio E

    2004-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Arina, Chiara; Sahu, Narendra

    2012-01-01

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

  1. Phenomenology of a Flavorful Two Higgs Doublet Model

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  2. Vacuum topology of the two Higgs doublet model

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

  3. 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.

  4. Microtubules self-repair in response to mechanical stress

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  5. Organization of neuronal microtubules in the nematode Caenorhabditis elegans

    PubMed Central

    1979-01-01

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

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

    PubMed

    Vorob'ev, V S; Portuganov, V V

    1980-10-01

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

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

    PubMed Central

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

    2016-01-01

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

  8. Microtubules self-repair in response to mechanical stress

    PubMed Central

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

    2015-01-01

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

  9. History-dependent catastrophes regulate axonal microtubule behavior.

    PubMed

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

    2010-06-08

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

  10. Phenomenology of the inert (2+1) and (4+2) Higgs doublet models

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

    We make a phenomenological study of a model with two inert doublets plus one Higgs doublet [I(2+1)HDM] which is symmetric under a Z2 group, preserved after electroweak symmetry breaking by the vacuum alignment (0,0,v). This model may be regarded as an extension to the model with one inert doublet plus one Higgs doublet [I(1+1)HDM], by the addition of an extra inert scalar doublet. The neutral fields from the two inert doublets provide a viable dark matter (DM) candidate which is stabilized by the conserved Z2 symmetry. We study the new Higgs decay channels offered by the scalar fields from the extra doublets and their effect on the standard model Higgs couplings, including a new decay channel into (off-shell) photon(s) plus missing energy, which distinguishes the I(2+1)HDM from the I(1+1)HDM. Motivated by supersymmetry, which requires an even number of doublets, we then extend this model into a model with four inert doublets plus two Higgs doublets [I(4+2)HDM] and study the phenomenology of the model with the vacuum alignment (0,0,0,0,v ,v). This scenario offers a wealth of Higgs signals, the most distinctive ones being cascade decays of heavy Higgs states into inert ones. Finally, we also remark that the smoking-gun signature of all the considered models is represented by invisible Higgs decays into the lightest inert Higgs bosons responsible for DM.

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

    PubMed

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

    2013-03-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. Non-centrosomal nucleation mediated by augmin organizes microtubules in post-mitotic neurons and controls axonal microtubule polarity

    PubMed Central

    Sánchez-Huertas, Carlos; Freixo, Francisco; Viais, Ricardo; Lacasa, Cristina; Soriano, Eduardo; Lüders, Jens

    2016-01-01

    Neurons display a highly polarized microtubule network that mediates trafficking throughout the extensive cytoplasm and is crucial for neuronal differentiation and function. In newborn migrating neurons, the microtubule network is organized by the centrosome. During neuron maturation, however, the centrosome gradually loses this activity, and how microtubules are organized in more mature neurons remains poorly understood. Here, we demonstrate that microtubule organization in post-mitotic neurons strongly depends on non-centrosomal nucleation mediated by augmin and by the nucleator γTuRC. Disruption of either complex not only reduces microtubule density but also microtubule bundling. These microtubule defects impair neurite formation, interfere with axon specification and growth, and disrupt axonal trafficking. In axons augmin does not merely mediate nucleation of microtubules but ensures their uniform plus end-out orientation. Thus, the augmin-γTuRC module, initially identified in mitotic cells, may be commonly used to generate and maintain microtubule configurations with specific polarity. PMID:27405868

  14. Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

    PubMed

    Margolis, R L; Rauch, C T; Pirollet, F; Job, D

    1990-12-01

    STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.

  15. 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

  16. A study of microtubule dipole lattices

    NASA Astrophysics Data System (ADS)

    Nandi, Shubhendu

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

  17. Physical basis of large microtubule aster growth

    PubMed Central

    Ishihara, Keisuke; Korolev, Kirill S; Mitchison, Timothy J

    2016-01-01

    Microtubule asters - radial arrays of microtubules organized by centrosomes - play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks. DOI: http://dx.doi.org/10.7554/eLife.19145.001 PMID:27892852

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

    PubMed Central

    1994-01-01

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

  19. Gauged Two Higgs Doublet Model confronts the LHC 750 GeV diphoton anomaly

    NASA Astrophysics Data System (ADS)

    Huang, Wei-Chih; Tsai, Yue-Lin Sming; Yuan, Tzu-Chiang

    2016-08-01

    In light of the recent 750 GeV diphoton anomaly observed at the LHC, we study the possibility of accommodating the deviation from the standard model prediction based on the recently proposed Gauged Two Higgs Doublet Model. The model embeds two Higgs doublets into a doublet of a non-abelian gauge group SU(2)H, while the standard model SU(2)L right-handed fermion singlets are paired up with new heavy fermions to form SU(2)H doublets, and SU(2)L left-handed fermion doublets are singlets under SU(2)H. An SU(2)H scalar doublet, which provides masses to the new heavy fermions as well as the SU(2)H gauge bosons, can be produced via gluon fusion and subsequently decays into two photons with the new fermions circulating the triangle loops to account for the deviation from the standard model prediction.

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

    PubMed Central

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

    1981-01-01

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

  1. Perturbative unitarity bounds in composite two-Higgs doublet models

    NASA Astrophysics Data System (ADS)

    De Curtis, Stefania; Moretti, Stefano; Yagyu, Kei; Yildirim, Emine

    2016-09-01

    We study bounds from perturbative unitarity in a composite two-Higgs doublet model (C2HDM) based on the spontaneous breakdown of a global symmetry S O (6 )→S O (4 )×S O (2 ) at the compositeness scale f . The eight pseudo-Nambu-Goldstone bosons (pNGBs) emerging from such a dynamics are identified as two isospin doublet Higgs fields. We calculate the S -wave amplitude for all possible two-to-two-body elastic (pseudo)scalar boson scatterings at energy scales √{s } reachable at the Large Hadron Collider (LHC) and beyond it, including the longitudinal components of weak gauge boson states as the corresponding pNGB states. In our calculation, the Higgs potential is assumed to have the same form as that in the elementary two-Higgs doublet model (E2HDM) with a discrete Z2 symmetry, which is expected to be generated at the one-loop level via the Coleman-Weinberg mechanism. We find that the S -wave amplitude matrix can be block-diagonalized with maximally 2 ×2 submatrices in a way similar to the E2HDM case as long as we only keep the contributions from O (ξ s ) and O (ξ0s0) in the amplitudes, where ξ =vSM2/f2 and vSM≃246 GeV , which is an appropriate approximation for our analysis. By requiring the C2HDM to satisfy perturbative unitarity at energies reachable by the LHC, we derive bounds on its parameters such as ξ and the masses of extra Higgs bosons present in the scenario alongside the Standard Model-like Higgs state discovered in 2012.

  2. Wafer-level fabrication of arrays of glass lens doublets

    NASA Astrophysics Data System (ADS)

    Passilly, Nicolas; Perrin, Stéphane; Albero, Jorge; Krauter, Johann; Gaiffe, Olivier; Gauthier-Manuel, Ludovic; Froehly, Luc; Lullin, Justine; Bargiel, Sylwester; Osten, Wolfgang; Gorecki, Christophe

    2016-04-01

    Systems for imaging require to employ high quality optical components in order to dispose of optical aberrations and thus reach sufficient resolution. However, well-known methods to get rid of optical aberrations, such as aspherical profiles or diffractive corrections are not easy to apply to micro-optics. In particular, some of these methods rely on polymers which cannot be associated when such lenses are to be used in integrated devices requiring high temperature process for their further assembly and separation. Among the different approaches, the most common is the lens splitting that consists in dividing the focusing power between two or more optical components. In here, we propose to take advantage of a wafer-level technique, devoted to the generation of glass lenses, which involves thermal reflow in silicon cavities to generate lens doublets. After the convex lens sides are generated, grinding and polishing of both stack sides allow, on the first hand, to form the planar lens backside and, on the other hand, to open the silicon cavity. Nevertheless, silicon frames are then kept and thinned down to form well-controlled and auto-aligned spacers between the lenses. Subsequent accurate vertical assembly of the glass lens arrays is performed by anodic bonding. The latter ensures a high level of alignment both laterally and axially since no additional material is required. Thanks to polishing, the generated lens doublets are then as thin as several hundreds of microns and compatible with micro-opto-electro-systems (MOEMS) technologies since they are only made of glass and silicon. The generated optical module is then robust and provide improved optical performances. Indeed, theoretically, two stacked lenses with similar features and spherical profiles can be almost diffraction limited whereas a single lens characterized by the same numerical aperture than the doublet presents five times higher wavefront error. To demonstrate such assumption, we fabricated glass

  3. Vacuum structure of the Higgs complex singlet-doublet model

    NASA Astrophysics Data System (ADS)

    Ferreira, P. M.

    2016-11-01

    The complex singlet-doublet model is a popular theory to account for dark matter and electroweak baryogenesis, wherein the Standard Model particle content is supplemented by a complex scalar gauge singlet, with certain discrete symmetries imposed. The scalar potential which results thereof can have seven different types of minima at tree level, which may coexist for specific choices of parameters. There is therefore the possibility that a given minimum is not global but rather a local one, and may tunnel to a deeper extremum, thus causing vacuum instability. This rich vacuum structure is explained and discussed in detail.

  4. Microtubules move the nucleus to quiescence.

    PubMed

    Laporte, Damien; Sagot, Isabelle

    2014-01-01

    The nucleus is a cellular compartment that hosts several macro-molecular machines displaying a highly complex spatial organization. This tight architectural orchestration determines not only DNA replication and repair but also regulates gene expression. In budding yeast microtubules play a key role in structuring the nucleus since they condition the Rabl arrangement in G1 and chromosome partitioning during mitosis through their attachment to centromeres via the kinetochore proteins. Recently, we have shown that upon quiescence entry, intranuclear microtubules emanating from the spindle pole body elongate to form a highly stable bundle that spans the entire nucleus. Here, we examine some molecular mechanisms that may underlie the formation of this structure. As the intranuclear microtubule bundle causes a profound re-organization of the yeast nucleus and is required for cell survival during quiescence, we discuss the possibility that the assembly of such a structure participates in quiescence establishment.

  5. Mechanical model of kinesin moving on microtubule

    NASA Astrophysics Data System (ADS)

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

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

  6. Orthotropic elastic shell model for buckling of microtubules.

    PubMed

    Wang, C Y; Ru, C Q; Mioduchowski, A

    2006-11-01

    In view of the fact that microtubules exhibit strong anisotropic elastic properties, an orthotropic elastic shell model for microtubules is developed to study buckling behavior of microtubules. The predicted critical pressure is found to agree well with recent unexplained experimental data on pressure-induced buckling of microtubules [Needleman, Phys. Rev. Lett. 93, 198104 (2004); Biophys. J. 89, 3410 (2005)] which are lower than that predicted by the isotropic shell model by four orders of magnitude. General buckling behavior of microtubules under axial compression or radial pressure is studied. The results show that the isotropic shell model greatly overestimates the bucking loads of microtubules, except columnlike axially compressed buckling of long microtubules (of length-to-diameter ratio larger than, say, 150). In particular, the present results also offer a plausible explanation for the length dependency of flexibility of microtubules reported in the literature.

  7. Understanding force-generating microtubule systems through in vitro reconstitution

    PubMed Central

    Kok, Maurits; Dogterom, Marileen

    2016-01-01

    ABSTRACT Microtubules switch between growing and shrinking states, a feature known as dynamic instability. The biochemical parameters underlying dynamic instability are modulated by a wide variety of microtubule-associated proteins that enable the strict control of microtubule dynamics in cells. The forces generated by controlled growth and shrinkage of microtubules drive a large range of processes, including organelle positioning, mitotic spindle assembly, and chromosome segregation. In the past decade, our understanding of microtubule dynamics and microtubule force generation has progressed significantly. Here, we review the microtubule-intrinsic process of dynamic instability, the effect of external factors on this process, and how the resulting forces act on various biological systems. Recently, reconstitution-based approaches have strongly benefited from extensive biochemical and biophysical characterization of individual components that are involved in regulating or transmitting microtubule-driven forces. We will focus on the current state of reconstituting increasingly complex biological systems and provide new directions for future developments. PMID:27715396

  8. Dynamic microtubules and the texture of plant cell walls.

    PubMed

    Lloyd, Clive

    2011-01-01

    The relationship between microtubules and cell-wall texture has had a fitful history in which progress in one area has not been matched by progress in the other. For example, the idea that wall texture arises entirely from self-assembly, independently of microtubules, originated with electron microscopic analyses of fixed cells that gave no clue to the ability of microtubules to reorganize. Since then, live-cell studies have established the surprising dynamicity of plant microtubules involving collisions, changes in angle, parallelization, and rotation of microtubule tracks. Combined with proof that cellulose synthases do track along shifting microtubules, this offers more realistic models for the dynamic influence of microtubules on wall texture than could have been imagined in the electron microscopic era-the era from which most ideas on wall texture originate. This review revisits the classical literature on wall organization from the vantage point of current knowledge of microtubule dynamics.

  9. 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

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

    PubMed

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

    2013-07-22

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

  11. Fluorescent microtubules break up under illumination

    PubMed Central

    1988-01-01

    We have synthesized three new fluorescent analogues of tubulin, using fluorescein or rhodamine groups attached to N-hydroxy-succinimidyl esters, and have partially characterized the properties of these analogues. We have also further characterized the tubulin derivatized with dichlorotriazinyl-aminofluorescein that has previously been used in this and other laboratories. Our results show that all four analogues assemble into microtubules which break up when exposed to light of the wavelengths that excite fluorescence. This sensitivity places severe constraints on the use of these analogues in studies of microtubule dynamics. PMID:3417772

  12. Colloidal Stabilization of Neurofilaments and Microtubules

    DTIC Science & Technology

    2002-06-01

    Alzheimer’s disease . To address this problem we used a set of biophysical methods, including atomic force microscopy, to investigate interfilament potentials. There are several main conclusions from the work under this award. First, microtubule associated proteins behave as though they are largely unstructured and can give rise to a long range repulsive force that is predominantly entropic in origin. This is an important finding that provides a biophysical mechanism that explains how microtubule spacing is maintained. Second, treating the unstructured proteins domains

  13. [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

  14. GDP-tubulin incorporation into growing microtubules modulates polymer stability.

    PubMed

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

    2010-06-04

    Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule "structural plasticity."

  15. Microtubules and the endoplasmic reticulum are highly interdependent structures

    PubMed Central

    1986-01-01

    The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system. PMID:3533956

  16. Probing the inert doublet dark matter model with Cherenkov telescopes

    SciTech Connect

    Garcia-Cely, Camilo; Gustafsson, Michael; Ibarra, Alejandro E-mail: michael.gustafsson@theorie.physik.uni-goettingen.de

    2016-02-01

    We present a detailed study of the annihilation signals of the inert dark matter doublet model in its high mass regime. Concretely, we study the prospects to observe gamma-ray signals of the model in current and projected Cherenkov telescopes taking into account the Sommerfeld effect and including the contribution to the spectrum from gamma-ray lines as well as from internal bremsstrahlung. We show that present observations of the galactic center by the H.E.S.S. instrument are able to exclude regions of the parameter space that give the correct dark matter relic abundance. In particular, models with the charged and the neutral components of the inert doublet nearly degenerate in mass have strong gamma-ray signals. Furthermore, for dark matter particle masses above 1 TeV, we find that the non-observation of the continuum of photons generated by the hadronization of the annihilation products typically give stronger constraints on the model parameters than the sharp spectral features associated to annihilation into monochromatic photons and the internal bremsstrahlung process. Lastly, we also analyze the interplay between indirect and direct detection searches for this model, concluding that the prospects for the former are more promising. In particular, we find that the upcoming Cherenkov Telescope Array will be able to probe a significant part of the high mass regime of the model.

  17. The CTA aims at the Inert Doublet Model

    SciTech Connect

    Queiroz, Farinaldo S.; Yaguna, Carlos E. E-mail: carlos.yaguna@mpi-hd.mpg.de

    2016-02-01

    We show that the Cherenkov Telescope Array (CTA) can realistically challenge the Inert Doublet Model, one of the simplest and best known models of dark matter. Specifically, the CTA may exclude its heavy regime up to dark matter masses of 800 GeV and probe a large fraction of the remaining viable parameter space at even higher masses. Two features of the Inert Doublet Model make it particularly suitable for CTA searches. First, the dark matter mass (in the heavy regime) must be larger than 500 GeV. Second, the dark matter annihilation cross section, σ v, is always larger than the thermal one, reaching values as high as 10{sup −25} cm{sup 3}s{sup −1}. This higher value of σv is the result of the unavoidable coannihilation effects that determine the relic density via thermal freeze-out in the early Universe. We find that with 100 hours of Galactic Center exposure, CTA's expected limit widely surpasses, even after the inclusion of systematic errors, current and projected bounds from Fermi-LAT and HESS on this model.

  18. Next-to-minimal two Higgs Doublet Model

    DOE PAGES

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

    2014-04-07

    The simplest extension of the Two Higgs Doublet Model is the addition of a real scalar singlet, S. The effects of mixing between the singlet and the doublets can be manifested in two ways. It can modify the couplings of the 126 GeV Higgs boson, h, and it can lead to direct detection of the heavy Higgs at the LHC. In this paper, we show that in the type-I Model, for heavy Higgs masses in the 200-600 GeV range, the latter effect will be detected earlier than the former for most of parameter space. Should no such Higgs be discoveredmore » in this mass range, then the upper limit on the mixing will be sufficiently strong such that there will be no significant effects on the couplings of the h for most of parameter space. Thus, the reverse is true in the type-II model, the limits from measurements of the couplings of the h will dominate over the limits from non-observation of the heavy Higgs.« less

  19. Next-to-minimal two Higgs Doublet Model

    SciTech Connect

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

    2014-04-07

    The simplest extension of the Two Higgs Doublet Model is the addition of a real scalar singlet, S. The effects of mixing between the singlet and the doublets can be manifested in two ways. It can modify the couplings of the 126 GeV Higgs boson, h, and it can lead to direct detection of the heavy Higgs at the LHC. In this paper, we show that in the type-I Model, for heavy Higgs masses in the 200-600 GeV range, the latter effect will be detected earlier than the former for most of parameter space. Should no such Higgs be discovered in this mass range, then the upper limit on the mixing will be sufficiently strong such that there will be no significant effects on the couplings of the h for most of parameter space. Thus, the reverse is true in the type-II model, the limits from measurements of the couplings of the h will dominate over the limits from non-observation of the heavy Higgs.

  20. Vesicle deformation by microtubules: A phase diagram

    NASA Astrophysics Data System (ADS)

    Emsellem, Virginie; Cardoso, Olivier; Tabeling, Patrick

    1998-10-01

    The experimental investigation of vesicles deformed by the growth of encapsulated microtubules shows that the axisymmetric morphologies can be classified into ovals, lemons, φ, cherries, dumbbells, and pearls. A geometrical phase diagram is established. Numerical minimization of the elastic energy of the membrane reproduces satisfactorily well the observed morphologies and the corresponding phase diagram.

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

    PubMed

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

    2003-03-27

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

  2. Microtubule Initiation from the Nuclear Surface Controls Cortical Microtubule Growth Polarity and Orientation in Arabidopsis thaliana

    PubMed Central

    Ambrose, Chris; Wasteneys, Geoffrey O.

    2014-01-01

    The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth. PMID:25008974

  3. Sliding of STOP proteins on microtubules: a model system for diffusion-dependent microtubule motility.

    PubMed

    Margolis, R L; Job, D; Pabion, M; Rauch, C T

    1986-01-01

    STOP proteins, of 145 kD, act substoichiometrically to block end-wise disassembly of microtubules. STOPs bind to microtubules either during microtubule assembly or when added at steady state, and when binding to the polymers is apparently irreversible. They are not measurably lost from polymers under competition conditions, and there is no measurable exchange between polymers. Nonetheless, STOP proteins exhibit an extraordinary behavior: they "slide" laterally on the surface of the microtubule. Displacement is assayed by forming hybrid microtubules in which cold stable or cold labile region subunits are labeled. Displacement of STOPs on the polymer with time will cause labeled subunits of cold-stable regions to become increasingly cold labile in a manner reciprocal to cold stabilization of previously cold-labile subunits. Because equilibrium exchange of STOP proteins onto and off the polymers can be ruled out, the displacement of STOPs relative to subunits can only be explained by lateral diffusion or "sliding." Axonal transport and mitotic mechanisms were discussed as implications of such a lateral translocation mechanism for microtubule-dependent motility.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  5. 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…

  6. Organelles are transported on sliding microtubules in Reticulomyxa.

    PubMed

    Orokos, D D; Cole, R W; Travis, J L

    2000-12-01

    Organelles and plasma membrane domains appear to be transported along Reticulomyxa's microtubule cytoskeleton. Previously we demonstrated that organelle and cell surface transport share the same enzymatic properties and suggested that both are powered by the same cytoplasmic dynein. Motility analysis in Reticulomyxa is complicated by the fact that the microtubules also are motile and appear to "slide" bidirectionally throughout the network. We have utilized laser ablation to address this frame-of-reference problem as to how each transport component (microtubule sliding vs. organelle translocations) contributes to reactivated bidirectional translocation of organelles along the microtubule cytoskeleton. Laser ablation was used to cut microtubule bundles from lysed networks into 4-15-microm segments. After examining these reactivated cut fragments, it appears that the majority of organelles did not move relative to microtubule fragments, but remained attached to microtubules and moved as the microtubules slid. Microtubule sliding stops after 1-2 min and cannot be reactivated even when perfused with fresh ATP. Furthermore, once sliding stops, organelle transport also stops. Our findings indicate that the majority of Reticulomyxa pseudopodial organelles do not move along the surface of the microtubules, rather it is the sliding of the microtubules to which they are attached that moves them.

  7. Microtubule Dynamics Control Tail Retraction in Migrating Vascular Endothelial Cells†

    PubMed Central

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

    2014-01-01

    Drugs that target microtubules are potent inhibitors of angiogenesis but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; but, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish rather than elongated cells, rapid but non-directional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells while more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay. PMID:24107446

  8. 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.

  9. Dual Role for Microtubules in Regulating Cortical Contractility during Cytokinesis

    PubMed Central

    Murthy, Kausalya; Wadsworth, Patricia

    2008-01-01

    Microtubules stimulate contractile ring formation in the equatorial cortex and simultaneously suppress contractility in the polar cortex; how they accomplish these differing activities is incompletely understood. We measured the behavior of GFP-actin in mammalian cells treated with nocodazole under conditions that either completely eliminate microtubules or selectively disassemble astral microtubules. Selective disassembly of astral microtubules resulted functional contractile rings that were wider than controls and had altered dynamic activity, as measured by FRAP. Complete microtubule disassembly or selective loss of astral microtubules resulted in wave-like contractile behavior of actin in the non-equatorial cortex and mislocalization of myosin II and Rho. FRAP experiments showed that both contractility and actin polymerization contributed to the wave-like behavior of actin. Wave-like, contractile behavior in anaphase cells was Rho-dependent. We conclude that dynamic astral microtubules function to suppress Rho activation in the nonequatorial cortex, limiting the contractile activity of the polar cortex. PMID:18559890

  10. Expansion and Polarity Sorting in Microtubule-Dynein Bundles

    NASA Astrophysics Data System (ADS)

    Zemel, A.; Mogilner, A.

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

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

    PubMed

    Kozlowski, Cleopatra; Srayko, Martin; Nedelec, Francois

    2007-05-04

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

  12. Mathematical modeling of microtubule dynamics: insights into physiology and disease.

    PubMed

    Buxton, Gavin A; Siedlak, Sandra L; Perry, George; Smith, Mark A

    2010-12-01

    Computer models of microtubule dynamics have provided the basis for many of the theories on the cellular mechanics of the microtubules, their polymerization kinetics, and the diffusion of tubulin and tau. In the three-dimensional model presented here, we include the effects of tau concentration and the hydrolysis of GTP-tubulin to GDP-tubulin and observe the emergence of microtubule dynamic instability. This integrated approach simulates the essential physics of microtubule dynamics in a cellular environment. The model captures the structure of the microtubules as they undergo steady state dynamic instabilities in this simplified geometry, and also yields the average number, length, and cap size of the microtubules. The model achieves realistic geometries and simulates cellular structures found in degenerating neurons in disease states such as Alzheimer disease. Further, this model can be used to simulate microtubule changes following the addition of antimitotic drugs which have recently attracted attention as chemotherapeutic agents.

  13. Characterizing and engineering microtubule properties for use in hybrid nanodevices

    NASA Astrophysics Data System (ADS)

    Jeune-Smith, Yolaine

    The emergence of nanotechnology in materials science research has had a major impact in biotechnology. Nature provides novel materials and structures that can be redesigned and reassembled for engineering purposes. One system in particular is the intracellular transport system consisting of the kinesin motor protein and microtubule. For synthetic devices, either the bead geometry (kinesin motors walking along a microtubule coated surface) or the gliding geometry (microtubules gliding over a kinesin-coated surface) is used. Molecular shuttles, utilizing the gliding geometry, have the potential for use in hybrid nanodevices such as biosensors. The kinesin-powered molecular shuttle has been extensively studied. Advances have been made in controlling activation of the kinesin motors, guiding movement of kinesin motors and cargo loading onto the molecular shuttles. In this dissertation the interest in molecular shuttle development is extended with a research focus on the microtubule filament. The microtubule is a central element in the molecular shuttle. The sensing capabilities and limitations of molecular shuttles are tied to the microtubules. It would be desired to have nanodevices with molecular shuttles of predictable size, speed and lifetime. Three materials properties of the microtubules are examined. First, the microtubule length distribution is measured and compared to the length distribution of synthetic polymers. Post polymerization processing techniques, shearing and annealing, are utilized to try to reduce the polydispersity index of the microtubule length distribution. Second, the effect of kinesin activity on the lifetime of the microtubules is observed and quantified. Degradation of microtubules is monitored as a function of kinesin activity and time. Lastly, the effect of cargo loading on microtubule gliding speed is measured to gain insight on the mechanism of cargo attachment. These property behaviors will play a role in the final development of

  14. Active doublet method for measuring small changes in physical properties

    DOEpatents

    Roberts, Peter M.; Fehler, Michael C.; Johnson, Paul A.; Phillips, W. Scott

    1994-01-01

    Small changes in material properties of a work piece are detected by measuring small changes in elastic wave velocity and attenuation within a work piece. Active, repeatable source generate coda wave responses from a work piece, where the coda wave responses are temporally displaced. By analyzing progressive relative phase and amplitude changes between the coda wave responses as a function of elapsed time, accurate determinations of velocity and attenuation changes are made. Thus, a small change in velocity occurring within a sample region during the time periods between excitation origin times (herein called "doublets") will produce a relative delay that changes with elapsed time over some portion of the scattered waves. This trend of changing delay is easier to detect than an isolated delay based on a single arrival and provides a direct measure of elastic wave velocity changes arising from changed material properties of the work piece.

  15. A two-Higgs-doublet model facing experimental hints

    NASA Astrophysics Data System (ADS)

    Crivellin, Andreas; Heeck, Julian; Stoffer, Peter

    2016-04-01

    Physics beyond the Standard Model has so far eluded our experimental probes. Nevertheless, a number of interesting anomalies have accumulated that can be taken as hints towards new physics: BaBar, Belle, and LHCb have found deviations of approximately 3:8σ in B → Dτν and B → D*τν; the anomalous magnetic moment of the muon differs by about 3σ from the theoretic prediction; the branching ratio for τ → μνν is about 2σ above the Standard Model expectation; and CMS and ATLAS found hints for a non-zero decay rate of h → μτ at 2.6σ. Here we consider these processes within a lepton-specific two-Higgs doublet model with additional non-standard Yukawa couplings and show how (and which of) these excesses can be accommodated.

  16. The electroweak phase transition in the Inert Doublet Model

    SciTech Connect

    Blinov, Nikita; Profumo, Stefano; Stefaniak, Tim

    2015-07-21

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

  17. The electroweak phase transition in the Inert Doublet Model

    SciTech Connect

    Blinov, Nikita; Profumo, Stefano; Stefaniak, Tim E-mail: profumo@ucsc.edu

    2015-07-01

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

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

    PubMed

    Gérard, J-M; Herquet, M

    2007-06-22

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

  19. Parity-doublet representation of Majorana fermions and neutron oscillation

    NASA Astrophysics Data System (ADS)

    Fujikawa, Kazuo; Tureanu, Anca

    2016-12-01

    We present a parity-doublet theorem for the representation of the intrinsic parity of Majorana fermions, which is expected to be useful also in condensed matter physics, and it is illustrated to provide a criterion of neutron-antineutron oscillation in a Bardeen-Cooper-Schrieffer type of effective theory with Δ B =2 baryon number-violating terms. The C P violation in the present effective theory causes no direct C P -violating effects in the oscillation itself, which is demonstrated by the exact solution, although it influences the neutron electric dipole moment in the leading order of small Δ B =2 parameters. An analog of Bogoliubov transformation, which preserves P and C P , is crucial in the analysis.

  20. Characteristics of the sperm tail of the velvet worm, Euperipatoides leuckarti (Onychophora).

    PubMed

    Dallai, R; Afzelius, B A

    1993-12-01

    The onychophoran sperm tail contains several kinds of microtubulcs; probably more than that of any other animal group. There are thus a peripheral manchette consisting of many tightly spaced microtubules, a ring of nine 'peripheral singlets' and a central axoneme of the classical 9 + 2 type (nine doublets and two central singlets). The protofilament organization of these various microtubules was examined and compared to the structure and mode of formation of the peripheral singlets with that of its analogues in other animal groups. The onychophoran peripheral singlets were found to differ in two respects from those in insects: they are formed from the manchette rather than from the axonemal doublets and their transient connection to the axoneme is to the A-subtubules of the doublet rather than to the B-subtubules. The manchette microtubules as well as the peripheral singlets consist of 13 protofilaments. The manchette may serve a mechanical function (to strengthen the unusually thick sperm tail) hut the role of the peripheral singlets remains unknown.

  1. Scalar doublet models confront τ and b anomalies

    NASA Astrophysics Data System (ADS)

    Cline, James M.

    2016-04-01

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

  2. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila

    PubMed Central

    Duncan, Jason E.; Lytle, Nikki K.; Zuniga, Alfredo; Goldstein, Lawrence S. B.

    2013-01-01

    Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila. The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila, which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila, we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport. PMID:23840848

  3. Microtubules Growth Rate Alteration in Human Endothelial Cells

    PubMed Central

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

    2010-01-01

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

  4. Microtubules growth rate alteration in human endothelial cells.

    PubMed

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

    2010-01-01

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

  5. Role of the microtubule cytoskeleton in gravisensing Chara rhizoids.

    PubMed

    Braun, M; Sievers, A

    1994-04-01

    The arrangement of the microtubule cytoskeleton in tip-growing and gravisensing Chara rhizoids has been documented by immunofluorescence microscopy. Predominantly axially oriented undulating bundles of cortical microtubules were found in the basal zone of the rhizoids and colocalized with the microfilament bundles underlying the cytoplasmic streaming. Microtubules penetrate the subapical zone, forming a three-dimensional network that envelops the nucleus and organelles. Microtubules are present up to 5 to 10 microns basal from the apical cytoplasmic region containing the statoliths. No microtubules were found in the apical zone of the rhizoid which is the site of tip growth and gravitropism. Depolymerization of microtubules by application of oryzalin does not affect cytoplasmic streaming and gravitropic growth until the relatively stationary and polarly organized apical and subapical cytoplasm is converted into streaming cytoplasm. When the statoliths and the apical cytoplasm are included in the cytoplasmic streaming, tip growth and gravitropism are stopped. Oryzalin-induced disruption of the microtubule cytoskeleton also results in a rearrangement of the dense network of apical and subapical microfilaments into thicker bundles, whereas disruption of the microfilament cytoskeleton by cytochalasin D had no effect on the organization of the microtubule cytoskeleton. It is, therefore, concluded that the arrangement of microtubules is essential for the polar cytoplasmic zonation and the functionally polar organization of the actin cytoskeleton which is responsible for the motile processes in rhizoids. Microtubules are not involved in the primary events of gravitropism in Chara rhizoids.

  6. Neurodegeneration and microtubule dynamics: death by a thousand cuts

    PubMed Central

    Dubey, Jyoti; Ratnakaran, Neena; Koushika, Sandhya P.

    2015-01-01

    Microtubules form important cytoskeletal structures that play a role in establishing and maintaining neuronal polarity, regulating neuronal morphology, transporting cargo, and scaffolding signaling molecules to form signaling hubs. Within a neuronal cell, microtubules are found to have variable lengths and can be both stable and dynamic. Microtubule associated proteins, post-translational modifications of tubulin subunits, microtubule severing enzymes, and signaling molecules are all known to influence both stable and dynamic pools of microtubules. Microtubule dynamics, the process of interconversion between stable and dynamic pools, and the proportions of these two pools have the potential to influence a wide variety of cellular processes. Reduced microtubule stability has been observed in several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), and tauopathies like Progressive Supranuclear Palsy. Hyperstable microtubules, as seen in Hereditary Spastic Paraplegia (HSP), also lead to neurodegeneration. Therefore, the ratio of stable and dynamic microtubules is likely to be important for neuronal function and perturbation in microtubule dynamics might contribute to disease progression. PMID:26441521

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

    PubMed

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

    2017-04-01

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

  8. Molecular architecture of the Dam1 complex–microtubule interaction

    PubMed Central

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

    2016-01-01

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

  9. Size scaling of microtubule asters in confinement

    NASA Astrophysics Data System (ADS)

    Pelletier, James; Field, Christine; Krutkramelis, Kaspars; Fakhri, Nikta; Oakey, John; Gatlin, Jay; Mitchison, Timothy

    Microtubule asters are radial arrays of microtubules (MTs) nucleated around organizing centers (MTOCs). Across a wide range of cell types and sizes, aster positioning influences cellular organization. To investigate aster size and positioning, we reconstituted dynamic asters in Xenopus cytoplasmic extract, confined in fluorous oil microfluidic emulsions. In large droplets, we observed centering of MTOCs. In small droplets, we observed a breakdown in natural positioning, with MTOCs at the droplet edge and buckled or bundled MTs along the interface. In different systems, asters are positioned by different forces, such as pushing due to MT polymerization, or pulling due to bulk or cortical dynein. To estimate different contributions to aster positioning, we biochemically perturbed dynactin function, or MT or actin polymerization. We used carbon nanotubes to measure molecular motions and forces in asters. These experimental results inform quantitative biophysical models of aster size and positioning in confinement. JFP was supported by a Fannie and John Hertz Graduate Fellowship.

  10. Dynamic Concentration of Motors in Microtubule Arrays

    NASA Astrophysics Data System (ADS)

    Nédélec, François; Surrey, Thomas; Maggs, A. C.

    2001-04-01

    We present experimental and theoretical studies of the dynamics of molecular motors in microtubule arrays and asters. By solving a convection-diffusion equation we find that the density profile of motors in a two-dimensional aster is characterized by continuously varying exponents. Simulations are used to verify the assumptions of the continuum model. We observe the concentration profiles of kinesin moving in quasi-two-dimensional artificial asters by fluorescent microscopy and compare with our theoretical results.

  11. Self-organization of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Ndlec, F. J.; Surrey, T.; Maggs, A. C.; Leibler, S.

    1997-09-01

    Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly `pathways'.

  12. Molecular Communication: Simulation of Microtubule Topology

    NASA Astrophysics Data System (ADS)

    Moore, Michael J.; Enomoto, Akihiro; Nakano, Tadashi; Kayasuga, Atsushi; Kojima, Hiroaki; Sakakibara, Hitoshi; Oiwa, Kazuhiro; Suda, Tatsuya

    Molecular communication is one method for communication among biological nanomachines. Nanomachines are artificial or biological nano-scale devices that perform simple computation, sensing, or actuation. Future applications using nanomachines may require various communication mechanisms. For example, broadcast is one primitive communication for transmission from one sender to many receivers. In this paper, we discuss preliminary work on designing a molecular communication system that is adapted from the molecular motor transport mechanism existing in biological cells. In the proposed molecular motor mechanism, a sender releases information molecules, and molecular motors transport the information molecules along microtubule filaments to receiver nanomachines up to hundreds of micrometers away. This paper describes some possible arrangements for microtubule filaments and simulations to evaluate sending of one information molecule to many receivers. The simulation results indicate that the proposed molecular motor system transports simulated information molecules (100nm radius spheres) more quickly than a diffusion-only communication and that placement of receivers at the plus-end of microtubules results in lower propagation delay.

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

    PubMed Central

    1992-01-01

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

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

    PubMed

    Hemphill, A; Affolter, M; Seebeck, T

    1992-04-01

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

  15. 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

  16. 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

  17. Dimer model for Tau proteins bound in microtubule bundles

    NASA Astrophysics Data System (ADS)

    Hall, Natalie; Kluber, Alexander; Hayre, N. Robert; Singh, Rajiv; Cox, Daniel

    2013-03-01

    The microtubule associated protein tau is important in nucleating and maintaining microtubule spacing and structure in neuronal axons. Modification of tau is implicated as a later stage process in Alzheimer's disease, but little is known about the structure of tau in microtubule bundles. We present preliminary work on a proposed model for tau dimers in microtubule bundles (dimers are the minimal units since there is one microtubule binding domain per tau). First, a model of tau monomer was created and its characteristics explored using implicit solvent molecular dynamics simulation. Multiple simulations yield a partially collapsed form with separate positively/negatively charged clumps, but which are a factor of two smaller than required by observed microtubule spacing. We argue that this will elongate in dimer form to lower electrostatic energy at a cost of entropic ``spring'' energy. We will present preliminary results on steered molecular dynamics runs on tau dimers to estimate the actual force constant. Supported by US NSF Grant DMR 1207624.

  18. General theory for the mechanics of confined microtubule asters

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  19. Design, manufacturing, performance and application of wide angle aspherical achromatic doublet

    NASA Astrophysics Data System (ADS)

    Melich, Radek; Procháska, František; Tomka, David; Rail, Zdeněk.; Bartoňíček, Jiří; Pleštil, Jan; Šrajer, Bohdan

    2016-11-01

    The paper describes an achromatic Steinhal type doublet that employs an aspherical surface to allow wide angle imaging. A design criteria, optimization techniques and tolerancing of the doublet are described. Further a manufacturing process of the system and achieved optical performance measurement is discussed. Benefits of the wide angle imaging doublet are recently planned to be used in automotive industry application, namely for optimizing of head-light performance and their final evaluation. The final device is planned to be part of the production line.

  20. 2HDMC — two-Higgs-doublet model calculator

    NASA Astrophysics Data System (ADS)

    Eriksson, David; Rathsman, Johan; Stål, Oscar

    2010-04-01

    We describe version 1.0.6 of the public C++ code 2HDMC, which can be used to perform calculations in a general, CP-conserving, two-Higgs-doublet model (2HDM). The program features simple conversion between different parametrizations of the 2HDM potential, a flexible Yukawa sector specification with choices of different Z-symmetries or more general couplings, a decay library including all two-body — and some three-body — decay modes for the Higgs bosons, and the possibility to calculate observables of interest for constraining the 2HDM parameter space, as well as theoretical constraints from positivity and unitarity. The latest version of the 2HDMC code and full documentation is available from: http://www.isv.uu.se/thep/MC/2HDMC. New version program summaryProgram title: 2HDMC Catalogue identifier: AEFI_v1_1 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEFI_v1_1.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU GPL No. of lines in distributed program, including test data, etc.: 12 110 No. of bytes in distributed program, including test data, etc.: 92 731 Distribution format: tar.gz Programming language: C++ Computer: Any computer running Linux Operating system: Linux RAM: 5 Mb Catalogue identifier of previous version: AEFI_v1_0 Journal reference of previous version: Comput. Phys. Comm. 180 (2010) 189 Classification: 11.1 External routines: GNU Scientific Library ( http://www.gnu.org/software/gsl/) Does the new version supersede the previous version?: Yes Nature of problem: Determining properties of the potential, calculation of mass spectrum, couplings, decay widths, oblique parameters, muon g-2, and collider constraints in a general two-Higgs-doublet model. Solution method: From arbitrary potential and Yukawa sector, tree-level relations are used to determine Higgs masses and couplings. Decay widths are calculated at leading order, including FCNC decays when applicable. Decays to off

  1. Cortical microtubule arrays are initiated from a nonrandom prepattern driven by atypical microtubule initiation.

    PubMed

    Lindeboom, Jelmer J; Lioutas, Antonios; Deinum, Eva E; Tindemans, Simon H; Ehrhardt, David W; Emons, Anne Mie C; Vos, Jan W; Mulder, Bela M

    2013-03-01

    The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.

  2. 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

  3. A divergent canonical WNT-signaling pathway regulates microtubule dynamics

    PubMed Central

    Ciani, Lorenza; Krylova, Olga; Smalley, Matthew J.; Dale, Trevor C.; Salinas, Patricia C.

    2004-01-01

    Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β). In the canonical WNT pathway, the negative regulator Axin forms a complex with β-catenin and GSK-3β, resulting in β-catenin degradation. Inhibition of GSK-3β by DVL increases β-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3β through a transcription- and β-catenin–independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3β–mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3β, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability. PMID:14734535

  4. 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).

  5. Vinblastine suppresses dynamics of individual microtubules in living interphase cells.

    PubMed Central

    Dhamodharan, R; Jordan, M A; Thrower, D; Wilson, L; Wadsworth, P

    1995-01-01

    We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells. Images PMID:8534917

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

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

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

  7. INSIGHTS INTO ANTI-PARALLEL MICROTUBULE CROSSLINKING BY PRC1, A CONSERVED NON-MOTOR MICROTUBULE BINDING PROTEIN

    PubMed Central

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

    2010-01-01

    SUMMARY Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish compliant crosslinks that selectively `mark' antiparallel overlap in dynamic cytoskeletal networks. PMID:20691902

  8. Muon g - 2 in the aligned two Higgs doublet model

    SciTech Connect

    Han, Tao; Kang, Sin Kyu; Sayre, Joshua

    2016-02-16

    In this paper, we study the Two-Higgs-Doublet Model with the aligned Yukawa sector (A2HDM) in light of the observed excess measured in the muon anomalous magnetic moment. We take into account the existing theoretical and experimental constraints with up-to-date values and demonstrate that a phenomenologically interesting region of parameter space exists. With a detailed parameter scan, we show a much larger region of viable parameter space in this model beyond the limiting case Type X 2HDM as obtained before. It features the existence of light scalar states with masses 3 GeV ≲ mH ≲ 50 GeV, or 10 GeV ≲ mA ≲ 130 GeV, with enhanced couplings to tau leptons. The charged Higgs boson is typically heavier, with 200 GeV ≲ mH+ ≲ 630 GeV. The surviving parameter space is forced into the CP-conserving limit by EDM constraints. Some Standard Model observables may be significantly modified, including a possible new decay mode of the SMlike Higgs boson to four taus. Lastly, we comment on future measurements and direct searches for those effects at the LHC as tests of the model.

  9. Higgs Doublet Decay as the Origin of the Baryon Asymmetry

    NASA Astrophysics Data System (ADS)

    Hambye, Thomas; Teresi, Daniele

    2016-08-01

    We consider a question that curiously had not been properly considered thus far: in the standard seesaw model, what is the minimum value the mass of a right-handed (RH) neutrino must have for allowing successful leptogenesis via C P -violating decays? Answering this question requires us to take into account a number of thermal effects. We show that, for low RH neutrino masses, and thanks to these effects, leptogenesis turns out to proceed efficiently from the decay of the standard model scalar doublet components into a RH neutrino and a lepton. Such decays produce the asymmetry at low temperatures, slightly before sphaleron decoupling. If the RH neutrino has thermalized prior to producing the asymmetry, this mechanism turns out to lead to the bound mN>2 GeV . If, instead, the RH neutrinos have not thermalized, leptogenesis from these decays is enhanced further and can be easily successful, even at lower scales. This Higgs-decay leptogenesis new mechanism works without requiring an interplay of flavor effects and/or cancellations of large Yukawa couplings in the neutrino mass matrix. Last but not least, such a scenario turns out to be testable, from direct production of the RH neutrino(s).

  10. Exploring collider signatures of the inert Higgs doublet model

    NASA Astrophysics Data System (ADS)

    Datta, Amitava; Ganguly, Nabanita; Khan, Najimuddin; Rakshit, Subhendu

    2017-01-01

    We revisit the multilepton (m l )+ ET+X signatures of the inert doublet model (IDM) of dark matter in future LHC experiments for m =3 , 4 and simulate, for the first time, the m =5 case. Here X stands for any number of jets. We illustrate these signals with benchmark points consistent with the usual constraints like unitarity, perturbativity, the precision electroweak data, the observed dark matter relic density of the Universe and, most importantly, the stringent LHC constraints from the post-Higgs (h ) discovery era like the measured Mh and the upper bound on the invisible width of h decay, which were not included in earlier analyses of multilepton signatures. We find that if the IDM is embedded in a grand desert scenario so that the unitarity constraint holds up to a very high scale, the whole of the highly restricted parameter space allowed by the above constraints can be probed at the LHC via the 3 l signal for an integrated luminosity ˜3000 fb-1 . On the other hand, if any new physics shows up at a scale ˜10 TeV , only a part of the enlarged allowed parameter space can be probed. The 4 l and 5 l signals can help to discriminate among different IDM scenarios as and when sufficient integrated luminosity accumulates.

  11. The doublet majoron model and solar neutrino oscillations

    NASA Astrophysics Data System (ADS)

    Bertolini, S.; Santamaria, A.

    1988-12-01

    We present a minimal extension of the standard electroweak theory which, as a consequence of the spontaneous breaking of lepton number and the radiative origin of the neutrino mass, offers a natural framework for the solution of the solar neutrino problem through matter-enhanced neutrino oscillations (Mikheyev-Smirnov-Wolfenstein mechanism). Indeed, we show that the presently available astrophysical bounds on the lepton-breaking vacuum expectation value naturally lead to neutrino masses in the required regime. The fact that the Majoron belongs to an SU(2)L doublet and not a triplet has relevant phenomenological implications. In particular, the scalar contribution to the Z0 width is four times smaller than in the triplet model and equivalent to 1/2 a neutrino-antineutrino mode. Relevant effects related to the presence of two physical singly charged scalars, both at the quantum and tree level, are studied. As a result we find that the model is tightly constrained by present data. In particular, for a wide range of parameters, the decay μ --> eγ is within two orders of magnitude from the present experimental limit. Also at Department de Física Teòrica, Universitat de València and IFIC, Universitat de València-CSIC, Spain.

  12. Muon g - 2 in the aligned two Higgs doublet model

    DOE PAGES

    Han, Tao; Kang, Sin Kyu; Sayre, Joshua

    2016-02-16

    In this paper, we study the Two-Higgs-Doublet Model with the aligned Yukawa sector (A2HDM) in light of the observed excess measured in the muon anomalous magnetic moment. We take into account the existing theoretical and experimental constraints with up-to-date values and demonstrate that a phenomenologically interesting region of parameter space exists. With a detailed parameter scan, we show a much larger region of viable parameter space in this model beyond the limiting case Type X 2HDM as obtained before. It features the existence of light scalar states with masses 3 GeV ≲ mH ≲ 50 GeV, or 10 GeV ≲more » mA ≲ 130 GeV, with enhanced couplings to tau leptons. The charged Higgs boson is typically heavier, with 200 GeV ≲ mH+ ≲ 630 GeV. The surviving parameter space is forced into the CP-conserving limit by EDM constraints. Some Standard Model observables may be significantly modified, including a possible new decay mode of the SMlike Higgs boson to four taus. Lastly, we comment on future measurements and direct searches for those effects at the LHC as tests of the model.« less

  13. Lausannevirus, a giant amoebal virus encoding histone doublets.

    PubMed

    Thomas, Vincent; Bertelli, Claire; Collyn, François; Casson, Nicola; Telenti, Amalio; Goesmann, Alexander; Croxatto, Antony; Greub, Gilbert

    2011-06-01

    Large viruses infecting algae or amoebae belong to the NucleoCytoplasmic Large DNA Viruses (NCLDV) and present genotypic and phenotypic characteristics that have raised major interest among microbiologists. Here, we describe a new large virus discovered in Acanthamoeba castellanii co-culture of an environmental sample. The virus, referred to as Lausannevirus, has a very limited host range, infecting Acanthamoeba spp. but being unable to infect other amoebae and mammalian cell lines tested. Within A. castellanii, this icosahedral virus of about 200 nm exhibits a development cycle similar to Mimivirus, with an eclipse phase 2 h post infection and a logarithmic growth leading to amoebal lysis in less than 24 h. The 346 kb Lausannevirus genome presents similarities with the recently described Marseillevirus, sharing 89% of genes, and thus belongs to the same family as confirmed by core gene phylogeny. Interestingly, Lausannevirus and Marseillevirus genomes both encode three proteins with predicted histone folds, including two histone doublets, that present similarities to eukaryotic and archaeal histones. The discovery of Lausannevirus and the analysis of its genome provide some insight in the evolution of these large amoebae-infecting viruses.

  14. Dark matter physics in neutrino specific two Higgs doublet model

    NASA Astrophysics Data System (ADS)

    Baek, Seungwon; Nomura, Takaaki

    2017-03-01

    Although the seesaw mechanism is a natural explanation for the small neutrino masses, there are cases when the Majorana mass terms for the right-handed neutrinos are not allowed due to symmetry. In that case, if neutrino-specific Higgs doublet is introduced, neutrinos become Dirac particles and their small masses can be explained by its small VEV. We show that the same symmetry, which we assume a global U(1) X , can also be used to explain the stability of dark matter. In our model, a new singlet scalar breaks the global symmetry spontaneously down to a discrete Z 2 symmetry. The dark matter particle, lightest Z 2-odd fermion, is stabilized. We discuss the phenomenology of dark matter: relic density, direct detection, and indirect detection. We find that the relic density can be explained by a novel Goldstone boson channel or by resonance channel. In the most region of parameter space considered, the direct detections is suppressed well below the current experimental bound. Our model can be further tested in indirect detection experiments such as FermiLAT gamma ray searches or neutrinoless double beta decay experiments.

  15. Diverse rupture processes in the 2015 Peru deep earthquake doublet

    PubMed Central

    Ye, Lingling; Lay, Thorne; Kanamori, Hiroo; Zhan, Zhongwen; Duputel, Zacharie

    2016-01-01

    Earthquakes in deeply subducted oceanic lithosphere can involve either brittle or dissipative ruptures. On 24 November 2015, two deep (606 and 622 km) magnitude 7.5 and 7.6 earthquakes occurred 316 s and 55 km apart. The first event (E1) was a brittle rupture with a sequence of comparable-size subevents extending unilaterally ~50 km southward with a rupture speed of ~4.5 km/s. This earthquake triggered several aftershocks to the north along with the other major event (E2), which had 40% larger seismic moment and the same duration (~20 s), but much smaller rupture area and lower rupture speed than E1, indicating a more dissipative rupture. A minor energy release ~12 s after E1 near the E2 hypocenter, possibly initiated by the S wave from E1, and a clear aftershock ~165 s after E1 also near the E2 hypocenter, suggest that E2 was likely dynamically triggered. Differences in deep earthquake rupture behavior are commonly attributed to variations in thermal state between subduction zones. However, the marked difference in rupture behavior of the nearby Peru doublet events suggests that local variations of stress state and material properties significantly contribute to diverse behavior of deep earthquakes. PMID:27386585

  16. Diverse rupture processes in the 2015 Peru deep earthquake doublet.

    PubMed

    Ye, Lingling; Lay, Thorne; Kanamori, Hiroo; Zhan, Zhongwen; Duputel, Zacharie

    2016-06-01

    Earthquakes in deeply subducted oceanic lithosphere can involve either brittle or dissipative ruptures. On 24 November 2015, two deep (606 and 622 km) magnitude 7.5 and 7.6 earthquakes occurred 316 s and 55 km apart. The first event (E1) was a brittle rupture with a sequence of comparable-size subevents extending unilaterally ~50 km southward with a rupture speed of ~4.5 km/s. This earthquake triggered several aftershocks to the north along with the other major event (E2), which had 40% larger seismic moment and the same duration (~20 s), but much smaller rupture area and lower rupture speed than E1, indicating a more dissipative rupture. A minor energy release ~12 s after E1 near the E2 hypocenter, possibly initiated by the S wave from E1, and a clear aftershock ~165 s after E1 also near the E2 hypocenter, suggest that E2 was likely dynamically triggered. Differences in deep earthquake rupture behavior are commonly attributed to variations in thermal state between subduction zones. However, the marked difference in rupture behavior of the nearby Peru doublet events suggests that local variations of stress state and material properties significantly contribute to diverse behavior of deep earthquakes.

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

    PubMed

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

    2015-10-20

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

  18. Twelve protofilament taxol-induced microtubules assembled from purified tublin. A synchrotron X-ray scattering study in comparison with glycerol- and map-induced microtubules

    NASA Astrophysics Data System (ADS)

    Andreu, J. M.; Garcia de Ancos, J.; Medrano, F. J.; Gil, R.; Diaz, J. F.; Nogales, E.; Towns-Andrews, E.; Pantos, E.; Bordas, J.

    1991-05-01

    The X-ray solution scattering profiles of taxol microtubules made of purified tubulin and control microtubules, assembled either from purified tubulin in glycerol buffer (a non-specific enhancer of the polymerization of tubulin) or from microtubule protein (a preparation containing tubulin plus microtubule associated proteins), were obtained to 3.3 nm resolution. These profiles show features of the microtubule wall structure which had not been observed in solution before. Comparison of the different profiles indicated that the structure of the microtubule wall is very similar in the three types of microtubules to the resolution of the measurements, however the mean diameter of the taxol microtubules is smaller than that of the control microtubules, by approximately one protofilament less. Actually, only 12 protofilament computer models of microtubules could fit the position of the maxima in the experimental scattering profile of the taxol microtubules. Having only 12 protofilaments implies a discontinuity on the microtubule wall, irrespective of whether the lateral contacts follow the A or B microtubule lattice, and also requires adjustment of the normal lattice to one protofilament axis with respect to the cylinder axis. The fact that the majority of these taxol microtubules assembled from purified tubulin have 12 protofilaments has been visualized by electron micrographs of tannic acid stained microtubule thin sections, and is fully consistent with the microtubule wall projections (fringe patterns) observed in negatively stained and cryo-electron microscopy specimens, which correspond to a 12 protofilament-three start lattice type.

  19. T violation in Kμ3 decay in a general two-Higgs-doublet model

    NASA Astrophysics Data System (ADS)

    Godina Nava, J. J.

    1996-02-01

    We calculate the transverse muon polarization in the K+μ3 process arising from the Yukawa couplings of charged Higgs bosons in a general two-Higgs-doublet model where spontaneous violation of CP invariance is present.

  20. 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.

  1. Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly.

    PubMed

    Hung, Liang-Yi; Chen, Hua-Ling; Chang, Ching-Wen; Li, Bor-Ran; Tang, Tang K

    2004-06-01

    We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.

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

    PubMed

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

    2017-01-01

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

  3. Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules

    PubMed Central

    1987-01-01

    The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti- centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole- resistant microtubules in MDCK cells contained detyrosinated alpha- tubulin (glu tubulin). On the other hand

  4. Effects of fluoro-doxorubicin (ME2303) on microtubules: influence of different classes of microtubule-associated proteins.

    PubMed

    Fromes, Y; Gounon, P; Tapiero, H; Fellous, A

    1996-08-01

    Anthracyclines are among the most useful agents for the treatment of neoplastic disease, but their clinical use is limited by progressive cardiomyopathy. A few studies have suggested the role of microtubules for the understanding of this toxicity. By using kinetic and structural studies, we demonstrate the disorganizing action of fluoro-doxorubicin, a novel anthracycline, on the microtubule system. Microtubules have a rich and complex composition in relation to their numerous functions in cells. In the present study, we investigate the role of two major microtubule-associated protein (MAP) families, Tau and MAP2. Both MAP families are responsible for the properties of different classes of microtubules. We show the differential effect of fluoro-doxorubicin on these two classes of microtubules. Furthermore, we show that fluoro-doxorubicin is able to affect the capacity of purified tubulin to form normal microtubules. This study confirms that anthracyclines may interfer with the microtubule organization. We suggest that some classes of microtubules, with regard to their MAP composition, may be affected more specifically in cardiac myocytes.

  5. Stability of the normal vacuum in multi-Higgs-doublet models

    SciTech Connect

    Barroso, A.; Ferreira, P. M.; Santos, R.; Silva, Joao P.

    2006-10-15

    We show that the vacuum structure of a generic multi-Higgs-doublet model shares several important features with the vacuum structure of the two and three Higgs-doublet model. In particular, one can still define the usual charge breaking, spontaneous CP breaking, and normal (charge and CP preserving) stationary points. We analyze the possibility of charge or spontaneous CP breaking by studying the relative depth of the potential in each of the possible stationary points.

  6. MARK4 is a novel microtubule-associated proteins/microtubule affinity-regulating kinase that binds to the cellular microtubule network and to centrosomes.

    PubMed

    Trinczek, Bernhard; Brajenovic, Miro; Ebneth, Andreas; Drewes, Gerard

    2004-02-13

    The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease.

  7. Characterization of the role of calcium in regulating the microtubule-destabilizing activity of MDP25.

    PubMed

    Qin, Tao; Li, Jiejie; Yuan, Ming; Mao, Tonglin

    2012-07-01

    Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins. Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.

  8. Video microscopy analysis of the polymerization dynamics of individual microtubules

    NASA Astrophysics Data System (ADS)

    Salmon, E. D.

    1991-05-01

    We have developed methods using video-enhanced differential interference contrast light microscopy (VE-DIC) to measure the association and dissociation rate constants and transition frequencies of microtubule dynamic instability for microtubules assembled from pure tubulin, plus brain microtubule assoicated proteins (MAPs), and for microtubule assembly in living cells and cytosol extracts. Following nucleation, a microtubule end is seen to elongate at constant velocity until it abruptly begins rapid shortening, a transition termed catastrophe. The microtubule either disappears, or converts back to the elongation phase, a transition termed rescue. Catastrophes and rescues occur stochastically and infrequently in comparison to the durations of the elongation and shortening phases. In purified tubulin preparations from both mammalian brain and sea urchin embryos, the elongation and shortening phases exhibit distinctly different association and dissociation rate constants; in particular, the rate of dissocation during rapid shortening can be 100 times or more greater than during elongation particularly at high Mg2+. Brain MAPs (MAP2 and Tau) promote faster elongation, but suppress dynamic instability mainly by decreasing the frequency of catastrophe and increasing the frequency of rescue. In contrast, there are unknown factors in living dividing cells and in extracts from dividing cells which enhance dynamic instability by producing high frequencies of catastrophe (.01-.05 sec-1) at fast elongation velocities (10 μm min-1). Using a microscope perfusion chamber, we have shown for microtubules assembled from pure tubulin that dilution induces rapid shortening within several seconds independent of the elongation velocity or microtubule length. Thus, the stabilizing cap at elongating microtubule ends is small and sensitive to transient changes in the rate of tubulin association, even at high elongation velocities. This means that substantial changes in microtubule

  9. Response of a doublet to a nearby dc electrode of uniform potential

    NASA Astrophysics Data System (ADS)

    Wirth, Christopher L.; Nuthalapati, Sri Harsha

    2016-10-01

    The electric-field-assisted directed assembly of spherical colloidal particles near an electrode has been studied for nearly three decades. Recently, focus has shifted to the electric-field-assisted assembly and propulsion of nonspherical (i.e., anisotropic) particles. This paper describes calculations and results for a doublet of asymmetric ζ potential and size responding to a dc electric field. The doublet experienced a net vertical force that depended on both the asymmetry in ζ potential and lobe size. In addition, the doublet experienced a net lateral force perpendicular to the applied electric field. The lateral force depended on the difference in ζ potential of the two lobes, the lobe size asymmetry, and also the angle of inclination of the doublet. The net force was used to predict an apparent lateral velocity, which was found to be perpendicular to the applied electric field. In addition, the particle experienced rotation from a net torque that depended on the lobe size asymmetry and also the angle of inclination of the doublet. The magnitude of the predicted velocity was of the same order of magnitude as has been observed for particles responding to ac electric fields in experiments. These results demonstrate that lobe ζ potential, lobe size, and orientation of a colloidal doublet can be tuned to achieve propulsion.

  10. Self-assembly of microtubules and motors

    NASA Astrophysics Data System (ADS)

    Aranson, Igor; Tsimring, Lev

    2005-03-01

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

  11. 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.

  12. Building the Microtubule Cytoskeleton Piece by Piece*

    PubMed Central

    Alfaro-Aco, Ray; Petry, Sabine

    2015-01-01

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

  13. Passive athermalization of doublets in 8-13 micron waveband

    NASA Astrophysics Data System (ADS)

    Schuster, Norbert

    2014-10-01

    Passive athermalization of lenses has become a key-technology for automotive and other outdoor applications using modern uncooled 25, 17 and 12 micron pixel pitch bolometer arrays. Typical pixel counts for thermal imaging are 384x288 (qVGA), 640x480 (VGA), and 1024x768 (XGA). Two lens arrangements (called Doublets) represent a cost effective way to satisfy resolution requirements of these detectors with F-numbers 1.4 or faster. Thermal drift of index of refraction and the geometrical changes (in lenses and housing) versus temperature defocus the initial image plane from the detector plane. The passive athermalization restricts this drop of spatial resolution in a wide temperature range (typically -40°C…+80°C) to an acceptable value without any additional external refocus. In particular, lenses with long focal lengths and high apertures claim athermalization. A careful choice of lens and housing materials and a sophistical dimensioning lead to three different principles of passivation: The Passive Mechanical Athermalization (PMA) shifts the complete lens cell, the Passive Optical and Mechanical Athermalization (POMA) shifts only one lens inside the housing, the Passive Optical Athermalization (POA) works without any mechanism. All three principles will be demonstrated for a typical narrow-field lens (HFOV about 12°) with high aperture (aperture based F-number 1.3) for the actual uncooled reference detector (17micron VGA). Six design examples using different combinations of lens materials show the impact on spatial lens resolution, on overall length, and on weight. First order relations are discussed. They give some hints for optimization solutions. Pros and cons of different passive athermalization principles are evaluated in regards of housing design, availability of materials and costing. Examples with a convergent GASIR®1-lens in front distinguish by best resolution, short overall length, and lowest weight.

  14. Competing Kondo Effects in Non-Kramers Doublet Systems

    NASA Astrophysics Data System (ADS)

    Kusunose, Hiroaki

    2016-06-01

    In non-Kramers Kondo systems with quadrupolar degrees of freedom, an ordinary magnetic Kondo effect can compete with the quadrupolar Kondo effect. We discuss such competition keeping PrT2Zn20 (T = Ir, Rh) and PrT2Al20 (T = V, Ti) in mind, where the Γ3 non-Kramers crystalline-electric-field (CEF) doublet ground state is realized in a Pr3+ ion with a (4f)2 configuration under cubic symmetry. The quadrupolar Kondo effect can be described by the two-channel Kondo model, which leads to the local non-Fermi-liquid (NFL) ground state, while the magnetic Kondo effect favors the ordinary local Fermi-liquid (FL) ground state. On the basis of the minimal extended two-channel Kondo model including the magnetic Kondo coupling as well, we investigate the competition and resulting thermodynamics, and orbital/magnetic and single-particle excitation spectra by Wilson's numerical renormalization group (NRG) method. There is a first-order transition between the NFL and FL ground states. In addition to these two states, the alternative FL state accompanied by a free magnetic spin appears in the intermediate temperature range, which eventually reaches the true NFL ground state, as a consequence of the stronger competition between the magnetic and quadrupolar Kondo effects. In this peculiar state, the magnetic susceptibility shows a Curie-like behavior, while the orbital fluctuation exhibits the FL behavior. Moreover, the single-particle spectra yield a more singular behavior. Implications to the Pr 1-2-20 systems are briefly discussed.

  15. Leading at the Front: How EB Proteins Regulate Microtubule Dynamics

    NASA Astrophysics Data System (ADS)

    Hawkins, Taviare

    2012-02-01

    Microtubules are the most rigid of the cytoskeletal filaments, they provide the cell's scaffolding, form the byways on which motor proteins transport intracellular cargo and reorganize to form the mitotic spindle when the cell needs to divide. These biopolymers are composed of alpha and beta tubulin monomers that create hollow cylindrical nanotubes with an outer diameter of 25 nm and an inner diameter of 17 nm. At steady state concentrations, microtubules undergo a process known as dynamic instability. During dynamic instability the length of individual microtubules is changing as the filament alternates between periods of growth to shrinkage (catastrophe) and shrinkage to growth (rescue). This process can be enhanced or diminished with the addition of microtubule associated proteins (MAPs). MAPs are microtubule binding proteins that stabilize, destabilize, or nucleate microtubules. We will discuss the effects of the stabilizing end-binding proteins (EB1, EB2 and EB3), on microtubule dynamics observed in vitro. The EBs are a unique family of MAPs known to tip track and enhance microtubule growth by stabilizing the ends. This is a different mechanism than those employed by structural MAPs such as tau or MAP4.

  16. Microtubule stabilising peptides rescue tau phenotypes in-vivo

    PubMed Central

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

    2016-01-01

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

  17. Microtubules: dynamically unstable stochastic phase-switching polymers

    NASA Astrophysics Data System (ADS)

    Zakharov, P. N.; Arzhanik, V. K.; Ulyanov, E. V.; Gudimchuk, N. B.; Ataullakhanov, F. I.

    2016-08-01

    One of the simplest molecular motors, a biological microtubule, is reviewed as an example of a highly nonequilibrium molecular machine capable of stochastic transitions between slow growth and rapid disassembly phases. Basic properties of microtubules are described, and various approaches to simulating their dynamics, from statistical chemical kinetics models to molecular dynamics models using the Metropolis Monte Carlo and Brownian dynamics methods, are outlined.

  18. Microtubule distribution in gravitropic protonemata of the moss Ceratodon

    NASA Technical Reports Server (NTRS)

    Schwuchow, J.; Sack, F. D.; Hartmann, E.

    1990-01-01

    Tip cells of dark-grown protonemata of the moss Ceratodon purpureus are negatively gravitropic (grow upward). They possess a unique longitudinal zonation: (1) a tip group of amylochloroplasts in the apical dome, (2) a plastid-free zone, (3) a zone of significant plastid sedimentation, and (4) a zone of mostly non-sedimenting plastids. Immunofluorescence of vertical cells showed microtubules distributed throughout the cytoplasm in a mostly axial orientation extending through all zones. Optical sectioning revealed a close spatial association between microtubules and plastids. A majority (two thirds) of protonemata gravistimulated for > 20 min had a higher density of microtubules near the lower flank compared to the upper flank in the plastid-free zone. This apparent enrichment of microtubules occurred just proximal to sedimented plastids and near the part of the tip that presumably elongates more to produce curvature. Fewer than 5% of gravistimulated protonemata had an enrichment in microtubules near the upper flank, whereas 14% of vertical protonemata were enriched near one of the side walls. Oryzalin and amiprophos-methyl (APM) disrupted microtubules, gravitropism, and normal tip growth and zonation, but did not prevent plastid sedimentation. We hypothesize that a microtubule redistribution plays a role in gravitropism in this protonema. This appears to be the first report of an effect of gravity on microtubule distribution in plants.

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

    PubMed

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

    2016-11-04

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

  20. Microtubule-severing enzymes at the cutting edge.

    PubMed

    Sharp, David J; Ross, Jennifer L

    2012-06-01

    ATP-dependent severing of microtubules was first reported in Xenopus laevis egg extracts in 1991. Two years later this observation led to the purification of the first known microtubule-severing enzyme, katanin. Katanin homologs have now been identified throughout the animal kingdom and in plants. Moreover, members of two closely related enzyme subfamilies, spastin and fidgetin, have been found to sever microtubules and might act alongside katanins in some contexts (Roll-Mecak and McNally, 2010; Yu et al., 2008; Zhang et al., 2007). Over the past few years, it has become clear that microtubule-severing enzymes contribute to a wide range of cellular activities including mitosis and meiosis, morphogenesis, cilia biogenesis and disassembly, and migration. Thus, this group of enzymes is revealing itself to be among the most important of the microtubule regulators. This Commentary focuses on our growing understanding of how microtubule-severing enzymes contribute to the organization and dynamics of diverse microtubule arrays, as well as the structural and biophysical characteristics that afford them the unique capacity to catalyze the removal of tubulin from the interior microtubule lattice. Our goal is to provide a broader perspective, focusing on a limited number of particularly informative, representative and/or timely findings.

  1. Assembly and Positioning of Microtubule Asters in Microfabricated Chambers

    NASA Astrophysics Data System (ADS)

    Holy, Timothy E.; Dogterom, Marileen; Yurke, Bernard; Leibler, Stanislas

    1997-06-01

    Intracellular organization depends on a variety of molecular assembly processes; while some of these have been studied in simplified cell-free systems, others depend on the confined geometry of cells and cannot be reconstructed using bulk techniques. To study the latter processes in vitro, we fabricated microscopic chambers that simulate the closed environment of cells. We used these chambers to study the positioning of microtubule asters. Microtubule assembly alone, without the action of molecular motors, is sufficient to position asters. Asters with short microtubules move toward the position expected from symmetry; however, once the microtubules become long enough to buckle, symmetry is broken. Calculations and experiments show that the bending-energy landscape has multiple minima. Microtubule dynamic instability modifies the landscape over time and allows asters to explore otherwise inaccessible configurations.

  2. Multimodal microtubule binding by the Ndc80 kinetochore complex.

    PubMed

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

    2012-11-01

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

  3. Tensile stress stimulates microtubule outgrowth in living cells

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  4. Molecular mechanism of action of microtubule-stabilizing anticancer agents.

    PubMed

    Prota, Andrea E; Bargsten, Katja; Zurwerra, Didier; Field, Jessica J; Díaz, José Fernando; Altmann, Karl-Heinz; Steinmetz, Michel O

    2013-02-01

    Microtubule-stabilizing agents (MSAs) are efficacious chemotherapeutic drugs widely used for the treatment of cancer. Despite the importance of MSAs for medical applications and basic research, their molecular mechanisms of action on tubulin and microtubules remain elusive. We determined high-resolution crystal structures of αβ-tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane pocket of β-tubulin and used their respective side chains to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. Further, our results offer fundamental structural insights into the control mechanisms of microtubule dynamics.

  5. The Ndc80 kinetochore complex forms oligomeric arrays along microtubules

    PubMed Central

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

    2010-01-01

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

  6. Multifunctional Microtubule-Associated Proteins in Plants

    PubMed Central

    Krtková, Jana; Benáková, Martina; Schwarzerová, Kateřina

    2016-01-01

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

  7. Transient Pinning and Pulling: A Mechanism for Bending Microtubules

    PubMed Central

    Kent, Ian A.; Rane, Parag S.; Dickinson, Richard B.; Ladd, Anthony J. C.; Lele, Tanmay P.

    2016-01-01

    Microtubules have a persistence length of the order of millimeters in vitro, but inside cells they bend over length scales of microns. It has been proposed that polymerization forces bend microtubules in the vicinity of the cell boundary or other obstacles, yet bends develop even when microtubules are polymerizing freely, unaffected by obstacles and cell boundaries. How these bends are formed remains unclear. By tracking the motions of microtubules marked by photobleaching, we found that in LLC-PK1 epithelial cells local bends develop primarily by plus-end directed transport of portions of the microtubule contour towards stationary locations (termed pinning points) along the length of the microtubule. The pinning points were transient in nature, and their eventual release allowed the bends to relax. The directionality of the transport as well as the overall incidence of local bends decreased when dynein was inhibited, while myosin inhibition had no observable effect. This suggests that dynein generates a tangential force that bends microtubules against stationary pinning points. Simulations of microtubule motion and polymerization accounting for filament mechanics and dynein forces predict the development of bends of size and shape similar to those observed in cells. Furthermore, simulations show that dynein-generated bends at a pinning point near the plus end can cause a persistent rotation of the tip consistent with the observation that bend formation near the tip can change the direction of microtubule growth. Collectively, these results suggest a simple physical mechanism for the bending of growing microtubules by dynein forces accumulating at pinning points. PMID:26974838

  8. Ultrastructure of the spermatozoon of the American Alligator, Alligator mississippiensis (Reptilia: Alligatoridae).

    PubMed

    Gribbins, Kevin M; Touzinsky, Katherine F; Siegel, Dustin S; Venable, Katherine J; Hester, Georgia L; Elsey, Ruth M

    2011-11-01

    This study details the ultrastructure of the spermatozoa of the American Alligator, Alligator mississippiensis. American Alligator spermatozoa are filiform and slightly curved. The acrosome is tapered at its anterior end and surrounded by the acrosome vesicle and an underlying subacrosomal cone, which rests just cephalic to the nuclear rostrum. One endonuclear canal extends from the subacrosomal cone through the rostral nucleus and deep into the nuclear body. The neck region separates the nucleus and midpiece and houses the proximal centriole and pericentriolar material. The distal centriole extends through the midpiece and has 9 × 3 sets of peripheral microtubules with a central doublet pair within the axoneme that is surrounded by a dense sheath. The midpiece is composed of seven to nine rings of mitochondria, which have combinations of concentrically and septate cristae. The principal piece has a dense fibrous sheath that surrounds an axoneme with a 9 + 2 microtubule arrangement. The sheath becomes significantly reduced in size caudally within the principal piece and is completely missing from the endpiece. Dense peripheral fibers, especially those associated with microtubule doublets 3 and 8, penetrate into the anterior portion of the principal piece axoneme. The data reported here hypothesize that sperm morphology is highly conserved in Crocodylia; however, specific morphological differences can exist between species.

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

    PubMed Central

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

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed

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

    2015-01-01

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

  12. Association of actin filaments with axonal microtubule tracts.

    PubMed

    Bearer, E L; Reese, T S

    1999-02-01

    Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 microm with some at least as long as 3.5 microm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.

  13. α-Synuclein is a Novel Microtubule Dynamase.

    PubMed

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

    2016-09-15

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

  14. Poleward microtubule flux mitotic spindles assembled in vitro

    PubMed Central

    1991-01-01

    In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase. PMID:1999464

  15. α-Synuclein is a Novel Microtubule Dynamase

    PubMed Central

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

    2016-01-01

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

  16. 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.

  17. Oscillatory fluid flow influences primary cilia and microtubule mechanics.

    PubMed

    Espinha, Lina C; Hoey, David A; Fernandes, Paulo R; Rodrigues, Hélder C; Jacobs, Christopher R

    2014-07-01

    Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues, such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity.

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

    PubMed

    Gozes, Illana

    2011-01-01

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

  19. Jupiter, a new Drosophila protein associated with microtubules.

    PubMed

    Karpova, Nina; Bobinnec, Yves; Fouix, Sylvaine; Huitorel, Philippe; Debec, Alain

    2006-05-01

    In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.

  20. A thermodynamic model of microtubule assembly and disassembly.

    PubMed

    Piette, Bernard M A G; Liu, Junli; Peeters, Kasper; Smertenko, Andrei; Hawkins, Timothy; Deeks, Michael; Quinlan, Roy; Zakrzewski, Wojciech J; Hussey, Patrick J

    2009-08-11

    Microtubules are self-assembling polymers whose dynamics are essential for the normal function of cellular processes including chromosome separation and cytokinesis. Therefore understanding what factors effect microtubule growth is fundamental to our understanding of the control of microtubule based processes. An important factor that determines the status of a microtubule, whether it is growing or shrinking, is the length of the GTP tubulin microtubule cap. Here, we derive a Monte Carlo model of the assembly and disassembly of microtubules. We use thermodynamic laws to reduce the number of parameters of our model and, in particular, we take into account the contribution of water to the entropy of the system. We fit all parameters of the model from published experimental data using the GTP tubulin dimer attachment rate and the lateral and longitudinal binding energies of GTP and GDP tubulin dimers at both ends. Also we calculate and incorporate the GTP hydrolysis rate. We have applied our model and can mimic published experimental data, which formerly suggested a single layer GTP tubulin dimer microtubule cap, to show that these data demonstrate that the GTP cap can fluctuate and can be several microns long.

  1. Selective adhesion of functional microtubules to patterned silane surfaces.

    PubMed Central

    Turner, D C; Chang, C; Fang, K; Brandow, S L; Murphy, D B

    1995-01-01

    We show that microtubule polymers can be immobilized selectively on lithographically patterned silane surfaces while retaining their native properties. Silane films were chemisorbed on polished silicon wafers or glass coverslips and patterned using a deep UV lithographic process developed at the Naval Research Laboratory. Hydrocarbon and fluorocarbon alkyl silanes, as well as amino and thiol terminal alkyl silanes, were investigated as substrates for microtubule adhesion with retention of biological activity. Microtubules were found to adhere strongly to amine terminal silanes while retaining the ability to act as substrates for the molecular motor protein kinesin. Aminosilane patterns with linewidths varying from 1 to 50 microns were produced lithographically and used to produce patterns of selectively adhered microtubules. Microtubules were partially aligned on the patterned lines by performing the immobilization in a fluid flow field. Patterns were imaged with atomic force microscopy and differential interference contrast microscopy. Motility assays were carried out using kinesin-coated beads and observed with differential interference contrast microscopy. Kinesin bead movement on the patterned microtubules was comparable to movement on microtubule control surfaces. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:8599684

  2. Nonlinear dynamics of C-terminal tails in cellular microtubules

    NASA Astrophysics Data System (ADS)

    Sekulic, Dalibor L.; Sataric, Bogdan M.; Zdravkovic, Slobodan; Bugay, Aleksandr N.; Sataric, Miljko V.

    2016-07-01

    The mechanical and electrical properties, and information processing capabilities of microtubules are the permanent subject of interest for carrying out experiments in vitro and in silico, as well as for theoretical attempts to elucidate the underlying processes. In this paper, we developed a new model of the mechano-electrical waves elicited in the rows of very flexible C-terminal tails which decorate the outer surface of each microtubule. The fact that C-terminal tails play very diverse roles in many cellular functions, such as recruitment of motor proteins and microtubule-associated proteins, motivated us to consider their collective dynamics as the source of localized waves aimed for communication between microtubule and associated proteins. Our approach is based on the ferroelectric liquid crystal model and it leads to the effective asymmetric double-well potential which brings about the conditions for the appearance of kink-waves conducted by intrinsic electric fields embedded in microtubules. These kinks can serve as the signals for control and regulation of intracellular traffic along microtubules performed by processive motions of motor proteins, primarly from kinesin and dynein families. On the other hand, they can be precursors for initiation of dynamical instability of microtubules by recruiting the proper proteins responsible for the depolymerization process.

  3. Modulation of Microtubule Interprotofilament Interactions by Modified Taxanes

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2000-08-25

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

  5. Tau mediates microtubule bundle architectures mimicking fascicles of microtubules found in the axon initial segment

    PubMed Central

    Chung, Peter J.; Song, Chaeyeon; Deek, Joanna; Miller, Herbert P.; Li, Youli; Choi, Myung Chul; Wilson, Leslie; Feinstein, Stuart C.; Safinya, Cyrus R.

    2016-01-01

    Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent ‘active' microtubule bundles at 37 °C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance Dw–w≈25–41 nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (Dw–w≈16–23 nm) is revealed under osmotic pressure. The wide spacing results from a balance between repulsive forces, due to Tau's projection domain (PD), and a stabilizing sum of transient sub-kBT cationic/anionic charge–charge attractions mediated by weakly penetrating opposing PDs. This landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration. PMID:27452526

  6. Tau mediates microtubule bundle architectures mimicking fascicles of microtubules found in the axon initial segment

    SciTech Connect

    Chung, Peter J.; Song, Chaeyeon; Deek, Joanna; Miller, Herbert P.; Li, Youli; Choi, Myung Chul; Wilson, Leslie; Feinstein, Stuart C.; Safinya, Cyrus R.

    2016-07-25

    Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent ‘active’ microtubule bundles at 37°C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance Dw–w≈25–41nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (Dw–w≈16–23nm) is revealed under osmotic pressure. The wide spacing results from a balance between repulsive forces, due to Tau’s projection domain (PD), and a stabilizing sum of transient sub-kBT cationic/anionic charge–charge attractions mediated by weakly penetrating opposing PDs. In the end, we find that this landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration.

  7. Tau mediates microtubule bundle architectures mimicking fascicles of microtubules found in the axon initial segment

    DOE PAGES

    Chung, Peter J.; Song, Chaeyeon; Deek, Joanna; ...

    2016-07-25

    Tau, an intrinsically disordered protein confined to neuronal axons, binds to and regulates microtubule dynamics. Although there have been observations of string-like microtubule fascicles in the axon initial segment (AIS) and hexagonal bundles in neurite-like processes in non-neuronal cells overexpressing Tau, cell-free reconstitutions have not replicated either geometry. Here we map out the energy landscape of Tau-mediated, GTP-dependent ‘active’ microtubule bundles at 37°C, as revealed by synchrotron SAXS and TEM. Widely spaced bundles (wall-to-wall distance Dw–w≈25–41nm) with hexagonal and string-like symmetry are observed, the latter mimicking bundles found in the AIS. A second energy minimum (Dw–w≈16–23nm) is revealed under osmoticmore » pressure. The wide spacing results from a balance between repulsive forces, due to Tau’s projection domain (PD), and a stabilizing sum of transient sub-kBT cationic/anionic charge–charge attractions mediated by weakly penetrating opposing PDs. In the end, we find that this landscape would be significantly affected by charge-altering modifications of Tau associated with neurodegeneration.« less

  8. Velocity modulation of microtubules in electric fields.

    PubMed

    Dujovne, Irene; van den Heuvel, Martin; Shen, Yi; de Graaff, Martijn; Dekker, Cees

    2008-12-01

    We show that the speed of microtubules gliding over a kinesin-coated surface can be controlled over a wide range of values by the application of an electric field. The speed can be increased by up to a factor of 5 compared to the speed at zero field when assisting forces are applied and slowed down to zero velocity for opposing fields. Sideways applied fields also induce significant motion. The kinesin surface density impacts the rate of velocity change, whereas the ATP concentration does not seem to play a major role, provided that it is nonzero. A simple grab-and-release model is presented that explains the velocity change with applied electric fields.

  9. Diffusion of dextran inside microtubule sample

    NASA Astrophysics Data System (ADS)

    Prodan, Camelia

    2005-03-01

    Microtubules (Mts) are the bones of the cell. Their exterior has been extensively studied but little is known about their interior. We have studied the diffusion of fluorescein labeled dextran in the presence of GDP Mts and taxol stabilized GDP Mts. The diffusion coefficient, D, of different size dextran (10 kD, 40 kD, 70 kD, 500 kD) was measured using fluorescence recovery after photo-bleaching (FRAP). If dextran was present during the assembling of Mts, D was smaller then free diffusion coefficient. When dextran was added after the assembling, D was the same as the free diffusion coefficient. For taxol stabilized Mts (0.90 fill ratio), D was also found the same as the free diffusion coefficient .

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

    PubMed

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

    2015-11-21

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

  11. 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.

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

    PubMed

    Li, Zhendong; Liu, Wenjian

    2016-01-12

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

  13. Acentrosomal Microtubule Assembly in Mitosis: The Where, When, and How.

    PubMed

    Meunier, Sylvain; Vernos, Isabelle

    2016-02-01

    In mitosis the cell assembles the bipolar spindle, a microtubule (MT)-based apparatus that segregates the duplicated chromosomes into two daughter cells. Most animal cells enter mitosis with duplicated centrosomes that provide an active source of dynamic MTs. However, it is now established that spindle assembly relies on the nucleation of acentrosomal MTs occurring around the chromosomes after nuclear envelope breakdown, and on pre-existing microtubules. Where chromosome-dependent MT nucleation occurs, when MT amplification takes place and how the two pathways function are still key questions that generate some controversies. We reconcile the data and present an integrated model accounting for acentrosomal microtubule assembly in the dividing cell.

  14. Mitochondrial inheritance is mediated by microtubules in mammalian cell division.

    PubMed

    Lawrence, Elizabeth; Mandato, Craig

    2013-11-01

    The mitochondrial network fragments and becomes uniformly dispersed within the cytoplasm when mammalian cells enter mitosis. Such morphology and distribution of mitochondria was previously thought to facilitate the stochastic inheritance of mitochondria by daughter cells. In contrast, we recently reported that mitochondria in dividing mammalian cells are inherited by an ordered mechanism of inheritance mediated by microtubules. We showed that mitochondria are progressively enriched at the cell equator and depleted at the poles throughout division. Furthermore, the mitochondrial distribution during division is dependent on microtubules, indicating an ordered inheritance strategy. The microtubule-mediated positioning of mitochondria in dividing mammalian cells may have functional consequences for cell division and/or mitochondrial inheritance.

  15. Microtubule plus-end tracking proteins and their roles in cell division.

    PubMed

    Ferreira, Jorge G; Pereira, Ana L; Maiato, Helder

    2014-01-01

    Microtubules are cellular components that are required for a variety of essential processes such as cell motility, mitosis, and intracellular transport. This is possible because of the inherent dynamic properties of microtubules. Many of these properties are tightly regulated by a number of microtubule plus-end-binding proteins or +TIPs. These proteins recognize the distal end of microtubules and are thus in the right context to control microtubule dynamics. In this review, we address how microtubule dynamics are regulated by different +TIP families, focusing on how functionally diverse +TIPs spatially and temporally regulate microtubule dynamics during animal cell division.

  16. Detection and quantification of microtubule detachment from centrosomes and spindle poles.

    PubMed

    Ganguly, Anutosh; Yang, Hailing; Cabral, Fernando

    2013-01-01

    Microtubule detachment from microtubule organizing centers is an important cellular process required for normal cell proliferation. When cells enter mitosis, microtubule turnover increases along with a concurrent increase in microtubule detachment. MCAK, a kinesin-related protein whose abundance is highest during the early stages of mitosis, has been shown to regulate microtubule detachment. Abnormal increases or decreases in the frequency of detachment interfere with spindle function and inhibit cell division. It has been shown that drugs able to promote microtubule assembly (e.g., paclitaxel, epothilones) prevent cell division by suppressing microtubule detachment from centrosomes. Conversely, cytotoxic concentrations of microtubule destabilizing drugs (e.g., vinblastine, nocodazole), tubulin mutations that cause paclitaxel resistance, and specific β-tubulin isotypes increase the frequency of microtubule detachment. In this chapter, we describe a method to calculate the frequency of microtubule detachment by transfecting cells with EGFP-MAP4 and directly observing detachment by live cell imaging.

  17. Higgs boson couplings in multi-doublet models with natural flavour conservation

    NASA Astrophysics Data System (ADS)

    Yagyu, Kei

    2016-12-01

    We investigate the deviation in the couplings of the standard model (SM) like Higgs boson (h) with a mass of 125 GeV from the prediction of the SM in multi-doublet models within the framework where flavour changing neutral currents at the tree level are naturally forbidden. After we present the general expressions for the modified gauge and Yukawa couplings for h, we show the correlation between the deviation in the Yukawa coupling for the tau lepton hτ+τ- and that for the bottom quark hb b bar under the assumption of a non-zero deviation in the hVV (V = W , Z) couplings in two Higgs doublet models (2HDMs) and three Higgs doublet models (3HDMs) as simple examples. We clarify the possible allowed prediction of the deviations in the 3HDMs which cannot be explained in the 2HDMs even taking into account the one-loop electroweak corrections to the Yukawa coupling.

  18. Special Mixing of the Neutral Higgs Bosons States in the Standard Model with Two Higgs Doublets

    SciTech Connect

    Juarez W, S. R.; Morales C, D.

    2008-07-02

    The Higgs sector of the Standard Model (SM) requires careful investigation in order to look for new physics. In the SM the masses of the physical particles arise, after the spontaneous symmetry breaking (SSB), through its couplings with a single Higgs doublet. In the simplest extension of the SM, called the Two Higgs Doublet Model (2HDM), a second Higgs doublet is introduced, with a potential dependent on seven parameters, which are related to five Higgs bosons, whose existence is predicted by the model. In this context, we obtain the masses and the physical eigenstates of the new scalar particles: two charged ones (H{sup {+-}}) and three neutral (A{sup 0}), (h{sup 0},H{sup 0}). We explore a particular situation in which very simple relations between the parameters and the masses are satisfied.

  19. Effects of the KIF2C neck peptide on microtubules: lateral disintegration of microtubules and β-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 β-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.

  20. Study of Opto-Electronic Properties of a Single Microtubule in the Microwave Regime

    DTIC Science & Technology

    2011-12-11

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

  1. Conceptual design summary for modifying Doublet III to a large dee-shaped configuration

    SciTech Connect

    Davis, L.G.; Gallix, R.; Luxon, J.L.; Mahdavi, M.A.; Puhn, F.A.; Rock, P.J.; Wesley, J.C.

    1983-05-01

    The Doublet III tokamak is to be reconfigured by replacing its indented (doublet) vacuum vessel with a larger one of a dee-shaped cross section. This change will permit significantly larger elongated plasmas than is presently possible and will allow higher plasma current (up to 5 MA) and anticipated longer confinement time. Reactor relevant values of stable beta and plasma pressure are predicted. This modification, while resulting in a significant change in capability, utilizes most of the existing coils, structure, systems and facility.

  2. Search for a lighter Higgs boson in Two Higgs Doublet Models

    NASA Astrophysics Data System (ADS)

    Cacciapaglia, Giacomo; Deandrea, Aldo; Gascon-Shotkin, Suzanne; Le Corre, Solène; Lethuillier, Morgan; Tao, Junquan

    2016-12-01

    We consider present constraints on Two Higgs Doublet Models, both from the LHC at Run 1 and from other sources in order to explore the possibility of constraining a neutral scalar or pseudo-scalar particle lighter than the 125 GeV Higgs boson. Such a lighter particle is not yet completely excluded by present data. We show with a simplified analysis that some new constraints could be obtained at the LHC if such a search is performed by the experimental collaborations, which we therefore encourage to continue carrying out light diphoton resonance searches at √{s}=13 TeV in the context of Two Higgs Doublet Models.

  3. Three Extra Mirror or Sequential Families: Case for a Heavy Higgs Boson and Inert Doublet

    SciTech Connect

    Martinez, Homero; Melfo, Alejandra; Nesti, Fabrizio; Senjanovic, Goran

    2011-05-13

    We study the possibility of the existence of extra fermion families and an extra Higgs doublet. We find that requiring the extra Higgs doublet to be inert leaves space for three extra families, allowing for mirror fermion families and a dark matter candidate at the same time. The emerging scenario is very predictive: It consists of a standard model Higgs boson, with a mass above 400 GeV, heavy new quarks between 340 and 500 GeV, light extra neutral leptons, and an inert scalar with a mass below M{sub Z}.

  4. Monocycle and doublet pulses generation via photon echo in rare-earth-doped optical crystal

    NASA Astrophysics Data System (ADS)

    Huang, Kai; Zhang, Shuanggen; Ma, Xiurong; Hu, Minglie; Wang, Qingyue

    2016-12-01

    All-optical pulse generation opens up a field for ultrawideband (UWB) applications. However, controllable pulse width and pulse type are still challenging. Here, we present a theoretical model and stimulated results of monocycle and doublet waveforms generation using programmable optical photon echo progress. We synthesized instantaneously monocycle and doublet waveforms by adjustment of pulse width, pulse amplitude, pulse position, and time interval of subpulses. We verified the possible application of the proposed method to design U.S. Federal Communications Commission-compliant UWB waveforms, and therefore, it may provide an avenue for waveform generation.

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

    PubMed

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

    2015-01-30

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

  6. 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.

  7. Mechanism and Dynamics of Breakage of Fluorescent Microtubules

    PubMed Central

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

    2006-01-01

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

  8. 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

  9. 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.

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

    DOE PAGES

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

    2015-01-27

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

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

    SciTech Connect

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

    2015-01-27

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

  12. The physical basis of microtubule structure and stability.

    PubMed

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

    2003-10-01

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

  13. A theory of microtubule catastrophes and their regulation.

    PubMed

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

    2009-12-15

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

  14. Mechanisms to Avoid and Correct Erroneous Kinetochore-Microtubule Attachments

    PubMed Central

    Lampson, Michael A.; Grishchuk, Ekaterina L.

    2017-01-01

    In dividing vertebrate cells multiple microtubules must connect to mitotic kinetochores in a highly stereotypical manner, with each sister kinetochore forming microtubule attachments to only one spindle pole. The exact sequence of events by which this goal is achieved varies considerably from cell to cell because of the variable locations of kinetochores and spindle poles, and randomness of initial microtubule attachments. These chance encounters with the kinetochores nonetheless ultimately lead to the desired outcome with high fidelity and in a limited time frame, providing one of the most startling examples of biological self-organization. This chapter discusses mechanisms that contribute to accurate chromosome segregation by helping dividing cells to avoid and resolve improper microtubule attachments. PMID:28067761

  15. Trichomonas vaginalis: microtubule cytoskeleton distribution using fluorescent taxoid.

    PubMed

    Lecke, Sheila B; Tasca, Tiana; Souto, André A; De Carli, Geraldo A

    2002-10-01

    Trichomonas vaginalis is a flagellated parasitic protist of the human urogenital tract. The parasite has a poorly known cytoskeleton formed by an axostyle and a pelta, which are formed by stable structures such as microtubules, essential for the maintenance of cell shape and organization. FLUTAX-2 is an active fluorescent derivative of Taxol, binds to alphabeta-tubulin dimer polymerized. In this paper we present the analysis of microtubule distribution in living trophozoites of T. vaginalis using FLUTAX-2.

  16. EML proteins in microtubule regulation and human disease.

    PubMed

    Fry, Andrew M; O'Regan, Laura; Montgomery, Jessica; Adib, Rozita; Bayliss, Richard

    2016-10-15

    The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy.

  17. 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

  18. Dynein prevents erroneous kinetochore-microtubule attachments in mitosis.

    PubMed

    Barisic, Marin; Maiato, Helder

    2015-01-01

    Equal distribution of the genetic material during cell division relies on efficient congression of chromosomes to the metaphase plate. Prior to their alignment, the Dynein motor recruited to kinetochores transports a fraction of laterally-attached chromosomes along microtubules toward the spindle poles. By doing that, Dynein not only contributes to chromosome movements, but also prevents premature stabilization of end-on kinetochore-microtubule attachments. This is achieved by 2 parallel mechanisms: 1) Dynein-mediated poleward movement of chromosomes counteracts opposite polar-ejection forces (PEFs) on chromosome arms by the microtubule plus-end-directed motors chromokinesins. Otherwise, they could stabilize erroneous syntelic kinetochore-microtubule attachments and lead to the random ejection of chromosomes away from the spindle poles; and 2) By transporting chromosomes to the spindle poles, Dynein brings the former to the zone of highest Aurora A kinase activity, further destabilizing kinetochore-microtubule attachments. Thus, Dynein plays an important role in keeping chromosome segregation error-free by preventing premature stabilization of kinetochore-microtubule attachments near the spindle poles.

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

    PubMed Central

    Staiger, CJ; Cande, WZ

    1991-01-01

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

  20. Elastic response, buckling, and instability of microtubules under radial indentation.

    PubMed

    Schaap, Iwan A T; Carrasco, Carolina; de Pablo, Pedro J; MacKintosh, Frederick C; Schmidt, Christoph F

    2006-08-15

    We tested the mechanical properties of single microtubules by lateral indentation with the tip of an atomic force microscope. Indentations up to approximately 3.6 nm, i.e., 15% of the microtubule diameter, resulted in an approximately linear elastic response, and indentations were reversible without hysteresis. At an indentation force of around 0.3 nN we observed an instability corresponding to an approximately 1-nm indentation step in the taxol-stabilized microtubules, which could be due to partial or complete rupture of a relatively small number of lateral or axial tubulin-tubulin bonds. These indentations were reversible with hysteresis when the tip was retracted and no trace of damage was observed in subsequent high-resolution images. Higher forces caused substantial damage to the microtubules, which either led to depolymerization or, occasionally, to slowly reannealing holes in the microtubule wall. We modeled the experimental results using finite-element methods and find that the simple assumption of a homogeneous isotropic material, albeit structured with the characteristic protofilament corrugations, is sufficient to explain the linear elastic response of microtubules.

  1. Accelerated actin filament polymerization from microtubule plus-ends

    PubMed Central

    Henty-Ridilla, Jessica L.; Rankova, Aneliya; Eskin, Julian A.; Kenny, Katelyn; Goode, Bruce L.

    2016-01-01

    Microtubules govern actin network remodeling in a wide range of biological processes, yet the mechanisms underlying this cytoskeletal crosstalk have remained obscure. Here we used single-molecule fluorescence microscopy to show that the microtubule plus-end associated protein CLIP-170 binds tightly to formins to accelerate actin filament elongation. Furthermore, we observed mDia1 dimers and CLIP-170 dimers co-tracking growing filament ends for minutes. CLIP-170-mDia1 complexes promoted actin polymerization approximately 18 times faster than free barbed end growth, while simultaneously enhancing protection from capping protein. We used a microtubule-actin dynamics co-reconstitution system to observe CLIP-170-mDia1 complexes being recruited to growing microtubule ends by EB1. The complexes triggered rapid growth of actin filaments that remained attached to the microtubule surface. These activities of CLIP-170 were required in primary neurons for normal dendritic morphology. Thus, our results reveal a cellular mechanism whereby growing microtubule plus-ends direct rapid actin assembly. PMID:27199431

  2. Fluorescent markers of the microtubule cytoskeleton in Zymoseptoria tritici

    PubMed Central

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

    2015-01-01

    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 α-tubulin (ZtTub2), to ZtPeb1, a homologue of the mammalian plus-end binding protein EB1, and to ZtGrc1, a component of the minus-end located γ-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

  3. Cooperative lattice dynamics and anomalous fluctuations of microtubules.

    PubMed

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

    2012-02-01

    Microtubules have been in the focus of biophysical research for several decades. However, the confusing and mutually contradictory results regarding their elasticity and fluctuations have cast doubt on their present understanding. In this paper, we present the empirical evidence for the existence of discrete guanosine diphosphate (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-sized 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 remarkable curved-helical appearance in general. We point out that the multistability and cooperative switching of tubulin dimers could participate in important cellular processes, and could in particular lead to efficient mechanochemical signaling along single microtubules.

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

    NASA Astrophysics Data System (ADS)

    Regmi, Chola; Cheng, Shengfeng

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

  5. 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.

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

    PubMed

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

    2006-11-07

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

  7. Both microtubule-stabilizing and microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accumulation and activity by disrupting microtubule function.

    PubMed

    Escuin, Daniel; Kline, Erik R; Giannakakou, Paraskevi

    2005-10-01

    We have recently identified a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the hypoxia-inducible factor-1 (HIF-1) pathway. Based on this model, we hypothesized that other microtubule-targeting drugs may have a similar effect on HIF-1alpha. To test that hypothesis, we studied the effects of different clinically relevant microtubule-disrupting agents, including taxotere, epothilone B, discodermolide, vincristine, 2-methoxyestradiol, and colchicine. In all cases, HIF-1alpha protein, but not mRNA, was down-regulated in a drug dose-dependent manner. In addition, HIF-1alpha transcriptional activity was also inhibited by all drugs tested. To further examine whether these effects were dependent on microtubule network disruption, we tested the ability of epothilone B to inhibit HIF-1alpha protein in the human ovarian cancer cell line 1A9 and its beta-tubulin mutant epothilone-resistant subclone 1A9/A8. Our data showed that epothilone B treatment down-regulated HIF-1alpha protein in the parental 1A9 cells but had no effect in the resistant 1A9/A8 cells. These observations were confirmed by confocal microscopy, which showed impaired nuclear accumulation of HIF-1alpha in parental 1A9 cells at epothilone B concentrations that induced extensive microtubule stabilization. In contrast, epothilone B treatment had no effect on either microtubules or HIF-1alpha nuclear accumulation in the resistant 1A9/A8 cells. Furthermore, epothilone B inhibited HIF-1 transcriptional activity in 1A9 cells, as evidenced by a hypoxia response element-luciferase reporter assay, but had no effect on HIF-1 activity in the resistant 1A9/A8 cells. These data directly link beta-tubulin drug binding with HIF-1alpha protein inhibition. Our results further provide a strong rationale for testing taxanes and epothilones in clinical trials targeting HIF-1 in cancer patients.

  8. Dendrites In Vitro and In Vivo Contain Microtubules of Opposite Polarity and Axon Formation Correlates with Uniform Plus-End-Out Microtubule Orientation

    PubMed Central

    Yau, Kah Wai; Schätzle, Philipp; Tortosa, Elena; Pagès, Stéphane; Holtmaat, Anthony

    2016-01-01

    In cultured vertebrate neurons, axons have a uniform arrangement of microtubules with plus-ends distal to the cell body (plus-end-out), whereas dendrites contain mixed polarity orientations with both plus-end-out and minus-end-out oriented microtubules. Rather than non-uniform microtubules, uniparallel minus-end-out microtubules are the signature of dendrites in Drosophila and Caenorhabditis elegans neurons. To determine whether mixed microtubule organization is a conserved feature of vertebrate dendrites, we used live-cell imaging to systematically analyze microtubule plus-end orientations in primary cultures of rat hippocampal and cortical neurons, dentate granule cells in mouse organotypic slices, and layer 2/3 pyramidal neurons in the somatosensory cortex of living mice. In vitro and in vivo, all microtubules had a plus-end-out orientation in axons, whereas microtubules in dendrites had mixed orientations. When dendritic microtubules were severed by laser-based microsurgery, we detected equal numbers of plus- and minus-end-out microtubule orientations throughout the dendritic processes. In dendrites, the minus-end-out microtubules were generally more stable and comparable with plus-end-out microtubules in axons. Interestingly, at early stages of neuronal development in nonpolarized cells, newly formed neurites already contained microtubules of opposite polarity, suggesting that the establishment of uniform plus-end-out microtubules occurs during axon formation. We propose a model in which the selective formation of uniform plus-end-out microtubules in the axon is a critical process underlying neuronal polarization. SIGNIFICANCE STATEMENT Live-cell imaging was used to systematically analyze microtubule organization in primary cultures of rat hippocampal neurons, dentate granule cells in mouse organotypic slices, and layer 2/3 pyramidal neuron in somatosensory cortex of living mice. In vitro and in vivo, all microtubules have a plus-end-out orientation in axons

  9. Product lambda-doublet ratios as an imprint of chemical reaction mechanism

    NASA Astrophysics Data System (ADS)

    Jambrina, P. G.; Zanchet, A.; Aldegunde, J.; Brouard, M.; Aoiz, F. J.

    2016-11-01

    In the last decade, the development of theoretical methods has allowed chemists to reproduce and explain almost all of the experimental data associated with elementary atom plus diatom collisions. However, there are still a few examples where theory cannot account yet for experimental results. This is the case for the preferential population of one of the Λ-doublet states produced by chemical reactions. In particular, recent measurements of the OD(2Π) product of the O(3P)+D2 reaction have shown a clear preference for the Π(A') Λ-doublet states, in apparent contradiction with ab initio calculations, which predict a larger reactivity on the A'' potential energy surface. Here we present a method to calculate the Λ-doublet ratio when concurrent potential energy surfaces participate in the reaction. It accounts for the experimental Λ-doublet populations via explicit consideration of the stereodynamics of the process. Furthermore, our results demonstrate that the propensity of the Π(A') state is a consequence of the different mechanisms of the reaction on the two concurrent potential energy surfaces

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

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

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

    SciTech Connect

    De, Madhab Ray, Debasis

    2015-05-15

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

  12. Product lambda-doublet ratios as an imprint of chemical reaction mechanism

    PubMed Central

    Jambrina, P. G.; Zanchet, A.; Aldegunde, J.; Brouard, M.; Aoiz, F. J.

    2016-01-01

    In the last decade, the development of theoretical methods has allowed chemists to reproduce and explain almost all of the experimental data associated with elementary atom plus diatom collisions. However, there are still a few examples where theory cannot account yet for experimental results. This is the case for the preferential population of one of the Λ-doublet states produced by chemical reactions. In particular, recent measurements of the OD(2Π) product of the O(3P)+D2 reaction have shown a clear preference for the Π(A′) Λ-doublet states, in apparent contradiction with ab initio calculations, which predict a larger reactivity on the A′′ potential energy surface. Here we present a method to calculate the Λ-doublet ratio when concurrent potential energy surfaces participate in the reaction. It accounts for the experimental Λ-doublet populations via explicit consideration of the stereodynamics of the process. Furthermore, our results demonstrate that the propensity of the Π(A′) state is a consequence of the different mechanisms of the reaction on the two concurrent potential energy surfaces PMID:27834381

  13. Product lambda-doublet ratios as an imprint of chemical reaction mechanism.

    PubMed

    Jambrina, P G; Zanchet, A; Aldegunde, J; Brouard, M; Aoiz, F J

    2016-11-11

    In the last decade, the development of theoretical methods has allowed chemists to reproduce and explain almost all of the experimental data associated with elementary atom plus diatom collisions. However, there are still a few examples where theory cannot account yet for experimental results. This is the case for the preferential population of one of the Λ-doublet states produced by chemical reactions. In particular, recent measurements of the OD((2)Π) product of the O((3)P)+D2 reaction have shown a clear preference for the Π(A') Λ-doublet states, in apparent contradiction with ab initio calculations, which predict a larger reactivity on the A'' potential energy surface. Here we present a method to calculate the Λ-doublet ratio when concurrent potential energy surfaces participate in the reaction. It accounts for the experimental Λ-doublet populations via explicit consideration of the stereodynamics of the process. Furthermore, our results demonstrate that the propensity of the Π(A') state is a consequence of the different mechanisms of the reaction on the two concurrent potential energy surfaces.

  14. THE Na 8200 Angstrom-Sign DOUBLET AS AN AGE INDICATOR IN LOW-MASS STARS

    SciTech Connect

    Schlieder, Joshua E.; Simon, Michal; Lepine, Sebastien; Rice, Emily; Fielding, Drummond; Tomasino, Rachael E-mail: schlieder@mpia-hd.mpg.de E-mail: erice@amnh.org E-mail: tomas1r@cmich.edu

    2012-05-15

    We investigate the use of the gravity sensitive neutral sodium (Na I) doublet at 8183 Angstrom-Sign and 8195 Angstrom-Sign (Na 8200 Angstrom-Sign doublet) as an age indicator for M dwarfs. We measured the Na doublet equivalent width (EW) in giants, old dwarfs, young dwarfs, and candidate members of the {beta} Pic moving group using medium-resolution spectra. Our Na 8200 A doublet EW analysis shows that the feature is useful as an approximate age indicator in M-type dwarfs with (V - K{sub s}) {>=} 5.0, reliably distinguishing stars older and younger than 100 Myr. A simple derivation of the dependence of the Na EW on temperature and gravity supports the observational results. An analysis of the effects of metallicity shows that this youth indicator is best used on samples with similar metallicity. The age estimation technique presented here becomes useful in a mass regime where traditional youth indicators are increasingly less reliable, is applicable to other alkali lines, and will help identify new low-mass members in other young clusters and associations.

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

    NASA Astrophysics Data System (ADS)

    De, Madhab; Ray, Debasis

    2015-05-01

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

  16. Symmetry enriched U(1) topological orders for dipole-octupole doublets on a pyrochlore lattice

    NASA Astrophysics Data System (ADS)

    Li, Yao-Dong; Chen, Gang

    2017-01-01

    Symmetry plays a fundamental role in our understanding of both conventional symmetry breaking phases and the more exotic quantum and topological phases of matter. We explore the experimental signatures of symmetry enriched U(1) quantum spin liquids (QSLs) on the pyrochlore lattice. We point out that the Ce local moment of the newly discovered pyrochlore QSL candidate Ce2Sn2O7 , is a dipole-octupole doublet. The generic model for these unusual doublets supports two distinct symmetry enriched U(1) QSL ground states in the corresponding quantum spin ice regimes. These two U(1) QSLs are dubbed dipolar U(1) QSL and octupolar U(1) QSL. While the dipolar U(1) QSL has been discussed in many contexts, the octupolar U(1) QSL is rather unique. Based on the symmetry properties of the dipole-octupole doublets, we predict the peculiar physical properties of the octupolar U(1) QSL, elucidating the unique spectroscopic properties in the external magnetic fields. We further predict the Anderson-Higgs transition from the octupolar U(1) QSL driven by the external magnetic fields. We identify the experimental relevance with the candidate material Ce2Sn2O7 and other dipole-octupole doublet systems.

  17. 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)

  18. MAP65/Ase1 promote microtubule flexibility

    PubMed Central

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

    2013-01-01

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

  19. Microtubules, polarity and vertebrate neural tube morphogenesis.

    PubMed

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

    2016-07-01

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

  20. The dual specificity phosphatase Cdc14B bundles and stabilizes microtubules

    SciTech Connect

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

    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.

  1. Wave propagation in protein microtubules modeled as orthotropic elastic shells including transverse shear deformations.

    PubMed

    Daneshmand, Farhang; Ghavanloo, Esmaeal; Amabili, Marco

    2011-07-07

    Wave propagation along the microtubules is one of the issues of major concern in various microtubule cellular functions. In this study, the general wave propagation behavior in protein microtubules is investigated based on a first-order shear deformation shell theory for orthotropic materials, with particular emphasis on the role of strongly anisotropic elastic properties of microtubules. According to experimental observation, the first-order shear deformation theory is used for the modeling of microtubule walls. A general displacement representation is introduced and a type of coupled polynomial eigenvalue problem is developed. Numerical examples describe the effects of shear deformation and rotary inertia on wave velocities in orthotropic microtubules. Finally, the influences of the microtubule shear modulus, axial external force, effective thickness and material temperature dependency on wave velocities along the microtubule protofilaments, helical pathway and radial directions are elucidated. Most results presented in the present investigation have been absent from the literature for the wave propagation in microtubules.

  2. Kinesin-1 heavy chain mediates microtubule sliding to drive changes in cell shape.

    PubMed

    Jolly, Amber L; Kim, Hwajin; Srinivasan, Divya; Lakonishok, Margot; Larson, Adam G; Gelfand, Vladimir I

    2010-07-06

    Microtubules are typically observed to buckle and loop during interphase in cultured cells by an unknown mechanism. We show that lateral microtubule movement and looping is a result of microtubules sliding against one another in interphase Drosophila S2 cells. RNAi of the kinesin-1 heavy chain (KHC), but not dynein or the kinesin-1 light chain, eliminates these movements. KHC-dependent microtubule sliding powers the formation of cellular processes filled with parallel microtubule bundles. The growth of these cellular processes is independent of the actin cytoskeleton. We further observe cytoplasmic microtubule sliding in Xenopus and Ptk2 cells, and show that antibody inhibition of KHC in mammalian cells prevents sliding. We therefore propose that, in addition to its well established role in organelle transport, an important universal function of kinesin-1 is to mediate cytoplasmic microtubule-microtubule sliding. This provides the cell with a dedicated mechanism to transport long and short microtubule filaments and drive changes in cell shape.

  3. On the significance of microtubule flexural behavior in cytoskeletal mechanics.

    PubMed

    Mehrbod, Mehrdad; Mofrad, Mohammad R K

    2011-01-01

    Quantitative description of cell mechanics has challenged biological scientists for the past two decades. Various structural models have been attempted to analyze the structure of the cytoskeleton. One important aspect that has been largely ignored in all these modeling approaches is related to the flexural and buckling behavior of microtubular filaments. The objective of this paper is to explore the influence of this flexural and buckling behavior in cytoskeletal mechanics.In vitro the microtubules are observed to buckle in the first mode, reminiscent of a free, simply-supported beam. In vivo images of microtubules, however, indicate that the buckling mostly occurs in higher modes. This buckling mode switch takes place mostly because of the lateral support of microtubules via their connections to actin and intermediate filaments. These lateral loads are exerted throughout the microtubule length and yield a considerable bending behavior that, unless properly accounted for, would produce erroneous results in the modeling and analysis of the cytoskeletal mechanics.One of the promising attempts towards mechanical modeling of the cytoskeleton is the tensegrity model, which simplifies the complex network of cytoskeletal filaments into a combination merely of tension-bearing actin filaments and compression-bearing microtubules. Interestingly, this discrete model can qualitatively explain many experimental observations in cell mechanics. However, evidence suggests that the simplicity of this model may undermine the accuracy of its predictions, given the model's underlying assumption that "every single member bears solely either tensile or compressive behavior," i.e. neglecting the flexural behavior of the microtubule filaments. We invoke an anisotropic continuum model for microtubules and compare the bending energy stored in a single microtubule with its axial strain energy at the verge of buckling. Our results suggest that the bending energy can exceed the axial energy

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

    PubMed

    Coffe, G; Raymond, M N

    1990-01-01

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

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

    PubMed Central

    Hamada, Takahiro

    2014-01-01

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

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

    PubMed

    Hamada, Takahiro

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  8. 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.

  9. Doublet Versus Single Agent as Second-Line Treatment for Advanced Gastric Cancer

    PubMed Central

    Zhang, Yong; Ma, Bing; Huang, Xiao-Tian; Li, Yan-Song; Wang, Yu; Liu, Zhou-Lu

    2016-01-01

    Abstract The purpose of this study was to perform a meta-analysis of randomized controlled trials (RCTs) to compare the efficacy and safety of doublet versus single agent as second-line treatment for advanced gastric cancer (AGC). A comprehensive literature search was performed to identify relevant RCTs. All clinical studies were independently identified by 2 authors for inclusion. Demographic data, treatment regimens, objective response rate (ORR), and progression-free survival (PFS) and overall survival (OS) were extracted and analyzed using Comprehensive Meta-Analysis software (Version 2.0). Ten RCTs involving 1698 pretreated AGC patients were ultimately identified. The pooled results demonstrated that doublet combination therapy as second-line treatment for AGC significantly improved OS (hazard ratio [HR] 0.87, 95% confidence interval [CI]: 0.78–0.97, P = 0.011), PFS (HR 0.79, 95% CI: 0.72–0.87, P < 0.001), and ORR (relative risk [RR] 1.57, 95% CI: 1.27–1.95, P < 0.001). Sub-group analysis according to treatment regimens also showed that targeted agent plus chemotherapy significantly improve OS, PFS, and ORR. However, no significant survival benefits had been observed in doublet cytotoxic chemotherapy when compared with single cytotoxic agent. Additionally, more incidences of grade 3 or 4 myelosuppression toxicities, diarrhea, and fatigue were observed in doublet combination groups, while equivalent frequencies of grade 3 or 4 thrombocytopenia and nausea were found between the 2 groups. In comparison with single cytotoxic agent alone, the addition of targeted agent to mono-chemotherapy as salvage treatment for pretreated AGC patients provide substantial survival benefits, while no significant survival benefits were observed in doublet cytotoxic chemotherapy regimens. PMID:26937908

  10. Experimental comparative study of doublet and triplet impinging atomization of gelled fuel based on PIV

    NASA Astrophysics Data System (ADS)

    Yang, Jian-lu; Li, Ning; Weng, Chun-sheng

    2016-10-01

    Gelled propellant is promising for future aerospace application because of its combination of the advantages of solid propellants and liquid propellants. An effort was made to reveal the atomization properties of gelled fuel by particle image velocimetry (PIV) system. The gelled fuel which was formed by gasoline and Nano-silica was atomized using a like-doublet impingement injector and an axisymmetric like-triplet impingement injector. The orifice diameter and length of the nozzle used in this work were of 0.8mm, 4.8mm, respectively. In the impinging spray process, the impingement angles were set at 90° and 120°, and the injection pressures were of 0.50MPa and 1.00MPa. The distance from the exit of the orifice to the impingement point was fixed at 9.6mm. In this study, high-speed visualization and temporal resolution particle image velocimetry techniques were employed to investigate the impingement atomization characteristics. The experimental investigation demonstrated that a long narrow high speed droplets belt formed around the axis of symmetry in the like-doublet impinging atomization area. However, there was no obvious high-speed belt with impingement angle 2θ = 90° and two high-speed belts appeared with impingement angle 2θ = 120° in the like-doublet impingement spray field. The high droplet velocity zone of the like-doublet impingement atomization symmetrically distributed around the central axis, and that of the like-triplet impingement spray deflected to the left of the central axis - opposite of injector. Although the droplets velocity distribution was asymmetry of like-triplet impingement atomization, the injectors were arranged like axisymmetric conical shape, and the cross section of spray area was similar to a circle rather than a narrow rectangle like the like-doublet impingement atomization.

  11. Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1.

    PubMed

    Tikhonenko, Irina; Irizarry, Karen; Khodjakov, Alexey; Koonce, Michael P

    2016-02-01

    It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm.

  12. Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1

    PubMed Central

    Tikhonenko, Irina; Irizarry, Karen; Khodjakov, Alexey; Koonce, Michael P.

    2015-01-01

    It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm. PMID:26298292

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

    PubMed

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

    1997-08-01

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

  14. 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.

  15. 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.

  16. Automated Stitching of Microtubule Centerlines across Serial Electron Tomograms

    PubMed Central

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

    2014-01-01

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

  17. Oscillatory Fluid Flow Influences Primary Cilia and Microtubule Mechanics

    PubMed Central

    Espinha, Lina C.; Hoey, David A.; Fernandes, Paulo R.; Rodrigues, Hélder C.; Jacobs, Christopher R.

    2014-01-01

    Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity. PMID:25044764

  18. Characterization of the role of calcium in regulating the microtubule-destabilizing activity of MDP25

    PubMed Central

    Qin, Tao; Li, Jiejie; Yuan, Ming; Mao, Tonglin

    2012-01-01

    Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins.1 Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.2 PMID:22751329

  19. 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.

  20. Biomolecular motor modulates mechanical property of microtubule.

    PubMed

    Kabir, Arif Md Rashedul; Inoue, Daisuke; Hamano, Yoshimi; Mayama, Hiroyuki; Sada, Kazuki; Kakugo, Akira

    2014-05-12

    The microtubule (MT) is the stiffest cytoskeletal filamentous protein that takes part in a wide range of cellular activities where its mechanical property plays a crucially significant role. How a single biological entity plays multiple roles in cell has been a mystery for long time. Over the recent years, it has been known that modulation of the mechanical property of MT by different cellular agents is the key to performing manifold in vivo activities by MT. Studying the mechanical property of MT thus has been a prerequisite in understanding how MT plays such diversified in vivo roles. However, the anisotropic structure of MT has been an impediment in obtaining a precise description of the mechanical property of MT along its longitudinal and lateral directions that requires employment of distinct experimental approach and has not been demonstrated yet. In this work, we have developed an experimental system that enabled us to investigate the effect of tensile stress on MT. By using our newly developed system, (1) we have determined the Young's modulus of MT considering its deformation under applied tensile stress and (2) a new role of MT associated motor protein kinesin in modulating the mechanical property of MT was revealed for the first time. Decrease in Young's modulus of MT with the increase in interaction with kinesin suggests that kinesin has a softening effect on MT and thereby can modulate the rigidity of MT. This work will be an aid in understanding the modulation of mechanical property of MTs by MT associated proteins and might also help obtain a clear insight of the endurance and mechanical instability of MTs under applied stress.

  1. Microtubule guidance tested through controlled cell geometry

    PubMed Central

    Huda, Sabil; Soh, Siowling; Pilans, Didzis; Byrska-Bishop, Marta; Kim, Jiwon; Wilk, Gary; Borisy, Gary G.; Kandere-Grzybowska, Kristiana; Grzybowski, Bartosz A.

    2012-01-01

    Summary In moving cells dynamic microtubules (MTs) target and disassemble substrate adhesion sites (focal adhesions; FAs) in a process that enables the cell to detach from the substrate and propel itself forward. The short-range interactions between FAs and MT plus ends have been observed in several experimental systems, but the spatial overlap of these structures within the cell has precluded analysis of the putative long-range mechanisms by which MTs growing through the cell body reach FAs in the periphery of the cell. In the work described here cell geometry was controlled to remove the spatial overlap of cellular structures thus allowing for unambiguous observation of MT guidance. Specifically, micropatterning of living cells was combined with high-resolution in-cell imaging and gene product depletion by means of RNA interference to study the long-range MT guidance in quantitative detail. Cells were confined on adhesive triangular microislands that determined cell shape and ensured that FAs localized exclusively at the vertices of the triangular cells. It is shown that initial MT nucleation at the centrosome is random in direction, while the alignment of MT trajectories with the targets (i.e. FAs at vertices) increases with an increasing distance from the centrosome, indicating that MT growth is a non-random, guided process. The guided MT growth is dependent on the presence of FAs at the vertices. The depletion of either myosin IIA or myosin IIB results in depletion of F-actin bundles and spatially unguided MT growth. Taken together our findings provide quantitative evidence of a role for long-range MT guidance in MT targeting of FAs. PMID:22992457

  2. Buckling behavior of individual and bundled microtubules.

    PubMed

    Soheilypour, Mohammad; Peyro, Mohaddeseh; Peter, Stephen J; Mofrad, Mohammad R K

    2015-04-07

    As the major structural constituent of the cytoskeleton, microtubules (MTs) serve a variety of biological functions that range from facilitating organelle transport to maintaining the mechanical integrity of the cell. Neuronal MTs exhibit a distinct configuration, hexagonally packed bundles of MT filaments, interconnected by MT-associated protein (MAP) tau. Building on our previous work on mechanical response of axonal MT bundles under uniaxial tension, this study is focused on exploring the compression scenarios. Intracellular MTs carry a large fraction of the compressive loads sensed by the cell and therefore, like any other column-like structure, are prone to substantial bending and buckling. Various biological activities, e.g., actomyosin contractility and many pathological conditions are driven or followed by bending, looping, and buckling of MT filaments. The coarse-grained model previously developed in our lab has been used to study the mechanical behavior of individual and bundled in vivo MT filaments under uniaxial compression. Both configurations show tip-localized, decaying, and short-wavelength buckling. This behavior highlights the role of the surrounding cytoplasm and MAP tau on MT buckling behavior, which allows MT filaments to bear much larger compressive forces. It is observed that MAP tau interconnections improve this effect by a factor of two. The enhanced ability of MT bundles to damp buckling waves relative to individual MT filaments, may be interpreted as a self-defense mechanism because it helps axonal MTs to endure harsher environments while maintaining their function. The results indicate that MT filaments in a bundle do not buckle simultaneously implying that the applied stress is not equally shared among the MT filaments, that is a consequence of the nonuniform distribution of MAP tau proteins along the bundle length. Furthermore, from a pathological perspective, it is observed that axonal MT bundles are more vulnerable to failure in

  3. Buckling Behavior of Individual and Bundled Microtubules

    PubMed Central

    Soheilypour, Mohammad; Peyro, Mohaddeseh; Peter, Stephen J.; Mofrad, Mohammad R.K.

    2015-01-01

    As the major structural constituent of the cytoskeleton, microtubules (MTs) serve a variety of biological functions that range from facilitating organelle transport to maintaining the mechanical integrity of the cell. Neuronal MTs exhibit a distinct configuration, hexagonally packed bundles of MT filaments, interconnected by MT-associated protein (MAP) tau. Building on our previous work on mechanical response of axonal MT bundles under uniaxial tension, this study is focused on exploring the compression scenarios. Intracellular MTs carry a large fraction of the compressive loads sensed by the cell and therefore, like any other column-like structure, are prone to substantial bending and buckling. Various biological activities, e.g., actomyosin contractility and many pathological conditions are driven or followed by bending, looping, and buckling of MT filaments. The coarse-grained model previously developed in our lab has been used to study the mechanical behavior of individual and bundled in vivo MT filaments under uniaxial compression. Both configurations show tip-localized, decaying, and short-wavelength buckling. This behavior highlights the role of the surrounding cytoplasm and MAP tau on MT buckling behavior, which allows MT filaments to bear much larger compressive forces. It is observed that MAP tau interconnections improve this effect by a factor of two. The enhanced ability of MT bundles to damp buckling waves relative to individual MT filaments, may be interpreted as a self-defense mechanism because it helps axonal MTs to endure harsher environments while maintaining their function. The results indicate that MT filaments in a bundle do not buckle simultaneously implying that the applied stress is not equally shared among the MT filaments, that is a consequence of the nonuniform distribution of MAP tau proteins along the bundle length. Furthermore, from a pathological perspective, it is observed that axonal MT bundles are more vulnerable to failure in

  4. Nonlinear amplitude dynamics in flagellar beating

    PubMed Central

    Casademunt, Jaume

    2017-01-01

    The physical basis of flagellar and ciliary beating is a major problem in biology which is still far from completely understood. The fundamental cytoskeleton structure of cilia and flagella is the axoneme, a cylindrical array of microtubule doublets connected by passive cross-linkers and dynein motor proteins. The complex interplay of these elements leads to the generation of self-organized bending waves. Although many mathematical models have been proposed to understand this process, few attempts have been made to assess the role of dyneins on the nonlinear nature of the axoneme. Here, we investigate the nonlinear dynamics of flagella by considering an axonemal sliding control mechanism for dynein activity. This approach unveils the nonlinear selection of the oscillation amplitudes, which are typically either missed or prescribed in mathematical models. The explicit set of nonlinear equations are derived and solved numerically. Our analysis reveals the spatio-temporal dynamics of dynein populations and flagellum shape for different regimes of motor activity, medium viscosity and flagellum elasticity. Unstable modes saturate via the coupling of dynein kinetics and flagellum shape without the need of invoking a nonlinear axonemal response. Hence, our work reveals a novel mechanism for the saturation of unstable modes in axonemal beating.

  5. Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins

    NASA Astrophysics Data System (ADS)

    Namdeo, S.; Onck, P. R.

    2016-10-01

    Flagella are hair-like projections from the surface of eukaryotic cells, and they play an important role in many cellular functions, such as cell-motility. The beating of flagella is enabled by their internal architecture, the axoneme, and is powered by a dense distribution of motor proteins, dyneins. The dyneins deliver the required mechanical work through the hydrolysis of ATP. Although the dynein-ATP cycle, the axoneme microstructure, and the flagellar-beating kinematics are well studied, their integration into a coherent picture of ATP-powered flagellar beating is still lacking. Here we show that a time-delayed negative-work-based switching mechanism is able to convert the individual sliding action of hundreds of dyneins into a regular overall beating pattern leading to propulsion. We developed a computational model based on a minimal representation of the axoneme consisting of two representative doublet microtubules connected by nexin links. The relative sliding of the microtubules is incorporated by modeling two groups of ATP-powered dyneins, each responsible for sliding in opposite directions. A time-delayed switching mechanism is postulated, which is key in converting the local individual sliding action of multiple dyneins into global beating. Our results demonstrate that an overall nonreciprocal beating pattern can emerge with time due to the spatial and temporal coordination of the individual dyneins. These findings provide insights in the fundamental working mechanism of axonemal dyneins and could possibly open new research directions in the field of flagellar motility.

  6. Molecular crowding creates traffic jams of kinesin motors on microtubules.

    PubMed

    Leduc, Cécile; Padberg-Gehle, Kathrin; Varga, Vladimír; Helbing, Dirk; Diez, Stefan; Howard, Jonathon

    2012-04-17

    Despite the crowdedness of the interior of cells, microtubule-based motor proteins are able to deliver cargoes rapidly and reliably throughout the cytoplasm. We hypothesize that motor proteins may be adapted to operate in crowded environments by having molecular properties that prevent them from forming traffic jams. To test this hypothesis, we reconstituted high-density traffic of purified kinesin-8 motor protein, a highly processive motor with long end-residency time, along microtubules in a total internal-reflection fluorescence microscopy assay. We found that traffic jams, characterized by an abrupt increase in the density of motors with an associated abrupt decrease in motor speed, form even in the absence of other obstructing proteins. To determine the molecular properties that lead to jamming, we altered the concentration of motors, their processivity, and their rate of dissociation from microtubule ends. Traffic jams occurred when the motor density exceeded a critical value (density-induced jams) or when motor dissociation from the microtubule ends was so slow that it resulted in a pileup (bottleneck-induced jams). Through comparison of our experimental results with theoretical models and stochastic simulations, we characterized in detail under which conditions density- and bottleneck-induced traffic jams form or do not form. Our results indicate that transport kinesins, such as kinesin-1, may be evolutionarily adapted to avoid the formation of traffic jams by moving only with moderate processivity and dissociating rapidly from microtubule ends.

  7. Tubulin tyrosine nitration regulates microtubule organization in plant cells

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. Microtubules contribute to maintain nucleus shape in epithelial cell monolayer

    NASA Astrophysics Data System (ADS)

    Tremblay, Dominique; Andrzejewski, Lukasz; Pelling, Andrew

    2013-03-01

    INTRODUCTION: Tissue strains can result in significant nuclear deformations and may regulate gene expression. However, the precise role of the cytoskeleton in regulating nuclear mechanics remains poorly understood. Here, we investigate the nuclear deformability of Madin-Darky canine kidney cells (MDCK) under various stretching conditions to clarify the role of the microtubules and actin network on the mechanical behavior of the nucleus. METHODS: A custom-built cell-stretching device allowing for real time imaging of MDCK nuclei was used. Cells were seeded on a silicone membrane coated with rat-tail collagen I. A nuclear stain, Hoechst-33342, was used to image nuclei during stretching. We exposed cells to a compressive and non-compressive stretching strain field of 25%. Nocodazole and cytochalasin-D were used to depolymerize the microtubules and actin network. RESULTS: Nuclei in control cells stretched more along their minor axis than major axis with a deformation of 5% and 2% respectively. This anisotropy vanished completely in microtubule-deprived cells and these cells showed a very high nuclear deformability along the minor axis when exposed to a compressive stretching strain field. CONCLUSIONS: The microtubules drive the anisotropic deformability of MDCK nuclei in a monolayer and maintain nuclear shape when exposed to compressive strain. Such intrinsic mechanical behavior indicates that microtubules are essential to maintain nuclear shape and may prevent down regulation of gene expression.

  10. SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

    PubMed Central

    Raff, Rudolf A.; Greenhouse, Gerald; Gross, Kenneth W.; Gross, Paul R.

    1971-01-01

    Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-3H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified. PMID:5165266

  11. Molecular crowding creates traffic jams of kinesin motors on microtubules

    PubMed Central

    Leduc, Cécile; Padberg-Gehle, Kathrin; Varga, Vladimír; Helbing, Dirk; Diez, Stefan; Howard, Jonathon

    2012-01-01

    Despite the crowdedness of the interior of cells, microtubule-based motor proteins are able to deliver cargoes rapidly and reliably throughout the cytoplasm. We hypothesize that motor proteins may be adapted to operate in crowded environments by having molecular properties that prevent them from forming traffic jams. To test this hypothesis, we reconstituted high-density traffic of purified kinesin-8 motor protein, a highly processive motor with long end-residency time, along microtubules in a total internal-reflection fluorescence microscopy assay. We found that traffic jams, characterized by an abrupt increase in the density of motors with an associated abrupt decrease in motor speed, form even in the absence of other obstructing proteins. To determine the molecular properties that lead to jamming, we altered the concentration of motors, their processivity, and their rate of dissociation from microtubule ends. Traffic jams occurred when the motor density exceeded a critical value (density-induced jams) or when motor dissociation from the microtubule ends was so slow that it resulted in a pileup (bottleneck-induced jams). Through comparison of our experimental results with theoretical models and stochastic simulations, we characterized in detail under which conditions density- and bottleneck-induced traffic jams form or do not form. Our results indicate that transport kinesins, such as kinesin-1, may be evolutionarily adapted to avoid the formation of traffic jams by moving only with moderate processivity and dissociating rapidly from microtubule ends. PMID:22431622

  12. Comparison of ciliature microtubule organelles in three hypotrichous ciliate species

    NASA Astrophysics Data System (ADS)

    Li, Yisong; Shi, Lei; Gu, Fukang

    2010-05-01

    We examined the structure and spatial organization of ciliature base-associated microtubules (BAM) in three hypotrichous ciliates ( Stylonychia mytilus, Pseudourostyla cristata, Euplotes woodruffi) in fluorescence microscopy. The results revealed that BAM, including the anterior (ALM), posterior longitudinal microtubule (PLM) and the transverse microtubule (TM) bands, are composed of tubulin. The respective microtubular bands have cytoplasmic polarization patterns that are significantly asymmetric. The BAM of the midventral files in P. cristata appear cord-shaped compared with the ALM bands of transverse cirri in both S. mytilus and E. woodruffi, which extend to the left anterior side of the cell before converging. The TM bands of the left marginal cirri (MC) in S. mytilus extend to the right side of the cell, while those of the right MC bands extend to the left. Our observations suggest that BAM traits are common in hypotrichous ciliates even though different species possess different microtubule arrangements related to the conserved cirral morphogenetic patterns in the respective species. The differing development of BAM in the three ciliate suggests that the microtubules may be conserved in different hypotrichs. We have also demonstrated that the BAM, which appear polar and asymmetric, are localized in specific cytoskeletal positions and extend in different orientations within the cortex to connect with other ciliature-associated structures and, thus, strengthen the cortex. These BAM features indicate that they are directly associated with cell motion.

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

    NASA Astrophysics Data System (ADS)

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

    2004-09-01

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

  14. Microtubule aging probed by microfluidics-assisted tubulin washout

    PubMed Central

    Duellberg, Christian; Cade, Nicholas Ian; Surrey, Thomas

    2016-01-01

    Microtubules switch stochastically between phases of growth and shrinkage. The molecular mechanism responsible for the end of a growth phase, an event called catastrophe, is still not understood. The probability for a catastrophe to occur increases with microtubule age, putting constraints on the possible molecular mechanism of catastrophe induction. Here we used microfluidics-assisted fast tubulin washout experiments to induce microtubule depolymerization in a controlled manner at different times after the start of growth. We found that aging can also be observed in this assay, providing valuable new constraints against which theoretical models of catastrophe induction can be tested. We found that the data can be quantitatively well explained by a simple kinetic threshold model that assumes an age-dependent broadening of the protective cap at the microtubule end as a result of an evolving tapered end structure; this leads to a decrease of the cap density and its stability. This analysis suggests an intuitive picture of the role of morphological changes of the protective cap for the age dependence of microtubule stability. PMID:27489342

  15. The feasibility of coherent energy transfer in microtubules.

    PubMed

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

    2014-11-06

    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.

  16. Diffusive Movement of Processive Kinesin-1 on Microtubules

    PubMed Central

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

    2009-01-01

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

  17. A Quantitative Method for Microtubule Analysis in Fluorescence Images.

    PubMed

    Lan, Xiaodong; Li, Lingfei; Hu, Jiongyu; Zhang, Qiong; Dang, Yongming; Huang, Yuesheng

    2015-12-01

    Microtubule analysis is of significant value for a better understanding of normal and pathological cellular processes. Although immunofluorescence microscopic techniques have proven useful in the study of microtubules, comparative results commonly rely on a descriptive and subjective visual analysis. We developed an objective and quantitative method based on image processing and analysis of fluorescently labeled microtubular patterns in cultured cells. We used a multi-parameter approach by analyzing four quantifiable characteristics to compose our quantitative feature set. Then we interpreted specific changes in the parameters and revealed the contribution of each feature set using principal component analysis. In addition, we verified that different treatment groups could be clearly discriminated using principal components of the multi-parameter model. High predictive accuracy of four commonly used multi-classification methods confirmed our method. These results demonstrated the effectiveness and efficiency of our method in the analysis of microtubules in fluorescence images. Application of the analytical methods presented here provides information concerning the organization and modification of microtubules, and could aid in the further understanding of structural and functional aspects of microtubules under normal and pathological conditions.

  18. Microtubule aging probed by microfluidics-assisted tubulin washout.

    PubMed

    Duellberg, Christian; Cade, Nicholas Ian; Surrey, Thomas

    2016-11-07

    Microtubules switch stochastically between phases of growth and shrinkage. The molecular mechanism responsible for the end of a growth phase, an event called catastrophe, is still not understood. The probability for a catastrophe to occur increases with microtubule age, putting constraints on the possible molecular mechanism of catastrophe induction. Here we used microfluidics-assisted fast tubulin washout experiments to induce microtubule depolymerization in a controlled manner at different times after the start of growth. We found that aging can also be observed in this assay, providing valuable new constraints against which theoretical models of catastrophe induction can be tested. We found that the data can be quantitatively well explained by a simple kinetic threshold model that assumes an age-dependent broadening of the protective cap at the microtubule end as a result of an evolving tapered end structure; this leads to a decrease of the cap density and its stability. This analysis suggests an intuitive picture of the role of morphological changes of the protective cap for the age dependence of microtubule stability.

  19. 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.

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

    PubMed

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

    1998-08-01

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

  1. 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.

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

    PubMed

    Jordan, M A

    2002-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  4. 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 γ-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 μm 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

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

    PubMed Central

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

    2016-01-01

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

  6. Recycling of cold-stable microtubules: evidence that cold stability is due to substoichiometric polymer blocks.

    PubMed

    Job, D; Rauch, C T; Fischer, E H; Margolis, R L

    1982-02-02

    A substantial subpopulation of mammalian brain crude extract microtubules is resistant to cold-temperature disassembly. We propose here that microtubules are rendered cold stable by rare substoichiometric blocks. Mild shearing of rat brain cold-stable microtubules makes them largely cold labile. In addition, cold-stable microtubules can be destabilized by exposure to low concentrations of calmodulin (5 microM) in the presence of calcium at 0 degree C. Cold-disassembled microtubule protein, obtained from sheared or calmodulin-treated cold-stable preparations, re-forms a cold-stable subpopulation upon reassembly. These observations allow strategies for the recycling purification of cold-stable microtubules. Comparison of purified cold-labile and cold-stable material by gel electrophoresis shows enrichment for a few unique polypeptides, of 135, 70-82, and 56 kilodaltons, in the cold-stable preparation. The 64-kilodalton "switch protein", previously identified as uniquely dephosphorylated in cold-stable microtubules, is equally represented in recycled cold-stable and cold-labile microtubule preparations. Furthermore, when disassembled, cold-stable microtubule proteins are passed through a calmodulin affinity column on which the polypeptides characteristic of cold-stable microtubules are specifically retained, the breakthrough (unbound) material repolymerizes into cold-labile microtubules only. Based on the above data, a model is presented in which microtubules are rendered cold stable by the presence of substoichiometric, calmodulin-sensitive blocks that randomly reshuffle upon reassembly of cold-stable microtubules.

  7. Wave equations, dispersion relations, and van Hove singularities for applications of doublet mechanics to ultrasound propagation in bio- and nanomaterials.

    PubMed

    Wu, Junru; Layman, Christopher; Liu, Jun

    2004-02-01

    A fundamental mathematical framework for applications of Doublet Mechanics to ultrasound propagation in a discrete material is introduced. A multiscale wave equation, dispersion relation for longitudinal waves, and shear waves are derived. The van Hove singularities and corresponding highest frequency limits for the Mth-order wave equations of longitudinal and shear waves are determined for a widely used microbundle structure. Doublet Mechanics is applied to soft tissue and low-density polyethylene. The experimental dispersion data for soft tissue and low-density polyethylene are compared with results predicted by Doublet Mechanics and an attenuation model based on a Kramers-Kronig relation in classical continuum mechanics.

  8. Wave equations, dispersion relations, and van Hove singularities for applications of doublet mechanics to ultrasound propagation in bio- and nanomaterials

    NASA Astrophysics Data System (ADS)

    Wu, Junru; Layman, Christopher; Liu, Jun

    2004-02-01

    A fundamental mathematical framework for applications of Doublet Mechanics to ultrasound propagation in a discrete material is introduced. A multiscale wave equation, dispersion relation for longitudinal waves, and shear waves are derived. The van Hove singularities and corresponding highest frequency limits for the Mth-order wave equations of longitudinal and shear waves are determined for a widely used microbundle structure. Doublet Mechanics is applied to soft tissue and low-density polyethylene. The experimental dispersion data for soft tissue and low-density polyethylene are compared with results predicted by Doublet Mechanics and an attenuation model based on a Kramers-Kronig relation in classical continuum mechanics.

  9. SCG10, a microtubule destabilizing factor, stimulates the neurite outgrowth by modulating microtubule dynamics in rat hippocampal primary cultured neurons.

    PubMed

    Morii, Hiroshi; Shiraishi-Yamaguchi, Yoko; Mori, Nozomu

    2006-09-01

    Microtubule dynamics, one of the key elements in neurite outgrowth, is regulated by various regulatory factors to determine the behavior of the neuronal growth cone and to form the specialized neuronal shape. SCG10 is a neuron-specific stathmin protein with a potent microtubule destabilizing factor and is enriched in the growth cones of the developing neurons. We investigated the functional role of SCG10 in neurite outgrowth using rat hippocampal primary cultured neurons. Genetic manipulation of SCG10 using a short-interfering RNA duplex markedly decreased the SCG10 expression level and significantly suppressed neurite outgrowth. This result was confirmed by immunodepletion experiments. On the other hand, the protein transduction of SCG10 using a polyarginine tag stimulated neurite outgrowth. Such manipulation of the SCG10 expression level affected microtubule morphology within the growth cones. A decrease in the SCG10 level converted the morphology to a more stable state, while an increase converted the morphology to a more dynamic state. However, an excess of SCG10 induced neurite retraction due to an excess of microtubule disassembly. These results suggest that SCG10 serves as an important regulatory factor of growth cone motility by enhancing microtubule dynamics, possibly through increasing the catastrophe frequency.

  10. Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation

    PubMed Central

    Winans, Amy M; Collins, Sean R; Meyer, Tobias

    2016-01-01

    Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon. DOI: http://dx.doi.org/10.7554/eLife.12387.001 PMID:26836307

  11. Mitochondrial inheritance is mediated by microtubules in mammalian cell division

    PubMed Central

    Lawrence, Elizabeth; Mandato, Craig

    2013-01-01

    The mitochondrial network fragments and becomes uniformly dispersed within the cytoplasm when mammalian cells enter mitosis. Such morphology and distribution of mitochondria was previously thought to facilitate the stochastic inheritance of mitochondria by daughter cells. In contrast, we recently reported that mitochondria in dividing mammalian cells are inherited by an ordered mechanism of inheritance mediated by microtubules. We showed that mitochondria are progressively enriched at the cell equator and depleted at the poles throughout division. Furthermore, the mitochondrial distribution during division is dependent on microtubules, indicating an ordered inheritance strategy. The microtubule-mediated positioning of mitochondria in dividing mammalian cells may have functional consequences for cell division and/or mitochondrial inheritance. PMID:24567781

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

    PubMed

    Nédélec, François

    2002-09-16

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

  13. Tubulin carbamoylation. Functional amino groups in microtubule assembly.

    PubMed Central

    Mellado, W; Slebe, J C; Maccioni, R B

    1982-01-01

    The characteristics of the carbamoylation of pig brain tubulin were examined by using the modification conditions with cyanate described previously [Mellado, Slebe + Maccioni (1980) Biochem. Int. I, 584--590]. The carbamoylation reaction resulted in an inhibition of microtubule assembly, which was dependent on the concentration of the modifying agent. This tubulin modification appears to inhibit the growth of microtubules. The presence of GTP did not protect tubulin against this inhibition. Electron microscopy showed a marked decrease in the number of tubules after carbamoylation, but no alterations were observed in the microtubule morphology. The incorporation of KN14CO into alpha- and beta-subunits with similar kinetics was also shown, and the carbamoylated residues were identified as epsilon-N-carbamoyl-lysine residues. Images PLATE 1 Fig. 4. PMID:7115308

  14. Microtubule motors involved in nuclear movement during skeletal muscle differentiation.

    PubMed

    Gache, V; Gomes, E R; Cadot, B

    2017-04-01

    Nuclear positioning is a determining event in several cellular processes, such as fertilization, cell migration, and cell differentiation. The structure and function of muscle cells, which contain hundreds of nuclei, have been shown to rely in part on proper nuclear positioning. Remarkably, in the course of muscle differentiation, nuclear movements along the myotube axis might represent the event required for the even positioning of nuclei in the mature myofiber. Here we analyze nuclear behavior, time in motion, speed, and alignment during myotube differentiation and temporal interference of cytoskeletal microtubule-related motors. Using specific inhibitors, we find that nuclear movement and alignment are microtubule dependent, with 19 microtubule motor proteins implicated in at least one nuclear behavior. We further focus on Kif1c, Kif5b, kif9, kif21b, and Kif1a, which affect nuclear alignment. These results emphasize the different roles of molecular motors in particular mechanisms.

  15. Disruption of Microtubule Integrity Initiates Mitosis during CNS Repair

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. Electric field-induced reversible trapping of microtubules along metallic glass microwire electrodes

    NASA Astrophysics Data System (ADS)

    Kim, Kyongwan; Sikora, Aurélien; Nakayama, Koji S.; Umetsu, Mitsuo; Hwang, Wonmuk; Teizer, Winfried

    2015-04-01

    Microtubules are among bio-polymers providing vital functions in dynamic cellular processes. Artificial organization of these bio-polymers is a requirement for transferring their native functions into device applications. Using electrophoresis, we achieve an accumulation of microtubules along a metallic glass (Pd42.5Cu30Ni7.5P20) microwire in solution. According to an estimate based on migration velocities of microtubules approaching the wire, the electrophoretic mobility of microtubules is around 10-12 m2/Vs. This value is four orders of magnitude smaller than the typical mobility reported previously. Fluorescence microscopy at the individual-microtubule level shows microtubules aligning along the wire axis during the electric field-induced migration. Casein-treated electrodes are effective to reversibly release trapped microtubules upon removal of the external field. An additional result is the condensation of secondary filamentous structures from oriented microtubules.

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

    PubMed

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

    2015-01-01

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

  19. An array of nuclear microtubules reorganizes the budding yeast nucleus during quiescence

    PubMed Central

    Laporte, Damien; Courtout, Fabien; Salin, Bénédicte; Ceschin, Johanna

    2013-01-01

    The microtubule cytoskeleton is a highly dynamic network. In dividing cells, its complex architecture not only influences cell shape and movement but is also crucial for chromosome segregation. Curiously, nothing is known about the behavior of this cellular machinery in quiescent cells. Here we show that, upon quiescence entry, the Saccharomyces cerevisiae microtubule cytoskeleton is drastically remodeled. Indeed, while cytoplasmic microtubules vanish, the spindle pole body (SPB) assembles a long and stable monopolar array of nuclear microtubules that spans the entire nucleus. Consequently, the nucleolus is displaced. Kinetochores remain attached to microtubule tips but lose SPB clustering and distribute along the microtubule array, leading to a large reorganization of the nucleus. When cells exit quiescence, the nuclear microtubule array slowly depolymerizes and, by pulling attached centromeres back to the SPB, allows the recovery of a typical Rabl-like configuration. Finally, mutants that do not assemble a nuclear array of microtubules are impaired for both quiescence survival and exit. PMID:24247429

  20. CLASP promotes microtubule rescue by recruiting tubulin dimers to the microtubule

    PubMed Central

    Al-Bassam, Jawdat; Kim, Hwajin; Brouhard, Gary; van Oijen, Antoine; Harrison, Stephen C.; Chang, Fred

    2010-01-01

    Spatial regulation of microtubule (MT) dynamics contributes to cell polarity and cell division. MT rescue, in which a MT stops shrinking and reinitiates growth, is the least understood aspect of MT dynamics. Cytoplasmic Linker Associated Proteins (CLASPs) are a conserved class of MT-associated proteins that contribute to MT stabilization and rescue in vivo. We show here that the Schizosaccharomyces pombe CLASP, Cls1p, is a homodimer that binds an αβ tubulin heterodimer through conserved TOG-like domains. In vitro, CLASP increases MT rescue frequency, decreases MT catastrophe frequency and moderately decreases MT disassembly rate. CLASP binds stably to the MT lattice, recruits tubulin and locally promotes rescues. Mutations in the CLASP TOG domains demonstrate that tubulin binding is critical for its rescue activity. We propose a mechanism for rescue in which CLASP-tubulin dimer complexes bind along the MT lattice and reverse MT depolymerization with their bound tubulin dimer. PMID:20708587

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

    NASA Astrophysics Data System (ADS)

    Johnson, Kenneth

    2007-03-01

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

  2. Disorders of Microtubule Function in Neurons: Imaging Correlates

    PubMed Central

    Mutch, Christopher A.; Poduri, Annapurdi; Sahin, Mustafa; Barry, Brenda; Walsh, Christopher A.; Barkovich, A. James

    2015-01-01

    Background and Significance A number of recent studies have described malformations of cortical development with mutations of components of microtubules and microtubule-associated proteins. Despite examinations of large numbers of MRIs, good phenotype-genotype correlations have been elusive. Additionally, most of these studies focused exclusively on cerebral cortical findings. Materials and Methods MRIs from18 patients with confirmed tubulin mutations (8 TUBA1A, 5 TUBB2B, and 5 TUBB3) and 15 patients with known mutations of the genes encoding microtubule-associated proteins (5 LIS1, 4 DCX, and 6 DYNC1H1) were carefully visually analyzed and compared. Specific note was made of cortical gyral pattern, basal ganglia and white matter to assess internal capsular size, cortical thickness, ventricular and cisternal size, and size and contours of the brain stem, cerebellar hemispheres and vermis, and the corpus callosum of patients with tubulin and microtubule-associated protein gene mutations. Results were determined by unanimous consensus of the authors. Results All patients had abnormal MRI scans. Large proportions of patients with tubulin gene mutations were found to have multiple cortical and subcortical abnormalities including microcephaly, ventriculomegaly, abnormal gyral and sulcal patterns (termed dysgyria), small or absent corpus callosum and small pons. All patients with microtubule-associated proteins mutations also had abnormal cerebral cortices (predominantly pachygyria and agyria), but fewer subcortical abnormalities were noted. Conclusion Comparison of MRIs from patients with known mutations of tubulin genes and microtubule-associated proteins allows for the establishment of some early correlations of phenotype with genotype and may assist in identification and diagnosis of these rare disorders. PMID:26564436

  3. Microtubule affinity-regulating kinase 4: structure, function, and regulation.

    PubMed

    Naz, Farha; Anjum, Farah; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2013-11-01

    MAP/Microtubule affinity-regulating kinase 4 (MARK4) belongs to the family of serine/threonine kinases that phosphorylate the microtubule-associated proteins (MAP) causing their detachment from the microtubules thereby increasing microtubule dynamics and facilitating cell division, cell cycle control, cell polarity determination, cell shape alterations, etc. The MARK4 gene encodes two alternatively spliced isoforms, L and S that differ in their C-terminal region. These isoforms are differentially regulated in human tissues including central nervous system. MARK4L is a 752-residue-long polypeptide that is divided into three distinct domains: (1) protein kinase domain (59-314), (2) ubiquitin-associated domain (322-369), and (3) kinase-associated domain (703-752) plus 54 residues (649-703) involved in the proper folding and function of the enzyme. In addition, residues 65-73 are considered to be the ATP-binding domain and Lys88 is considered as ATP-binding site. Asp181 has been proposed to be the active site of MARK4 that is activated by phosphorylation of Thr214 side chain. The isoform MARK4S is highly expressed in the normal brain and is presumably involved in neuronal differentiation. On the other hand, the isoform MARK4L is upregulated in hepatocarcinoma cells and gliomas suggesting its involvement in cell cycle. Several biological functions are also associated with MARK4 including microtubule bundle formation, nervous system development, and positive regulation of programmed cell death. Therefore, MARK4 is considered as the most suitable target for structure-based rational drug design. Our sequence, structure- and function-based analysis should be helpful for better understanding of mechanisms of regulation of microtubule dynamics and MARK4 associated diseases.

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

    PubMed

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Fang, Fang; Betterton, Meredith; Shelley, Michael

    2014-11-01

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

  6. The engine of microtubule dynamics comes into focus.

    PubMed

    Mitchison, T J

    2014-05-22

    In this issue, Alushin et al. report high-resolution structures of three states of the microtubule lattice: GTP-bound, which is stable to depolymerization; unstable GDP-bound; and stable Taxol and GDP-bound. By comparing these structures at near-atomic resolution, they are able to propose a detailed model for how GTP hydrolysis destabilizes the microtubule and thus powers dynamic instability and chromosome movement. Destabilization of cytoskeleton filaments by nucleotide hydrolysis is an important general principle in cell dynamics, and this work represents a major step forward on a problem with a long history.

  7. Physical aspects of the assembly and function of microtubules

    NASA Astrophysics Data System (ADS)

    Holy, Timothy Eric

    Living cells contain polymers called microtubules. Microtubules are used to control cell shape, to generate force for movement, to transport vesicles, and to separate chromosomes during cell division. Microtubule polymerization is governed by a unique, energy-consuming phenomenon called dynamic instability, which leads to large fluctuations in length. This dissertation reports on physical studies (theory and experiment) of microtubules and their roles in living cells. To understand dynamic instability we need to know its mechanism and its function. Experiments on dynamic instability have led to a seeming contradiction as to its mechanism. By introducing a phenomenological model that unites the existing data, we show that this contradiction can be resolved. The model describes the stochastic dynamics of a stabilizing cap which promotes growth, but whose loss leads to disassembly. The theory matches experiments over time scales from seconds to minutes. We address the biological role of dynamic instability, also from a theoretical standpoint. We show that these large length fluctuations are useful: they lead to a rapid search of intracellular space. This search may be an essential step in organizing the cell interior, forging connections between widely-separated components. We show that dynamic instability speeds a search by several orders of magnitude. We also find that the parameters which govern dynamic instability appear to be chosen by the cell so as to minimize the search time. This thesis also reports experiments showing that microtubule polymerization may generate forces for movement and organization. Archetypal movements in living cells (e.g., the movement of the sperm nucleus from the periphery to the center of the egg) were reconstructed in an artificial system. The components of our system are purified proteins and two specially-designed materials: latex beads coated so as to nucleate microtubules, and microscopic chambers fabricated to mimic the confined

  8. 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

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

    SciTech Connect

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

    2006-04-15

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

  10. Miniature optical planar camera based on a wide-angle metasurface doublet corrected for monochromatic aberrations

    NASA Astrophysics Data System (ADS)

    Arbabi, Amir; Arbabi, Ehsan; Kamali, Seyedeh Mahsa; Horie, Yu; Han, Seunghoon; Faraon, Andrei

    2016-11-01

    Optical metasurfaces are two-dimensional arrays of nano-scatterers that modify optical wavefronts at subwavelength spatial resolution. They are poised to revolutionize optics by enabling complex low-cost systems where multiple metasurfaces are lithographically stacked and integrated with electronics. For imaging applications, metasurface stacks can perform sophisticated image corrections and can be directly integrated with image sensors. Here we demonstrate this concept with a miniature flat camera integrating a monolithic metasurface lens doublet corrected for monochromatic aberrations, and an image sensor. The doublet lens, which acts as a fisheye photographic objective, has a small f-number of 0.9, an angle-of-view larger than 60° × 60°, and operates at 850 nm wavelength with 70% focusing efficiency. The camera exhibits nearly diffraction-limited image quality, which indicates the potential of this technology in the development of optical systems for microscopy, photography, and computer vision.

  11. A rotated transmission grating spectrometer for detecting spectral separation of doublet Na

    SciTech Connect

    Santosa, Ignatius Edi

    2015-04-16

    Transmission gratings are usually used in a spectrometer for measuring the wavelength of light. In the common design, the position of the grating is perpendicular to the incident light. In order to increase the angular dispersion, in contrary to the common design, in this experiment the transmission grating was rotated. Due to the non-zero incident angle, the diffracted light was shifted. This rotated transmission grating spectrometer has been used to determine the separation of doublet Na. In this experiment, the diffraction angle was measured at various incident angles. The spectral separation of doublet Na was identified from the difference in the diffraction angle of two spectral lines. This spectral separation depends on the incident angle, the grating constant and the order of diffraction. As the effect of increasing the incident angle, a significant increase of the spectral separation can be achieved up to three fold.

  12. Miniature optical planar camera based on a wide-angle metasurface doublet corrected for monochromatic aberrations

    PubMed Central

    Arbabi, Amir; Arbabi, Ehsan; Kamali, Seyedeh Mahsa; Horie, Yu; Han, Seunghoon; Faraon, Andrei

    2016-01-01

    Optical metasurfaces are two-dimensional arrays of nano-scatterers that modify optical wavefronts at subwavelength spatial resolution. They are poised to revolutionize optics by enabling complex low-cost systems where multiple metasurfaces are lithographically stacked and integrated with electronics. For imaging applications, metasurface stacks can perform sophisticated image corrections and can be directly integrated with image sensors. Here we demonstrate this concept with a miniature flat camera integrating a monolithic metasurface lens doublet corrected for monochromatic aberrations, and an image sensor. The doublet lens, which acts as a fisheye photographic objective, has a small f-number of 0.9, an angle-of-view larger than 60° × 60°, and operates at 850 nm wavelength with 70% focusing efficiency. The camera exhibits nearly diffraction-limited image quality, which indicates the potential of this technology in the development of optical systems for microscopy, photography, and computer vision. PMID:27892454

  13. CP violating Two-Higgs-Doublet Model: constraints and LHC predictions

    NASA Astrophysics Data System (ADS)

    Keus, Venus; King, Stephen F.; Moretti, Stefano; Yagyu, Kei

    2016-04-01

    Two-Higgs-Doublet Models (2HDMs) are amongst the simplest extensions of the Standard Model. Such models allow for tree-level CP Violation (CPV) in the Higgs sector. We analyse a class of CPV 2HDM (of Type-I) in which only one of the two Higgs doublets couples to quarks and leptons, avoiding dangerous Flavour Changing Neutral Currents. We provide an up to date and comprehensive analysis of the constraints and Large Hadron Collider (LHC) predictions of such a model. Of immediate interest to the LHC Run 2 is the golden channel where all three neutral Higgs bosons are observed to decay into gauge boson pairs, WW and ZZ, providing a smoking gun signature of the CPV 2HDM.

  14. Sliding of microtubules by a team of dynein motors: Understanding the effect of spatial distribution of motor tails and mutual exclusion of motor heads on microtubules

    NASA Astrophysics Data System (ADS)

    Singh, Hanumant Pratap; Takshak, Anjneya; Mall, Utkarsh; Kunwar, Ambarish

    2016-06-01

    Molecular motors are natural nanomachines that use the free energy released from ATP hydrolysis to generate mechanical forces. Cytoplasmic dynein motors often work collectively as a team to drive important processes such as axonal growth, proplatelet formation and mitosis, as forces generated by single motors are insufficient. A large team of dynein motors is used to slide cytoskeletal microtubules with respect to one another during the process of proplatelet formation and axonal growth. These motors attach to a cargo microtubule via their tail domains, undergo the process of detachment and reattachment of their head domains on another track microtubule, while sliding the cargo microtubule along the track. Traditional continuum/mean-field approaches used in the past are not ideal for studying the sliding mechanism of microtubules, as they ignore spatial and temporal fluctuations due to different possible distributions of motor tails on cargo filament, as well as binding/unbinding of motors from their track. Therefore, these models cannot be used to address important questions such as how the distribution of motor tails on microtubules, or how the mutual exclusion of motor heads on microtubule tracks affects the sliding velocity of cargo microtubule. To answer these, here we use a computational stochastic model where we model each dynein motor explicitly. In our model, we use both random as well as uniform distributions of dynein motors on cargo microtubule, as well as mutual exclusion of motors on microtubule tracks. We find that sliding velocities are least affected by the distribution of motor tails on microtubules, whereas they are greatly affected by mutual exclusion of motor heads on microtubule tracks. We also find that sliding velocity depends on the length of cargo microtubule if mutual exclusion among motor heads is considered.

  15. Development of integrated superconducting quadrupole doublet modules for operation in the SIS100 accelerator

    NASA Astrophysics Data System (ADS)

    Meier, J.; Bleile, A.; Ceballos Velasco, J.; Fischer, E.; Hess, G.; Macavei, J.; Spiller, P.

    2015-12-01

    The FAIR project (Facility for Antiproton and Ion Research) evolves and builds an international accelerator- and experimental facility for basic research activities in various fields of modern physics. Within the course of this project, integrated quadrupole doublet modules are in development. The quadrupole doublet modules provide a pair of superconducting main quadrupoles (focusing and defocusing), corrector magnets, cryogenic collimators and beam position monitors as integrated sets of ion-optical elements. Furthermore LHe cooled beam pipes and vacuum cold-warm transitions are used as ultra-high vacuum components for beam transportation. Superconducting bus bars are used for 13 kA current supply of the main quadrupole magnets. All components are integrated as one common cold mass into one cryostat. High temperature super conductor local current leads will be applied for the low current supply of corrector magnets. The quadrupole doublet modules will be operated in the SIS100 heavy ion accelerator, the core component of the FAIR project. A first version of a corrector magnet has already been manufactured at the Joint Institute for Nuclear Research (JINR), Russia, and is now ready for testing. The ion-optical lattice structure of SIS100 requires multiple configurations of named components. Eleven different configurations, organized in four categories, provide the required quadrupole doublet module setups. The high integration level of multiple ion-optical, mechanical and cryogenic functions, based on requirements of operation safety, is leading towards a sophisticated mechanical structure and cooling solution, to satisfy the demanding requirements on position preservation during thermal cycling. The mechanical and cryogenic design solutions will be discussed.

  16. Photoluminescence Detected Doublet Structure in the Integer and Fractional Quantum Hall Regime

    SciTech Connect

    Kim, Yongmin; Munteanu, F.M.; Perry, C.H.; Reno, J.L.; Rickel, D.G.; Simmons, J.A.

    1999-05-25

    We present here the results of polarized magneto-photoluminescence measurements on a high mobility single-heterojunction. The presence of a doublet structure over a large magnetic field range (2>v>l/6) is interpreted as possible evidence for the existence of a magneto-roton minima of the charged density waves. This is understood as an indication of strong electronic correlation even in the case of the IQHE limit.

  17. Selection of doublet cellular patterns in directional solidification through spatially periodic perturbations

    SciTech Connect

    Losert, W.; Stillman, D.A.; Cummins, H.Z.; Kopczynski, P.; Rappel, W.; Karma, A.

    1998-12-01

    Pattern formation at the solid-liquid interface of a growing crystal was studied in directional solidification using a perturbation technique. We analyzed both experimentally and numerically the stability range and dynamical selection of cellular arrays of {open_quotes}doublets{close_quotes} with asymmetric tip shapes, separated by alternate deep and shallow grooves. Applying an initial periodic perturbation of arbitrary wavelength to the unstable planar interface allowed us to force the interface to evolve into doublet states that would not otherwise be dynamically accessible from a planar interface. We determined systematically the ranges of wavelength corresponding to stable singlets, stable doublets, and transient unstable patterns. Experimentally, this was accomplished by applying a brief UV light pulse of a desired spatial periodicity to the planar interface during the planar-cellular transient using the model alloy Succinonitrile-Coumarin 152. Numerical simulations of the nonlinear evolution of the interface were performed starting from a small sinusoidal perturbation of the steady-state planar interface. These simulations were carried out using a computationally efficient phase-field symmetric model of directional solidification with recently reformulated asymptotics and vanishing kinetics [A. Karma and W.-J. Rappel, Phys. Rev. E {bold 53} R3017 (1996); Phys. Rev. Lett. {bold 77}, 4050 (1996); Phys. Rev. E {bold 57}, 4323 (1998)], which allowed us to simulate spatially extended arrays that can be meaningfully compared to experiments. Simulations and experiments show remarkable qualitative agreement in the dynamic evolution, steady-state structure, and instability mechanisms of doublet cellular arrays. {copyright} {ital 1998} {ital The American Physical Society}

  18. 125 GeV Higgs state in the context of four generations with two Higgs doublets

    NASA Astrophysics Data System (ADS)

    Geller, Michael; Bar-Shalom, Shaouly; Eilam, Gad; Soni, Amarjit

    2012-12-01

    We interpret the recent discovery of a 125 GeV Higgs-like state in the context of a two-Higgs-doublet model with a heavy fourth sequential generation of fermions, in which one Higgs doublet couples only to the fourth-generation fermions, while the second doublet couples to the lighter fermions of the first three families. This model is designed to accommodate the apparent heaviness of the fourth-generation fermions and to effectively address the low-energy phenomenology of a dynamical electroweak-symmetry-breaking scenario. The physical Higgs states of the model are, therefore, viewed as composites primarily of the fourth-generation fermions. We find that the lightest Higgs, h, is a good candidate for the recently discovered 125 GeV spin-zero particle, when tan⁡β˜O(1), for typical fourth-generation fermion masses of M4G=400-600GeV, and with a large t-t' mixing in the right-handed quark sector. This, in turn, leads to BR(t'→th)˜O(1), which drastically changes the t' decay pattern. We also find that, based on the current Higgs data, this two-Higgs-doublet model generically predicts an enhanced production rate (compared to the Standard Model) in the pp→h→ττ channel, and reduced rates in the VV→h→γγ and pp¯/pp→V→hV→Vbb channels. Finally, the heavier CP-even Higgs is excluded by the current data up to mH˜500GeV, while the pseudoscalar state, A, can be as light as 130 GeV. These heavier Higgs states and the expected deviations from the Standard Model din some of the Higgs production channels can be further excluded or discovered with more data.

  19. Spatial regulation of astral microtubule dynamics by Kif18B in PtK cells.

    PubMed

    Walczak, Claire E; Zong, Hailing; Jain, Sachin; Stout, Jane R

    2016-10-15

    The spatial and temporal control of microtubule dynamics is fundamentally important for proper spindle assembly and chromosome segregation. This is achieved, in part, by the multitude of proteins that bind to and regulate spindle microtubules, including kinesin superfamily members, which act as microtubule-destabilizing enzymes. These fall into two general classes: the kinesin-13 proteins, which directly depolymerize microtubules, and the kinesin-8 proteins, which are plus end-directed motors that either destabilize microtubules or cap the microtubule plus ends. Here we analyze the contribution of a PtK kinesin-8 protein, Kif18B, in the control of mitotic microtubule dynamics. Knockdown of Kif18B causes defects in spindle microtubule organization and a dramatic increase in astral microtubules. Kif18B-knockdown cells had defects in chromosome alignment, but there were no defects in chromosome segregation. The long astral microtubules that occur in the absence of Kif18B are limited in length by the cell cortex. Using EB1 tracking, we show that Kif18B activity is spatially controlled, as loss of Kif18B has the most dramatic effect on the lifetimes of astral microtubules that extend toward the cell cortex. Together our studies provide new insight into how diverse kinesins contribute to spatial microtubule organization in the spindle.

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

    SciTech Connect

    Scaife, R.M. ); Wilson, L. ); Purich, D.L. )

    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.

  1. Spatial regulation of astral microtubule dynamics by Kif18B in PtK cells

    PubMed Central

    Walczak, Claire E.; Zong, Hailing; Jain, Sachin; Stout, Jane R.

    2016-01-01

    The spatial and temporal control of microtubule dynamics is fundamentally important for proper spindle assembly and chromosome segregation. This is achieved, in part, by the multitude of proteins that bind to and regulate spindle microtubules, including kinesin superfamily members, which act as microtubule-destabilizing enzymes. These fall into two general classes: the kinesin-13 proteins, which directly depolymerize microtubules, and the kinesin-8 proteins, which are plus end–directed motors that either destabilize microtubules or cap the microtubule plus ends. Here we analyze the contribution of a PtK kinesin-8 protein, Kif18B, in the control of mitotic microtubule dynamics. Knockdown of Kif18B causes defects in spindle microtubule organization and a dramatic increase in astral microtubules. Kif18B-knockdown cells had defects in chromosome alignment, but there were no defects in chromosome segregation. The long astral microtubules that occur in the absence of Kif18B are limited in length by the cell cortex. Using EB1 tracking, we show that Kif18B activity is spatially controlled, as loss of Kif18B has the most dramatic effect on the lifetimes of astral microtubules that extend toward the cell cortex. Together our studies provide new insight into how diverse kinesins contribute to spatial microtubule organization in the spindle. PMID:27559136

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

    PubMed Central

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

    2000-01-01

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

  3. Interplay between kinesin-1 and cortical dynein during axonal outgrowth and microtubule organization in Drosophila neurons

    PubMed Central

    del Castillo, Urko; Winding, Michael; Lu, Wen; Gelfand, Vladimir I

    2015-01-01

    In this study, we investigated how microtubule motors organize microtubules in Drosophila neurons. We showed that, during the initial stages of axon outgrowth, microtubules display mixed polarity and minus-end-out microtubules push the tip of the axon, consistent with kinesin-1 driving outgrowth by sliding antiparallel microtubules. At later stages, the microtubule orientation in the axon switches from mixed to uniform polarity with plus-end-out. Dynein knockdown prevents this rearrangement and results in microtubules of mixed orientation in axons and accumulation of microtubule minus-ends at axon tips. Microtubule reorganization requires recruitment of dynein to the actin cortex, as actin depolymerization phenocopies dynein depletion, and direct recruitment of dynein to the membrane bypasses the actin requirement. Our results show that cortical dynein slides ‘minus-end-out’ microtubules from the axon, generating uniform microtubule arrays. We speculate that differences in microtubule orientation between axons and dendrites could be dictated by differential activity of cortical dynein. DOI: http://dx.doi.org/10.7554/eLife.10140.001 PMID:26615019

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

    PubMed Central

    Hunt, Alan J.; McIntosh, J. Richard

    1998-01-01

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

  5. Photonic generation of ultra-wideband doublet pulse using a semiconductor-optical-amplifier based polarization-diversified loop.

    PubMed

    Luo, Bowen; Dong, Jianji; Yu, Yuan; Yang, Ting; Zhang, Xinliang

    2012-06-15

    We propose and demonstrate a novel scheme of ultra-wideband (UWB) doublet pulse generation using a semiconductor optical amplifier (SOA) based polarization-diversified loop (PDL) without any assistant light. In our scheme, the incoming gaussian pulse is split into two parts by the PDL, and each of them is intensity modulated by the other due to cross-gain modulation (XGM) in the SOA. Then, both parts are recombined with incoherent summation to form a UWB doublet pulse. Bi-polar UWB doublet pulse generation is demonstrated using an inverted gaussian pulse injection. Moreover, pulse amplitude modulation of UWB doublet is also experimentally demonstrated. Our scheme shows some advantages, such as simple implementation without assistant light and single optical carrier operation with good fiber dispersion tolerance.

  6. Cortical Microtubule Arrays Are Initiated from a Nonrandom Prepattern Driven by Atypical Microtubule Initiation1[W][OA

    PubMed Central

    Lindeboom, Jelmer J.; Lioutas, Antonios; Deinum, Eva E.; Tindemans, Simon H.; Ehrhardt, David W.; Emons, Anne Mie C.; Vos, Jan W.; Mulder, Bela M.

    2013-01-01

    The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation. PMID:23300168

  7. Protein Kinase C Activation Promotes Microtubule Advance in Neuronal Growth Cones by Increasing Average Microtubule Growth Lifetimes

    PubMed Central

    Kabir, Nurul; Schaefer, Andrew W.; Nakhost, Arash; Sossin, Wayne S.; Forscher, Paul

    2001-01-01

    We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow—a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance. PMID:11238458

  8. A new classification of the amino acid side chains based on doublet acceptor energy levels.

    PubMed Central

    Sneddon, S F; Morgan, R S; Brooks, C L

    1988-01-01

    We describe a new classification of the amino acid side chains based on the potential energy level at which each will accept an extra (doublet) electron. The doublet acceptor energy level, and the doublet acceptor orbital were calculated using semiempirical INDO/2-UHF molecular orbital theory. The results of these calculations show that the side chains fall into four groups. We have termed these groups repulsive, insulating, semiconducting, and attractive in accordance with where each lies on the relative energy scale. We use this classification to examine the role of residues between the donor and acceptor in modulating the rate and mechanism of electron transfer in proteins. With the calculated acceptor levels, we construct a potential barrier for those residues between the donor and acceptor. It is the area beneath this barrier that determines the decay of electronic coupling between donor and acceptor, and thus the transfer rate. We have used this schematic approach to characterize the four electron transfer pathways in myoglobin recently studied by Mayo et al. (Mayo, S.L., W.R. Ellis, R.J. Crutchley, and H.B. Gray. 1986. Science [Wash. DC]. 233:948-952). PMID:3342271

  9. The two Suvasvesi impact structures, Finland: Argon isotopic evidence for a "false" impact crater doublet

    NASA Astrophysics Data System (ADS)

    Schmieder, Martin; Schwarz, Winfried H.; Trieloff, Mario; Buchner, Elmar; Hopp, Jens; Tohver, Eric; Pesonen, Lauri J.; Lehtinen, Martti; Moilanen, Jarmo; Werner, Stephanie C.; Öhman, Teemu

    2016-05-01

    The two neighboring Suvasvesi North and South impact structures in central-east Finland have been discussed as a possible impact crater doublet produced by the impact of a binary asteroid. This study presents 40Ar/39Ar geochronologic data for impact melt rocks recovered from the drilling into the center of the Suvasvesi North impact structure and melt rock from glacially transported boulders linked to Suvasvesi South. 40Ar/39Ar step-heating analysis yielded two essentially flat age spectra indicating a Late Cretaceous age of ~85 Ma for the Suvasvesi North melt rock, whereas the Suvasvesi South melt sample gave a Neoproterozoic minimum (alteration) age of ~710 Ma. Although the statistical likelihood for two independent meteorite strikes in close proximity to each other is rather low, the remarkable difference in 40Ar/39Ar ages of >600 Myr for the two Suvasvesi impact melt samples is interpreted as evidence for two temporally separate, but geographically closely spaced, impacts into the Fennoscandian Shield. The Suvasvesi North and South impact structures are, thus, interpreted as a "false" crater doublet, similar to the larger East and West Clearwater Lake impact structures in Québec, Canada, recently shown to be unrelated. Our findings have implications for the reliable recognition of impact crater doublets and the apparent rate of binary asteroid impacts on Earth and other planetary bodies in the inner solar system.

  10. Axion-Higgs interplay in the two-Higgs-doublet model

    NASA Astrophysics Data System (ADS)

    Espriu, Domènec; Mescia, Federico; Renau, Albert

    2015-11-01

    The Zhitnitsky and Dine, Fischler and Srednicki (DFSZ) model is a natural extension of the two-Higgs-doublet model containing an additional singlet, endowed with a Peccei-Quinn symmetry, and leading to a physically acceptable axion. In this paper we reexamine this model in the light of some new developments. For generic couplings the model reproduces the minimal Standard Model showing only tiny deviations (extreme decoupling scenario) and all additional degrees of freedom (with the exception of the axion) are very heavy. Recently it has been remarked that the limit where the coupling between the singlet and the two doublets becomes very small is technically natural. Combining this limit with the requirement of exact or approximate custodial symmetry, we may obtain an additional 0+ Higgs at the weak scale, accompanied by relatively light charged and neutral pseudoscalars. The mass spectrum would then resemble that of a generic two-Higgs-doublet model, with naturally adjustable masses in spite of the large scale that the axion introduces. However, the couplings are nongeneric in this model. We use the recent constraints derived from the Higgs-W W coupling together with oblique corrections to constrain the model as much as possible. As an additional result, we work out the nonlinear parametrization of the DFSZ model in the generic case where all scalars except the lightest Higgs and the axion have masses at or beyond the TeV scale.

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

    SciTech Connect

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

    2009-07-01

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

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

    NASA Astrophysics Data System (ADS)

    Cvetič, G.; Kim, C. S.

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

  13. Vapor deposition on doublet airfoil substrates: Control of coating thickness and microstructure

    SciTech Connect

    Rodgers, Theron M.; Zhao, Hengbei; Wadley, Haydn N. G.

    2015-11-15

    Gas jet assisted vapor deposition processes for depositing coatings are conducted at higher pressures than conventional physical vapor deposition methods, and have shown promise for coating complex shaped substrates including those with non-line-of-sight (NLS) regions on their surface. These regions typically receive vapor atoms at a lower rate and with a wider incident angular distribution than substrate regions in line-of-sight (LS) of the vapor source. To investigate the coating of such substrates, the thickness and microstructure variation along the inner (curved) surfaces of a model doublet airfoil containing both LS and NLS regions has been investigated. Results from atomistic simulations and experiments confirm that the coating's thickness is thinner in flux-shadowed regions than in other regions for all the coating processes investigated. They also indicated that the coatings columnar microstructure and pore volume fraction vary with surface location through the LS to NLS transition zone. A substrate rotation strategy for optimizing the thickness over the entire doublet airfoil surface was investigated, and led to the identification of a process that resulted in only small variation of coating thickness, columnar growth angle, and pore volume fraction on all doublet airfoil surfaces.

  14. Lewis Carroll's Doublets Net of English Words: Network Heterogeneity in a Complex System

    PubMed Central

    Fushing, Hsieh; Chen, Chen; Hsieh, Yin-Chen; Farrell, Patrick

    2014-01-01

    Lewis Carroll's English word game Doublets is represented as a system of networks with each node being an English word and each connectivity edge confirming that its two ending words are equal in letter length, but different by exactly one letter. We show that this system, which we call the Doublets net, constitutes a complex body of linguistic knowledge concerning English word structure that has computable multiscale features. Distributed morphological, phonological and orthographic constraints and the language's local redundancy are seen at the node level. Phonological communities are seen at the network level. And a balancing act between the language's global efficiency and redundancy is seen at the system level. We develop a new measure of intrinsic node-to-node distance and a computational algorithm, called community geometry, which reveal the implicit multiscale structure within binary networks. Because the Doublets net is a modular complex cognitive system, the community geometry and computable multi-scale structural information may provide a foundation for understanding computational learning in many systems whose network structure has yet to be fully analyzed. PMID:25517974

  15. Influence of an embedded quantum dot on the Josephson effect in the topological superconducting junction with Majorana doublets

    NASA Astrophysics Data System (ADS)

    Gong, Wei-Jiang; Gao, Zhen; Shan, Wan-Fei; Yi, Guang-Yu

    2016-03-01

    One Majorana doublet can be realized at each end of the time-reversal-invariant Majorana nanowires. We investigate the Josephson effect in the Majorana-doublet-presented junction modified by different inter-doublet coupling manners. It is found that when the Majorana doublets couple indirectly via a non-magnetic quantum dot, only the normal Josephson effect occurs, and the fermion parity in the system just affects the current direction and amplitude. However, one magnetic field applied on the dot can induce the fractional Josephson effect in the odd-parity case. Next if the direct and indirect couplings between the Majorana doublets coexist, no fractional Josephson effect takes place, regardless of the presence of magnetic field. Instead, there almost appears the π-period-like current in some special cases. All the results are clarified by analyzing the influence of the fermion occupation in the quantum dot on the parity conservation in the whole system. We ascertain that this work will be helpful for describing the dot-assisted Josephson effect between the Majorana doublets.

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

    PubMed Central

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

    2014-01-01

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

  17. 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

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

    PubMed Central

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

    2012-01-01

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

  19. Analysis of Shallow and Deep Earthquake Doublets in the Fiji-Tonga-Kermadec Region

    NASA Astrophysics Data System (ADS)

    Gibowicz, Slawomir J.; Lasocki, Stanislaw

    2007-01-01

    For the Fiji-Tonga-Kermadec area and for the period from January 1977 to July 2003, the Harvard CMT catalogue lists 1022 shallow, 410 intermediate and 633 deep earthquakes of moment magnitude from 4.9 to 8.0. The magnitude threshold, above which the catalogue is complete, is 5.3 5.4, and the number of earthquakes of magnitude above this value is 691 for shallow, 329 for intermediate and 476 for deep events, respectively. The proportion of earthquakes associated with doublets and multiplets against the total number of earthquakes is approximately the same in both data sets and therefore all earthquake pairs were considered regardless of their magnitude. We investigated all the pairs of earthquakes that occurred at a centroid distance of less than 40, 60 or 90 km from each other and within a time interval of 200, 300 or 450 days, depending on their magnitude. We found 208 pairs of shallow, 31 of intermediate and 92 of deep events. To ascertain whether these earthquakes in pairs are not connected by chance, the possibility of their occurrence in an uncorrelated Poissonian catalogue was considered. It was assumed that in such a catalogue the inter-event time is exponentially distributed, the earthquake magnitude follows the Gutenberg-Richter relation, and the distribution of centroid distances between the events in pairs is controlled by its non-parametric kernel estimate. The probability of the appearance of the observed proportion of doublets of shallow earthquakes in the Poissonian catalogue was found to be very low. The low probability of occurrence in a semi-random catalogue, created by randomising centroid locations in the actual data set, also indicates major importance of the distance criterion used for a doublet specification. In general, shallow earthquakes tend to form pairs at shorter distances and within shorter time intervals than deep earthquakes. Both the distance and the time intervals do not depend on the magnitude of involved events. The largest

  20. Fission Yeast Scp3 Potentially Maintains Microtubule Orientation through Bundling

    PubMed Central

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

    2015-01-01

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

  1. The size of the EB cap determines instantaneous microtubule stability

    PubMed Central

    Duellberg, Christian; Cade, Nicholas I; Holmes, David; Surrey, Thomas

    2016-01-01

    The function of microtubules relies on their ability to switch between phases of growth and shrinkage. A nucleotide-dependent stabilising cap at microtubule ends is thought to be lost before this switch can occur; however, the nature and size of this protective cap are unknown. Using a microfluidics-assisted multi-colour TIRF microscopy assay with close-to-nm and sub-second precision, we measured the sizes of the stabilizing cap of individual microtubules. We find that the protective caps are formed by the extended binding regions of EB proteins. Cap lengths vary considerably and longer caps are more stable. Nevertheless, the trigger of instability lies in a short region at the end of the cap, as a quantitative model of cap stability demonstrates. Our study establishes the spatial and kinetic characteristics of the protective cap and provides an insight into the molecular mechanism by which its loss leads to the switch from microtubule growth to shrinkage. DOI: http://dx.doi.org/10.7554/eLife.13470.001 PMID:27050486

  2. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

    SciTech Connect

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

    2006-10-13

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

  3. Microtubules: 50 years on from the discovery of tubulin

    PubMed Central

    Borisy, Gary; Heald, Rebecca; Howard, Jonathon; Janke, Carsten; Musacchio, Andrea; Nogales, Eva

    2016-01-01

    Next year will be the 50th anniversary of the discovery of tubulin. To celebrate this discovery, six leaders in the field of microtubule research reflect on key findings and technological breakthroughs over the past five decades, discuss implications for therapeutic applications and provide their thoughts on what questions need to be addressed in the near future. PMID:27103327

  4. Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles

    PubMed Central

    Asbury, Charles L.

    2017-01-01

    The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through loss of tubulin subunits from the kinetochore-attached plus ends. In addition, kinetochore-fiber disassembly in many cells occurs partly through ‘flux’, where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive these disassembly-coupled movements, are discussed. PMID:28218660

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

    PubMed

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

    2008-09-01

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

  6. Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation

    PubMed Central

    1991-01-01

    A novel protein has been identified which may serve a key function in nucleating spindle microtubule growth in mitosis. This protein, called centrophilin, is sequentially relocated from the centromeres to the centrosomes to the midbody in a manner dependent on the mitotic phase. Centrophilin was initially detected by immunofluorescence with a monoclonal, primate-specific antibody (2D3) raised against kinetochore- enriched chromosome extract from HeLa cells (Valdivia, M. M., and B. R. Brinkley. 1985. J. Cell Biol. 101:1124-1134). Centrophilin forms prominent crescents at the poles of the metaphase spindle, gradually diminishes during anaphase, and bands the equatorial ends of midbody microtubules in telophase. The formation and breakdown of the spindle and midbody correlates in time and space with the aggregation and disaggregation of centrophilin foci. Immunogold EM reveals that centrophilin is a major component of pericentriolar material in metaphase. During recovery from microtubule inhibition, centrophilin foci act as nucleation sites for the assembly of spindle tubules. The 2D3 probe recognizes two high molecular mass polypeptides, 180 and 210 kD, on immunoblots of whole HeLa cell extract. Take