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1

Molecular architecture of axonemal microtubule doublets revealedby cryo-electron tomography  

SciTech Connect

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.

Sui, Haixin; Downing, Kenneth H.

2006-05-22

2

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

PubMed Central

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

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

3

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

PubMed

The axoneme-the conserved core of eukaryotic cilia and flagella-contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy shows that the mutant axonemes lack the IJ, and cryo-electron tomography combined with a structural labeling method reveals that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of the axoneme and may be involved in planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component, because uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beak's localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra-B-tubular substructures required for a planar asymmetrical waveform. PMID:24574454

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

2014-05-01

4

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

PubMed Central

The axoneme—the conserved core of eukaryotic cilia and flagella—contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy shows that the mutant axonemes lack the IJ, and cryo–electron tomography combined with a structural labeling method reveals that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of the axoneme and may be involved in planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component, because uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beak's localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra–B-tubular substructures required for a planar asymmetrical waveform. PMID:24574454

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

2014-01-01

5

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

PubMed Central

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

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

2014-01-01

6

Space-Dependent Formation of Central Pair Microtubules and Their Interactions with Radial Spokes  

PubMed Central

Cilia and flagella contain nine outer doublet microtubules and a pair of central microtubules. The central pair of microtubules (CP) is important for cilia/flagella beating, as clearly shown by primary ciliary dyskinesia resulting from the loss of the CP. The CP is thought to regulate axonemal dyneins through interaction with radial spokes (RSs). However, the nature of the CP-RS interaction is poorly understood. Here we examine the appearance of CPs in the axonemes of a Chlamydomonas mutant, bld12, which produces axonemes with 8 to 11 outer-doublets. Most of its 8-doublet axonemes lack CPs. However, in the double mutant of bld12 and pf14, a mutant lacking the RS, most 8-doublet axonemes contain the CP. Thus formation of the CP apparently depends on the internal space limited by the outer doublets and RSs. In 10- or 11-doublet axonemes, only 3–5 RSs are attached to the CP and the doublet arrangement is distorted most likely because the RSs attached to the CP pull the outer doublets toward the axonemal center. The CP orientation in the axonemes varies in double mutants formed between bld12 and mutants lacking particular CP projections. The mutant bld12 thus provides the first direct and visual information about the CP-RS interaction, as well as about the mechanism of CP formation. PMID:25333940

Nakazawa, Yuki; Ariyoshi, Tetsuro; Noga, Akira; Kamiya, Ritsu; Hirono, Masafumi

2014-01-01

7

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

PubMed

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

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

2014-06-20

8

Integrated control of axonemal dynein AAA(+) motors.  

PubMed

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

King, Stephen M

2012-08-01

9

An axonemal PP2A B-subunit is required for PP2A localization and flagellar motility  

PubMed Central

Analysis of Chlamydomonas axonemes revealed that the protein phosphatase, PP2A, is localized to the outer doublet microtubules and is implicated in regulation of dynein-driven motility. We tested the hypothesis that PP2A is localized to the axoneme by a specialized, highly conserved 55-kDa B-type subunit identified in the Chlamydomonas flagellar proteome. The B-subunit gene is defective in the motility mutant pf4. Consistent with our hypothesis, both the B- and C-subunits of PP2A fail to assemble in pf4 axonemes, while the dyneins and other axonemal structures are fully assembled in pf4 axonemes. Two pf4 intragenic revertants were recovered that restore PP2A to the axonemes and re-establish nearly wild-type motility. The revertants confirmed that the slow-swimming Pf4 phenotype is a result of the defective PP2A B-subunit. These results demonstrate that the axonemal B-subunit is, in part, an anchor protein required for PP2A localization and that PP2A is required for normal ciliary motility. PMID:21692192

Elam, Candice A.; Wirschell, Maureen; Yamamoto, Ryosuke; Fox, Laura. A.; York, Kerry; Kamiya, Ritsu; Dutcher, Susan K.; Sale, Winfield S.

2011-01-01

10

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

PubMed

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

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

2014-06-01

11

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

PubMed Central

Dark-field microscopy with a high-powered light source revealed that the outer doublet microtubules (DMTs) from sea urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella assume helically coiled configurations (Miki-Noumura, T., and R. Kamiya. 1976. Exp. Cell Res. 97: 451.). We report here that the DMTs change shape when the pH or Ca-ion concentration is changed. The DMTs assumed a left- handed helical shape with a diameter of 3.7 +/- 0.5 micron and a pitch of 2.8 +/- 0.7 micron at pH 7.4 in the presence of 0.1 mM CaCl2, 1 mM MgSO4, and 10 mM Tris-HCl. When the pH was raised to 8.3, the helical diameter and pitch decreased to 2.1 +/- 0.1 micron and 1.3 +/- 0.3 micron, respectively. This transformation was a rapid and reversible process and was completed within 1 min. Between pH 7.2 and 8.3, the DMTs assumed intermediate shapes. When the Ca-ion concentration was depleted with EGTA, the helical structure became significantly larger in both pitch and diameter. For instance, the diameter was 3.8 +/- 0.4 micron at pH 8.3 in the presence of 1 mM EGTA and 2 mM MgSO4. Using a Ca-buffer system, we obtained results which suggested that this Ca- induced transformation took place at a Ca concentration of approximately 10(-7) M. These results were highly reproducible. The conformational changes in the DMT may play some role in the bending wave form of flagellar movement. PMID:38258

1979-01-01

12

Ultrastructural evidence that motility changes caused by variations in ATP, Mg(2+) , and ADP correlate to conformational changes in reactivated bull sperm axonemes.  

PubMed

We report the results of an ultrastructural study of Triton X-100-extracted, Mg-adenosine triphosphate (ATP)-reactivated bull sperm. We utilized a rapid fixation method to look for differences in the flagellar apparatus that correlate to the state of motility of reactivated sperm models. In a companion article we examined the motility characteristics induced in bull sperm models by varying the concentration ratio of ATP and Mg(2+) and the stabilizing effect of adenosine diphosphate (ADP) on coordinated beating. Based on the results of that report we selected four dissimilar states that appeared to represent extremes. One reactivation condition produces vigorous motility similar to live sperm, another produces large amplitude, low frequency beating while the remaining two conditions produce small amplitude vibrations of the flagellum with little coordinated beating. Morphometric analysis of transmission electron micrographs of sperm from these four treatment conditions revealed statistically significant differences between the samples in regard to axoneme diameter, inter-microtubule doublet spacing, and outer dense fiber spacing. Our results show that Mg(2+) decreases the axoneme diameter and reduces interdoublet spacing, while ATP, un-complexed with Mg(2+) , had the opposite effect. We also provide supporting evidence that this may be due to Mg(2+) increasing, and ATP decreasing, the inter-doublet adhesion of dynein. We also found that 4 mM ADP significantly increases the separation between the outer dense fibers (ODFs) and the space between the ODFs and the central axoneme within the middle piece. We present a hypothetical explanation that is consistent with our results to explain how ATP, ADP and Mg(2+) act to regulate the beat cycle. This article is protected by copyright. All rights reserved. PMID:25430605

Lesich, Kathleen A; DePinho, Tania G; Dang, Loan; Lindemann, Charles B

2014-11-27

13

The flagellar beat of rat sperm is organized by the interaction of two functionally distinct populations of dynein bridges with a stable central axonemal partition.  

PubMed

Two distinct patterns of microtubular sliding were observed in rat sperm flagellar axonemes. The particular pattern of sliding was determined by the extraction conditions used to prepare the sperm for axoneme disintegration. Sperm prepared by incubating concentrated suspensions of Triton X-100-extracted sperm at pH 9.0 disintegrated by extruding the doublets and outer dense fibers numbered 4 through 7 in response to Mg-ATP. Sperm prepared by incubating motile Triton X-100-extracted models at 37 degrees C for 1 to 3 hours extruded doublets and outer dense fibers 9, 1 and 2. Axonemes disintegrated by both regimens tended to have doublets 3 and 8 (with their corresponding outer dense fibers), as well as the central pair, in place. In numerous instances, the 3-central-8 complex with outer dense fibers 3 and 8 could be found isolated in midpiece sections prepared from both methods. The 3-central-8 partition was also sometimes seen in isolation in cross-sections of the principal piece where it remained attached to the fibrous sheath. The flagellar remnant produced by extrusion of fibers 4 through 7 under high pH conditions was generally straight or randomly curved. In contrast, the flagellar remnant produced by extrusion of the 9-1-2 bundle of fibers was most often curved into a hook in the midpiece region. While the hook-like configuration was not Ca(2+)-dependent, it may be based on a related mechanism. The sliding of the 9-1-2 group of fibers is a consequence of dynein-tubulin sliding between the 2 and 3 doublets. This sliding pattern appears to be preferentially activated in the motile sperm models in EGTA, but seldom if ever produced sliding in the high-pH-extracted models. We conclude that the 3-central pair-8 complex and associated outer dense fibers form an I-beam-like partition that does not participate in sliding, but acts as a structural foundation for organizing a planar beat. In addition, it is clear that preferential activation of certain dynein arms can be evoked, depending on the treatment regimen employed. This shows definitively that the types of microtubule sliding in the two bend directions are not identical. PMID:1400632

Lindemann, C B; Orlando, A; Kanous, K S

1992-06-01

14

Isolation of a sixth dynein subunit adenosine triphosphatase of Chlamydomonas axonemes  

PubMed Central

This study of the axoneme led to the identification of a previously unknown adenosine triphosphatase (ATPase), which is likely a major component of inner dynein arms. The ATPase was isolated from a soluble fraction of axonemes obtained from pf 28, a Chlamydomonas mutant lacking the outer dynein arms. The activity hydrolyzed up to 2.3 mumol of ATP.min-1.mg-1 of protein (at pH 7.2, in the presence of both Ca++ and Mg++), had a sedimentation coefficient of 11S in sucrose gradient, and cosedimented with four polypeptides of apparent molecular weight 325,000, 315,000 140,000, and 42,000. Several arguments indicate that the new ATPase is a component of the inner dynein arms. Three or four polypeptides cosedimenting with the activity belong to a group of axonemal components that are deficient in the axonemes of pf 23 and pf 30, two mutants that display different levels of inner dynein arm deficiency. The 42,000 component is axonemal actin, a subunit of two other inner dynein ATPases. The two polypeptides of molecular weight greater than 300,000 have electrophoretic mobility similar to that of high molecular weight components of outer and inner dynein arms. In spite of some similarities each ATPase isolated from inner or outer arms is composed of a different set of polypeptides. Different ATPases may be required for the modulation of localized sliding of adjacent outer double microtubules in the axoneme. PMID:2963009

1988-01-01

15

Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein.  

PubMed

Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a critical regulator of ciliary and flagellar beating. To understand I1 architecture and function better, we analyzed the 3D structure and composition of the I1 dynein in Chlamydomonas axonemes by cryoelectron tomography and subtomogram averaging. Our data revealed several connections from the I1 dynein to neighboring structures that are likely to be important for assembly and/or regulation, including a tether linking one I1 motor domain to the doublet microtubule and doublet-specific differences potentially contributing to the asymmetrical distribution of dynein activity required for ciliary beating. We also imaged three I1 mutants and analyzed their polypeptide composition using 2D gel-based proteomics. Structural and biochemical comparisons revealed the likely location of the regulatory IC138 phosphoprotein and its associated subcomplex. Overall, our studies demonstrate that I1 dynein is connected to multiple structures within the axoneme, and therefore ideally positioned to integrate signals that regulate ciliary motility. PMID:22733763

Heuser, Thomas; Barber, Cynthia F; Lin, Jianfeng; Krell, Jeremy; Rebesco, Matthew; Porter, Mary E; Nicastro, Daniela

2012-07-24

16

Regulation of axonemal motility in demembranated equine sperm.  

PubMed

Equine in vitro fertilization is not yet successful because equine sperm do not effectively capacitate in vitro. Results of previous studies suggest that this may be due to failure of induction of hyperactivated motility in equine sperm under standard capacitating conditions. To evaluate factors directly affecting axonemal motility in equine sperm, we developed a demembranated sperm model and analyzed motility parameters in this model under different conditions using computer-assisted sperm analysis. Treatment of ejaculated equine sperm with 0.02% Triton X-100 for 30 sec maximized both permeabilization and total motility after reactivation. The presence of ATP was required for motility of demembranated sperm after reactivation, but cAMP was not. The calculated intracellular pH of intact equine sperm was 7.14 ± 0.07. Demembranated sperm showed maximal total motility at pH 7. Neither increasing pH nor increasing calcium levels, nor any interaction of the two, induced hyperactivated motility in demembranated equine sperm. Motility of demembranated sperm was maintained at free calcium concentrations as low as 27 pM, and calcium arrested sperm motility at much lower concentrations than those reported in other species. Calcium arrest of sperm motility was not accompanied by flagellar curvature, suggesting a failure of calcium to induce the tonic bend seen in other species and thought to support hyperactivated motility. This indicated an absence, or difference in calcium sensitivity, of the related asymmetric doublet-sliding proteins. These studies show a difference in response to calcium of the equine sperm axoneme to that reported in other species that may be related to the failure of equine sperm to penetrate oocytes in vitro under standard capacitating conditions. Further work is needed to determine the factors that stimulate hyperactivated motility at the axonemal level in equine sperm. PMID:25339104

Loux, Shavahn C; Macías-Garcia, Beatríz; González-Fernández, Lauro; Canesin, Heloisa DeSiqueira; Varner, Dickson D; Hinrichs, Katrin

2014-12-01

17

Microtubule polarity and distribution in teleost photoreceptors.  

PubMed

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

Troutt, L L; Burnside, B

1988-07-01

18

Decoration of spindle microtubules with Dynein: evidence for uniform polarity  

Microsoft Academic Search

Studies were conducted to determine whether the microtubules present within native spindles isolated from eggs of the surf clam, Spisula solidissima, could bind dynein obtained from axonemes of Tetrahymena thermophila . SDS gel electrophoresis revealed that the high molecular weight polypeptides that make up dynein cosedimented with the isolated spindles . Moreover, the ATPase activity of dynein bound to the

BRUCE R. TELZER; LEAH T. HAIMO

1981-01-01

19

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

PubMed Central

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. PMID:2921286

1989-01-01

20

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

SciTech Connect

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.

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

1989-03-01

21

Modified axonemes and ciliary membranes in three polychaete species  

Microsoft Academic Search

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

H.-D. Pfannenstiel

1982-01-01

22

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

PubMed

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. PMID:24611953

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

23

A BBSome subunit links ciliogenesis, microtubule stability, and acetylation.  

PubMed

Primary cilium dysfunction affects the development and homeostasis of many organs in Bardet-Biedl syndrome (BBS). We recently showed that seven highly conserved BBS proteins form a stable complex, the BBSome, that functions in membrane trafficking to and inside the primary cilium. We have now discovered a BBSome subunit that we named BBIP10. Similar to other BBSome subunits, BBIP10 localizes to the primary cilium, BBIP10 is present exclusively in ciliated organisms, and depletion of BBIP10 yields characteristic BBS phenotypes in zebrafish. Unexpectedly, BBIP10 is required for cytoplasmic microtubule polymerization and acetylation, two functions not shared with any other BBSome subunits. Strikingly, inhibition of the tubulin deacetylase HDAC6 restores microtubule acetylation in BBIP10-depleted cells, and BBIP10 physically interacts with HDAC6. BBSome-bound BBIP10 may therefore function to couple acetylation of axonemal microtubules and ciliary membrane growth. PMID:19081074

Loktev, Alexander V; Zhang, Qihong; Beck, John S; Searby, Charles C; Scheetz, Todd E; Bazan, J Fernando; Slusarski, Diane C; Sheffield, Val C; Jackson, Peter K; Nachury, Maxence V

2008-12-01

24

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

Microsoft Academic Search

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

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

1998-01-01

25

Ciliary microtubule capping structures contain a mammalian kinetochore antigen  

PubMed Central

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

1990-01-01

26

The role of microtubules and microtubule-organising centres during the migration of mitochondria.  

PubMed Central

The translocation of mitochondria towards the primitive inner segment of the cones in the tree shrew Tupaia belangeri was investigated by transmission electron microscopy. Throughout ontogeny the migrating mitochondria were codistributed with cytoplasmic microtubules which were preserved after the application of conventional preparation techniques for transmission electron microscopy. Both the basal body of the connecting cilium and the second centriole located in the vicinity of the basal body were demonstrated to act as microtubule-organising centres (MTOCs) from which axonemal and cytoplasmic microtubules originated. The megamitochondria in the inner segment of the retinal cones of Tupaia are unique among mammals with respect to their extraordinary size and to their ordered distribution characterised by longitudinal and radial size-gradients within developing and mature cone inner segments. Thus the consistent finding of microtubules and MTOCs in the structurally polarised cones represents an extreme example of the capacity of cells to regulate the transport and distribution of organelles. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8886960

Knabe, W; Kuhn, H J

1996-01-01

27

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

PubMed

The ?-?-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. PMID:25646500

Sloboda, Roger D

2015-01-01

28

Binding of Tetrahymena dynein to axonemes of Marsilea vestita lacking the outer dynein arm.  

PubMed

Axonemes from the heterosporous water fern Marsilea vestita were fixed in the presence of tannic acid and examined by thin-section electron microscopy. Transverse sections revealed the normal 9+2 configuration except for the absence of the outer of the two dynein arms. Both arms were normally preserved in parallel preparations of Chlamydomonas axonemes. Isolated dynein from the ciliated protozoon Tetrahymena bound to Marsilea axonemes at the site normally occupied by the outer arm. Dynein binding was partially reversed by ATP as judged by both electron microscopy and polyacrylamide gel electrophoresis. This system should provide a valuable insight into the biochemistry and function of the inner dynein arm and the relationship of the two arms to motility in more conventionally equipped axonemes. PMID:3160715

Hyams, J S

1985-02-01

29

Isolation of a sixth dynein subunit adenosine triphosphatase of Chlamydomonas axonemes  

Microsoft Academic Search

This study of the axoneme led to the identification of a previously unknown adenosine triphosphatase (ATPase), which is likely a major com- ponent of inner dynein arms. The ATPase was isolated from a soluble fraction of axonemes obtained from pf28, a Chlamydomonas mutant lacking the outer dynein arms. The activity hydrolyzed up to 2.3 Ixmol of ATP.min-t.mg -~ of protein

Gianni Piperno

1988-01-01

30

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

PubMed Central

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

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

2009-01-01

31

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

PubMed Central

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

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

2013-01-01

32

Doublet craters on Venus  

NASA Astrophysics Data System (ADS)

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.

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

2003-09-01

33

FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.  

PubMed

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo-electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule. PMID:25540426

Vasudevan, Krishna Kumar; Song, Kangkang; Alford, Lea M; Sale, Winfield S; Dymek, Erin E; Smith, Elizabeth F; Hennessey, Todd; Joachimiak, Ewa; Urbanska, Paulina; Wloga, Dorota; Dentler, William; Nicastro, Daniela; Gaertig, Jacek

2015-02-15

34

Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components  

PubMed Central

The fine structure, protein composition, and roles in flagellar movement of specific axonemal components were studied in wild-type Chlamydomonas and paralyzed mutants pf-14, pf-15A, and pf-19. Electron microscope examination of the isolated axoneme of pf-14 showed that it lacks the radial spokes but is otherwise structurally normal. Comparison of isolated axonemes of wild type and pf-14 by sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the mutant is missing a protein of 118,000 mol wt; this protein is apparently a major component of the spokes. Pf-15A and pf-19 lack the central tubules and sheath; axonemes of these mutants are missing three high molecular weight proteins which are probably components of the central tubule-central sheath complex. Under conditions where wild-type axonemes reactivated, axonemes of the three mutants remained intact but did not form bends. However, mutant and wild-type axonemes underwent identical adenosine triphosphate-induced disintegration after treatment with trypsin; the dynein arms of the mutants are therefore capable of generating interdoublet shearing forces. These findings indicated that both the radial spokes and the central tubule-central sheath complex are essential for conversion of interdoublet sliding into axonemal bending. Moreover, because axonemes of pf-14 remained intact under reactivating conditions, the nexin links alone are sufficient to limit the amount of interdoublet sliding that occurs. The axial periodicities of the central sheath, dynein arms, radial spokes, and nexin links of Chlamydomonas were determined by electron microscopy using the lattice- spacing of crystalline catalase as an internal standard. Some new ultrastructural details of the components are described. PMID:632325

1978-01-01

35

Microtubules, Tubulins and Associated Proteins.  

ERIC Educational Resources Information Center

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)

Raxworthy, Michael J.

1988-01-01

36

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

PubMed Central

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

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

2014-01-01

37

Nanomechanics of Microtubules  

Microsoft Academic Search

We have determined the mechanical anisotropy of a single microtubule by simultaneously measuring the Young's and the shear moduli in vitro. This was achieved by elastically deforming the microtubule deposited on a substrate tailored by electron-beam lithography with a tip of an atomic force microscope. The shear modulus is 2orders of magnitude lower than the Young's, giving rise to a

A. Kis; S. Kasas; B. Babic; A. J. Kulik; W. Benoît; G. A. Briggs; C. Schönenberger; S. Catsicas; L. Forró

2002-01-01

38

Ofd1 Controls Dorso-Ventral Patterning and Axoneme Elongation during Embryonic Brain Development  

PubMed Central

Oral-facial-digital type I syndrome (OFDI) is a human X-linked dominant-male-lethal developmental disorder caused by mutations in the OFD1 gene. Similar to other inherited disorders associated to ciliary dysfunction OFD type I patients display neurological abnormalities. We characterized the neuronal phenotype that results from Ofd1 inactivation in early phases of mouse embryonic development and at post-natal stages. We determined that Ofd1 plays a crucial role in forebrain development, and in particular, in the control of dorso-ventral patterning and early corticogenesis. We observed abnormal activation of Sonic hedgehog (Shh), a major pathway modulating brain development. Ultrastructural studies demonstrated that early Ofd1 inactivation results in the absence of ciliary axonemes despite the presence of mature basal bodies that are correctly orientated and docked. Ofd1 inducible-mediated inactivation at birth does not affect ciliogenesis in the cortex, suggesting a developmental stage-dependent role for a basal body protein in ciliogenesis. Moreover, we showed defects in cytoskeletal organization and apical-basal polarity in Ofd1 mutant embryos, most likely due to lack of ciliary axonemes. Thus, the present study identifies Ofd1 as a developmental disease gene that is critical for forebrain development and ciliogenesis in embryonic life, and indicates that Ofd1 functions after docking and before elaboration of the axoneme in vivo. PMID:23300826

Avallone, Bice; Piscopo, Immacolata; Tammaro, Roberta; Studer, Michèle; Franco, Brunella

2012-01-01

39

Dark matter with two inert doublets plus one Higgs doublet  

NASA Astrophysics Data System (ADS)

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

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

2014-11-01

40

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

PubMed Central

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

1992-01-01

41

Do prokaryotes contain microtubules?  

NASA Technical Reports Server (NTRS)

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.

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

1994-01-01

42

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

PubMed

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

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

2014-10-01

43

Isolation of microtubules and microtubule-associated proteins using Paclitaxel.  

PubMed

Paclitaxel binds to tubulin, strongly promotes microtubule assembly from subunits, and stabilizes the assembled polymer against disassembly. Because of its ability to drive the assembly reaction almost completely toward microtubules, the paclitaxel-dependent procedure outlined here is particularly useful for the isolation of microtubules from tissues in which the intracellular concentration of tubulin is low (e.g., nonneuronal sources, cultured cells, and invertebrate tissues). The microtubule-associated proteins (MAPs) remain bound to the paclitaxel-stabilized microtubules. The isolation of these MAPs by salt extraction is also described here. PMID:25561621

Sloboda, Roger D

2015-01-01

44

Chlamydomonas mutants display reversible deficiencies in flagellar beating and axonemal assembly  

PubMed Central

Axonemal complexes in flagella are largely pre-packaged in the cell body. As such, one mutation often results in the absence of the co-assembled components and permanent motility deficiencies. For example, a Chlamydomonas mutant defective in RSP4 in the radial spoke (RS), which is critical for bend propagation, has paralyzed flagella that also lack the paralogue RSP6 and three additional RS proteins. Intriguingly, recent studies showed that several mutant strains contain a mixed population of swimmers and paralyzed cells despite their identical genetic background. Here we report a cause underlying these variations. Two new mutants lacking RSP6 swim processively and other components appear normally assembled in early log phase indicating that, unlike RSP4, this paralogue is dispensable. However, swimmers cannot maintain the typical helical trajectory and reactivated cell models tend to spin. Interestingly the motile fraction and the spokehead content dwindle during stationary phase. These results suggest that 1) intact RS is critical for maintaining the rhythm of oscillatory beating and thus the helical trajectory; 2) assembly of the axonemal complex with subtle defects is less efficient and the inefficiency is accentuated in compromised conditions, leading to reversible dyskinesia. Consistently, several organisms only possess one RSP4/6 gene. Gene duplication in Chlamydomonas enhances RS assembly to maintain optimal motility in various environments. PMID:20169531

Wei, Mei; Sivadas, Priyanka; Owen, Heather A.; Mitchell, David R.; Yang, Pinfen

2010-01-01

45

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

PubMed Central

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

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

2013-01-01

46

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

SciTech Connect

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

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

1987-10-23

47

Microtubules and the tax payer.  

PubMed

Plant microtubules have evolved into a versatile tool to link environmental signals into flexible morphogenesis. Cortical microtubules define the axiality of cell expansion by control of cellulose orientation. Plant-specific microtubule structures such as preprophase band and phragmoplast determine symmetry and axiality of cell divisions. In addition, microtubules act as sensors and integrators for stimuli such as mechanic load, gravity, but also osmotic stress, cold and pathogen attack. Many of these functions are specific for plants and involve specific proteins or the recruitment of proteins to new functions. The review aims to ventilate the potential of microtubule-based strategies for biotechnological application by highlighting representative case studies. These include reorientation of cortical microtubules to increase lodging resistance, control of microtubule dynamics to alter the gravity-dependent orientation of leaves, the use of microtubules as sensitive thermometers to improve adaptive cold tolerance of chilling and freezing sensitive plants, the reduction of microtubule treadmilling to inhibit cell-to-cell transport of plant viruses, or the modulation of plant defence genes by pharmacological manipulation of microtubules. The specificity of these responses is controlled by a great variety of specific associated proteins opening a wide field for biotechnological manipulation of plant architecture and stress tolerance. PMID:22006077

Nick, Peter

2012-06-01

48

Energy Consumption of Actively Beating Flagella  

NASA Astrophysics Data System (ADS)

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.

Chen, Daniel; Nicastro, Daniela; Dogic, Zvonimir

2012-02-01

49

Molecular mechanisms of kinetochore microtubule attachment  

E-print Network

To ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive poleward chromosome movement. Microtubules are highly dynamic structures ...

Schmidt, Jens C. (Jens Christopher)

2012-01-01

50

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

PubMed Central

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

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

2012-01-01

51

Microtubules and angiosperm bordered pit formation  

Microsoft Academic Search

Microtubules have been shown to run around the perimeter of the pit aperture of developing bordered pits of Salix fragilis, L. These microtubules are parallel to the microfibrils being produced and do not converge with them. Although microtubules may be parallel to microfibrils in differentiating vessel elements as well as in fibres, many cases have been found where microtubules are

A. W. Robards; P. G. Humpherson

1967-01-01

52

Mutation of an axonemal dynein affects left–right asymmetry in inversus viscerum mice  

PubMed Central

The development of characteristic visceral asymmetries along the left–right (LR) axis in an initially bilaterally symmetrical embryo is an essential feature of vertebrate patterning. The allelic mouse mutations inversus viscerum (iv)1,2 and legless (lgl)3,4 produce LR inversion, or situs inversus, in half of live-born homozygotes. This suggests that the iv gene product drives correct LR determination, and in its absence this process is randomized2. These mutations provide tools for studying the development of LR-handed asymmetry and provide mouse models of human lateralization defects. At the molecular level, the normally LR asymmetric expression patterns of nodal5 and lefty6 are randomized in iv/iv embryos, suggesting that iv functions early in the genetic hierarchy of LR specification. Here we report the positional cloning of an axonemal dynein heavy-chain gene, left/right-dynein (lrd), that is mutated in both lgl and iv. lrd is expressed in the node of the embryo at embryonic day 7.5, consistent with its having a role in LR development7. Our findings indicate that dynein, a micro-tubule-based motor, is involved in the determination of LR-handed asymmetry and provide insight into the early molecular mechanisms of this process. PMID:9353118

Supp, Dorothy M.; Witte, David P.; Potter, S. Steven; Brueckner, Martina

2007-01-01

53

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

PubMed Central

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

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

1994-01-01

54

Ultrastructural study of the male gamete of Glossobothrium sp. (Cestoda: Bothriocephalidea: Triaenophoridae) a parasite of Schedophilus velaini (Perciformes: Centrolophidae) in Senegal.  

PubMed

This paper describes the ultrastructure of the male gamete of Glossobothrium sp. (Bothriocephalidea: Triaenophoridae). The mature spermatozoon of Glossobothrium sp. is filiform and possesses two axonemes, a single helicoidal crested body, a parallel nucleus, parallel cortical microtubules and granules of glycogen. In Glossobothrium sp. we describe for first time a 200-250 nm thick crest-like body in the Bothriocephalidean. The anterior part of the spermatozoon exhibits a ring of 27 electron-dense cortical microtubules encircling the first axoneme. This structure persists until the appearance of the second axoneme. When the ring of electron-dense cortical microtubules disappears, the spermatozoon exhibits two bundles of thin cortical microtubules. The posterior part of the spermatozoon contains the posterior extremity of the second axoneme, the posterior extremity of the nucleus and few cortical microtubules. Soon nucleus disappears and the axoneme is disorganized. Thus the posterior extremity of the spermatozoon of Glossobothrium sp. exhibits only singlets produced by the disorganization of the doublets of the second axoneme and few cortical microtubules. This type of posterior extremity of the mature spermatozoon has never been described previously in the Triaenophoridae. PMID:22633207

Ndiaye, P I; Quilichini, Y; Bâ, A; Bâ, C T; Marchand, B

2012-10-01

55

Teamwork in microtubule motors.  

PubMed

Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport. PMID:23877011

Mallik, Roop; Rai, Arpan K; Barak, Pradeep; Rai, Ashim; Kunwar, Ambarish

2013-11-01

56

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

PubMed Central

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

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

2014-01-01

57

Persistence Length of Stable Microtubules  

NASA Astrophysics Data System (ADS)

Microtubules are a vital component of the cytoskeleton. As the most rigid of the cytoskeleton filaments, they give shape and support to the cell. They are also essential for intracellular traffic by providing the roadways onto which organelles are transported, and they are required to reorganize during cellular division. To perform its function in the cell, the microtubule must be rigid yet dynamic. We are interested in how the mechanical properties of stable microtubules change over time. Some "stable" microtubules of the cell are recycled after days, such as in the axons of neurons or the cilia and flagella. We measured the persistence length of freely fluctuating taxol-stabilized microtubules over the span of a week and analyzed them via Fourier decomposition. As measured on a daily basis, the persistence length is independent of the contour length. Although measured over the span of the week, the accuracy of the measurement and the persistence length varies. We also studied how fluorescently-labeling the microtubule affects the persistence length and observed that a higher labeling ratio corresponded to greater flexibility.

Hawkins, Taviare; Mirigian, Matthew; Selcuk Yasar, M.; Ross, Jennifer

2011-03-01

58

Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate KinaseD?  

PubMed Central

Of the uncloned ODA genes required for outer dynein arm assembly in Chlamydomonas, ODA5 and ODA10 are of particular interest because they do not encode known subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC), and because genetic studies suggest their products interact. Beginning with a tagged oda5 allele, we isolated genomic and cDNA clones of the wild-type gene. ODA5 predicts a novel, 66-kDa coiled-coil protein. Immunoblotting indicates Oda5p is an axonemal component that assembles onto the axoneme independently of the outer arm and ODA-DC and is uniquely missing in oda5 and oda10 axonemes. Oda5p is released from the axoneme by extraction with 0.6 M KCl, but the soluble Oda5p does not cosediment with the outer dynein arm/ODA-DC in sucrose gradients. Quantitative mass spectrometry by using isotope coded affinity tagging revealed that a previously unidentified adenylate kinase is reduced 35–50% in oda5 flagella. Direct enzymatic assays demonstrated a comparable reduction in adenylate kinase activity in oda5 flagella, and also in oda10 flagella, but not in flagella of other oda mutants. We propose that Oda5p is part of a novel axonemal complex that is required for outer arm assembly and anchors adenylate kinase in proximity to the arm. PMID:15064350

Wirschell, Maureen; Pazour, Gregory; Yoda, Akinori; Hirono, Masafumi; Kamiya, Ritsu; Witman, George B.

2004-01-01

59

Dark two Higgs doublet model  

NASA Astrophysics Data System (ADS)

We perform a detailed study of a specific two Higgs doublet model with a U(1) gauge symmetry, instead of a typical Z2 discrete symmetry, containing a very light gauge boson Z' (GeV scale or below). The Standard Model (SM) fermions do not carry U(1) charges, but induced couplings to the Z' (called the dark Z) are generated through mixing with the SM neutral gauge bosons. Such a light Z' could explain some astrophysical anomalies as well as the muon g-2 deviation, and has been the subject of great experimental interest. We consider the scenario in which the 125 GeV SM-like Higgs (H) is the heavier scalar state, and focus on the lighter neutral state (h) as well as charged Higgs. We analyze the constraints on the model from various experiments and predict novel channels to search for these Higgs scalars at the LHC. In particular, experiments looking for lepton jets are among potentially important searches.

Lee, Hye-Sung; Sher, Marc

2013-06-01

60

Dark Two Higgs Doublet Model  

SciTech Connect

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

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

2013-06-01

61

Dark Two Higgs Doublet Model  

E-print Network

We perform a detailed study of a specific Two Higgs Doublet Model (2HDM) with a U(1) gauge symmetry, instead of a typical Z2 discrete symmetry, containing a very light gauge boson Z' (GeV scale or below). The Standard Model (SM) fermions do not carry U(1) charges, but induced couplings to the Z' (called the dark Z) are generated through mixing with the SM neutral gauge bosons. Such a light Z' could explain some astrophysical anomalies as well as the muon g-2 deviation, and has been the subject of great experimental interest. We consider the scenario in which the 125 GeV SM-like Higgs (H) is the heavier scalar state, and focus on the lighter neutral state (h) as well as charged Higgs. We analyze the constraints on the model from various experiments and predict novel channels to search for these Higgs scalars at the LHC. In particular, experiments looking for lepton-jets are among potentially important searches.

Hye-Sung Lee; Marc Sher

2013-03-26

62

Inert doublet model and LEP II limits  

SciTech Connect

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.

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

2009-02-01

63

An earthquake doublet in Ometepec, Guerrero, Mexico  

Microsoft Academic Search

On June 7, 1982 an earthquake doublet occurred in a gap near Ometepec, Guerrero, Mexico which had been given a high seismic potential. The two earthquakes (first event: Ms = 6.9, mb = 6.0, 16.3°N, 98.4°W, d = 25 km; second event Ms = 7.0, mb = 6.3, 16.4°N, 98.5°W, d = 8 km) of the doublet occurred within five

Luciana Astiz; Hiroo Kanamori

1984-01-01

64

Disruption of cytoplasmic microtubules by ultraviolet radiation  

SciTech Connect

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

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

1991-07-01

65

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

PubMed Central

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

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

2014-01-01

66

Length-dependent dynamics of microtubules  

NASA Astrophysics Data System (ADS)

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

Yadav, Vandana; Mukherji, Sutapa

2011-12-01

67

Microtubule defects & Neurodegeneration  

PubMed Central

One of the major challenges facing the long term survival of neurons is their requirement to maintain efficient axonal transport over long distances. In humans as large, long-lived vertebrates, the machinery maintaining neuronal transport must remain efficient despite the slow accumulation of cell damage during aging. Mutations in genes encoding proteins which function in the transport system feature prominently in neurologic disorders. Genes known to cause such disorders and showing traditional Mendelian inheritance have been more readily identified. It has been more difficult, however, to isolate factors underlying the complex genetics contributing to the more common idiopathic forms of neurodegenerative disease. At the heart of neuronal transport is the rail network or scaffolding provided by neuron specific microtubules (MTs). The importance of MT dynamics and stability is underscored by the critical role tau protein plays in MT-associated stabilization versus the dysfunction seen in Alzheimer’s disease, frontotemporal dementia and other tauopathies. Another example of the requirement for tight regulation of MT dynamics is the need to maintain balanced levels of post-translational modification of key MT building-blocks such as ?-tubulin. Tubulins require extensive polyglutamylation at their carboxyl-terminus as part of a novel post-translational modification mechanism to signal MT growth versus destabilization. Dramatically, knock-out of a gene encoding a deglutamylation family member causes an extremely rapid cell death of Purkinje cells in the ataxic mouse model, pcd. This review will examine a range of neurodegenerative conditions where current molecular understanding points to defects in the stability of MTs and axonal transport to emphasize the central role of MTs in neuron survival. PMID:24563812

Baird, Fiona J.; Bennett, Craig L

2014-01-01

68

Tau Protein Diffuses along the Microtubule Lattice*  

PubMed Central

Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised. PMID:23019339

Hinrichs, Maike H.; Jalal, Avesta; Brenner, Bernhard; Mandelkow, Eckhard; Kumar, Satish; Scholz, Tim

2012-01-01

69

Microtubules and angiosperm bordered pit formation.  

PubMed

Microtubules have been shown to run around the perimeter of the pit aperture of developing bordered pits of Salix fragilis, L. These microtubules are parallel to the microfibrils being produced and do not converge with them. Although microtubules may be parallel to microfibrils in differentiating vessel elements as well as in fibres, many cases have been found where microtubules are not so orientated. There is no direct evidence for the involvement of microtubules in the orientation of microfibrils in the secondary wall, although their position during wall synthesis suggests some ancillary role. PMID:24522541

Robards, A W; Humpherson, P G

1967-09-01

70

Microtubule catastrophe from protofilament dynamics  

NASA Astrophysics Data System (ADS)

The disappearance of the guanosine triphosphate- (GTP) tubulin cap is widely believed to be the forerunner event for the growth-shrinkage transition (“catastrophe”) in microtubule filaments in eukaryotic cells. We study a discrete version of a stochastic model of the GTP cap dynamics, originally proposed by Flyvbjerg, Holy, and Leibler [Phys. Rev. Lett.PRLTAO0031-900710.1103/PhysRevLett.73.2372 73, 2372 (1994)]. Our model includes both spontaneous and vectorial hydrolysis, as well as dissociation of a nonhydrolyzed dimer from the filament after incorporation. In the first part of the paper, we apply this model to a single protofilament of a microtubule. A catastrophe transition is defined for each protofilament, similarly to the earlier one-dimensional models, the frequency of occurrence of which is then calculated under various conditions but without explicit assumption of steady-state conditions. Using a perturbative approach, we show that the leading asymptotic behavior of the protofilament catastrophe in the limit of large growth velocities is remarkably similar across different models. In the second part of the paper, we extend our analysis to the entire filament by making a conjecture that a minimum number of such transitions are required to occur for the onset of microtubule catastrophe. The frequency of microtubule catastrophe is then determined using numerical simulations and compared with analytical and semianalytical estimates made under steady-state and quasi-steady-state assumptions, respectively, for the protofilament dynamics. A few relevant experimental results are analyzed in detail and compared with predictions from the model. Our results indicate that loss of GTP cap in two to three protofilaments is necessary to trigger catastrophe in a microtubule.

Jemseena, V.; Gopalakrishnan, Manoj

2013-09-01

71

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

PubMed

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

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

2013-07-23

72

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

PubMed Central

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

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

2013-01-01

73

PURIFICATION OF INTACT MICROTUBULES FROM BRAIN  

PubMed Central

Hundred-fold purification of intact microtubules from homogenates of rat brain is reported. The success of purification depends on stabilizing the microtubule structure by the combined effects of hexylene glycol, acidic Ph, and low temperature. A practical, negative stain, electron microscopic assay is used to study purity and stability of microtubule fractions. The purified fractions show a major band which migrates like purified tubulin in the SDS gel electrophoresis system. PMID:19866738

Kirkpatrick, Joel B.; Hyams, Lyon; Thomas, Virginia L.; Howley, Peter M.

1970-01-01

74

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

PubMed Central

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

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

2013-01-01

75

Microtubule Bundling and Shape Transitions  

NASA Astrophysics Data System (ADS)

Microtubules (MTs) are hollow cylindrical polymers composed of heterodimers of the protein tubulin that align end-to-end in the MT wall, forming linear protofilaments that interact laterally. Placing MTs under osmotic pressure causes them to reversibly buckle to a noncircular shape and pack into rectangular bundles at a critical osmotic pressure; further increases in pressure continue to distort MTs elastically. At higher osmotic pressures stressing polymers may be forced into the MT lumen causing the MTs to revert to a circle cross-section and pack into hexagonal bundles. This SAXRD-osmotic stress study provides a probe of the inter-protofilament bond strength and gives insight into the mechanisms by which microtubule associated proteins and the cancer chemotherapeutic drug Taxol stabilize MTs. We present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the microtubule lumen. Supported by NSF DMR- 0203755, NIH GM-59288 and NS-13560, and CTS-0103516. SSRL is supported by the U.S. DOE.

Needleman, Daniel

2005-03-01

76

Ectopic A-lattice seams destabilize microtubules  

PubMed Central

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe. PMID:24463734

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

2014-01-01

77

Antivascular actions of microtubule-binding drugs.  

PubMed

Microtubule-binding drugs (MBD) are widely used in cancer chemotherapy and also have clinically relevant antiangiogenic and vascular-disrupting properties. These antivascular actions are due in part to direct effects on endothelial cells, and all MBDs (both microtubule-stabilizing and microtubule-destabilizing) inhibit endothelial cell proliferation, migration, and tube formation in vitro, actions that are thought to correspond to therapeutic antiangiogenic actions. In addition, the microtubule-destabilizing agents cause prominent changes in endothelial cell morphology, an action associated with rapid vascular collapse in vivo. The effects on endothelial cells occur in vitro at low drug concentrations, which do not affect microtubule gross morphology, do not cause microtubule bundling or microtubule loss and do not induce cell cycle arrest, apoptosis, or cell death. Rather, it has been hypothesized that, at low concentrations, MBDs produce more subtle effects on microtubule dynamics, block critical cell signaling pathways, and prevent the microtubules from properly interacting with transient subcellular assemblies (focal adhesions and adherens junctions) whose subsequent stabilization and/or maturation are required for cell motility and cell-cell interactions. This review will focus on recent studies to define the molecular mechanisms for the antivascular actions of the MBDs, information that could be useful in the identification or design of agents whose actions more selectively target the tumor vasculature. PMID:19351751

Schwartz, Edward L

2009-04-15

78

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

PubMed Central

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

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

2014-01-01

79

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

PubMed

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

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

2014-12-01

80

Dark Matter from the Inert Doublet Model  

E-print Network

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

Laura Lopez Honorez

2007-06-01

81

Design and realization of an aspherical doublet  

NASA Astrophysics Data System (ADS)

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

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

2015-01-01

82

The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella  

Microsoft Academic Search

The gene product of EFHC1 recently implicated in juvenile myoclonic epilepsy (JME) was found to be a homolog of Chlamydomonas axonemal protein Rib72, whose homologs are present in a wide variety of organisms that have motile cilia and flagella. Western blot analyses and immunofluorescence localization of the mouse ortholog mRib72-1\\/Efhc1 indicated that it is indeed abundantly present in sperm flagella

Takashi Ikeda; Kazuho Ikeda; Masahiro Enomoto; Min Kyun Park; Masafumi Hirono; Ritsu Kamiya

2005-01-01

83

Mechanical Properties of Doubly Stabilized Microtubule Filaments  

PubMed Central

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? [(?, ?)-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

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

2013-01-01

84

Microtubule-targeted anticancer agents and apoptosis  

Microsoft Academic Search

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

Kapil N Bhalla

2003-01-01

85

Microtubule Motors in Microfluidics Maruti Uppalapati,  

E-print Network

of being able to work against concentration gradients and bulk fluid flow, and microtubules can kinesin-driven transport in engineered microchannels. Protocols are described for expressing and purifying and microtubules into microchannels, followed by a detailed description of a microfluidic device prototype

Hancock, William O.

86

Buckling and force propagation along intracellular microtubules  

E-print Network

stresses in the cytoplasm. These stresses can be highly inhomogeneous, with compressive/tensile forces by recent experiments showing the compressive buckling of microtubules in cells, we study theoretically. We find that embedded microtubules buckle when their compressive load exceeds a critical value fc

MacKintosh, F.C.

87

Tubulin Bistability and Polymorphic Dynamics of Microtubules  

NASA Astrophysics Data System (ADS)

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.

Mohrbach, Hervé; Johner, Albert; Kuli?, Igor M.

2010-12-01

88

Microtubule networks for plant cell division.  

PubMed

During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants. PMID:25136380

de Keijzer, Jeroen; Mulder, Bela M; Janson, Marcel E

2014-09-01

89

Cortical Dynein Controls Microtubule Dynamics to Generate Pulling Forces that Reliably Position Microtubule Asters  

PubMed Central

Dynein-mediated pulling forces generated on dynamic microtubule ends at the cortex contribute to cellular positioning processes such as spindle positioning during embryonic cell division and centrosome positioning during fibroblast migration. The details of dynein’s interaction with microtubule ends, and its consequences for positioning processes remain however unclear. We have reconstituted the ‘cortical’ interaction between dynein and dynamic microtubule ends in an in vitro system using microfabricated barriers. We show that barrier-attached dynein captures microtubule ends, inhibits growth, and triggers microtubule catastrophes, thereby controlling microtubule length. The subsequent interaction with shrinking microtubule ends generates pulling forces up to several pN. By combining experiments in microchambers with a theoretical description of aster mechanics, we show that dynein-mediated pulling forces lead to the reliable centering of microtubule asters in simple confining geometries. Our results demonstrate the intrinsic ability of cortical microtubule-dynein interactions to regulate microtubule dynamics and drive positioning processes in living cells. PMID:22304918

Laan, Liedewij; Pavin, Nenad; Husson, Julien; Romet-Lemonne, Guillaume; van Duijn, Martijn; López, Magdalena Preciado; Vale, Ronald D.; Jülicher, Frank; Reck-Peterson, Samara L.; Dogterom, Marileen

2012-01-01

90

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

Microsoft Academic Search

Heterotrimeric kinesin-II is a plus end- directed microtubule (MT) motor protein consisting of distinct heterodimerized motor subunits associated with an accessory subunit. To probe the intracellular transport functions of kinesin-II, we microinjected fer- tilized sea urchin eggs with an anti-kinesin-II mono- clonal antibody, and we observed a dramatic inhibition of ciliogenesis at the blastula stage characterized by the assembly of

Robert L. Morris; Jonathan M. Scholey

1997-01-01

91

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C)  

PubMed Central

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein- labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo. PMID:8421058

1993-01-01

92

Mechanical and geometrical constraints control kinesin-based microtubule guidance.  

PubMed

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

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

2014-02-01

93

Dynamics and mechanics of the microtubule plus end  

Microsoft Academic Search

An important function of microtubules is to move cellular structures such as chromosomes, mitotic spindles and other organelles around inside cells. This is achieved by attaching the ends of microtubules to cellular structures; as the microtubules grow and shrink, the structures are pushed or pulled around the cell. How do the ends of microtubules couple to cellular structures, and how

Joe Howard; Anthony A. Hyman

2003-01-01

94

Reovirus Cell Entry Requires Functional Microtubules  

PubMed Central

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

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

2013-01-01

95

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

SciTech Connect

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.

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

2010-09-03

96

Discrete and continuous symmetries in multi-Higgs-doublet models  

SciTech Connect

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.

Ferreira, P. M. [Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emidio Navarro, 1900 Lisboa (Portugal); Centro de Fisica Teorica e Computacional, Faculdade de Ciencias, Universidade de Lisboa, Avenida Professor Gama Pinto 2, 1649-003 Lisboa (Portugal); Silva, Joao P. [Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emidio Navarro, 1900 Lisboa (Portugal); Centro de Fisica Teorica de Particulas, Instituto Superior Tecnico, P-1049-001 Lisboa (Portugal)

2008-12-01

97

Doublet-triplet fermionic dark matter  

NASA Astrophysics Data System (ADS)

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

Dedes, Athanasios; Karamitros, Dimitrios

2014-06-01

98

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

PubMed Central

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

Atkinson, Samantha; Kirik, Angela; Kirik, Viktor

2014-01-01

99

Multiple chiral doublet bands of identical configuration in 103Rh  

E-print Network

Three sets of chiral doublet band structures have been identified in the 103Rh nucleus. The properties of the observed chiral doublet bands are in good agreement with theoretical results obtained using constrained covariant density functional theory and particle rotor model calculations. Two of them belong to an identical configuration, and provide the first experimental evidence for a novel type of multiple chiral doublets, where an "excited" chiral doublet of a configuration is seen together with the "yrast" one. This observation shows that the chiral geometry in nuclei can be robust against the increase of the intrinsic excitation energy.

I. Kuti; Q. B. Chen; J. Timar; D. Sohler; S. Q. Zhang; Z. H. Zhang; P. W. Zhao; J. Meng; K. Starosta; T. Koike; E. S. Paul; D. B. Fossan; C. Vaman

2014-07-10

100

Inert Higgs doublet extension of the NMSSM  

NASA Astrophysics Data System (ADS)

We introduce one pair of inert Higgs doublets {Hd,Hu} and singlets {Nc,N}, and consider their couplings with the Higgs doublets of the minimal supersymmetric standard model (MSSM), W ?yNNchuHd+yN'NhdHu. We assign extra U(1)Z' gauge charges only to the extra vectorlike superfields, and so all the MSSM superfields remain neutral under the new U(1)Z'. They can be an extension of the "? term," W ??Shuhd in the next-to MSSM (NMSSM). Because of the U(1)Z', the maximally allowed low energy value of yN can be lifted up to 0.85, avoiding a Landau pole (LP) below the grand unification scale. Such colorless vectorlike superfields remarkably enhance the radiative MSSM Higgs mass particularly for large tan? through the yN term and the corresponding holomorphic soft term. As a result, the lower bound of ? and the upper bound of tan? can be relaxed to disappear from the restricted parameter space of the original NMSSM, 0.6?? ?0.7 and 1

Kyae, Bumseok

2014-04-01

101

A protein factor essential for microtubule assembly.  

PubMed Central

A heat stable protein essentail for microtubule assembly has been isolated. This protein, which we designate tau (tau), is present in association with tubulin purified from porcine brain by repeated cycles of polymerization. Tau is separated from tubulin by ion exchange chromatography on phosphocellulose. In the absence of tau, tubulin exists entirely as a 6S dimer of two polypeptide chains (alpha and beta tubulin) with a molecular weight of 120,000, which will not assemble into microtubules in vitro. Addition of tau completely restores tubule-forming capacity. Under nonpolymerizing conditions, tau converts 6S dimers to 36S rings-structures which have been implicated as intermediates in tubule formation. Hence, tau appears to act on the 6S tubulin dimer, activating it for polymerization. The unique ability of tau to restore the normal features of in vitro microtubule assembly makes it likely that tau is a major regulator of microtubule formation in cells. Images PMID:1057175

Weingarten, M D; Lockwood, A H; Hwo, S Y; Kirschner, M W

1975-01-01

102

Measuring kinetochore-microtubule interaction in vitro  

PubMed Central

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

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

2014-01-01

103

Kinetochore–microtubule interactions during cell division  

Microsoft Academic Search

Proper segregation of chromosomes during cell division is essential for the maintenance of genetic stability. During this\\u000a process chromosomes must establish stable functional interactions with microtubules through the kinetochore, a specialized\\u000a protein structure located on the surface of the centromeric heterochromatin. Stable attachment of kinetochores to a number\\u000a of microtubules results in the formation of a kinetochore fibre that mediates

Helder Maiato; Claudio E. Sunkel

2004-01-01

104

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

PubMed

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. PMID:7447728

Vorob'ev, V S; Portuganov, V V

1980-10-01

105

Mobility of Taxol in Microtubule Bundles  

NASA Astrophysics Data System (ADS)

Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching (FRAP) on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With > 63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, k_{off}. It was found that 10*k_{off} (GMPCPP) ~ k_{off} (GTP), consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With < 63% sites occupied, recovery times decreased as ~ [Tax]^{-1/5} for both types of microtubules. We conclude that the diffusion of taxol along the microtubule interior is hindered by rebinding events when open sites are within ~7 nm of each other.

Ross, J.

2003-06-01

106

Why earthquake doublets in the Ometepec, Guerrero, Mexico subduction area?  

Microsoft Academic Search

We have studied the aftershock sequence following the major earthquakes of 7 June 1982 that occurred in the Ometepec, Guerrero in southern Mexico, a region of high seismic potential. Earthquakes in this region frequently occur in pairs (called earthquake doublets in seismology). Earthquake doublet appearance in other regions has been related to the presence of large isolated asperities, that is,

Jaime Yamamoto; Luis Quintanar; Zenón Jiménez

2002-01-01

107

Doublet craters and the tidal disruption of binary asteroids  

Microsoft Academic Search

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

H. J. Melosh; J. A. Stansberry

1991-01-01

108

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

E-print Network

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

Paris-Sud XI, Université de

109

Liquid-phase mixing of bipropellant doublets  

NASA Technical Reports Server (NTRS)

Experimental results of unlike doublet mixing are correlated with an analytically derived equation predicting fluid cavitation. The correlation relates the minimum orifice pressure drop required to initiate cavitation, with the system back pressure, cold flow simulant vapor pressure, and the orifice flow discharge and contraction coefficients. Stream flow instabilities are also visually correlated with the onset of cavitation and orifice discharge coefficient measurements. The influence of cavitation on the characteristic phenomenon of hydraulic flip is observed for both circular and noncircular shaped orifices. For certain intermediate orifice lengths, some noncircular shapes are shown to produce more fully developed flows (shorter recovery lengths) and therefore a more cohesive jet, which in turn yields slightly higher cold flow mixing uniformities than circular shaped orifices of equal absolute length. The particular noncircular shaped elements evaluated are shown to be more sensitive to liquid stream misimpingement than the corresponding circular orifices.

Hoehn, F. W.; Rupe, J. H.; Sotter, J. G.

1972-01-01

110

Two Higgs Doublets from Fermion Condensation  

E-print Network

We consider the most generic situation in models where the electroweak symmetry is broken by the condensation of a strongly coupled fermion sector, such as for instance a fourth generation. We study the scalar content resulting from the condensation of both the up and the down type fermions, corresponding to a two-Higgs doublet model. We estimate the scalar spectrum using the Nambu--Jona-Lasinio model, improved by the renormalization group. We show that the scalar spectrum is generically lighter than for the case with only one right-handed fermion condensing and that due to a remnant Peccei-Quinn symmetry the lightest state is the pseudo-scalar, with masses ranging typically from 10 GeV to 120 GeV. We discuss the phenomenological consequences of this distinct spectrum.

Gustavo Burdman; Carlos E. F. Haluch

2011-09-18

111

Constraining the Doublet Left-Right Model  

E-print Network

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

Silva, Luiz Vale

2015-01-01

112

[A functional flagella with a 6 + 0 pattern  

PubMed Central

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

1975-01-01

113

MAPs: cellular navigators for microtubule array orientations in Arabidopsis.  

PubMed

Microtubules are subcellular nanotubes composed of ?- and ?-tubulin that arise from microtubule nucleation sites, mainly composed of ?-tubulin complexes [corrected]. Cell wall encased plant cells have evolved four distinct microtubule arrays that regulate cell division and expansion. Microtubule-associated proteins, the so called MAPs, construct, destruct and reorganize microtubule arrays thus regulating their spatiotemporal transitions during the cell cycle. By physically binding to microtubules and/or modulating their functions, MAPs control microtubule dynamic instability and/or interfilament cross talk. We survey the recent analyses of Arabidopsis MAPs such as MAP65, MOR1, CLASP, katanin, TON1, FASS, TRM, TAN1 and kinesins in terms of their effects on microtubule array organizations and plant development. PMID:23903948

Struk, Sylwia; Dhonukshe, Pankaj

2014-01-01

114

Ferritin associates with marginal band microtubules  

SciTech Connect

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

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

2007-05-01

115

Large aperture adaptive doublet polymer lens for imaging applications.  

PubMed

We report a full design process-finite element modeling, fabrication, and characterization-of adaptive doublet polymer lenses. A first-order model was developed and used to design fluidic doublets, analogous to their glass counterparts. Two constant-volume fluidic chambers were enclosed by three flexible membranes, resulting in a variable focal length doublet with a clear aperture of 19.0 mm. Chromatic focal shift was then used to compare numerical modeling to experimentally measured results over a positive focal length range of 55-200 mm (f/2.89 to f/10.5). PMID:25121541

Santiago, Freddie; Bagwell, Brett; Martinez, Ty; Restaino, Sergio; Krishna, Sanjay

2014-08-01

116

Doublet craters and the tidal disruption of binary asteroids  

NASA Technical Reports Server (NTRS)

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.

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

1991-01-01

117

GDP-Tubulin Incorporation into Growing Microtubules Modulates Polymer Stability*  

PubMed Central

Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule “structural plasticity.” PMID:20371874

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

2010-01-01

118

Dielectric Measurement of Individual Microtubules Using the Electroorientation Method  

PubMed Central

Little is known about the electrostatic/dynamic properties of microtubules, which are considered to underlie their electrostatic interactions with various proteins such as motor proteins, microtubule-associated proteins, and microtubules themselves (lateral association of microtubules). To measure the dielectric properties of microtubules, we developed an experiment system in which the electroorientation of microtubules was observed under a dark-field microscope. Upon application of an alternating electric field (0.5–1.9 × 105 V/m, 10 kHz–3 MHz), the microtubules were oriented parallel to the field line in a few seconds because of the dipole moment induced along their long axes. The process of this orientation was analyzed based on a dielectric ellipsoid model, and the conductivity and dielectric constant of each microtubule were calculated. The analyses revealed that the microtubules were highly conductive, which is consistent with the counterion polarization model—counterions bound to highly negatively charged microtubules can move along the long axis, and this mobility might be the origin of the high conductivity. Our experiment system provides a useful tool to quantitatively evaluate the polyelectrolyte nature of microtubules, thus paving the way for future studies aiming to understand the physicochemical mechanism underlying the electrostatic interactions of microtubules with various proteins. PMID:16500962

Minoura, Itsushi; Muto, Etsuko

2006-01-01

119

Putative microtubule-associated proteins from the Arabidopsis genome  

Microsoft Academic Search

Summary. Plant microtubule-associated proteins (MAPs) are important in modulating the function of the microtubule cytoskeleton. Various plant MAPs have already been described. However, because of the complexity of the plant microtubule cytoskeleton and its responses to developmental and environmental stimuli, there are undoubtedly many more MAPs to be discovered. We have used a literature search and the BLAST protein comparison

J. Gardiner; J. Marc

2003-01-01

120

Analysis of the microtubule-binding domain of MAP-2  

PubMed Central

We examined the microtubule-binding domain of the microtubule- associated protein (MAP), MAP-2, using rabbit antibodies that specifically bind to the microtubule-binding region ("stub") and the projection portion ("arm") of MAP-2. We found that (a) microtubules decorated with arm antibody look similar to those labeled with whole unfractionated MAP antibody, though microtubules are not labeled with stub antibody; (b) incubation of depolymerized microtubule protein with stub antibody prior to assembly partially inhibits the rate of microtubule elongation, presumably because MAPs that are complexed with antibody cannot bind to microtubules and stabilize elongating polymers; (c) the rate of appearance and amounts of 36- and 40-kD microtubule- binding peptides produced by digestion with chymotrypsin are distinct for MAPs associated with microtubules vs. MAPs free in solution. The enhanced stability of the 40-kD peptide when associated with microtubules suggests that this domain of the protein is closely associated with, or partially buried in, the microtubule surface; (d) MAP-2 is a slender, elongate molecule as determined by unidirectional platinum shadowing (90 +/- 30 nm), which is in approximate agreement with previous observations. Stub antibody labels MAP-2 in the terminal one-quarter of the extended protein, indicating an intrinsic asymmetry in the molecule. PMID:4055896

1985-01-01

121

Microtubules: from classical properties to quantum effects in human cognition  

E-print Network

Microtubules: from classical properties to quantum effects in human cognition Ivan Kukuljan properties, higher order assemblies and goes on to discuss the role of microtubules in human cognition. Here to be the medium for the quantum effects in human cognition. The microtubules turned out to be an amazing structure

Â?umer, Slobodan

122

Microtubule dynamics in vivo: a test of mechanisms of turnover  

PubMed Central

Clarification of the mechanism of microtubule dynamics requires an analysis of the microtubule pattern at two time points in the same cell with single fiber resolution. Single microtubule resolution was obtained by microinjection of haptenized tubulin (fluorescein-tubulin) and subsequent indirect immunofluorescence with an antifluorescein antibody. The two time points in a single cell were, first, the time of photobleaching fluorescein-tubulin, and second, the time of fixation. The pattern of fluorescence replacement in the bleached zone during this time interval revealed the relevant mechanisms. In fibroblasts, microtubule domains in the bleached zone are replaced microtubule by microtubule and not by mechanisms that affect all microtubules simultaneously. Of the models we consider, treadmilling and subunit exchange along the length do not account for this observation, but dynamic instability can since it suggests that growing and shrinking microtubules coexist. In addition, we show that the half-time for microtubule replacement is shortest at the leading edge. Dynamic instability accounts for this observation if in general microtubules do not catastrophically disassemble from the plus end, but instead have a significant probability of undergoing a transition to the growing phase before they depolymerize completely. This type of instability we call tempered rather than catastrophic because, through limited disassembly followed by regrowth, it will preferentially replace polymer domains at the ends of microtubules, thus accounting for the observation that the half-time of microtubule domain replacement is shorter with proximity to the leading edge. PMID:3546333

1987-01-01

123

Association between Endocrine Pancreatic Secretory Granules and In-vitro-assembled Microtubules IsDependent upon Microtubule-associated Proteins  

Microsoft Academic Search

By use of dark-fieldlightmicroscopy, secretorygranules isolatedfrom the anglerfish endocrine pancreas were observed to attach to and release from microtubules assembled in vitrofrom brain homogenates .Secretory granules only bound to microtubules assembled in the presence of microtubule-associated proteins (MAPS) and not to microtubules assembled from purified tubulin.The addition of a MAP fraction to purified tubulin restored secretory granule binding .The secretory

K. A. SUPRENANT; W. L. DENTLER

124

Fluorescent microtubules break up under illumination  

PubMed Central

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

1988-01-01

125

Limiting two-Higgs-doublet models  

NASA Astrophysics Data System (ADS)

We update the constraints on two-Higgs-doublet models (2HDMs) focusing on the parameter space relevant to explain the present muon g -2 anomaly, ? a ?, in four different types of models, type I, II, "lepton specific" (or X) and "flipped" (or Y). We show that the strong constraints provided by the electroweak precision data on the mass of the pseudoscalar Higgs, whose contribution may account for ? a ?, are evaded in regions where the charged scalar is degenerate with the heavy neutral one and the mixing angles ? and ? satisfy the Standard Model limit ? - ? ? ?/2. We combine theoretical constraints from vacuum stability and perturbativity with direct and indirect bounds arising from collider and B physics. Possible future constraints from the electron g -2 are also considered. If the 126 GeV resonance discovered at the LHC is interpreted as the light CP-even Higgs boson of the 2HDM, we find that only models of type X can satisfy all the considered theoretical and experimental constraints.

Broggio, Alessandro; Chun, Eung Jin; Passera, Massimo; Patel, Ketan M.; Vempati, Sudhir K.

2014-11-01

126

Analysis of microtubule polymerization dynamics in live cells  

PubMed Central

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

Gierke, Sarah; Kumar, Praveen; Wittmann, Torsten

2012-01-01

127

Inert Doublet Model with a 125 GeV Higgs  

E-print Network

A 125 GeV Higgs-like particle discovered at the LHC in 2012 has properties expected for it in the Standard Model (SM), with a possible enhancement in the two-photon channel. Such SM-like Higgs scenario can be realized within the Inert Doublet Model (IDM) - a version of the Two Higgs Doublet Model with an exact discrete D (Z_2-type) symmetry. In this model one SU(2) doublet plays the role of the SM Higgs doublet with one SM-like Higgs boson. The second doublet has no vacuum expectation value and does not interact with fermions. Among four scalars constituting this D-odd doublet the lightest one is stable, being if neutral a good DM candidate with the right relic density. In this paper an analysis of the two-photon Higgs decay rate in IDM, respecting theoretical and other experimental constraints, is presented. The enhancement in the two-photon channel is possible only if invisible channels are closed, with the enhancement R_{\\gamma \\gamma}>1.2 for masses of DM and charged scalars below 154 GeV. The temperature...

Krawczyk, Maria; Swiezewska, Bogumila

2013-01-01

128

Microtubule Dynamics Control Tail Retraction in Migrating Vascular Endothelial Cells†  

PubMed Central

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

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

2014-01-01

129

Structural basis of EB1 effects on microtubule dynamics.  

PubMed

+TIPs (plus-end tracking proteins) are an increasing group of molecules that localize preferentially to the end of growing microtubules. +TIPs regulate microtubule dynamics and contribute to the organization of the microtubular network within the cell. Thus they participate in a wide range of cellular processes including cell division, motility and morphogenesis. EB1 (end-binding 1) is a highly conserved key member of the +TIP group that has been shown to modulate microtubule dynamics both in vitro and in cells. EB1 is involved in accurate chromosome segregation during mitosis and in the polarization of the microtubule cytoskeleton in migrating cells. Here, we review recent in vitro studies that have started to reveal a regulating activity of EB1, and its yeast orthologue Mal3p, on microtubule structure. In particular, we examine how EB1-mediated changes in the microtubule architecture may explain its effects on microtubule dynamics. PMID:19754439

Coquelle, Frédéric M; Vitre, Benjamin; Arnal, Isabelle

2009-10-01

130

GTP regulates the microtubule nucleation activity of ?-tubulin.  

PubMed

Both subunits of ??-tubulin that comprise the core components of microtubules bind GTP. GTP binding to ?-tubulin has a structural role, whereas ?-tubulin binds and hydrolyses GTP to regulate microtubule dynamics. ?-tubulin, another member of the tubulin superfamily that seeds microtubule nucleation at microtubule-organizing centres, also binds GTP; however, the importance of this association remains elusive. To address the role of GTP binding to ?-tubulin, we systematically mutagenized the GTP contact residues in the yeast ?-tubulin Tub4. Tub4(GTP)-mutant proteins that exhibited greatly reduced GTP affinity still assembled into the small ?-tubulin complex. However, tub4(GTP) mutants were no longer viable, and had defects in interaction between ?-tubulin and ??-tubulin, decreased microtubule nucleation and defects in microtubule organization. In vitro and in vivo data show that only ?-tubulin loaded with GTP nucleates microtubules. Our results suggest that GTP recruitment to ?-tubulin enhances its interaction with ??-tubulin similarly to GTP recruitment to ?-tubulin. PMID:24161932

Gombos, Linda; Neuner, Annett; Berynskyy, Mykhaylo; Fava, Luca L; Wade, Rebecca C; Sachse, Carsten; Schiebel, Elmar

2013-11-01

131

Spindle Assembly and Architecture: From Laser Ablation to Microtubule Nucleation  

NASA Astrophysics Data System (ADS)

Spindles are arrays of microtubules that segregate chromosomes during cell division. It has been difficult to validate models of spindle assembly due to a lack of information on the organization of microtubules in these structures. Here we present a novel method, based on femtosecond laser ablation, capable of measuring the detailed architecture of spindles. We used this method to study the metaphase spindle and find that microtubules are shortest near poles and become progressively longer towards the center of the spindle. These data, in combination with mathematical modeling, high resolution imaging, and biochemical perturbations, are sufficient to reject previously proposed mechanisms of spindle assembly. Our results support a new model of spindle assembly in which microtubule polymerization dynamics are not spatially regulated, microtubule transport locally sorts microtubules -- determining their proper organization in the spindle without moving them appreciable distances --, and the profile of microtubule nucleation controls the length of the spindle.

Needleman, Daniel; Brugues, Jan; Nuzzo, Valeria; Mazur, Eric

2012-02-01

132

Effects of Tau on Flow-Aligned Microtubule Bundles  

NASA Astrophysics Data System (ADS)

Microtubules are cylindrical crystals of the protein tubulin with 17nm inner diameter and 25nm outer diameter. Recent structural studies suggest that the microtubule wall may be porous to small molecules. We have investigated the mobility of molecules in bundles of flow aligned microtubules. We find the spacing between the microtubules in the bundle is increased by the addition of tau, a microtubule associated protein. In the absence of tau, flow can be used to make tightly packed bundles of microtubules. Adding tau causes the tight bundles to swell and separate. We use fluorescence recovery after photobleaching (FRAP) to quantify the mobility of a taxol, a small drug that binds to the microtubule interior.

Ross, Jennifer L.; Kuchnir Fygenson, D.

2003-03-01

133

Isolation and analysis of microtubules and associated proteins.  

PubMed

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

Sloboda, Roger D

2015-01-01

134

How dynein and microtubules rotate the nucleus.  

PubMed

In living cells, a fluctuating torque is exerted on the nuclear surface but the origin of the torque is unclear. In this study, we found that the nuclear rotation angle is directionally persistent on a time scale of tens of minutes, but rotationally diffusive on longer time scales. Rotation required the activity of the microtubule motor dynein. We formulated a model based on microtubules undergoing dynamic instability, with tensional forces between a stationary centrosome and the nuclear surface mediated by dynein. Model simulations suggest that the persistence in rotation angle is due to the transient asymmetric configuration of microtubules exerting a net torque in one direction until the configuration is again randomized by dynamic instability. The model predicts that the rotational magnitude must depend on the distance between the nucleus and the centrosome. To test this prediction, rotation was quantified in patterned cells in which the cell's centrosome was close to the projected nuclear centroid. Consistent with the prediction, the angular displacement was found to decrease in these cells relative to unpatterned cells. This work provides the first mechanistic explanation for how nuclear dynein interactions with discrete microtubules emanating from a stationary centrosome cause rotational torque on the nucleus. PMID:21792925

Wu, Jun; Lee, Kristen C; Dickinson, Richard B; Lele, Tanmay P

2011-10-01

135

New one-dimensional conductors: Graphitic microtubules  

Microsoft Academic Search

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

Noriaki Hamada; Shin-Ichi Sawada; Atsushi Oshiyama

1992-01-01

136

Doublets and other allied well patterns  

SciTech Connect

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.

Brigham, W.E.

1997-06-01

137

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

PubMed Central

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

Farnum, Cornelia E.; Wilsman, Norman J.

2012-01-01

138

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

PubMed Central

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

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

1998-01-01

139

INF1 Is a Novel Microtubule-associated Formin  

PubMed Central

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

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

2008-01-01

140

An assay to image neuronal microtubule dynamics in mice.  

PubMed

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

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

2014-01-01

141

Effects of dynein on microtubule mechanics and centrosome positioning  

PubMed Central

To determine forces on intracellular microtubules, we measured shape changes of individual microtubules following laser severing in bovine capillary endothelial cells. Surprisingly, regions near newly created minus ends increased in curvature following severing, whereas regions near new microtubule plus ends depolymerized without any observable change in shape. With dynein inhibited, regions near severed minus ends straightened rapidly following severing. These observations suggest that dynein exerts a pulling force on the microtubule that buckles the newly created minus end. Moreover, the lack of any observable straightening suggests that dynein prevents lateral motion of microtubules. To explain these results, we developed a model for intracellular microtubule mechanics that predicts the enhanced buckling at the minus end of a severed microtubule. Our results show that microtubule shapes reflect a dynamic force balance in which dynein motor and friction forces dominate elastic forces arising from bending moments. A centrosomal array of microtubules subjected to dynein pulling forces and resisted by dynein friction is predicted to center on the experimentally observed time scale, with or without the pushing forces derived from microtubule buckling at the cell periphery. PMID:22013075

Wu, Jun; Misra, Gaurav; Russell, Robert J.; Ladd, Anthony J. C.; Lele, Tanmay P.; Dickinson, Richard B.

2011-01-01

142

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

PubMed Central

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

1992-01-01

143

Microtubules stabilize cell polarity by localizing rear signals.  

PubMed

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

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

2014-11-18

144

Dynamic instability of microtubules: effect of catastrophe-suppressing drugs  

E-print Network

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

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

2003-10-23

145

The unusual microtubule polarity in teleost retinal pigment epithelial cells  

PubMed Central

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

1988-01-01

146

Dynamic instability of microtubules: Effect of catastrophe-suppressing drugs  

NASA Astrophysics Data System (ADS)

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

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

2005-11-01

147

MICROBIOLOGY: Bacterial Bushwacking Through a Microtubule Jungle  

NSDL National Science Digital Library

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

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

2006-11-10

148

The Human Kinetochore Ska1 Complex Facilitates Microtubule Depolymerization-Coupled Motility  

E-print Network

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

Cheeseman, Iain McPherson

149

Gache et al Purification and mass-spectrometry identification of microtubule-binding proteins  

E-print Network

of functions including to generate force to move along microtubules (5). The minimal definition of a MAP co-localize with microtubules in the cell (6), co-precipitate with microtubules (7) and/or affect

Paris-Sud XI, Université de

150

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

E-print Network

microtubules ( ). The5 minimal definition of a MAP is a protein which can bind to microtubules, but more often by MAPs we understand proteins whichin vitro also co-localize with microtubules in the cell ( ), co-precipitate

Paris-Sud XI, Université de

151

Microtubules in cardiac toxicity and disease  

Microsoft Academic Search

Microtubules (MTs) are dynamic, cytoskeletal fibers that are found in every eukaryotic cell type. MTs serve a wide range of\\u000a functions, including cell division, membrane and vesicle transport, and motility. As such, MTs play pivotal roles in cardiac\\u000a development and function. Agents that disrupt normal MT function, including such therapeutic agents as vincristine and paclitaxel,\\u000a have also been shown to

Daniel R. Webster

2002-01-01

152

Centaurin-?? interacts with ?-tubulin and stabilizes microtubules.  

PubMed

Centaurin-?? is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-?? can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-?? interacting protein, ?-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of ?-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-?? overexpression in HeLa cells and extraction of soluble (?? dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-?? mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-?? remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-?? and tubulin confirmed that Centaurin-?? promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-?? overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-?? role on MT stabilization. Centaurin-?? interacts with ?-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-?? as a new microtubule-associated protein (MAP) increasing MT stability. PMID:23285209

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

2012-01-01

153

Gibberellin stabilizes microtubules in onion leaf sheath cells  

Microsoft Academic Search

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

T. Mita; H. Shibaoka

1984-01-01

154

Direct observation of single kinesin molecules moving along microtubules  

Microsoft Academic Search

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

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

1996-01-01

155

Microtubules are organized independently of the centrosome in Drosophila neurons  

PubMed Central

Background The best-studied arrangement of microtubules is that organized by the centrosome, a cloud of microtubule nucleating and anchoring proteins is clustered around centrioles. However, noncentrosomal microtubule arrays are common in many differentiated cells, including neurons. Although microtubules are not anchored at neuronal centrosomes, it remains unclear whether the centrosome plays a role in organizing neuronal microtubules. We use Drosophila as a model system to determine whether centrosomal microtubule nucleation is important in mature neurons. Results In developing and mature neurons, centrioles were not surrounded by the core nucleation protein ?-tubulin. This suggests that the centrioles do not organize functional centrosomes in Drosophila neurons in vivo. Consistent with this idea, centriole position was not correlated with a specific region of the cell body in neurons, and growing microtubules did not cluster around the centriole, even after axon severing when the number of growing plus ends is dramatically increased. To determine whether the centrosome was required for microtubule organization in mature neurons, we used two approaches. First, we used DSas-4 centriole duplication mutants. In these mutants, centrioles were present in many larval sensory neurons, but they were not fully functional. Despite reduced centriole function, microtubule orientation was normal in axons and dendrites. Second, we used laser ablation to eliminate the centriole, and again found that microtubule polarity in axons and dendrites was normal, even 3 days after treatment. Conclusion We conclude that the centrosome is not a major site of microtubule nucleation in Drosophila neurons, and is not required for maintenance of neuronal microtubule organization in these cells. PMID:22145670

2011-01-01

156

Stability transitions in the microtubule system and their possible significance  

NASA Astrophysics Data System (ADS)

In living organisms microtubules exist as highly labile or very stable structures. In higher eucaryotes, the main, and probably the only, physiological effectors capable of triggering microtubule transtions between lability and stability, are specialized proteins. In the present paper, general methods to assay and isolate these effectors are proposed. The properties of a particular protein (STOP protein) are shown. Finally the potential significance of such effectors in the generation of organelle movement and microtubule spatial self organization are discussed.

Job, D.

1991-05-01

157

Microtubule-Actin Cross-talk at Focal Adhesions  

NSDL National Science Digital Library

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

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

2002-07-02

158

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)

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.

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

159

Singlet scalar Dark Matter in Dark Two Higgs Doublet Model  

E-print Network

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

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

2014-10-20

160

ARRANGEMENT OF SUBUNITS IN FLAGELLAR MICROTUBULES  

Microsoft Academic Search

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

LINDA A. AMOS; A. KLUG

161

Insights into the mechanism of microtubule stabilization by Taxol  

PubMed Central

The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the ?/?-tubulin heterodimer, GTP bound to ?-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to ?-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dimers, even in the absence of exogenous GTP. However, there is little biochemical insight into the mechanism(s) by which Taxol stabilizes microtubules. Here, we analyze the structural changes occurring in both ?- and ?-tubulin upon microtubule stabilization by Taxol. Hydrogen/deuterium exchange (HDX) coupled to liquid chromatography–electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both ?-and ?-tubulin when Taxol was present. Decreased local HDX in peptic peptides was mapped on the tubulin structure and revealed both expected and new dimer–dimer interactions. The increased rigidity in Taxol microtubules was distinct from and complementary to that due to GTP-induced polymerization. The Taxol-induced changes in tubulin conformation act against microtubule depolymerization in a precise directional way. These results demonstrate that HDX coupled to liquid chromatography–electrospray ionization MS can be effectively used to study conformational effects induced by small ligands on microtubules. The present study also opens avenues for locating drug and protein binding sites and for deciphering the mechanisms by which their interactions alter the conformation of microtubules and tubulin dimers. PMID:16801540

Xiao, Hui; Verdier-Pinard, Pascal; Fernandez-Fuentes, Narcis; Burd, Berta; Angeletti, Ruth; Fiser, Andras; Horwitz, Susan Band; Orr, George A.

2006-01-01

162

The Ndc80 kinetochore complex directly modulates microtubule dynamics  

PubMed Central

The conserved Ndc80 complex is an essential microtubule-binding component of the kinetochore. Recent findings suggest that the Ndc80 complex influences microtubule dynamics at kinetochores in vivo. However, it was unclear if the Ndc80 complex mediates these effects directly, or by affecting other factors localized at the kinetochore. Using a reconstituted system in vitro, we show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). In vivo, an N-terminal domain in the Ndc80 complex is phosphorylated by the Aurora B kinase. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment, and mutations that block phosphorylation damp kinetochore oscillations. We find that the Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even when robustly coupled to disassembling microtubule tips. This impaired ability to affect dynamics is not simply because of weakened microtubule binding, as an N-terminally truncated complex with similar binding affinity is able to promote rescue. Taken together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the Ndc80 complex. PMID:22908300

Umbreit, Neil T.; Gestaut, Daniel R.; Tien, Jerry F.; Vollmar, Breanna S.; Gonen, Tamir; Asbury, Charles L.; Davis, Trisha N.

2012-01-01

163

Microtubule Elasticity: Connecting All-Atom Simulations with Continuum Mechanics  

NASA Astrophysics Data System (ADS)

The mechanical properties of microtubules have been extensively studied using a wide range of biophysical techniques, seeking to understand the mechanics of these cylindrical polymers. Here we develop a method for connecting all-atom molecular dynamics simulations with continuum mechanics and show how this can be applied to understand microtubule mechanics. Our coarse-graining technique applied to the microscopic simulation system yields consistent predictions for the Young’s modulus and persistence length of microtubules, while clearly demonstrating how binding of the drug Taxol decreases the stiffness of microtubules. The techniques we develop should be widely applicable to other macromolecular systems.

Sept, David; Mackintosh, Fred C.

2010-01-01

164

Kinetochore-microtubule interactions: the means to the end.  

PubMed

Kinetochores are proteinaceous complexes containing dozens of components; they are assembled at centromeric DNA regions and provide the major microtubule attachment site on chromosomes during cell division. Recent studies have defined the kinetochore components comprising the direct interface with spindle microtubules, primarily through structural and functional analysis of the Ndc80 and Dam1 complexes. These studies have facilitated our understanding of how kinetochores remain attached to the end of dynamic microtubules and how proper orientation of a kinetochore-microtubule attachment is promoted on the mitotic spindle. In this article, we review these recent studies and summarize their key findings. PMID:18182282

Tanaka, Tomoyuki U; Desai, Arshad

2008-02-01

165

Vinblastine suppresses dynamics of individual microtubules in living interphase cells.  

PubMed Central

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

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

1995-01-01

166

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

E-print Network

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

Backer, Chelsea B.

167

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

PubMed

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

Eng, Ryan Christopher; Wasteneys, Geoffrey O

2014-08-01

168

Extending the Microtubule/Microfibril paradigm. Cellulose synthesis is required for normal cortical microtubule alignment in elongating cells  

PubMed

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25-2.5 m) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 g mL-1 fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information. PMID:9501137

Fisher; Cyr

1998-03-01

169

Dark Matter with Topological Defects in the Inert Doublet Model  

E-print Network

We examine the production of dark matter by decaying topological defects in the high mass region $m_{\\mathrm{DM}} \\gg m_W$ of the Inert Doublet Model, extended with an extra U(1) gauge symmetry. The density of dark matter states (the neutral Higgs states of the inert doublet) is determined by the interplay of the freeze-out mechanism and the additional production of dark matter states from the decays of topological defects, in this case cosmic strings. These decays increase the predicted relic abundance compared to the standard freeze-out only case, and as a consequence the viable parameter space of the Inert Doublet Model can be widened substantially. In particular, for a given dark matter annihilation rate lower dark matter masses become viable. We investigate the allowed mass range taking into account constraints on the energy injection rate from the diffuse $\\gamma$-ray background and Big Bang Nucleosynthesis, together with constraints on the dark matter properties coming from direct and indirect detection limits. For the Inert Doublet Model high-mass region, an inert Higgs mass as low as $\\sim 200$ 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 $10^{12}$ GeV to $10^{13}$ GeV.

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

2014-12-15

170

Masses of a Fourth Generation with Two Higgs Doublets  

SciTech Connect

We use sampling techniques to find robust constraints on the masses of a possible fourth sequential fermion generation from electroweak oblique variables. We find that in the case of a light (115 GeV) Higgs from a single electroweak symmetry breaking doublet, inverted mass hierarchies are possible for both quarks and leptons, but a mass splitting more than MW in the quark sector is unlikely. We also find constraints in the case of a heavy (600 GeV) Higgs in a single doublet model. As recent data from the Large Hadron Collider hints at the existence of a resonance at 124.5 GeV and a single Higgs doublet at that mass is inconsistent with a fourth fermion generation, we examine a Type II two Higgs doublet model. In this model, there are ranges of parameter space where the Higgs sector can potentially counteract the effects of the fourth generation. Even so, we find that such scenarios produce qualitatively similar fermion mass distributions.

Bellantoni, Leo; /Fermilab; Erler, Jens; /UNAM, Mexico; Heckman, Jonathan J.; /Princeton, Inst. Advanced Study; Ramirez-Homs, Enrique; /Texas U., El Paso

2012-05-01

171

CP violation conditions in N-Higgs-doublet potentials  

E-print Network

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

C. C. Nishi

2007-10-26

172

Linking axonal degeneration to microtubule remodeling by Spastin-mediated microtubule severing.  

PubMed

Mutations in the AAA adenosine triphosphatase (ATPase) Spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia, which is a retrograde axonopathy primarily characterized pathologically by the degeneration of long spinal neurons in the corticospinal tracts and the dorsal columns. Using recombinant Spastin, we find that six mutant forms of Spastin, including three disease-associated forms, are severely impaired in ATPase activity. In contrast to a mutation designed to prevent adenosine triphosphate (ATP) binding, an ATP hydrolysis-deficient Spastin mutant predicted to remain kinetically trapped on target proteins decorates microtubules in transfected cells. Analysis of disease-associated missense mutations shows that some more closely resemble the canonical hydrolysis mutant, whereas others resemble the ATP-binding mutant. Using real-time imaging, we show that Spastin severs microtubules when added to permeabilized, cytosol-depleted cells stably expressing GFP-tubulin. Using purified components, we also show that Spastin interacts directly with microtubules and is sufficient for severing. These studies suggest that defects in microtubule severing are a cause of axonal degeneration in human disease. PMID:15716377

Evans, Katia J; Gomes, Edgar R; Reisenweber, Steven M; Gundersen, Gregg G; Lauring, Brett P

2005-02-14

173

Linking axonal degeneration to microtubule remodeling by Spastin-mediated microtubule severing  

PubMed Central

Mutations in the AAA adenosine triphosphatase (ATPase) Spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia, which is a retrograde axonopathy primarily characterized pathologically by the degeneration of long spinal neurons in the corticospinal tracts and the dorsal columns. Using recombinant Spastin, we find that six mutant forms of Spastin, including three disease-associated forms, are severely impaired in ATPase activity. In contrast to a mutation designed to prevent adenosine triphosphate (ATP) binding, an ATP hydrolysis–deficient Spastin mutant predicted to remain kinetically trapped on target proteins decorates microtubules in transfected cells. Analysis of disease-associated missense mutations shows that some more closely resemble the canonical hydrolysis mutant, whereas others resemble the ATP-binding mutant. Using real-time imaging, we show that Spastin severs microtubules when added to permeabilized, cytosol-depleted cells stably expressing GFP-tubulin. Using purified components, we also show that Spastin interacts directly with microtubules and is sufficient for severing. These studies suggest that defects in microtubule severing are a cause of axonal degeneration in human disease. PMID:15716377

Evans, Katia J.; Gomes, Edgar R.; Reisenweber, Steven M.; Gundersen, Gregg G.; Lauring, Brett P.

2005-01-01

174

The Leading Role of Microtubules in Endothelial Barrier Dysfunction: Disassembly of Peripheral Microtubules Leaves Behind the Cytoskeletal Reorganization  

PubMed Central

Disturbance of the endothelial barrier is characterized by dramatic cytoskeleton reorganization, activation of actomyosin contraction and, finally, leads to intercellular gap formation. Here we demonstrate that the edemagenic agent, thrombin, causes a rapid increase in the human pulmonary artery endothelial cell (EC) barrier permeability accompanied by fast decreasing in the peripheral microtubules quantity and reorganization of the microtubule system in the internal cytoplasm of the EC within 5 min of the treatment. The actin stress-fibers formation occurs gradually and the maximal effect is observed relatively later, 30 min of the thrombin treatment. Thus, microtubules reaction develops faster than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction development. Direct microtubules depolymerization by nocodazole initiates the cascade of barrier dysfunction reactions. Nocodazole-induced barrier disruption is connected directly with the degree of peripheral microtubules depolymerization. Short-term loss of endothelial barrier function occurs at the minimal destruction of peripheral microtubules, when actin filament system is still intact. Specifically, we demonstrate that the EC microtubule dynamics examined by time-lapse imaging of EB3-GFP comets movement has changed under these conditions: microtubule plus ends growth rate significantly decreased near the cell periphery. The microtubules, apparently, are the first target in the circuit of reactions leading to the pulmonary EC barrier compromise. Our results show that dynamic microtubules play an essential role in the barrier function in vitro: peripheral microtubules depolymerization is necessary and sufficient condition for initiation of endothelial barrier dysfunction. PMID:23606375

Alieva, Irina B.; Zemskov, Evgeny A.; Smurova, Ksenija M.; Kaverina, Irina N.; Verin, Alexander D.

2014-01-01

175

The Role of Microtubules in Guard Cell Function1  

Microsoft Academic Search

Guard cells are able to sense a multitude of environmental signals and appropriately adjust the stomatal pore to regulate gas exchange in and out of the leaf. The role of the microtubule cytoskeleton during these stomatal movements has been debated. To help resolve this debate, in vivo stomatal aperture assays with different microtubule inhibitors were performed. We observed that guard

Adam I. Marcus; Richard C. Moore; Richard J. Cyr

2001-01-01

176

A new directionality tool for assessing microtubule pattern alterations  

PubMed Central

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

Liu, Wenhua; Ralston, Evelyn

2014-01-01

177

Mathematics and biophysics of cortical microtubules in plants  

E-print Network

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

Allard, Jun

178

A new directionality tool for assessing microtubule pattern alterations.  

PubMed

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

Liu, Wenhua; Ralston, Evelyn

2014-04-01

179

Microtubule-severing enzymes at the cutting edge.  

PubMed

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. PMID:22595526

Sharp, David J; Ross, Jennifer L

2012-06-01

180

Stall force of polymerizing microtubules and filament bundles  

E-print Network

OFFPRINT Stall force of polymerizing microtubules and filament bundles J. Krawczyk and J. Kierfeld, 93 (2011) 28006 www.epljournal.org doi: 10.1209/0295-5075/93/28006 Stall force of polymerizing investigate stall force and polymerization kinetics of rigid protofilaments in a microtubule or interacting

Kierfeld, Jan

181

Microtubule distribution in gravitropic protonemata of the moss Ceratodon  

NASA Technical Reports Server (NTRS)

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.

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

1990-01-01

182

Jupiter, a New Drosophila Protein Associated With Microtubules  

E-print Network

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

Villefranche sur mer

183

?-Tubulin and microtubule organization during microsporogenesis in Ginkgo biloba  

Microsoft Academic Search

This is the first report on ?-tubulin and microtubule arrays during microsporogenesis in a gymnosperm. Meiosis in Ginkgo biloba is polyplastidic, as is typical of the spermatophyte clade, and microtubule arrays are organized at various sites during meiosis and cytokinesis. In early prophase, a cluster of ?-tubulin globules occurs in the central cytoplasm adjacent to the off-center nucleus. These globules

R. C. Brown; B. E. Lemmon

2005-01-01

184

A novel direct interaction of endoplasmic reticulum with microtubules.  

PubMed Central

The positioning and dynamics of organelles in eukaryotic cells critically depend on membrane-cytoskeleton interactions. Motor proteins play an important role in the directed movement of organelle membranes along microtubules, but the basic mechanism by which membranes stably interact with the microtubule cytoskeleton is largely unknown. Here we report that p63, an integral membrane protein of the reticular subdomain of the rough endoplasmic reticulum (ER), binds microtubules in vivo and in vitro. Overexpression of p63 in cell culture led to a striking rearrangement of the ER and to concomitant bundling of microtubules along the altered ER. Mutational analysis of the cytoplasmic domain of p63 revealed two determinants responsible for these changes: an ER rearrangement determinant near the N-terminus and a central microtubule-binding region. The two determinants function independently of one another as indicated by deletion experiments. A peptide corresponding to the cytoplasmic tail of p63 promoted microtubule polymerization in vitro. p63 is the first identified integral membrane protein that can link a membrane organelle directly to microtubules. By doing so, it may contribute to the positioning of the ER along microtubules. PMID:9799226

Klopfenstein, D R; Kappeler, F; Hauri, H P

1998-01-01

185

MODEL BASED DYNAMICS ANALYSIS IN MICROTUBULE VIDEOS A. Altinok1  

E-print Network

and Developmental Biology3 University of California Santa Barbara, Santa Barbara, CA 93106 ABSTRACT Microtubule databases. 1. INTRODUCTION Microtubules (MT) are filamentous subcellular structures in- volved in cell dissimilarities between regulatory agents. Furthermore, this measure can be used for querying MT video databases

California at Santa Barbara, University of

186

Cryo-electron tomography of microtubule-kinesin motor complexes  

PubMed Central

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

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

2010-01-01

187

Effect on Microtubule Dynamics of XMAP230, a Microtubule-associated Protein Present in Xenopus laevis Eggs and Dividing Cells  

Microsoft Academic Search

The reorganization from a radical inter- phase microtubule (MT) network into a bipolar spin- dle at the onset of mitosis involves a dramatic change in MT dynamics. Microtubule-associated proteins (MAPs) and other factors are thought to regulate MT dynamics both in interphase and in mitosis. In this study we report the purification and functional in vitro characterization of a 230-KD

Seren S. L. Andersen; Brigitte Buendia; Jorge E. Domfnguez; Alan Sawyer; Eric Karsenti

1994-01-01

188

1. What is the "dynamic instability" of microtubules? How does this property influence the function(s) of microtubules? (10%)  

E-print Network

1. What is the "dynamic instability" of microtubules? How does this property influence the function(s) of microtubules? (10%) 2. Describe the roles of tropomyosin and troponin in muscle contraction. (5%) 3. What%) 5. There are two basic kind of EndoplasmicReticulum (ER). What are they? What are the differences

Huang, Haimei

189

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

PubMed Central

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

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

2012-01-01

190

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

Microsoft Academic Search

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

R. C. Brown; B. E. Lemmon

1983-01-01

191

Protein Arms in the Kinetochore-Microtubule Interface of the Yeast DASH Complex  

Microsoft Academic Search

The yeast DASH complex is a heterodecameric component of the kinetochore necessary for accurate chromosome segregation. DASH forms closed rings around microtubules with a large gap between the DASH ring and the microtubule cylinder. We characterized the microtubule-binding properties of limited proteolysis products and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic

JJ L. Miranda; David S. King; Stephen C. Harrison

2007-01-01

192

Microtubule-targeted anticancer agents and apoptosis.  

PubMed

Over the past decade, significant progress has been made in our understanding of the biology of microtubule (MT) assembly into the mitotic spindle during mitosis and the molecular signaling and execution of the various pathways to apoptosis. In the same period, the microtubule-targeted tubulin-polymerizing agents (MTPAs), notably paclitaxel and taxotere, have come to occupy a central role in the treatment of a variety of human epithelial cancers. Following their binding to B-tubulin, MTPAs inhibit MT dynamic instability, cell cycle G2/M phase transition and mitotic arrest of cancer cells. MTPA-induced anti-MT and cell cycle effects trigger the molecular signaling for the mitochondrial pathway of apoptosis. This triggering is orchestrated through different molecular links and determined by the threshold for apoptosis that is set and controlled diversely in various cancer types. The complexity and regulatory potential of the links and the apoptosis threshold are integral to the transformed biology of the cancer cell. The emerging understanding of this biology and how it is influenced by treatment with MTPAs has highlighted novel strategies to further enhance the antitumor activity and overcome resistance to MTPA-induced apoptosis in cancer cells. PMID:14663486

Bhalla, Kapil N

2003-12-01

193

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

PubMed Central

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

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

2012-01-01

194

Simple Description of Doublet Bands with Mass of Approximately 100  

NASA Astrophysics Data System (ADS)

A simple collective model is applied to understand the structure of the ? I = 1 doublet bands with the ? h_{11/2} ? ? g_{9/2} configuration in the doubly-odd nuclei, ^{98-104}Tc and ^{100-106}Rh. In the model, a doubly-odd nucleus is realized when there is one neutron in the 0h_{11/2} orbital and one proton in the 0g_{9/2} orbital, and the collective core represents the even-even part of the nucleus. The calculation reproduces the energy spectra and the ratios B(M1; I ? I-1) / B(E2; I ? I-2) for the doublet bands quite well. The analysis of the wave functions reveals new band structure, showing various angular momentum configurations of the neutron and the proton, weakly coupled with the quadrupole collective excitations of the core.

Higashiyama, K.; Yoshinaga, N.

2008-09-01

195

Two-Higgs doublet models confront the naturalness problem  

NASA Astrophysics Data System (ADS)

The conjecture that some unknown symmetry is responsible for keeping the Higgs boson light at 125 GeV does not hold for the Standard Model, where the coefficient of the quadratic divergence of Higgs boson self-energy is far from zero. We show that such a cancellation can be achieved in two-Higgs doublet models, by virtue of which all the scalars remain at the electroweak scale and the naturalness problem is avoided. We explore the consequences of such cancellations in different two-Higgs doublet models with no flavor-changing neutral current, and show that the parameter space becomes tightly constrained; in particular, the ratio of two vacuum expectation values, tan ? , no longer remains a free parameter but turns out to be a function of the quartic couplings.

Chakraborty, Indrani; Kundu, Anirban

2014-12-01

196

A search for close-mass lepton doublet  

SciTech Connect

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.

Riles, J.K.

1989-04-01

197

A simple description of doublet bands in mass around 100  

NASA Astrophysics Data System (ADS)

The structure of doublet bands associated with neutron 0h11/2 and proton 0g9/2 orbital in the doubly-odd nuclei, 98-104Tc and 100-106Rh is studied theoretically using the quadrupole coupling model. The calculated energy levels and electromagnetic transitions are in excellent agreement with experimental data. The internal structure of the yrast states is discussed in terms of the QCM wave functions.

Yoshinaga, N.; Higashiyama, K.

2009-05-01

198

CP-odd invariants in models with several Higgs doublets  

E-print Network

We present CP-odd Higgs-basis invariants, which can be used to signal CP violation in a multi-Higgs system, written in an arbitrary Higgs basis. It is shown through specific examples how these CP-odd invariants can also be useful to determine the character of CP breaking (i.e. whether it is hard or soft CP breaking) in a given Higgs Lagrangian. We analyse in detail the cases of two and three Higgs doublets.

Gustavo C. Branco; M. N. Rebelo; J. I. Silva-Marcos

2005-02-13

199

Theoretical analysis of microtubule dynamics at all times.  

PubMed

Microtubules are biopolymers consisting of tubulin dimer subunits. As a major component of cytoskeleton they are essential for supporting most important cellular processes such as cell division, signaling, intracellular transport and cell locomotion. The hydrolysis of guanosine triphosphate (GTP) molecules attached to each tubulin subunit supports the nonequilibrium nature of microtubule dynamics. One of the most spectacular properties of microtubules is their dynamic instability when their growth from continuous attachment of tubulin dimers stochastically alternates with periods of shrinking. Despite the critical importance of this process to all cellular activities, its mechanism remains not fully understood. We investigated theoretically microtubule dynamics at all times by analyzing explicitly temporal evolution of various length clusters of unhydrolyzed subunits. It is found that the dynamic behavior of microtubules depends strongly on initial conditions. Our theoretical findings provide a microscopic explanation for recent experiments which found that the frequency of catastrophes increases with the lifetime of microtubules. It is argued that most growing microtubule configurations cannot transit in one step into a shrinking state, leading to a complex overall temporal behavior. Theoretical calculations combined with Monte Carlo computer simulations are also directly compared with experimental observations, and good agreement is found. PMID:25390471

Li, Xin; Kolomeisky, Anatoly B

2014-12-01

200

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

PubMed Central

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

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

1994-01-01

201

An Improved Quantitative Analysis Method for Plant Cortical Microtubules  

PubMed Central

The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD) algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1) image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM) algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies. PMID:24744684

Lu, Yi; Huang, Chenyang; Wang, Jia; Shang, Peng

2014-01-01

202

A Thermodynamic Model of Microtubule Assembly and Disassembly  

PubMed Central

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. PMID:19668378

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

2009-01-01

203

Two-Higgs-doublet type-II seesaw model  

NASA Astrophysics Data System (ADS)

Motivated by the new observed scalar boson of 126 GeV at ATLAS and CMS, various phenomena in the two-Higgs-doublet model are investigated broadly in the literature. For considering the model that possesses a solution to the massive neutrinos, we study the simplest extension of conventional type-II seesaw model to two Higgs doublets. We find that the new interactions in the scalar potential cause the sizable mixture of charged Higgses in a triplet and doublets. As a result, we have a completely different decay pattern for doubly charged Higgs (?±±); even the vacuum expectation value of a Higgs triplet is at GeV level, which is limited by the precision measurement for the ? parameter. For illustrating the new characters of the model, we study the influence of new interactions on the new open channels ?++?(H1+W+(*),H1+H1+) with H1+ being the lightest charged Higgs. Additionally, due to the new mixing effect, the triplet charged Higgs could couple to quarks in the model; therefore, the search for ?++ via ?++?tbW+?bb¯W+W+ by mediated H1+ becomes significant.

Chen, Chuan-Hung; Nomura, Takaaki

2014-10-01

204

Dark Matter with Topological Defects in the Inert Doublet Model  

E-print Network

We examine the production of dark matter by decaying topological defects in the high mass region $m_{\\mathrm{DM}} \\gg m_W$ of the Inert Doublet Model, extended with an extra U(1) gauge symmetry. The density of dark matter states (the neutral Higgs states of the inert doublet) is determined by the interplay of the freeze-out mechanism and the additional production of dark matter states from the decays of topological defects, in this case cosmic strings. These decays increase the predicted relic abundance compared to the standard freeze-out only case, and as a consequence the viable parameter space of the Inert Doublet Model can be widened substantially. In particular, for a given dark matter annihilation rate lower dark matter masses become viable. We investigate the allowed mass range taking into account constraints on the energy injection rate from the diffuse $\\gamma$-ray background and Big Bang Nucleosynthesis, together with constraints on the dark matter properties coming from direct and indirect detectio...

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

2014-01-01

205

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

E-print Network

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

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

2010-01-01

206

Regulation of Microtubule Severing by Katanin Subunits during Neuronal Development  

PubMed Central

Katanin, the microtubule-severing protein, consists of a subunit termed P60 that breaks the lattice of the microtubule and another subunit termed P80, the functions of which are not well understood. Data presented here show that the ratio of P60 to P80 varies markedly in different tissues, at different phases of development, and regionally within the neuron. P80 is more concentrated in the cell body and less variable during development, whereas P60 often shows concentrations in the distal tips of processes as well as dramatic spikes in expression at certain developmental stages. Overexpression of P60 at various stages in the differentiation of cultured hippocampal neurons results in substantial loss of microtubule mass and a diminution in total process length. In comparison, overexpression of P80, which is thought to augment the severing of microtubules by P60, results in a milder loss of microtubule mass and diminution in process length. At the developmental stage corresponding to axogenesis, overexpression of P60 decreases the total number of processes extended by the neuron, whereas overexpression of P80 produces the opposite result, suggesting that the effects on neuronal morphology are dependent on the degree of microtubule severing and loss of polymer. The microtubules that occupy the axon are notably more resistant to depolymerization in response to excess P60 or P80 than microtubules elsewhere in the neuron, suggesting that regional differences in the susceptibility of microtubules to severing proteins may be a critical factor in the generation and maintenance of neuronal polarity. PMID:15944385

Yu, Wenqian; Solowska, Joanna M.; Qiang, Liang; Karabay, Arzu; Baird, Douglas; Baas, Peter W.

2005-01-01

207

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

E-print Network

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

Welburn, Julie P. I.

208

Microtubule attachment and spindle assembly checkpoint signaling at the kinetochore  

PubMed Central

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

Foley, Emily A.; Kapoor, Tarun M.

2013-01-01

209

The segmentation of microtubules in electron tomograms using Amira.  

PubMed

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

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

2014-01-01

210

Transcriptional regulation of an axonemal central apparatus gene, sperm-associated antigen 6, by a SRY-related high mobility group transcription factor, S-SOX5.  

PubMed

SOX5 is a transcription factor with homology to the high mobility group box region of the testis-determining factor, SRY. Both the mouse and human SOX5 genes encode a 48-kDa SOX5 protein (S-SOX5) that is only present in tissues containing cells with motile cilia/flagella. The mammalian sperm-associated antigen 6 gene (SPAG6) encodes an axoneme central apparatus protein. Because human and mouse SPAG6 gene promoters contain multiple potential binding sites for SOX5, SPAG6 gene regulation by S-SOX5 was investigated in BEAS-2B cells, a line derived from human bronchial cells. Like FOXJ1, a transcription factor known to be essential for motile ciliogenesis, S-SOX5 stimulated mouse and human SPAG6 promoter function in BEAS-2B cells, but the effect was abrogated when the SOX5 binding sites were mutated or deleted. S-SOX5 and FOXJ1 functioned cooperatively in stimulating SPAG6 promoter activity. The SPAG6 message was up-regulated when S-SOX5 was overexpressed in BEAS-2B cells, and silencing of S-SOX5 by RNA interference down-regulated SPAG6 transcripts. Chromatin immunoprecipitation and EMSA experiments demonstrated that S-SOX5 associates with the SPAG6 promoter directly. The present study demonstrates that SPAG6 is a S-SOX5 target gene, indicating a key role for S-SOX5 in the formation and function of motile cilia. PMID:20668334

Kiselak, Elizabeth Anne; Shen, Xuening; Song, Jingmei; Gude, David Roberto; Wang, Jiannan; Brody, Steven L; Strauss, Jerome F; Zhang, Zhibing

2010-10-01

211

Crowding of molecular motors determines microtubule depolymerization  

E-print Network

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

Reese, Louis; Frey, Erwin

2011-01-01

212

Tau Induces Cooperative Taxol Binding to Microtubules  

NASA Astrophysics Data System (ADS)

Taxol and tau are two ligands which stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds ? tubulin in the MT interior. Tau is a MT-associated protein that binds both ? and ? tubulin on the MT exterior. Both taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts as a buttress to bundle, stiffen, and space MTs. A structural study recently suggested that taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau and yields a measure of taxol cooperativity when tau is present.

Ross, Jennifer; Santangelo, Christian; Victoria, Makrides; Fygenson, Deborah

2004-03-01

213

Diffusion of dextran inside microtubule sample  

NASA Astrophysics Data System (ADS)

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 .

Prodan, Camelia

2005-03-01

214

Synthesis of neolignans as microtubule stabilisers.  

PubMed

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

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

2014-02-15

215

High-Resolution Microtubule Structures Reveal the Structural Transitions  

E-print Network

, CA 94720, USA 2Life Sciences Division, Lawrence Berkeley National Lab, Berkeley, CA 94720, USA 3., 2010; Rieder and Salmon, 1994). Highlighting this fact, many successful anti- proliferative drugs bind- mational cycle accompanying tubulin polymerization, nucleotide hydrolysis, and microtubule depolymerization

Baker, David

216

Dynamics of an Idealized Model of Microtubule Growth and Catastrophe  

PubMed Central

We investigate a simple dynamical model of a microtubule that evolves by attachment of guanosine triphosphate (GTP) tubulin to its end, irreversible conversion of GTP to guanosine diphosphate (GDP) tubulin by hydrolysis, and detachment of GDP at the end of a microtubule. As a function of rates of these processes, the microtubule can grow steadily or its length can fluctuate wildly. In the regime where detachment can be neglected, we find exact expressions for the tubule and GTP cap length distributions, as well as power-law length distributions of GTP and GDP islands. In the opposite limit of instantaneous detachment, we find the time between catastrophes, where the microtubule shrinks to zero length, and determine the size distribution of avalanches (sequence of consecutive GDP detachment events). We obtain the phase diagram for general rates and verify our predictions by numerical simulations. PMID:17995026

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

2008-01-01

217

Nanotube interactions with microtubules: implications for cancer medicine.  

PubMed

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

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

2014-07-01

218

Stochastic Modeling and Analysis of Plant Microtubule System Characteristics  

E-print Network

In this dissertation, we consider a complex biological system known as cortical microtubule (CMT) system, where stochastic dynamics of the components (i.e., the CMTs) are defined in both space and time. CMTs have an inherent spatial dimension...

Eren, Ezgi

2012-07-16

219

Multiscale Polar Theory of Microtubule and Motor-Protein Assemblies  

NASA Astrophysics Data System (ADS)

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.

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

2015-01-01

220

Multiscale polar theory of microtubule and motor-protein assemblies  

E-print Network

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

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

2015-01-27

221

Mechanical Models of Microtubule Bundle Collapse in Alzheimer's Disease  

NASA Astrophysics Data System (ADS)

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

Sendek, Austin; Singh, Rajiv; Cox, Daniel

2013-03-01

222

Motion observation and SPR measurements of kinesin motility on microtubules  

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

223

Microtubules and control of macronuclear 'amitosis' in Paramecium.  

PubMed

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

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

1980-08-01

224

Structure of cortical microtubule arrays in plant cells  

Microsoft Academic Search

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

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

1978-01-01

225

Contrasting models for kinetochore microtubule attachment in mammalian cells  

Microsoft Academic Search

Kinetochore function is mediated through its interaction with microtubule plus ends embedded in the kinetochore outer plate.\\u000a Here, we compare and evaluate current models for kinetochore microtubule attachment, beginning with a brief review of the\\u000a molecular, biochemical, cellular, and structural studies upon which these models are based. The majority of these studies\\u000a strongly support a model in which the kinetochore

Bruce F. McEwen; Yimin Dong

2010-01-01

226

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

PubMed

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

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

2014-02-01

227

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

PubMed Central

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

Hamada, Takahiro

2014-01-01

228

Dimer model for Tau proteins bound in microtubule bundles  

NASA Astrophysics Data System (ADS)

The microtubule associated protein tau is important in nucleating and maintaining microtubule spacing and structure in neuronal axons. Modification of tau is implicated as a later stage process in Alzheimer's disease, but little is known about the structure of tau in microtubule bundles. We present preliminary work on a proposed model [1] for tau dimers in microtubule bundles (dimers are the minimal units since there is one microtubule binding domain per tau). First, a model of tau monomer was created and its characteristics explored using implicit solvent molecular dynamics simulation. Multiple simulations yield a partially collapsed form with separate positively/negatively charged clumps, but which are a factor of two smaller than required by observed microtubule spacing. We argue that this will elongate in dimer form to lower electrostatic energy at a cost of entropic ``spring'' energy. We will present preliminary results on steered molecular dynamics runs on tau dimers to estimate the actual force constant.[4pt] [1] Rosenberg, K. J. Ross, J. L. Feinstein, H. E., Feinstein, S. C. Israelachvili, J., PNAS (USA) 105, 2008, 7445-50.

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

2013-03-01

229

Direct interaction of microtubule- and actin-based transport motors  

NASA Technical Reports Server (NTRS)

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.

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

1999-01-01

230

Evidence for Opposing Effects of Calmodulin on Cortical Microtubules.  

PubMed Central

Microtubule integrity within the cortical array was visualized in detergent-lysed carrot (Daucus carota L.) protoplasts that were exposed to various exogenous levels of Ca2+ and calmodulin (CaM). CaM appears to help stabilize cortical microtubules against the destabilizing action of Ca2+/CaM complexes at low Ca2+ concentrations, but not at higher Ca2+ concentrations. The hypothesis that CaM interacts with microtubules at two different sites, determined by the concentration of Ca2+, is supported by the effects of the CaM antagonists N-(6-aminohexyl)-1-naphthalene-sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfanamide (20 [mu]M) and by affinity chromatography. Two classes of proteins were identified that interact with tubulin and bind to CaM. One class required Ca2+ for CaM binding, whereas the second class bound only when Ca2+ concentrations were low (<320 nM). Thus, CaM's ability to have two opposing effects upon microtubules may be regulated by the concentration of intracellular Ca2+ and its differential interactions with microtubule-associated proteins. Experimental manipulation of intracellular Ca2+ concentrations, as monitored by Indo-1, revealed that the effect of Ca2+ is specific to the cortical microtubules and does not affect actin microfilaments in these cells. PMID:12226434

Fisher, D. D.; Gilroy, S.; Cyr, R. J.

1996-01-01

231

Mammalian end binding proteins control persistent microtubule growth  

PubMed Central

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

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

2009-01-01

232

Lucky 13-microtubule depolymerisation by kinesin-13 motors.  

PubMed

The kinesin-13 class of motors catalyses microtubule depolymerisation by bending tubulins at microtubule ends. Depolymerisation activity is intrinsic to the kinesin-13 motor core but the activity of the core alone is very low compared with that of constructs that also contain a conserved neck sequence. The full-length dimeric motor is an efficient depolymeriser and also diffuses along the microtubule lattice, which helps it to find microtubule ends. Current evidence supports the idea of a generic mechanism for kinesin-13-catalysed depolymerisation. However, the activity of kinesin-13 motors is precisely localised and regulated in vivo to enable a wide range of cellular roles. The proteins are involved in global control of microtubule dynamics. They also localise to mitotic and meiotic spindles, where they contribute to formation and maintenance of spindle bipolarity, chromosomal congression, attachment correction and chromatid separation. In interphase cells, intricate and subtle mechanisms appear to allow kinesin-13 motors to act on specific populations of microtubules. Such carefully controlled localisation and regulation makes these kinesins efficient, multi-tasking molecular motors. PMID:16988025

Moores, Carolyn A; Milligan, Ronald A

2006-10-01

233

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

E-print Network

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

Mirny, Leonid A.

234

Two-loop correction to weak-interaction parameters due to a heavy fermion doublet  

Microsoft Academic Search

The two-loop corrections to the varrho-parameter and the vector boson mass-shifts due to a heavy fermion doublet were calculated. For very heavy (~ TeV) quarks we find improved limits on the allowed mass difference within a doublet. The limit on the top quark mass (300 GeV) is unaffected. For a degenerate doublet there is a Higgs mass dependent limit on

J. J. van der Bij; F. Hoogeveen

1987-01-01

235

Pole in k cotdelta for doublet, s-wave, n-d scattering  

Microsoft Academic Search

The position of the pole in k cotdelta, for doublet s-wave n-d scattering, and its residue are shown to be correlated with the doublet scattering length. An approximate, analytic solution of the N\\/D equations of Barton and Phillips indicates a linear dependence on the doublet scattering length for the pole position, and a quadratic dependence for the residue. These relationships

James Whiting; Michael Fuda

1976-01-01

236

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

NASA Technical Reports Server (NTRS)

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

Jukes, T. H.

1977-01-01

237

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

PubMed Central

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

Baas, Peter W.; Lin, Shen

2011-01-01

238

Kinesin-13s in mitosis: Key players in the spatial and temporal organization of spindle microtubules.  

PubMed

Dynamic microtubules are essential for the process of mitosis. Thus, elucidating when, where, and how microtubule dynamics are regulated is key to understanding this process. One important class of proteins that directly regulates microtubule dynamics is the Kinesin-13 family. Kinesin-13 proteins induce depolymerization uniquely from both ends of the microtubule. This activity coincides with their cellular localization and with their ability to regulate microtubule dynamics to control spindle assembly and kinetochore-microtubule attachments. In this review, we highlight recent findings that dissect the important actions of Kinesin-13 family members and summarize important studies on the regulation of their activity by phosphorylation and by protein-protein interactions. PMID:20109574

Ems-McClung, Stephanie C; Walczak, Claire E

2010-05-01

239

Quantum computation in brain microtubules: Decoherence and biological feasibility  

NASA Astrophysics Data System (ADS)

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

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

2002-06-01

240

Associations of elements of the Golgi apparatus with microtubules  

PubMed Central

The intracellular spatial relationships between elements of the Golgi apparatus (GA) and microtubules in interphase cells have been explored by double immunofluorescence microscopy. By using cultured cells infected with the temperature-sensitive Orsay-45 mutant of vesicular stomatitis virus and a temperature shift-down protocol, we visualized functional elements of the GA by immunolabeling of the G protein of the virus that was arrested in the GA during its intracellular passage to the plasma membrane 13 min after the temperature shift-down. Complete disassembly of the cytoplasmic microtubules by nocodazole at the nonpermissive temperature before the temperature shift led to the dispersal of the GA elements, from their normal compact perinuclear configuration close to the microtubule-organizing center (MTOC) into the cell periphery. Washout of the nocodazole that led to the reassembly of the microtubules from the MTOC also led to the recompaction of the GA elements to their normal configuration. During this recompaction process, GA elements were seen in close lateral apposition to microtubules. In cells treated with nocodazole followed by taxol, an MTOC developed, but most of the microtubules were free of the MTOC and were assembled into bundles in the cell periphery. Under these circumstances, the GA elements that had been dispersed into the cell periphery by the nocodazole treatment remained dispersed despite the presence of an MTOC. In cells treated directly with taxol, free microtubules were seen in the cytoplasm in widely different, bundled configurations from one cell to another, but, in each case, elements of the GA appeared to be associated with one of the two end regions of the microtubule bundles, and to be uncorrelated with the locations of the vimentin intermediate filaments in these cells. These results are interpreted to suggest two types of associations of elements of the GA with microtubules: one lateral, and the other (more stable) end-on. The end-on association is suggested to involve the minus-end regions of microtubules, and it is proposed that this accounts for the GA-MTOC association in normal cells. PMID:6381504

1984-01-01

241

On the significance of microtubule flexural behavior in cytoskeletal mechanics.  

PubMed

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 of microtubules by 40 folds. A modification to tensegrity model is, therefore, proved necessary in order to take into account the flexural response of microtubules. The concept of "bendo-tensegrity" is proposed as a modification to contemporary cytoskeletal tensegrity models. PMID:21998675

Mehrbod, Mehrdad; Mofrad, Mohammad R K

2011-01-01

242

MCAK-Independent Functions of ch-Tog/XMAP215 in Microtubule Plus-End Dynamics? †  

PubMed Central

The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells. PMID:18809577

Barr, Alexis R.; Gergely, Fanni

2008-01-01

243

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

PubMed

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

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

2009-10-01

244

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

PubMed Central

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

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

2013-01-01

245

Fission yeast mtr1p regulates interphase microtubule cortical dwell-time  

PubMed Central

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

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

2014-01-01

246

Electron/muon specific two Higgs doublet model  

NASA Astrophysics Data System (ADS)

We discuss two Higgs doublet models with a softly-broken discrete S3 symmetry, where the mass matrix for charged-leptons is predicted as the diagonal form in the weak eigenbasis of lepton fields. Similarly to an introduction of Z2 symmetry, the tree level flavor changing neutral current can be forbidden by imposing the S3 symmetry to the model. Under the S3 symmetry, there are four types of Yukawa interactions depending on the S3 charge assignment to right-handed fermions. We find that extra Higgs bosons can be muon and electron specific in one of four types of the Yukawa interaction. This property does not appear in any other two Higgs doublet models with a softly-broken Z2 symmetry. We discuss the phenomenology of the muon and electron specific Higgs bosons at the Large Hadron Collider; namely we evaluate allowed parameter regions from the current Higgs boson search data and discovery potential of such a Higgs boson at the 14 TeV run.

Kajiyama, Yuji; Okada, Hiroshi; Yagyu, Kei

2014-10-01

247

Next-to-Minimal Two Higgs Doublet Model  

DOE PAGESBeta

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

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

2014-04-01

248

Spinning Janus doublets driven in uniform ac electric fields  

NASA Astrophysics Data System (ADS)

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

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

2014-01-01

249

Role of microtubules in the contractile dysfunction of hypertrophied myocardium  

NASA Technical Reports Server (NTRS)

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.

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

1999-01-01

250

Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters  

PubMed Central

Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and Ptdlns3K-dependent formation of LC3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A1, but not nutrient deprivation or rapamycin treatment, further increased LC3-II generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with SQSTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. Efficient autophagy by hypertonic stress required microtubule remodeling and was DYNC/dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (EHNA) or genetic [DCTN2/dynactin 2 (p50) overexpression] interference of DYNC activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response. PMID:23380587

Nunes, Paula; Ernandez, Thomas; Roth, Isabelle; Qiao, Xiaomu; Strebel, Déborah; Bouley, Richard; Charollais, Anne; Ramadori, Pierluigi; Foti, Michelangelo; Meda, Paolo; Féraille, Eric; Brown, Dennis; Hasler, Udo

2013-01-01

251

Transcription factor NF-?B associates with microtubules and stimulates apoptosis in response to suppression of microtubule dynamics in MCF-7 cells.  

PubMed

NF-?B, a master regulator of several signaling cascades, is known to be actively transported in the nucleus in response to various stimuli. Here, we found that NF-?B is associated with polymeric tubulin and co-localized with microtubules in MCF-7 cells. Using TN16, a known microtubule targeting agent, we found that microtubule dynamics plays a critical role in NF-?B-microtubule interaction. Treatment of cells with low concentrations of TN16 (25 and 50nM) that suppressed microtubule dynamics without visibly affecting microtubule organization enhanced the association of NF-?B with microtubules and facilitated nuclear translocation of NF-?B. Colchicine and vinblastine also produced similar nuclear translocation of NF-?B. Further, nuclear import of NF-?B activated apoptotic pathway in the cells that were blocked in mitosis by TN16 treatment suggesting that NF-?B acts as a pro-apoptotic protein in response to the suppression of microtubule dynamics. Interestingly, in the presence of high concentrations of TN16 that extensively disrupted the microtubule network, though there was an increase in the apoptotic cell death, the interaction of NF-?B with microtubules and its nuclear import were significantly reduced. Under these conditions, we detected an increase in the level of phosphorylation and nuclear accumulation of ERK, a MAP kinase, suggesting that the induction of apoptosis was caused by ERK signaling. The results indicate that the interaction of NF-?B with microtubules, its nuclear accumulation and subsequent gene transcription are critically dependent on microtubule dynamics. The data suggest a correlation between the functional status of microtubules and different apoptotic mechanisms invoked in response to microtubule inhibitors. PMID:25536174

Rai, Ankit; Kapoor, Sonia; Singh, Shalini; Chatterji, Biswa Prasun; Panda, Dulal

2015-02-01

252

Hook1, microtubules, and Rab22  

PubMed Central

Clathrin-independent endocytosis (CIE) mediates the internalization of many plasma membrane (PM) proteins involved in homeostasis, immune response, and signaling. CIE cargo molecules are internalized independent of clathrin, and dynamin, and modulated by the small G protein Arf6. After internalization the CIE cargo proteins either follow a default pathway of trafficking to lysosomes for degradation or follow a pathway where they are routed directly to the recycling endosomes for return to the PM. The selective endosomal sorting of molecules like CD44, CD98, and CD147, which are involved in cell-cell and cell-extracellular interactions, indicates that sorting mechanisms dictate the post-endocytic fate of CIE cargo proteins. In a recent study, we identified sorting signals that specify the endosomal trafficking of CIE cargo proteins and uncover a role for Hook1 as an endosomal cargo adaptor that routes CIE cargo to the recycling endosomes. Furthermore, we found that Hook1, microtubules, and Rab22a work in coordination to directly recycle the cargo and facilitate cell spreading. Here, we discuss our current view on the endosomal sorting of CIE cargo proteins and their molecular regulators. PMID:24284901

Maldonado-Báez, Lymarie; Donaldson, Julie G

2013-01-01

253

Reconstitution of microtubule-dependent organelle transport.  

PubMed

Microtubule (MT)-based motor proteins transport many cellular factors to their functionally relevant locations within cells, and defects in transport are linked to human disease. Understanding the mechanism and regulation of this transport process in living cells is difficult because of the complex in vivo environment and limited means to manipulate the system. On the other hand, in vitro motility assays using purified motors attached to beads does not recapitulate the full complexity of cargo transport in vivo. Assaying motility of organelles in cell extracts is therefore attractive, as natural cargoes are being examined, but in an environment that is more amenable to manipulation. Here, we describe the purification and in vitro MT-based motility of phagosomes from Dictyostelium and lipid droplets from rat liver. These assays have the potential to address diverse questions related to endosome/phagosome maturation, fatty acid regulation, and could also serve as a starting point for reconstituting the motility of other types of organelles. PMID:24630110

Barak, Pradeep; Rai, Ashim; Dubey, Alok Kumar; Rai, Priyanka; Mallik, Roop

2014-01-01

254

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

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

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

2014-01-01

255

Functional architecture of the outer arm dynein conformational switch.  

PubMed

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

King, Stephen M; Patel-King, Ramila S

2012-01-27

256

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

Microsoft Academic Search

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

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

1997-01-01

257

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

PubMed

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

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

2014-04-01

258

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

PubMed Central

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

Cheerambathur, Dhanya K.; Desai, Arshad

2014-01-01

259

An array of nuclear microtubules reorganizes the budding yeast nucleus during quiescence  

PubMed Central

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

Laporte, Damien; Courtout, Fabien; Salin, Bénédicte; Ceschin, Johanna

2013-01-01

260

Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase.  

PubMed

Acetylation of ?-tubulin Lys40 by tubulin acetyltransferase (TAT) is the only known posttranslational modification in the microtubule lumen. It marks stable microtubules and is required for polarity establishment and directional migration. Here, we elucidate the mechanistic underpinnings for TAT activity and its preference for microtubules with slow turnover. 1.35 Å TAT cocrystal structures with bisubstrate analogs constrain TAT action to the microtubule lumen and reveal Lys40 engaged in a suboptimal active site. Assays with diverse tubulin polymers show that TAT is stimulated by microtubule interprotofilament contacts. Unexpectedly, despite the confined intraluminal location of Lys40, TAT efficiently scans the microtubule bidirectionally and acetylates stochastically without preference for ends. First-principles modeling and single-molecule measurements demonstrate that TAT catalytic activity, not constrained luminal diffusion, is rate limiting for acetylation. Thus, because of its preference for microtubules over free tubulin and its modest catalytic rate, TAT can function as a slow clock for microtubule lifetimes. PMID:24906155

Szyk, Agnieszka; Deaconescu, Alexandra M; Spector, Jeffrey; Goodman, Benjamin; Valenstein, Max L; Ziolkowska, Natasza E; Kormendi, Vasilisa; Grigorieff, Nikolaus; Roll-Mecak, Antonina

2014-06-01

261

Unusual ciliary abnormalities in three 9/11 response workers.  

PubMed

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

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

2011-01-01

262

Microtubules contribute to maintain nucleus shape in epithelial cell monolayer  

NASA Astrophysics Data System (ADS)

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.

Tremblay, Dominique; Andrzejewski, Lukasz; Pelling, Andrew

2013-03-01

263

Quantitative Analysis of Tau-Microtubule Interaction Using FRET  

PubMed Central

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

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

2014-01-01

264

SUBPLASMALEMMAL MICROFILAMENTS AND MICROTUBULES IN RESTING AND PHAGOCYTIZING CULTIVATED MACROPHAGES  

PubMed Central

The subplasmalemmal organization of the free and glass-attached surfaces of resting and phagocytizing cultivated macrophages were examined in an attempt to define specific membrane-associated structures related to phagocytosis. From analysis of serial thin sections of oriented cells it was found that the subplasmalemmal region of the attached cell surface has a complex microfilament and microtubule organization relative to the subplasmalemmal area of the free surface. A filamentous network composed of 40–50-Å microfilaments extended for a depth of 400–600 Å from the attached plasma membrane. Immediately subjacent to the filamentous network was a zone of oriented bundles of 40–50-Å microfilaments and a zone of microtubules. Additional microtubules were found to extend from the plasma membrane to the interior of the cell in close association with electron-dense, channellike structures. In contrast, the free aspect of the cultivated macrophage contained only the subplasmalemmal filamentous network. However, after a phagocytic pulse with polystyrene particles (14 µm diam) microtubules and oriented filaments similar to those found on the attached surface were observed surrounding the ingested particles. The observations reported in this paper provide support for the hypothesis that microfilaments and/or microtubules play a role in the translocation of plasma membrane required for the functionally similar processes of phagocytosis and cell attachment to glass. PMID:4356569

Reaven, Eve P.; Axline, Stanton G.

1973-01-01

265

Microtubules and Their Role in Cellular Stress in Cancer  

PubMed Central

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

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

2014-01-01

266

Beyond taxanes: the next generation of microtubule-targeting agents.  

PubMed

Taxanes are a standard first-line option for metastatic breast cancer (MBC), but their utility may be limited by primary or acquired resistance. New microtubule-targeting agents have been developed to overcome taxane resistance and provide additional options for improving patient outcomes. This article reviews these alternative microtubule-targeting agents and their potential clinical benefits for MBC patients. Relevant clinical data were compiled through searches within PubMed and congress abstract databases. Ixabepilone, a novel microtubule-stabilizing drug approved by the US Food and Drug Administration (FDA), has proven efficacy across multiple lines of therapy, including patients with taxane-resistant/refractory disease. In phase III trials, ixabepilone plus capecitabine significantly improved progression-free survival compared with capecitabine alone in anthracycline/taxane-pretreated patients. Eribulin has recently been approved by the FDA and by the European Medicines Agency for the treatment of patients with MBC who have received at least two prior chemotherapy regimens for late-stage disease. In a phase III trial, eribulin extended overall survival compared with the physician's treatment choice in heavily pretreated MBC patients. In addition, several investigational microtubule-targeting agents may have therapeutic potential in MBC. The development of new microtubule-targeting agents helps to address the need for additional effective regimens for patients progressing after standard treatment with anthracycline- and taxane-containing regimens. PMID:22113255

Cortes, Javier; Vidal, Maria

2012-06-01

267

Microtubule motors mediate endosomal sorting by maintaining functional domain organization  

PubMed Central

Summary Many microtubule motors have been shown to couple to endosomal membranes. These motors include dynein in addition to many different kinesin family members. Sorting nexins (SNXs) are central to the organization and function of endosomes. These proteins can actively shape endosomal membranes and couple directly or indirectly to the minus-end microtubule motor dynein. Motor proteins acting on endosomes drive their motility, dictate their morphology and affect cargo segregation. We have used well-characterized members of the SNX family to elucidate motor coupling using high-resolution light microscopy coupled with depletion of specific microtubule motors. Endosomal domains labelled with SNX1, SNX4 and SNX8 couple to discrete combinations of dynein and kinesin motors. These specific combinations govern the structure and motility of each SNX-coated membrane in addition to the segregation of distinct functional endosomal subdomains. Taken together, our data show that these key features of endosome dynamics are governed by the same set of opposing microtubule motors. Thus, microtubule motors help to define the mosaic layout of endosomes that underpins cargo sorting. PMID:23549789

Hunt, Sylvie D.; Townley, Anna K.; Danson, Chris M.; Cullen, Peter J.; Stephens, David J.

2013-01-01

268

The feasibility of coherent energy transfer in microtubules.  

PubMed

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

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

2014-11-01

269

Anillin promotes astral microtubule-directed cortical myosin polarization  

PubMed Central

Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle–directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex. PMID:21737681

Tse, Yu Chung; Piekny, Alisa; Glotzer, Michael

2011-01-01

270

Tubulin tyrosine nitration regulates microtubule organization in plant cells  

PubMed Central

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

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

2013-01-01

271

Comparison of ciliature microtubule organelles in three hypotrichous ciliate species  

NASA Astrophysics Data System (ADS)

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.

Li, Yisong; Shi, Lei; Gu, Fukang

2010-05-01

272

Spastin's Microtubule-Binding Properties and Comparison to Katanin  

PubMed Central

Spastin and katanin are ring-shaped hexameric AAA ATPases that sever microtubules, and thus crucially depend on a physical interaction with microtubules. For the first time, we report here the microtubule binding properties of spastin at the single-molecule level, and compare them to katanin. Microscopic fluorescence assays showed that human spastin bound to microtubules by ionic interactions, and diffused along microtubules with a diffusion coefficient comparable to katanin. The microscopic measurement of landing and dissociation rates demonstrated the ionic character of the interaction, which could be mapped to a patch of three lysine residues outside of the catalytic domain of human spastin. This motif is not conserved in Drosophila spastin or katanin, which also bound by non-catalytic parts of the protein. The binding affinities of spastin and katanin were nucleotide-sensitive, with the lowest affinities under ADP,, the highest under ATP-?S conditions. These changes correlated with the formation of higher oligomeric states, as shown in biochemical experiments and electron microscopic images. Vice versa, the artificial dimerization of human spastin by addition of a coiled coil led to a constitutively active enzyme. These observations suggest that dimer formation is a crucial step in the formation of the active complex, and thus the severing process by spastin. PMID:23272056

Eckert, Thomas; Le, Doan Tuong-Van; Link, Susanne; Friedmann, Lena; Woehlke, Günther

2012-01-01

273

Properties of the general N-Higgs-doublet model. I. The orbit space  

SciTech Connect

We study the scalar sector of the general N-Higgs-doublet model via geometric constructions in the space of gauge orbits. We give a detailed description of the shape of the orbit space both for general N and, in more detail, for N=3. We also comment on remarkable analogies between the N-Higgs-doublet model and quantum information theory.

Ivanov, I. P. [IFPA, Universite de Liege, Allee du 6 Aout 17, batiment B5a, 4000 Liege (Belgium); Sobolev Institute of Mathematics, Koptyug Avenue 4, 630090, Novosibirsk (Russian Federation); Nishi, C. [Universidade Federal do ABC, Rua Santa Adelia, 166, 09.210-170, Santo Andre, Sao Paulo (Brazil)

2010-07-01

274

Spelling Dutch Doublets: Children's Learning of a Phonological and Morphological Spelling Rule  

ERIC Educational Resources Information Center

This study addresses the question of why spellings determined by morphology are relatively hard to acquire by presenting a latent class model of children's acquisition of a doublet of consonants in the spelling of Dutch verbs. This spelling pattern can be determined either by a phonological rule (after a short vowel, a doublet is spelled) or a…

Notenboom, Annelise; Reitsma, Pieter

2007-01-01

275

A renormalizable supersymmetric SO(10) model with natural doublet-triplet splitting  

NASA Astrophysics Data System (ADS)

We propose a renormalizable supersymmetric SO(10) model where the doublet-triplet splitting problem is solved using the Dimopoulos-Wilczek mechanism. An unwanted coupling is forbidden through a filter sector. To suppress proton decay without spoiling gauge coupling unification, there is a problem in the weak doublets which requires further improvements.

Chen, Ying-Kang; Zhang, Da-Xin

2015-01-01

276

Attachment of the cap to the central microtubules of tetrahymena cilia  

E-print Network

) ends. Further studies of the sites of central microtubule assembly in regenerating flagella will be necessary to determine the sites of central microtubule assembly. Since the plug structures are present at the apparent sites of microtubule assembly... (Allen & Wolf, 1979; Scott & Hufnagel, 1983) and with secretory cells in planarians (Dent- ler, unpublished data). Characterization of this dense material and its comparison with proteins associated with the plugs in ciliary microtubules will depend...

Dentler, William L., Jr

1984-03-01

277

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

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

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

278

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

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

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

2014-11-01

279

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

PubMed Central

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

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

2014-01-01

280

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

E-print Network

Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules Peter Hinow1 in the possible presence of a microtubule associated drug. As an example for the latter, we both experimentally that among drugs that act locally at the microtubule tip, primary inhibition of the loss of GDP tubulin

Hinow, Peter

281

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

282

Fibrils Connect Microtubule Tips with Kinetochores: A Mechanism to Couple Tubulin Dynamics to Chromosome Motion  

Microsoft Academic Search

SUMMARY Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving ''protofilaments,'' strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore ''plate'' and an encircling, ring- shaped protein complex have been proposed to link protofilament bending to

J. Richard McIntosh; Ekaterina L. Grishchuk; Mary K. Morphew; Artem K. Efremov; Kirill Zhudenkov; Vladimir A. Volkov; Iain M. Cheeseman; Arshad Desai; David N. Mastronarde; Fazly I. Ataullakhanov

2008-01-01

283

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

Microsoft Academic Search

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

Richard D. Irons; Douglas A. Neptun

1980-01-01

284

Effects of microtubule mechanics on hydrolysis and catastrophes  

E-print Network

We introduce a model for microtubule mechanics containing lateral bonds between dimers in neighboring protofilaments, bending rigidity of dimers, and repulsive interactions between protofilaments modeling steric constraints to investigate the influence of mechanical forces on hydrolysis and catastrophes. We use the allosteric dimer model, where tubulin dimers are characterized by an equilibrium bending angle, which changes from $0^\\circ$ to $22^\\circ$ by hydrolysis of a dimer. This also affects the lateral interaction and bending energies and, thus, the mechanical equilibrium state of the microtubule. As hydrolysis gives rise to conformational changes in dimers, mechanical forces also influence the hydrolysis rates by mechanical energy changes modulating the hydrolysis rate. The interaction via the microtubule mechanics then gives rise to correlation effects in the hydrolysis dynamics, which have not been taken into account before. Assuming a dominant influence of mechanical energies on hydrolysis rates, we i...

Müller, Nina

2014-01-01

285

Disruption of Microtubule Integrity Initiates Mitosis during CNS Repair  

PubMed Central

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

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

2012-01-01

286

Effect of microtubule disruption on cell adhesion and spreading.  

PubMed

Microtubules have been involved in a variety of cellular processes. In this study, we examined the role of the microtubular system in the adhesion and spreading of the adenocarcinoma cell line HT29-D4. Disruption of microtubules by nocodazole or navelbine resulted in an increase in cell adhesion to purified ECM proteins. This enhanced cell adhesion is mediated by integrins, but is not attributable to quantitative changes in the number of integrin receptors at the cell surface, as determined by flow cytometric analysis. In contrast to attachment, spreading of HT29-D4 cells was reduced by nocodazole treatment in a dose-dependent manner. Thus, microtubule depolymerization appears to increase initial attachment of cells to extracellular matrix, while impeding subsequent cell spreading. PMID:9618274

Kadi, A; Pichard, V; Lehmann, M; Briand, C; Braguer, D; Marvaldi, J; Rognoni, J B; Luis, J

1998-05-29

287

Possible regulation of microtubules through destabilization of tubulin.  

PubMed

Chaperones and folding cofactors are known to mediate posttranslational folding of nascent tubulin, thus forming functional dimers. Based on sequence likeness, a novel protein similar to cofactor E, E-like protein (El), was identified. In overexpression experiments El, similar to a subset of folding factors (i.e. cofactors D and E), appears to disrupt functional dimers and target them for destruction by the proteasome. El apparently does not interact with microtubules directly and has no function in the tubulin folding pathway. Suppression of El expression seems to increase the cellular content of stable, posttranslationally modified microtubules by an unknown mechanism. Degradation of functional tubulin dimers as well as the alteration of the cellular content of stable microtubules through El might regulate the distribution and organization of organelles in vivo. PMID:16202601

Keller, Christian E; Lauring, Brett P

2005-11-01

288

Cell prestress. II. Contribution of microtubules  

NASA Technical Reports Server (NTRS)

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.

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

2002-01-01

289

Cold Electroweak Baryogenesis in the Two Higgs-Doublet Model  

E-print Network

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

Anders Tranberg; Bin Wu

2012-03-22

290

Active doublet method for measuring small changes in physical properties  

DOEpatents

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.

Roberts, Peter M. (Los Alamos, NM); Fehler, Michael C. (Los Alamos, NM); Johnson, Paul A. (Santa Fe, NM); Phillips, W. Scott (Santa Fe, NM)

1994-01-01

291

Tracer transport modeling of the doublet well system  

SciTech Connect

Steady-state flow and tracer transport between an injection well and a pumping we in a heterogeneous confined aquifer were investigated with numerical modeling. Calculation of transport was based on the advective model for heterogeneous aquifers. Dispersion was assumed to be controlled by microscale velocity variation. An effective parameter of dispersion evaluated on the breakthrough curves was defined to account for the influences of heterogeneity. Breakthrough curves were calculated by using numerical modeling of transport in a strongly heterogeneous aquifer with spatial heterogeneous transmissivity fields. The results of modeling were processed by comparison with analytical solutions of doublet systems to obtain the effective parameters. A special solution was developed for advective transport in aquifers with a layered structure. Examples of real field heterogeneity were given to show its influence on breakthrough cures and the resulting impact on the effective macroscopic parameters.

Pozdniakov, S.P. [Moscow State Univ., Moscow (Russian Federation). Dept. of Geographical]|[Lawrence Berkeley Lab., CA (United States); Tsang, Chin-Fu [Lawrence Berkeley Lab., CA (United States)

1994-07-01

292

CPV Phenomenology of Flavor Conserving Two Higgs Doublet Models  

E-print Network

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

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

2014-03-17

293

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

E-print Network

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

Cheng-Wei Chiang; Takaaki Nomura; Kei Yagyu

2015-02-03

294

Regulation of processive motion and microtubule localization of cytoplasmic dynein.  

PubMed

The cytoplasmic dynein complex is the major minus-end-directed microtubule motor. Although its directionality is evolutionary well conserved, differences exist among cytoplasmic dyneins from different species in their stepping behaviour, maximum velocity and force production. Recent experiments also suggest differences in processivity regulation. In the present article, we give an overview of dynein's motile properties, with a special emphasis on processivity and its regulation. Furthermore, we summarize recent findings of different pathways for microtubule plus-end loading of dynein. The present review highlights how distinct functions in different cell types or organisms appear to require different mechanochemical dynein properties and localization pathways. PMID:25619245

Jha, Rupam; Surrey, Thomas

2015-02-01

295

Development of microtubule capping structures in ciliated epithelial-cells  

E-print Network

Development of microtubule capping structures in ciliated epithelial cells R. W. PORTMAN, E. L. LeCLUYSE and W. L. DENTLER Department of Physiology and Cell Biology, University of Kansas, Lawrence, KS 66045, USA Summary Although capping structures... are present at the tips of microtubules in both growing cilia and mature cilia, previous work has not determined the time of cap formation. The results reported here reveal that the large caps of mature palate cilia appear in cilia with lengths as short as 1...

Dentler, William L., Jr; Portman, R. W.; LeCluyse, E. L.

1987-02-01

296

Non-equilibrium microtubule fluctuations in a model cytoskeleton  

E-print Network

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

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

2007-09-19

297

Visualization of cellulose synthase demonstrates functional association with microtubules.  

PubMed

Expression of a functional yellow fluorescent protein fusion to cellulose synthase (CESA) in transgenic Arabidopsis plants allowed the process of cellulose deposition to be visualized in living cells. Spinning disk confocal microscopy revealed that CESA complexes in the plasma membrane moved at constant rates in linear tracks that were aligned and were coincident with cortical microtubules. Within each observed linear track, complex movement was bidirectional. Inhibition of microtubule polymerization changed the fine-scale distribution and pattern of moving CESA complexes in the membrane, indicating a relatively direct mechanism for guidance of cellulose deposition by the cytoskeleton. PMID:16627697

Paredez, Alexander R; Somerville, Christopher R; Ehrhardt, David W

2006-06-01

298

Cortical Microtubule Arrays Are Initiated from a Nonrandom Prepattern Driven by Atypical Microtubule Initiation1[W][OA  

PubMed Central

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-?-tubulin complex protein2-tagged ?-nucleation complexes (?-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving ?-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

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

299

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

PubMed

Synthesis and maintenance of primary cilia are regulated by the von Hippel-Lindau (VHL) tumour suppressor protein. Recent studies indicate that this regulation is linked to microtubule-dependent functions of pVHL such as orienting microtubule growth and increasing plus-end microtubule stability, however little is known how this occurs. We have identified the kinesin-2 motor complex, known to regulate cilia, as a novel and endogenous pVHL binding partner. The interaction with kinesin-2 facilitates pVHL binding to microtubules. These data suggest that microtubule-dependent functions of pVHL are influenced by kinesin-2. PMID:17825299

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

2007-10-01

300

Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure  

PubMed Central

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on ?-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, ?TAT1, with microtubules and find that ?TAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of ?TAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments. PMID:24227885

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

2014-01-01

301

Taccalonolide binding to tubulin imparts microtubule stability and potent in vivo activity  

PubMed Central

The taccalonolides are highly acetylated steroids that stabilize cellular microtubules and overcome multiple mechanisms of taxane resistance. Recently, two potent taccalonolides, AF and AJ, were identified that bind tubulin directly and enhance microtubule polymerization. Extensive studies were conducted to characterize these new taccalonolides. AF and AJ caused aberrant mitotic spindles and bundling of interphase microtubules that differed from the effects of either paclitaxel or laulimalide. AJ also distinctly affected microtubule polymerization in that it enhanced the rate and extent of polymerization in the absence of any noticeable effect on microtubule nucleation. Additionally, the resulting microtubules were found to be profoundly cold stable. These data, along with studies showing synergistic antiproliferative effects between AJ and either paclitaxel or laulimalide, suggest a distinct binding site. Direct binding studies demonstrated that AJ could not be displaced from microtubules by paclitaxel, laulimalide or denaturing conditions, suggesting irreversible binding of AJ to microtubules. Mass spectrometry confirmed a covalent interaction of AJ with a peptide of ?-tubulin containing the cyclostreptin binding sites. Importantly, AJ imparts strong inter-protofilament stability in a manner different from other microtubule stabilizers that covalently bind tubulin, consistent with the distinct effects of the taccalonolides as compared to other stabilizers. AF was found to be a potent and effective antitumor agent that caused tumor regression in the MDA-MB-231 breast cancer xenograft model. The antitumor efficacy of some taccalonolides, which stabilize microtubules in a manner different from other microtubule stabilizers, provides the impetus to explore the therapeutic potential of this site. PMID:24048820

Risinger, AL; Li, J; Bennett, MJ; Rohena, CC; Peng, J; Schriemer, DC; Mooberry, SL

2013-01-01

302

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

PubMed

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

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

2015-01-01

303

The kinetochore-bound Ska1 complex tracks depolymerizing microtubules and binds to curved protofilaments  

PubMed Central

Summary To ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive chromosome movements. How kinetochores associate with depolymerizing microtubules, which undergo dramatic structural changes forming curved protofilaments, has yet to be defined in vertebrates. Here, we demonstrate that the conserved kinetochore-localized Ska1 complex tracks with depolymerizing microtubule ends and associates with both the microtubule lattice and curved protofilaments. In contrast, the Ndc80 complex, a central player in the kinetochore-microtubule interface, binds only to the straight microtubule lattice and lacks tracking activity. We demonstrate that the Ska1 complex imparts its tracking capability to the Ndc80 complex. Finally, we present a structure of the Ska1 microtubule binding domain that reveals its interaction with microtubules and its regulation by Aurora B. This work defines an integrated kinetochore-microtubule interface formed by the Ska1 and Ndc80 complexes that associates with depolymerizing microtubules, potentially by interacting with curved microtubule protofilaments. PMID:23085020

Schmidt, Jens C.; Arthanari, Haribabu; Boeszoermenyi, Andras; Dashkevich, Natalia M.; Wilson-Kubalek, Elizabeth M.; Monnier, Nilah; Markus, Michelle; Oberer, Monika; Milligan, Ron A.; Bathe, Mark; Wagner, Gerhard; Grishchuk, Ekaterina L.; Cheeseman, Iain M.

2012-01-01

304

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

PubMed Central

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

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

2011-01-01

305

Tubulin bond energies and microtubule biomechanics determined from nanoindentation in silico  

E-print Network

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

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

2015-01-01

306

Microtubules are required for efficient epithelial tight junction homeostasis and restoration.  

PubMed

Epithelial tight junctions are critical for creating a barrier yet allowing paracellular transport. Although it is well established that the actin cytoskeleton is critical for preserving the dynamic organization of the tight junction and maintaining normal tight junction protein recycling, contributions of microtubules to tight junction organization and function remain undefined. The aim of this study is to determine the role of microtubules in tight junction homeostasis and restoration. Our data demonstrate that occludin traffics on microtubules and that microtubule disruption perturbs tight junction structure and function. Microtubules are also shown to be required for restoring barrier function following Ca(2+) chelation and repletion. These processes are mediated by proteins participating in microtubule minus-end-directed trafficking but not plus-end-directed trafficking. These studies show that microtubules participate in the preservation of epithelial tight junction structure and function and play a vital role in tight junction restoration, thus expanding our understanding of the regulation of tight junction physiology. PMID:24920678

Glotfelty, Lila G; Zahs, Anita; Iancu, Catalin; Shen, Le; Hecht, Gail A

2014-08-01

307

Transglutaminase and Polyamination of Tubulin: Posttranslational Modification for Stabilizing Axonal Microtubules  

PubMed Central

SUMMARY Neuronal microtubules support intracellular transport, facilitate axon growth, and form a basis for neuronal morphology. While microtubules in non-neuronal cells are depolymerized by cold, Ca2+ or antimitotic drugs, neuronal microtubules are unusually stable. Such stability is important for normal axon growth and maintenance, while hyperstability may compromise neuronal function in aging and degeneration. Though mechanisms for stability were unclear, studies suggested that stable microtubules contain biochemically distinct tubulins that are more basic than conventional tubulins. Transglutaminase-catalyzed posttranslational incorporation of polyamines is one of the few modifications of intracellular proteins that add positive charges. Here we show that neuronal tubulin can be polyaminated by transglutaminase. Endogenous brain transglutaminase-catalyzed polyaminated tubulins have the biochemical characteristics of neuronal stable microtubules. Inhibiting polyamine synthesis or transglutaminase activity significantly decreases microtubule stability in vitro and in vivo. Together, this suggests that transglutaminase-catalyzed polyamination of tubulins stabilizes neuronal microtubules essential for unique neuronal structures and functions. PMID:23583110

Song, Yuyu; Kirkpatrick, Laura L.; Schilling, Alexander B.; Helseth, Donald L.; Chabot, Nicolas; Keillor, Jeffrey W.; Johnson, Gail V.W.; Brady, Scott T.

2013-01-01

308

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

PubMed Central

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

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

2014-01-01

309

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

PubMed Central

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

Ambrose, Chris; Wasteneys, Geoffrey O.

2011-01-01

310

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

PubMed Central

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

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

2014-01-01

311

Arf family GTPases: roles in membrane traffic and microtubule dynamics.  

PubMed

Database mining and phylogenetic analysis of the Arf (ADP-ribosylation factor) superfamily revealed the presence in mammals of at least 22 members, including the six Arfs, two Sars and 14 Arl (Arf-like) proteins. At least six Arf family members were found in very early eukaryotes, including orthologues of Arf, Sar, Arl2, Arl3, Arl6 and Arl8. While roles for Arfs in membrane traffic are well known, those for most of the Arls remain unknown. Depletion in cells of the most closely related human Arf proteins, Arf1-Arf5, reveals specificities among their cellular roles and suggests that they may function in pairs at different steps in endocytic and secretory membrane traffic. In addition, recent results from a number of laboratories suggest that several of the Arl proteins may be involved in different aspects of microtubule-dependent functions. Thus, a second major role for Arf family GTPases, that of regulating microtubules, is emerging. Because membrane traffic is often dependent upon movement of vesicles along microtubules this raises the possibility that these two fundamental functions of Arf family members, regulation of vesicle traffic and microtubule dynamics, diverged from one function of Arfs in the earliest cells that has continued to branch and allow additional levels of regulation. PMID:16246095

Kahn, R A; Volpicelli-Daley, L; Bowzard, B; Shrivastava-Ranjan, P; Li, Y; Zhou, C; Cunningham, L

2005-12-01

312

Lymphocyte cytotoxicity: tug-of-war on microtubules.  

PubMed

Lymphocyte cytotoxicity is essential in immune defense. In this issue of Blood, Kurowska and colleagues define a Rab27a/Slp3/kinesin-1 complex that facilitates anterograde microtubule transport of lytic granules, representing a critical step in lymphocyte granule exocytosis and cytotoxicity. PMID:22538494

Wood, Stephanie M; Bryceson, Yenan T

2012-04-26

313

Analysis of Microtubule Dynamics Using Growth Curve Models  

E-print Network

, providing structural support as well as taking part in many of the cellular processes. A large body of data isoforms of a protein tau on micro- tubule dynamics using growth curve models. The results show of 3-repeat tau protein has a similar effect as a 4-repeat tau protein on microtubule dynamics

Jammalamadaka, S. Rao

314

Microtubule transport DOI: 10.1002/smll.200600410  

E-print Network

Williams* Control of the transport and organization of nano- and mi- croscale materials and molecules­16] and reorienting microtubules by fluid flow[17] or electric fields.[18­20] These approaches all work to fabri- cate (for example, microchannels), or they nonselectively exert forces on all components

Hancock, William O.

315

Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia  

PubMed Central

Recently, we identified 1-aminoanthracene as a fluorescent general anesthetic. To investigate the mechanism of action, a photoactive analogue, 1-azidoanthracene, was synthesized. Administration of 1-azidoanthracene to albino stage 40–47 tadpoles was found to immobilize animals upon near-UV irradiation of the forebrain region. The immobilization was often reversible, but it was characterized by a longer duration consistent with covalent attachment of the ligand to functionally important targets. IEF/SDS-PAGE examination of irradiated tadpole brain homogenate revealed labeled protein, identified by mass spectrometry as ?-tubulin. In vitro assays with aminoanthracene-cross-linked tubulin indicated inhibition of microtubule polymerization, similar to colchicine. Tandem mass spectrometry confirmed anthracene binding near the colchicine site. Stage 40–47 tadpoles were also incubated 1 h with microtubule stabilizing agents, epothilone D or discodermolide, followed by dosing with 1-aminoanthracene. The effective concentration of 1-aminoanthracene required to immobilize the tadpoles was significantly increased in the presence of either microtubule stabilizing agent. Epothilone D similarly mitigated the effects of a clinical neurosteroid general anesthetic, allopregnanolone, believed to occupy the colchicine site in tubulin. We conclude that neuronal microtubules are “on-pathway” targets for anthracene general anesthetics and may also represent functional targets for some neurosteroid general anesthetics. PMID:23484901

Emerson, Daniel J.; Weiser, Brian P.; Psonis, John; Liao, Zhengzheng; Taratula, Olena; Fiamengo, Ashley; Wang, Xiaozhao; Sugasawa, Keizo; Smith, Amos B.; Eckenhoff, Roderic G; Dmochowski, Ivan J.

2013-01-01

316

A targeted multi-enzyme mechanism for selective microtubule  

E-print Network

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

Paris-Sud XI, Université de

317

Microtubule architecture: inspiration for novel carbon nanotube-based  

E-print Network

for cell motility, cell division and intra- cellular trafficking. Microtubules have outstanding mechanical, provide oriented tracks for intracellular trafficking of organelles and support the cell's shape during, with applications ranging from `super-tough' composite fibers [3] to drug-delivery systems [4]. Molecular control

Texas at Austin. University of

318

Effects of microtubule mechanics on hydrolysis and catastrophes  

E-print Network

Effects of microtubule mechanics on hydrolysis and catastrophes N Müller and J Kierfeld Department modeling steric constraints to investigate the influence of mechanical forces on hydrolysis bending angle, which changes from °0 to °22 by hydrolysis of a dimer. This also affects the lateral

Kierfeld, Jan

319

Random Hydrolysis Controls the Dynamic Instability of Microtubules Ranjith Padinhateeri,  

E-print Network

Random Hydrolysis Controls the Dynamic Instability of Microtubules Ranjith Padinhateeri, * Anatoly hydrolysis. Despite decades of experimental work in this field, the precise mechanism of hydrolysis to the vectorial model, hydrolysis occurs only at the unique interface between units bound to GTP/ATP and units

Lacoste, David

320

Force on spindle microtubule minus ends moves chromosomes  

PubMed Central

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

Elting, Mary Williard; Hueschen, Christina L.; Udy, Dylan B.

2014-01-01

321

On the Significance of Microtubule Flexural Behavior in Cytoskeletal Mechanics  

E-print Network

of the promising attempts towards mechanical modeling of the cytoskeleton is the tensegrity model, which simplifies to tensegrity model is, therefore, proved necessary in order to take into account the flexural response of microtubules. The concept of ``bendo-tensegrity'' is proposed as a modification to contemporary cytoskeletal

Mofrad, Mohammad R. K.

322

Prion protein inhibits microtubule assembly by inducing tubulin oligomerization  

SciTech Connect

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.

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

2006-10-13

323

Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes.  

PubMed

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes. PMID:14758368

Rebollo, Elena; Llamazares, Salud; Reina, José; Gonzalez, Cayetano

2004-01-01

324

Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos  

NASA Technical Reports Server (NTRS)

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

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

2003-01-01

325

Force fluctuations and polymerization dynamics of intracellular microtubules  

NASA Astrophysics Data System (ADS)

Microtubules are dynamic biopolymers within the cytoskeleton of living cells. They play a central role in many biological processes including cell division, migration, and cargo transport. Microtubules are significantly more rigid than other cytoskeletal biopolymers, such as actin filaments, and are insensitive to thermal fluctuations on cellular length scales. However, we show that intracellular microtubules exhibit bending amplitudes with a surprisingly thermal-like wavevector dependence, but with an apparent persistence length about 100 times smaller than that measured in vitro. By studying the time-dependent bending fluctuations of individual filaments, we find that the thermal-like bends are fluctuating significantly only on short length scales, while they are frozen-in on longer length scales [1], reminiscent of non-ergodic behavior seen in systems far from equilibrium. Long wavelength bends are suppressed by the surrounding elastic cytoskeleton, which confines bending to short length scales on the order of a few microns [2]. These short wavelength bending fluctuations naturally cause fluctuations in the orientation of the microtubule tip. Tip fluctuations result in a persistent random walk trajectory of microtubule growth, but with a small non-equilibrium persistence length, explaining the origin of quenched thermal-like bends. These results suggest that intracellular motor activity has a highly fluctuating character that dominates over thermal fluctuations, with important consequences for fundamental biological processes. [1] CP Brangwynne, FC MacKintosh, DA Weitz, PNAS, 104:16128 (2007). [2] CP Brangwynne, FC MacKintosh, S Kumar, NA Geisse, J Talbot, L. Mahadevan, KK Parker, DE Ingber, DA Weitz, JCB, 173:733 (2006).

Brangwynne, Clifford

2008-03-01

326

Microtubule-Dependent Modulation of Adhesion Complex Composition  

PubMed Central

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

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

2014-01-01

327

A planar microtubule-organizing zone in guard cells of Allium : experimental depolymerization and reassembly of microtubules  

Microsoft Academic Search

The generation of the unique radial array of microtubules (MTs) in stomatal guard cells raises questions about the location and activities of relevant MT-organizing centers. By using tubulin immunofluorescence microscopy, we studied the pattern of depolymerization and reassembly of MTs in guard cells of Allium cepa L. Chilling at 0°C reduces the MTs to small remnants that surround the nuclear

J. Marc; Y. Mineyuki; B. A. Palevitz

1989-01-01

328

The doublet majoron model and solar neutrino oscillations  

NASA Astrophysics Data System (ADS)

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 ? --> e? 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.

Bertolini, S.; Santamaria, A.

1988-12-01

329

Photonic generation of ultra-wideband monocycle and doublet pulses using simplex semiconductor optical amplifier  

NASA Astrophysics Data System (ADS)

We demonstrate two all-optical methods for UWB pulse generation based on various nonlinearities of the semiconductor optical amplifier (SOA), namely, self phase modulation (SPM), and cross gain modulation (XGM). In the first method, we present UWB doublet generation based on SPM. The monocycle pulse is generated from dark return-to-zero (RZ) signal, and then converted to doublet pulse by injecting an additional probe signal with the SMF transmission. For the first time to best of our knowledge, we report that the generated doublet pulses are transmitted over 5km SMF by proper dispersion compensation without distortion. Second, we present UWB doublet generation by XGM of two cascaded SOAs. The configuration of our all-optical methods is compact and simple.

Dong, Jianji; Zhang, Xinliang; Huang, Dexiu; Zhou, Enbo

2008-11-01

330

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

SciTech Connect

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.

Barroso, A.; Ferreira, P. M.; Santos, R. [Centro de Fisica Teorica e Computacional, Faculdade de Ciencias, Universidade de Lisboa, Avenida Professor Gama Pinto 2, 1649-003 Lisbon (Portugal); Silva, Joao P. [Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emidio Navarro, 1900 Lisbon (Portugal); Centro de Fisica Teorica de Particulas, Instituto Superior Tecnico, P-1049-001 Lisbon (Portugal)

2006-10-15

331

Localized RanGTP Accumulation Promotes Microtubule Nucleation at Kinetochores in Somatic Mammalian Cells  

PubMed Central

Centrosomes are the major sites for microtubule nucleation in mammalian cells, although both chromatin- and kinetochore-mediated microtubule nucleation have been observed during spindle assembly. As yet, it is still unclear whether these pathways are coregulated, and the molecular requirements for microtubule nucleation at kinetochore are not fully understood. This work demonstrates that kinetochores are initial sites for microtubule nucleation during spindle reassembly after nocodazole. This process requires local RanGTP accumulation concomitant with delocalization from kinetochores of the hydrolysis factor RanGAP1. Kinetochore-driven microtubule nucleation is also activated after cold-induced microtubule disassembly when centrosome nucleation is impaired, e.g., after Polo-like kinase 1 depletion, indicating that dominant centrosome activity normally masks the kinetochore-driven pathway. In cells with unperturbed centrosome nucleation, defective RanGAP1 recruitment at kinetochores after treatment with the Crm1 inhibitor leptomycin B activates kinetochore microtubule nucleation after cold. Finally, nascent microtubules associate with the RanGTP-regulated microtubule-stabilizing protein HURP in both cold- and nocodazole-treated cells. These data support a model for spindle assembly in which RanGTP-dependent abundance of nucleation/stabilization factors at centrosomes and kinetochores orchestrates the contribution of the two spindle assembly pathways in mammalian cells. The complex of RanGTP, the export receptor Crm1, and nuclear export signal-bearing proteins regulates microtubule nucleation at kinetochores. PMID:18287525

Torosantucci, Liliana; De Luca, Maria; Guarguaglini, Giulia; Lavia, Patrizia

2008-01-01

332

Microtubule behavior during guidance of pioneer neuron growth cones in situ  

PubMed Central

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

1991-01-01

333

Profiles of the Resonance Doublets Formed in Bipolar Winds in Symbiotic Stars  

E-print Network

We compute the profiles of resonance doublet lines formed in bipolar winds with velocity greater than the doublet separation in symbiotic stars. Particular attention has been paid on the doublet line ratio, where the conversion of the short wavelength component arising from the $2S_{1/2}-2P_{3/2}$ transition into the long wavelengh component for the transition $2S_{1/2}-2P_{1/2}$ plays an essential role in determining the flux line ratio of the doublet. We adopted a Monte Carlo technique and the Sobolev approximation. Our bipolar winds take the form of a cone and are characterised by the terminal wind velocity, mass loss rate and the opening angle of the cone. When the observer is in the polar direction and the Sobolev optical depth is moderate $\\tau_{Sob}\\simeq 1$, then we mainly obtain profiles with inverted flux line ratios, where the short wavelength component is weaker than the long wavelength component. When the observer is in the equatorial direction, we find that the profiles are characterised by two broad components, where the long wavelength component is the broader and stronger of the two. We conclude that the profiles obtained in our model provide qualitative understanding of broad and inverted intensity ratio of the doublets in symbiotic stars and that the bipolarity of the stellar winds can be inferred from the broader and stronger long wavelength components in the doublet.

Jerry Jaiyul Yoo; Hee-Won Lee; Sang-Hyeon Ahn

2002-05-23

334

NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents.  

PubMed

Nicotinamide adenine dinucleotide (NAD(+)) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD(+)-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD(+). Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD(+) levels. We find that these effects of NAD(+) are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD(+) on microtubule polymers. Taken together, these data demonstrate that NAD(+) and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606

Harkcom, William T; Ghosh, Ananda K; Sung, Matthew S; Matov, Alexandre; Brown, Kevin D; Giannakakou, Paraskevi; Jaffrey, Samie R

2014-06-17

335

NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents  

PubMed Central

Nicotinamide adenine dinucleotide (NAD+) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD+. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD+ levels. We find that these effects of NAD+ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD+ on microtubule polymers. Taken together, these data demonstrate that NAD+ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606

Harkcom, William T.; Ghosh, Ananda K.; Sung, Matthew S.; Matov, Alexandre; Brown, Kevin D.; Giannakakou, Paraskevi; Jaffrey, Samie R.

2014-01-01

336

Electron-microscopic and immunochemical analysis of kinetochore microtubules after ultraviolet microbeam irradiation of kinetochores.  

PubMed

We used an ultraviolet microbeam to irradiate kinetochores of chromosomes in crane-fly spermatocytes. We used one of two doses, low (0.106 erg microns-2) or high (0.301 erg microns-2), and then studied the microtubules in those spindles using electron microscopy or immunofluorescence microscopy. After irradiation with low doses microtubules are present as usual, with normal fluorescence and in normal numbers. After irradiation with high doses microtubules are no longer associated with the irradiated kinetochore. After irradiation with either dose, non-kinetochore microtubules are in smaller numbers in the irradiated half-spindle than in the non-irradiated half-spindle or in non-irradiated cells. Since irradiation with low doses alters interchromosomal 'signals', but microtubules remain attached to the kinetochore, we argue that low doses of ultraviolet light damage a signal-related function of kinetochores without altering the ability of the kinetochores to bind microtubules. PMID:1757487

Swedak, J A; Leggiadro, C; Forer, A

1991-10-01

337

Cell cycle-dependent behavior of microtubules in hybrids of mouse oocytes and blastomeres.  

PubMed

The behavior of microtubules was studied in hybrids formed between mouse oocytes arrested in metaphase II or activated parthenogenetically and mouse embryo interphase blastomeres. In all cases the interphase blastomere's network of microtubules disassembles rapidly after fusion with oocytes. Introduction of interphase cytoplasm and nuclei to metaphase oocytes during fusion induces the polymerization of new microtubules in the cytoplasm and in the meiotic spindle. The degree and the duration of this facilitated polymerization of microtubules was positively correlated with the volume of blastomeres used for fusion. The blastomere nuclei induce the formation of microtubular frames, which become more evident when the chromatin undergoes premature condensation. Finally, spindle-like structures are formed around the prematurely condensed chromosomes. In hybrids activated around the time of fusion, the blastomere nuclei undergo pronuclear-like transformation. These hybrids develop an interphase network of microtubules typical for activated oocytes. These results are discussed with regards to the cell cycle control of microtubule behavior. PMID:1801868

Kubiak, J Z

1991-12-01

338

Effect of microtubules and intermediate filaments on mitochondrial distribution.  

PubMed

The laser dye rhodamine 123 specifically stains mitochondria in living cells and facilitates the observation of changes in mitochondrial distribution in single cells under a variety of experimental conditions. Visualization of mitochondria in a number of cell lines followed by processing of these cells to study different cytoskeletal elements by indirect immunofluorescence, revealed good but not absolute correlation between mitochondria and microtubules or intermediate filaments. Mitochondria and microfilament distribution within the same cell did not show such a correlation. On the basis of observations made by various experimental approaches, we suggest that mitochondrial distribution is under the strong influence of the two systems, microtubules and intermediate filaments. Neither plays an absolute role but one seems able to play a more dominant role in the absence of the other. PMID:6350334

Summerhayes, I C; Wong, D; Chen, L B

1983-05-01

339

A common pharmacophore for cytotoxic natural products that stabilize microtubules.  

PubMed

Taxol (paclitaxel), a complex diterpene obtained from the Pacific yew, Taxus brevifolia, is arguably the most important new drug in cancer chemotherapy. The mechanism of cytotoxic action for paclitaxel-i.e., the stabilization of microtubules leading to mitotic arrest-is now shared by four recently identified natural products, eleutherobin, epothilones A and B, and discodermolide. Their ability to competitively inhibit [3H]paclitaxel binding to microtubules strongly suggests the existence of a common binding site. Recently, we have developed nonaromatic analogues of paclitaxel that maintain high cytotoxicity and tubulin binding (e.g., nonataxel). We now propose a common pharmacophore that unites paclitaxel, nonataxel, the epothilones, eleutherobin, and discodermolide, and rationalizes the extensive structure-activity relationship data pertinent to these compounds. Insights from the common pharmacophore have enabled the development of a hybrid construct with demonstrated cytotoxic and tubulin-binding activity. PMID:10200249

Ojima, I; Chakravarty, S; Inoue, T; Lin, S; He, L; Horwitz, S B; Kuduk, S D; Danishefsky, S J

1999-04-13

340

Regulation of microtubule motors by tubulin isotypes and posttranslational modifications  

PubMed Central

The ‘tubulin-code’ hypothesis proposes that different tubulin genes or posttranslational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogenous tubulin species has hindered biochemical testing of this hypothesis. Here, we have engineered 25 ?/? tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single molecule assays. Our results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, we show that kinesin-1 motility on neuronal ?-tubulin (TUBB3) is increased by polyglutamylation and that robust kinesin-2 motility requires detyrosination of ?-tubulin. Our results also show that different molecular motors recognize distinctive tubulin “signatures”, which supports the premise of tubulin-code hypothesis. PMID:24633327

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

2014-01-01

341

Effects of 3-repeat tau on taxol mobility through microtubules  

NASA Astrophysics Data System (ADS)

Both the anti-cancer drug taxol and the microtubule-associated protein tau suppress dynamics of microtubules (MT). We have observed taxol mobility with full-length 3-repeat tau, one of six tau isoforms, using fluorescence recovery after photobleaching (FRAP) on MTs and compare with earlier results on recombinant full-length adult 4-repeat tau. Taxol mobility becomes highly sensitive to taxol concentration in the presence of 3-repeat tau (up to 1:1 molar ratio) as it does in the presence of 4-repeat tau, but is 2 to 3 times faster at low taxol concentrations. Fitting to a mean-field binding reaction model [J.L. Ross et.al, PNAS 101:12910-5 (2004)] suggests that the presence of 3-repeat tau enhances taxol movement through pores in the MT walls.

Park, Hyunjoo; Fygenson, Deborah; Kim, Mahn Won

2005-03-01

342

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles  

NASA Astrophysics Data System (ADS)

Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment.

Hoffmann, Céline; Mazari, Elsa; Lallet, Sylvie; Le Borgne, Roland; Marchi, Valérie; Gosse, Charlie; Gueroui, Zoher

2013-03-01

343

Tetrahydroisoquinolinone-based Steroidomimetic and Chimeric Microtubule Disruptors  

PubMed Central

A SAR translation strategy was used for the discovery of tetrahydroisoquinoline (THIQ)-based steroidomimetic and chimeric microtubule disruptors based upon a steroidal starting point. A steroid A,B-ring-mimicking THIQ core was connected to methoxy aryl D-ring ring mimics through methylene, carbonyl and sulfonyl linkers to afford a number of steroidomimetic hits (e.g. 20c GI50 2.1 ?M). Optimisation and control experiments demonstrate the complementary SAR of this series and the steroid derivatives that inspired its design. Linkage of the THIQ-based A,B-mimic with the trimethoxy aryl motif prevalent in colchicine site binding microtubule disruptors delivered a series of chimeric molecules whose activity (to GI50 40 nM) surpasses that of the parent steroid derivatives. Validation of this strategy was obtained from the excellent oral activity of 20z relative to a benchmark steroidal bis-sulfamate in an in vivo model of multiple myeloma. PMID:24124095

Leese, Mathew P.; Jourdan, Fabrice L.; Major, Meriel R.; Dohle, Wolfgang; Hamel, Ernest; Ferrandis, Eric; Fiore, Ann; Kasprzyk, Philip G.; Potter, Barry V. L.

2013-01-01

344

Centaurin-?2 Interacts with ?-Tubulin and Stabilizes Microtubules  

PubMed Central

Centaurin-?2 is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-?2 can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-?2 interacting protein, ?-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of ?-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-?2 overexpression in HeLa cells and extraction of soluble (?? dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-?2 mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-?2 remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-?2 and tubulin confirmed that Centaurin-?2 promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-?2 overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-?2 role on MT stabilization. Centaurin-?2 interacts with ?-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-?2 as a new microtubule-associated protein (MAP) increasing MT stability. PMID:23285209

Pandini, Vittorio; Venturin, Marco; Aliverti, Alessandro; Battaglioli, Elena; Cappelletti, Graziella; Riva, Paola

2012-01-01

345

A Common Pharmacophore for Cytotoxic Natural Products That Stabilize Microtubules  

Microsoft Academic Search

Taxol (paclitaxel), a complex diterpene obtained from the Pacific yew, Taxus brevifolia, is arguably the most important new drug in cancer chemotherapy. The mechanism of cytotoxic action for paclitaxel--i.e., the stabilization of microtubules leading to mitotic arrest--is now shared by four recently identified natural products, eleutherobin, epothilones A and B, and discodermolide. Their ability to competitively inhibit [3H]paclitaxel binding to

Iwao Ojima; Subrata Chakravarty; Tadashi Inoue; Songnian Lin; Lifeng He; Susan Band Horwitz; Scott D. Kuduk; Samuel J. Danishefsky

1999-01-01

346

Hallmarks of Molecular Action of Microtubule Stabilizing Agents  

PubMed Central

Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, whereas others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Recent studies from our laboratory utilizing hydrogen-deuterium exchange in combination with mass spectrometry (MS) provide new information on the conformational effects of Taxol and discodermolide on microtubules isolated from chicken erythrocytes (CET). We report here a comprehensive analysis of the effects of epothilone B, ixabepilone (IXEMPRATM), laulimalide, and peloruside A on CET conformation. The results of our comparative hydrogen-deuterium exchange MS studies indicate that all MSAs have significant conformational effects on the C-terminal H12 helix of ?-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent ??-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, the lateral interactions between the adjacent protofilaments in CET are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site binding drugs are used. PMID:21245138

Khrapunovich-Baine, Marina; Menon, Vilas; Yang, Chia-Ping Huang; Northcote, Peter T.; Miller, John H.; Angeletti, Ruth Hogue; Fiser, Andras; Horwitz, Susan Band; Xiao, Hui

2011-01-01

347

The role of microtubules in contractile ring function  

NASA Technical Reports Server (NTRS)

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.

Conrad, A. H.; Paulsen, A. Q.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1992-01-01

348

Abnormal fibroblast microtubule response in familial Alzheimer disease  

Microsoft Academic Search

There is increasing evidence that cytoskeletal changes, in particular perturbation of the microtubule (MT) system, play a\\u000a role in the pathogenesis of Alzheimer disease (AD). The reappearance of the cytoplasmic MT network following treatment with\\u000a the MT disrupting agent colchicine was investigated in commercially available (Human Genetic Cell Repository, Camden, NJ)\\u000a fibroblasts derived from 11 patients with familial AD and

Steven S. Matsuyama; Lissy F. Jarvik

1993-01-01

349

Pentagons, heptagons and negative curvature in graphite microtubule growth  

Microsoft Academic Search

THE standard carbon-arc synthesis for fullerenes also produces graphitic microtubules with helical structures1. In most cases the cylindrical tubes are closed by polyhedral caps, some being first transformed into a conical shape before closure2. Here we present images from transmission electron microscopy of a further kind of growth morphology, in which cone-like growth is transformed into cylindrical growth by the

Sumio Lijima; Toshinari Ichihashi; Yoshinori Ando

1992-01-01

350

Microtubule distribution in gravitropic protonemata of the moss Ceratodon  

Microsoft Academic Search

Summary Tip cells of dark-grown protonemata of the mossCeratodon 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

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

1990-01-01

351

Regulation of the inward K + -channels in stomatal guard cells by cytoskeletal microtubules  

Microsoft Academic Search

Patch clamp techniques were applied to investigating the regulation of the inward K+-channels inVicia stomatal guard cells by cytoskeletal microtubules. The intracellular addition of either microtubule-disassembling reagent\\u000a amprophos-methyl (APM) or microtubule-stabilizing reagent taxol resulted in significant inhibition of the inward K+-currents across the plasma membranes ofVicia stomatal guard cells. The results suggest that the activation of the inward K+-channels in

Ximing Zhou; Weihua Wu; Ming Yuan; Xuechen Wang

1999-01-01

352

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

Microsoft Academic Search

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

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

2007-01-01

353

A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules  

SciTech Connect

Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

Iimori, Makoto; Ozaki, Kanako [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan)] [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Chikashige, Yuji [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan)] [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Habu, Toshiyuki [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan) [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan); Hiraoka, Yasushi [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan) [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, 565-0871 (Japan); Maki, Takahisa; Hayashi, Ikuko [Graduate School of Nanobioscience, Yokohama City University, Tsurumi, Yokohama, 230-0045 (Japan)] [Graduate School of Nanobioscience, Yokohama City University, Tsurumi, Yokohama, 230-0045 (Japan); Obuse, Chikashi [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan)] [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan); Matsumoto, Tomohiro, E-mail: tmatsumo@house.rbc.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan) [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan)

2012-02-01

354

Targeting Microtubules by Natural Agents for Cancer Therapy  

PubMed Central

Natural compounds that target microtubules and disrupt the normal function of the mitotic spindle have proven to be one of the best classes of cancer chemotherapeutic drugs available in clinics to date. There is increasing evidence showing that even minor alteration of microtubule dynamics can engage the spindle checkpoint, arresting cell cycle progression at mitosis and subsequently leading to cell death. Our improved understanding of tumor biology and our continued appreciation for what the microtubule target agents (MTAs) can do has helped pave the way for a new era in the treatment of cancer. The effectiveness of these agents for cancer therapy has been impaired, however, by various side effects and drug resistance. Several new MTAs have shown potent activity against the proliferation of various cancer cells, including resistance to the existing MTAs. Sustained investigation of the mechanisms of action of MTAs, development and discovery of new drugs, and exploring new treatment strategies that reduce side effects and circumvent drug resistance could provide more effective therapeutic options for cancer patients. This review focuses on the successful cancer chemotherapy from natural compounds in clinical settings and the challenges that may abort their usefulness. PMID:24435445

Mukhtar, Eiman; Adhami, Vaqar Mustafa; Mukhtar, Hasan

2014-01-01

355

Elevated polar ejection forces stabilize kinetochore–microtubule attachments  

PubMed Central

Chromosome biorientation promotes congression and generates tension that stabilizes kinetochore–microtubule (kt-MT) interactions. Forces produced by molecular motors also contribute to chromosome alignment, but their impact on kt-MT attachment stability is unclear. A critical force that acts on chromosomes is the kinesin-10–dependent polar ejection force (PEF). PEFs are proposed to facilitate congression by pushing chromosomes away from spindle poles, although knowledge of the molecular mechanisms underpinning PEF generation is incomplete. Here, we describe a live-cell PEF assay in which tension was applied to chromosomes by manipulating levels of the chromokinesin NOD (no distributive disjunction; Drosophila melanogaster kinesin-10). NOD stabilized syntelic kt-MT attachments in a dose- and motor-dependent manner by overwhelming the ability of Aurora B to mediate error correction. NOD-coated chromatin stretched away from the pole via lateral and end-on interactions with microtubules, and NOD chimeras with either plus end–directed motility or tip-tracking activity produced PEFs. Thus, kt-MT attachment stability is modulated by PEFs, which can be generated by distinct force-producing interactions between chromosomes and dynamic spindle microtubules. PMID:23337118

Cane, Stuart; Ye, Anna A.; Luks-Morgan, Sasha J.

2013-01-01

356

Effects of microtubule mechanics on hydrolysis and catastrophes  

E-print Network

We introduce a model for microtubule mechanics containing lateral bonds between dimers in neighboring protofilaments, bending rigidity of dimers, and repulsive interactions between protofilaments modeling steric constraints to investigate the influence of mechanical forces on hydrolysis and catastrophes. We use the allosteric dimer model, where tubulin dimers are characterized by an equilibrium bending angle, which changes from $0^\\circ$ to $22^\\circ$ by hydrolysis of a dimer. This also affects the lateral interaction and bending energies and, thus, the mechanical equilibrium state of the microtubule. As hydrolysis gives rise to conformational changes in dimers, mechanical forces also influence the hydrolysis rates by mechanical energy changes modulating the hydrolysis rate. The interaction via the microtubule mechanics then gives rise to correlation effects in the hydrolysis dynamics, which have not been taken into account before. Assuming a dominant influence of mechanical energies on hydrolysis rates, we investigate the most probable hydrolysis pathways both for vectorial and random hydrolysis. Investigating the stability with respect to lateral bond rupture, we identify initiation configurations for catastrophes along the hydrolysis pathways and values for a lateral bond rupture force. If we allow for rupturing of lateral bonds between dimers in neighboring protofilaments above this threshold force, our model exhibits avalanche-like catastrophe events.

Nina Müller; Jan Kierfeld

2014-06-05

357

3-D structure and dynamics of microtubule self-organization  

NASA Astrophysics Data System (ADS)

Laser scanning confocal microscopy was used to study the dynamics of 3D assemblies spontaneously formed in microtubule (MT) solutions. Microtubule solutions prepared by mixing and incubating tubulin in the presence of GTP and Oregon Green conjugated taxol in PM buffer were placed in long, sub-millimeter thin glass cells by the capillary action. Within 24 hours, starting with a uniform distribution, microtubules were found to be gradually separated into a few large ``buckled'' bundles along the long direction, and in the middle plane, of the sample cell. A well-defined wavelength of the buckling sinusoids was around 510 ?m. The cross section of these round bundles was approximately 40 ?m in diameter and the lengths were several centimeters. Detailed analysis of the 3-D image within the bundles revealed that each bundle seemed to consist of loosely packed MTs. It appeared that MTs were phase separated resulting from attractive interactions between charged MT fibers. The ``buckling'' behavior could be the result of geometrical constraints of the repulsive cell walls and the repulsive interaction between bundles. Detailed 3-D observations of the dynamic evolution of MT assembly could provide insight to the mechanisms of cellular MT organization and phase separation of charged colloidal rods.

Wang, Jing; Ou-Yang, H. Daniel

2008-03-01

358

Elevated polar ejection forces stabilize kinetochore-microtubule attachments.  

PubMed

Chromosome biorientation promotes congression and generates tension that stabilizes kinetochore-microtubule (kt-MT) interactions. Forces produced by molecular motors also contribute to chromosome alignment, but their impact on kt-MT attachment stability is unclear. A critical force that acts on chromosomes is the kinesin-10-dependent polar ejection force (PEF). PEFs are proposed to facilitate congression by pushing chromosomes away from spindle poles, although knowledge of the molecular mechanisms underpinning PEF generation is incomplete. Here, we describe a live-cell PEF assay in which tension was applied to chromosomes by manipulating levels of the chromokinesin NOD (no distributive disjunction; Drosophila melanogaster kinesin-10). NOD stabilized syntelic kt-MT attachments in a dose- and motor-dependent manner by overwhelming the ability of Aurora B to mediate error correction. NOD-coated chromatin stretched away from the pole via lateral and end-on interactions with microtubules, and NOD chimeras with either plus end-directed motility or tip-tracking activity produced PEFs. Thus, kt-MT attachment stability is modulated by PEFs, which can be generated by distinct force-producing interactions between chromosomes and dynamic spindle microtubules. PMID:23337118

Cane, Stuart; Ye, Anna A; Luks-Morgan, Sasha J; Maresca, Thomas J

2013-01-21

359

Tubulin hooks as probes for microtubule polarity: an analysis of the method and an evaluation of data on microtubule polarity in the mitotic spindle  

PubMed Central

The structural polarity of cellular microtubules can be visualized in situ by lysing cells in special buffers containing tubulin. Under these conditions, the tubulin polymerizes to form curved sheets which attach to the walls of the endogenous microtubules. When such decorated microtubules are cut in cross section and viewed in the electron microscope, they appear to bear hooks curving clockwise or counter- clockwise. The direction of hook curvature is defined by the orientation of the decorated microtubule and thus serves as a probe for microtubule polarity. In this paper we describe a way to analyze the relative frequencies of hooks of different curvatures so as to measure the fidelity of the relation between hook curvature and microtubule polarity. The assumptions of the method are tested and found to be valid to a reasonable accuracy. The correlation between hook curvature and microtubule orientation is shown to be at least 0.98 for the spindles of PtK cells and Haemanthus endosperm at all stages of division and at all places in the spindle. The correlation is shown to be valid for each hook that forms, so the polarity of those microtubules that bear multiple hooks is specified with even better certainty than 0.98. This property of hook decoration is used to reinvestigate the possibility that some of the microtubules of the kinetochore fiber might be oriented with their plus ends distal to the kinetochore (opposite to the direction previously shown to predominate). Close analysis fails to identify such oppositely oriented microtubules. The scoring of tubules bearing multiple hooks also shows that individual interzone fibers at anaphase are constructed from clusters of antiparallel microtubules. The method for estimating the correlation between hook decoration and microtubule polarity is shown to be applicable to many structures and circumstances, but we find that the hook decoration assay for microtubule polarity is not uniformly accurate. We suggest that future studies using hook decorations should employ the method of data analysis presented here to assess the accuracy of the results obtained. PMID:6693493

1984-01-01

360

EB1 regulates microtubule dynamics and tubulin sheet closure in vitro.  

PubMed

End binding 1 (EB1) is a plus-end-tracking protein (+TIP) that localizes to microtubule plus ends where it modulates their dynamics and interactions with intracellular organelles. Although the regulating activity of EB1 on microtubule dynamics has been studied in cells and purified systems, the molecular mechanisms involved in its specific activity are still unclear. Here, we describe how EB1 regulates the dynamics and structure of microtubules assembled from pure tubulin. We found that EB1 stimulates spontaneous nucleation and growth of microtubules, and promotes both catastrophes (transitions from growth to shrinkage) and rescues (reverse events). Electron cryomicroscopy showed that EB1 induces the initial formation of tubulin sheets, which rapidly close into the common 13-protofilament-microtubule architecture. Our results suggest that EB1 favours the lateral association of free tubulin at microtubule-sheet edges, thereby stimulating nucleation, sheet growth and closure. The reduction of sheet length at microtubule growing-ends together with the elimination of stressed microtubule lattices may account for catastrophes. Conversely, occasional binding of EB1 to the microtubule lattice may induce rescues. PMID:18364701

Vitre, Benjamin; Coquelle, Frédéric M; Heichette, Claire; Garnier, Cyrille; Chrétien, Denis; Arnal, Isabelle

2008-04-01

361

The pool of map kinase associated with microtubules is small but constitutively active.  

PubMed Central

Mitogen-activated protein kinase (MAPK) is activated by many kinds of stimuli and plays an important role in integrating signal transduction cascades. MAPK is present abundantly in brain, where we have studied its association with microtubules. Immunofluorescence of primary hippocampal neurons revealed that MAPK staining co-localized with microtubules and biochemical analyses showed that MAPK co-purified with microtubules. Approximately 4% of MAPK in cytosolic extracts was associated with microtubules, where it was associated with both tubulin and microtubule-associated proteins (MAPs) fractions. Further fractionation of MAPs suggested that a portion of MAPK is associated with MAP2. An association with MAP2 was also demonstrated by co-immunoprecipitation and in vitro binding experiments. A similar association was shown for the juvenile MAP2 isoform, MAP2C. The pool of MAPK associated with microtubules had a higher activity relative to the nonassociated pool in both brain and proliferating PC12 cells. Although MAPK was activated by nerve growth factor in PC12 cells, the activity of microtubule-associated MAPK did not further increase. These results raise the possibility that microtubule-associated MAPK operates through constitutive phosphorylation activity to regulate microtubule function in neurons. Images PMID:8816996

Morishima-Kawashima, M; Kosik, K S

1996-01-01

362

Notch signalling in adult neurons: a potential target for microtubule stabilization  

PubMed Central

Cytoskeletal dysfunction has been proposed during the last decade as one of the main mechanisms involved in the aetiology of several neurodegenerative diseases. Microtubules are basic elements of the cytoskeleton and the dysregulation of microtubule stability has been demonstrated to be causative for axonal transport impairment, synaptic contact degeneration, impaired neuronal function leading finally to neuronal loss. Several pathways are implicated in the microtubule assembly/disassembly process. Emerging evidence is focusing on Notch as a microtubule dynamics regulator. We demonstrated that activation of Notch signalling results in increased microtubule stability and changes in axonal morphology and branching. By contrast, Notch inhibition leads to an increase in cytoskeleton plasticity with intense neurite remodelling. Until now, several microtubule-binding compounds have been tested and the results have provided proof of concept that microtubule-binding agents or compounds with the ability to stabilize microtubules may have therapeutic potential for the treatment of Alzheimer’s disease and other neurodegenerative diseases. In this review, based on its key role in cytoskeletal dynamics modulation, we propose Notch as a new potential target for microtubule stabilization. PMID:24228073

Bonini, Sara Anna; Ferrari-Toninelli, Giulia; Montinaro, Mery

2013-01-01

363

Dynamic Change of Cellular Localization of Microtubule-Organizing Center During Conjugation of Ciliate Tetrahymena thermophila.  

PubMed

To obtain a comprehensive picture of microtubule dynamics during conjugation, the mode of sexual reproduction in ciliates, we combined indirect immunofluorescence and three-dimensional imaging using confocal laser-scanning microscope to visualize the cellular localization of DNA, microtubules, and ?-tubulin, the main component of the microtubule-organizing center in mating Tetrahymena cells. As the conjugational stages proceeded, the distribution of ?-tubulin changed drastically and microtubules showed dynamic appearance and disappearance during meiosis, nuclear selection, nuclear exchange, and the development of new macronuclei. This study highlights the involvement of cytoskeletal regulation in the modulation of germline nuclear motilities required for ciliate reproduction. PMID:25660693

Kushida, Yasuharu; Takaine, Masak; Nakano, Kentaro; Sugai, Toshiro; Numata, Osamu

2015-01-01

364

The conserved KMN network constitutes the core microtubule-binding site of the kinetochore.  

PubMed

The microtubule-binding interface of the kinetochore is of central importance in chromosome segregation. Although kinetochore components that stabilize, translocate on, and affect the polymerization state of microtubules have been identified, none have proven essential for kinetochore-microtubule interactions. Here, we examined the conserved KNL-1/Mis12 complex/Ndc80 complex (KMN) network, which is essential for kinetochore-microtubule interactions in vivo. We identified two distinct microtubule-binding activities within the KMN network: one associated with the Ndc80/Nuf2 subunits of the Ndc80 complex, and a second in KNL-1. Formation of the complete KMN network, which additionally requires the Mis12 complex and the Spc24/Spc25 subunits of the Ndc80 complex, synergistically enhances microtubule-binding activity. Phosphorylation by Aurora B, which corrects improper kinetochore-microtubule connections in vivo, reduces the affinity of the Ndc80 complex for microtubules in vitro. Based on these findings, we propose that the conserved KMN network constitutes the core microtubule-binding site of the kinetochore. PMID:17129783

Cheeseman, Iain M; Chappie, Joshua S; Wilson-Kubalek, Elizabeth M; Desai, Arshad

2006-12-01

365

Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface  

E-print Network

Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate ...

Welburn, Julie P. I.

366

Electrical control of kinesin-microtubule motility using a transparent functionalized-graphene substrate.  

PubMed

We report a new strategy to selectively localize and control microtubule translocation via electrical control of microtubules using a microfabricated channel on a functionalized-graphene electrode with high transparency and conductivity. A patterned SU-8 film acts as an insulation layer which shields the electrical field generated by the graphene underneath while the localized electric field on the exposed graphene surface guides the negatively charged microtubules. This is the first report showing that functionalized graphene can support and control microtubule motility. PMID:23594920

Kim, Eunji; Byun, Kyung-Eun; Choi, Dong Shin; Lee, Dong Jun; Cho, Duck Hyung; Lee, Byung Yang; Yang, Heejun; Heo, Jinseong; Chung, Hyun-Jong; Seo, Sunae; Hong, Seunghun

2013-05-17

367

Coupled oscillations of a protein microtubule immersed in cytoplasm: an orthotropic elastic shell modeling.  

PubMed

Revealing vibration characteristics of sub-cellular structural components such as membranes and microtubules has a principal role in obtaining a deeper understanding of their biological functions. Nevertheless, limitations and challenges in biological experiments at this scale necessitates the use of mathematical and computational models as an alternative solution. As one of the three major cytoskeletal filaments, microtubules are highly anisotropic structures built from tubulin heterodimers. They are hollow cylindrical shells with a ? 25 nm outer diameter and are tens of microns long. In this study, a mechanical model including the effects of the viscous cytosol and surrounding filaments is developed for predicting the coupled oscillations of a single microtubule immersed in cytoplasm. The first-order shear deformation shell theory for orthotropic materials is used to model the microtubule, whereas the motion of the cytosol is analyzed by considering the Stokes flow. The viscous cytosol and the microtubule are coupled through the continuity condition across the microtubule-cytosol interface. The stress and velocity fields in the cytosol induced by vibrating microtubule are analytically determined. Finally, the influences of the dynamic viscosity of the cytosol, filament network elasticity, microtubule shear modulus, and circumferential wave-number on longitudinal, radial, and torsional modes of microtubule vibration are elucidated. PMID:23729907

Daneshmand, Farhang; Amabili, Marco

2012-06-01

368

DHTP is an allosteric inhibitor of the kinesin-13 family of microtubule depolymerases.  

PubMed

The kinesin-13 family of microtubule depolymerases is a major regulator of microtubule dynamics. RNA interference-induced knockdown studies have highlighted their importance in many cell division processes including spindle assembly and chromosome segregation. Since microtubule turnovers and most mitotic events are relatively rapid (in minutes or seconds), developing tools that offer faster control over protein functions is therefore essential to more effectively interrogate kinesin-13 activities in living cells. Here, we report the identification and characterization of a selective allosteric kinesin-13 inhibitor, DHTP. Using high resolution microscopy, we show that DHTP is cell permeable and can modulate microtubule dynamics in cells. PMID:24859087

Talje, Lama; Ben El Kadhi, Khaled; Atchia, Kaleem; Tremblay-Boudreault, Thierry; Carreno, Sébastien; Kwok, Benjamin H

2014-06-27

369

Phase-change kinetics for a microtubule with two free ends.  

PubMed Central

The two-phase macroscopic kinetic model of the end of a microtubule is extended to microtubules in solution, with two free ends. The theoretical treatment of this system is complicated by the possibility of microtubules shortening all the way to disappearance. Another possibility, if a microtubule is shortening from one end only (and has a GTP cap on the other end), is that completed shortening will leave a residual cap from which growth can then take place at both ends. Two approximations are introduced. PMID:3855561

Hill, T L

1985-01-01

370

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

PubMed Central

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

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

2013-01-01

371

The microtubule-binding fragment of microtubule-associated protein-2: location of the protease-accessible site and identification of an assembly-promoting peptide  

PubMed Central

Thrombin cleavage of bovine brain microtubule-associated protein (MAP- 2) yields two stable limit polypeptide fragments (28,000 and 240,000 Mr). The smaller cleavage product contains the microtubule-binding domain and is derived from the carboxyl terminus of MAP-2 while the 240,000 Mr fragment is derived from the amino terminus. The amino terminal sequence of the smaller cleavage product is homologous with the microtubule-binding fragment of tau in sequence and in a similar location relative to three imperfect octadecapeptide repeats implicated in microtubule binding. Peptides corresponding to the cleavage site and the three repeats of MAP-2 were synthesized. Only the second octadecapeptide repeat (VTSKCGSLKNIRHRPGGG) was capable of stimulating microtubule nucleation and elongation. Microtubules formed in the presence of this peptide displayed normal morphology and retained the inhibition properties of calcium ion, podophyllotoxin, and colchicine. Our result indicates that a region comprising only approximately 1% of the MAP-2 sequence can promote microtubule assembly. PMID:2808529

1989-01-01

372

Abstract--The purpose of this project was to determine if the properties of doublet force twitches change with  

E-print Network

change with recruitment level. Isometric, human tibialis anterior force twitches were measured, the results suggest that caution must be exercised when extending doublet properties at full recruitment

Durfee, William K.

373

Abstract--Stimulation trains using doublets, two closely spaced stimulation pulses, are thought to reduce the rapid  

E-print Network

including exercising paralyzed muscle to convert fibers into slow fatiguing units [5], cyclic stimulation and doublets. Bigland- Ritchie et. al. [10] stimulated isometric thenar muscles with conventional trains

Durfee, William K.

374

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

PubMed

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

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

2014-11-01

375

Inert doublet dark matter with an additional scalar singlet and 125 GeV Higgs boson  

NASA Astrophysics Data System (ADS)

In this work we consider a model for particle dark matter where an extra inert Higgs doublet and an additional scalar singlet is added to the Standard Model (SM) Lagrangian. The dark matter candidate is obtained from only the inert doublet. The stability of this one component dark matter is ensured by imposing a symmetry on this additional inert doublet. The additional singlet scalar has a vacuum expectation value (VEV) and mixes with the Standard Model Higgs doublet, resulting in two CP even scalars and . We treat one of these scalars, , to be consistent with the SM Higgs-like boson of mass around 125 GeV reported by the LHC experiment. These two CP even scalars contribute to the annihilation cross section of this inert doublet dark matter, resulting in a larger dark matter mass region that satisfies the observed relic density. We also investigate the and processes and compared these with LHC results. This is also used to constrain the dark matter parameter space in the present model. We find that the dark matter candidate in the mass region 60-80 GeV ( GeV, mass of ) satisfies the recent bound from LUX direct detection experiment.

Dutta Banik, Amit; Majumdar, Debasish

2014-11-01

376

Use of a heat-stable microtubule-associated protein class-specific antibody to investigate the mechanism of microtubule binding.  

PubMed

Members of the heat-stable family of microtubule-associated proteins (MAPs), MAP 2, tau, and MAP 4, contain three or four tandem imperfect repeated sequences close to their carboxyl termini. These sequences lie within the microtubule-binding domains of the MAPs; they have been proposed to be responsible for microtubule binding and the ability of these MAPs to lower the critical concentration for microtubule assembly. Their spacing may reflect that of the regularly arrayed tubulin subunits on the microtubule surface. We here characterize the 32- and 34-kDa chymotryptic microtubule-binding fragments of MAP 2 identified in earlier work. We identify the primary chymotryptic cleavage site in high molecular weight MAP 2 as between Phe1525 and Lys1526, within 13 amino acids of the known MAP 2 splice junction. We have raised a monoclonal antibody to the 32- and 34-kDa fragments and find that it reacts with all members of the heat-stable MAPs class. To determine where it reacts, we sequenced immunoreactive subfragments of the 32- and 34-kDa fragments, selected several cDNA clones with the antibody, and tested for antibody reactivity against a series of synthetic MAP 2 and tau peptides. We identify the epitope sequence as HHVPGGG (His-His-Val-Pro-Gly-Gly-Gly). The antibody also recognized several other MAP 2 and tau repeats. Despite reacting with this highly conserved element, we find that the antibody does not block microtubule binding, but binds to the MAPs and co-sediments with microtubules. These results suggest that there are other regions besides the repeated elements which are essential for microtubule binding. PMID:1717454

Dingus, J; Obar, R A; Hyams, J S; Goedert, M; Vallee, R B

1991-10-01

377

She1-mediated inhibition of dynein motility along astral microtubules promotes polarized spindle movements  

PubMed Central

SUMMARY Background Cytoplasmic dynein motility along microtubules is critical for diverse cellular processes ranging from vesicular transport to nuclear envelope breakdown to mitotic spindle alignment. In yeast, we have proposed a regulated-offloading model to explain how dynein motility drives microtubule sliding along the cortex, powering transport of the nucleus into the mother-bud neck [1, 2]: the dynein regulator She1 limits dynein offloading by gating the recruitment of dynactin to the astral microtubule plus end, a prerequisite for offloading to the cortex. However, whether She1 subsequently affects cortically anchored dynein activity during microtubule sliding is unclear. Results Using single-molecule motility assays, we show that She1 strongly inhibits dynein movement along microtubules, acting directly on the motor domain in a manner independent of dynactin. She1 has no effect on the motility of either Kip2, a kinesin that utilizes the same microtubule track as dynein, or human kinesin-1, demonstrating the specificity of She1 for the dynein motor. At single-molecule resolution, She1 binds tightly to and exhibits diffusional behavior along microtubules. Diffusive She1 collides with and pauses motile dynein motors, prolonging their attachment to the microtubule. Furthermore, Aurora B/Ipl1 directly phosphorylates She1 and this modification appears to enhance the diffusive behavior of She1 along microtubules and its potency against dynein. In cells, She1 dampens productive microtubule-cortex interactions specifically in the mother compartment, polarizing spindle movements toward the bud cell. Conclusions Our data reveal how inhibitory microtubule-associated proteins selectively regulate motor activity to achieve unidirectional nuclear transport, and demonstrate a direct link between cell cycle machinery and dynein pathway activity. PMID:23142046

Markus, Steven M.; Kalutkiewicz, Katelyn A.; Lee, Wei-Lih

2012-01-01

378

Molecular Mechanisms for Microtubule Length Regulation by Kinesin-8 and XMAP215 Proteins  

E-print Network

The cytoskeleton is regulated by a plethora of enzymes that influence the stability and dynamics of cytoskeletal filaments. Molecular motors of the kinesin-8 protein family depolymerise microtubules in a length-dependent manner, and experimental and theoretical evidence suggest a role for kinesin-8 in the dynamic regulation of microtubules. However, so far the detailed molecular mechanisms how these molecular motors interact with the growing microtubule tip remain elusive. Here we investigate two interaction scenarios for kinesin-8 and the microtubule tip. We give a comprehensive analysis of regimes where length-regulation is possible and characterise how the stationary length depends on the biochemical rates and the bulk concentrations of the various proteins. For a neutral scenario, where microtubules grow irrespective of whether the microtubule tip is occupied by a molecular motor, length regulation is possible only for a narrow range of biochemical rates and limited to small polymerisation rates. In contrast, for an inhibition scenario, where the presence of a motor at the microtubule tip inhibits microtubule growth, the regime of length regulation is extremely broad and includes high growth rates. These results also apply to situations where polymerising enzymes, like XMAP215, and kinesin-8 mutually exclude each other from the microtubule tip. We also investigate the stochastic dynamics of the two scenarios. While for the neutral scenario length is tightly controlled, length dynamics is intermittent for the inhibition scenario and exhibits extended periods of microtubule growth and shrinkage, reminiscent of microtubule dynamic instability. On a broader perspective, the set of models established in this work quite generally suggests that mutual exclusion of molecules at the ends of cytoskeletal filaments is an important factor for filament dynamics and regulation.

Louis Reese; Anna Melbinger; Erwin Frey

2014-05-22

379

beta-tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast  

E-print Network

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast beta-tubulin C354, which is located near the ...

Bode, Claudia Janelle; Himes, Richard H.; Bloom, K. S.; Suprenant, Kathy A.; Pearson, C. G.; Thrower, D. A.; Gupta, M. L.

2002-08-01

380

Loop suppression of Dirac neutrino mass in the neutrinophilic two-Higgs-doublet model  

NASA Astrophysics Data System (ADS)

We extend the scalar sector of the neutrinophilic two-Higgs-doublet model, where small masses of Dirac neutrinos are obtained via a small vacuum expectation value v? of the neutrinophilic SU(2-doublet scalar field which has a Yukawa interaction with only right-handed neutrinos. A global U(1 symmetry is used for the neutrinophilic nature of the second SU(2-doublet scalar field and also for eliminating Majorana mass terms of neutrinos. By virtue of an appropriate assignment of the U(1-charges to new particles, our model has an unbroken Z2 symmetry, under which the lightest Z2-odd scalar boson can be a dark matter candidate. In our model, v? is generated by the one-loop diagram to which Z2-odd particles contribute. We briefly discuss a possible signature of our model at the LHC.

Kanemura, Shinya; Matsui, Toshinori; Sugiyama, Hiroaki

2013-11-01

381

Evidence for initiation of microtubules in discrete regions of the cell cortex in Azolla root-tip cells, and an hypothesis on the development of cortical arrays of microtubules  

Microsoft Academic Search

Complexes of microtubules, vesicles, and (to varying degrees) dense matrix material around the microtubules were seen along the edges of cells in root apices of Azolla pinnata R.Br. (viewing the cells as polyhedra with faces, vertices and edges). They are best developed after cytokinesis has been completed, when the daughter cells are reinstating their interphase arrays of microtubules. They are

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

1978-01-01

382

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

NASA Astrophysics Data System (ADS)

Microtubules are hollow cylinders composed of tubulin heterodimers that stack into linear protofilaments that interact laterally to form the microtubule wall. Synchrotron x-ray diffraction of microtubules under increasing osmotic stress shows they transition to rectangular bundles with noncircular buckled cross sections, followed by hexagonally packed bundles. This new technique probes the strength of interprotofilamen bonds, yielding insight into the mechanism by which associated proteins and the chemotherapy drug taxol stabilize microtubules.

Needleman, Daniel J.; Ojeda-Lopez, Miguel A.; Raviv, Uri; Ewert, Kai; Jones, Jayna B.; Miller, Herbert P.; Wilson, Leslie; Safinya, Cyrus R.

2004-11-01

383

CFEOM1-associated kinesin KIF21A is a cortical microtubule growth inhibitor.  

PubMed

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-?1/?1 and the components of the LL5?-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis. PMID:24120883

van der Vaart, Babet; van Riel, Wilhelmina E; Doodhi, Harinath; Kevenaar, Josta T; Katrukha, Eugene A; Gumy, Laura; Bouchet, Benjamin P; Grigoriev, Ilya; Spangler, Samantha A; Yu, Ka Lou; Wulf, Phebe S; Wu, Jingchao; Lansbergen, Gideon; van Battum, Eljo Y; Pasterkamp, R Jeroen; Mimori-Kiyosue, Yuko; Demmers, Jeroen; Olieric, Natacha; Maly, Ivan V; Hoogenraad, Casper C; Akhmanova, Anna

2013-10-28

384

Large Microtubule-Associated Protein of T. brucei Has Tandemly Repeated, Near-Identical Sequences  

Microsoft Academic Search

The parasitic protozoon Trypanosoma brucei contains a highly organized membrane skeleton, consisting of a dense array of parallel, singlet microtubules that are laterally interconnected and that are also in tight contact with the overlying cell membrane. A high molecular weight, heat-stable protein from this membrane skeleton was isolated that is localized along the microtubules. Protease digestion experiments and sequencing of

Andre Schneider; Andrew Hemphill; Toni Wyler; Thomas Seebeck

1988-01-01

385

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

PubMed Central

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

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

2012-01-01

386

Microtubule reorganization accompanying preprophase band formation in guard mother cells of Avena sativa L  

Microsoft Academic Search

Summary In order to study developmental changes in microtubule organization attending the formation of a longitudinally oriented preprophase band, the guard mother cells ofAvena were examined using a new procedure for anti-tubulin immunocytochemistry on large epidermal segments. We found that the interphase band (IMB) of transverse cortical microtubules present in these cells following asymmetric division is replaced after subsidiary cell

J. B. Mullinax; B. A. Palevitz

1989-01-01

387

Small scale effects on the mechanical behaviors of protein microtubules based on the nonlocal elasticity theory  

SciTech Connect

Based on the nonlocal elastic theory, small scale effects are considered in the investigation of the mechanical properties of protein microtubules. A new prediction formula for the persistence lengths of microtubules with the consideration of the small scale effect is presented. Subsequently, the buckling of microtubules is studied based on a nonlocal elastic beam model. The predicted results of our model indicate that the length-dependence of persistence length is related not only to the shear terms, but also to the small scale effect. The Eular beam model, which is always considered unable to explain the length-dependence of microtubules, can capture the length-dependence of the persistence length of microtubules with the consideration of the small scale effect. The elastic buckling behaviors of microtubules in viscoelastic surrounding cytoplasm are also considered using the nonlocal Timoshenko beam model in this paper, and the results indicate that the small scale effect of microtubules also plays an important role in the buckling of microtubules.

Gao, Yuanwen, E-mail: ywgao@lzu.edu.cn [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China)] [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China); Lei, Fang-Ming [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China)] [Key Laboratory of Mechanics on Western Disaster and Environment, Ministry of Education, Department of Mechanics and Engineering Science, College of Civil Engineering and Mechanics, Lanzhou University, Lanzhou 730000 (China)

2009-09-25

388

KINETIC ANALYSIS OF TUBULIN ASSEMBLY IN THE PRESENCE OF THE MICROTUBULE-ASSOCIATED PROTEIN TOGp.  

E-print Network

of microtubules is further modified in the cell by numerous protein factors that favour alternatively elongation targets that could affect spindle integrity. A promising approach is to identify the protein regulators1 KINETIC ANALYSIS OF TUBULIN ASSEMBLY IN THE PRESENCE OF THE MICROTUBULE-ASSOCIATED PROTEIN TOGp

Paris-Sud XI, Université de

389

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

PubMed Central

The diverse populations of microtubule polymers in cells are functionally distinguished by different posttranslational modifications, including polyglutamylation. Polyglutamylation is enriched on subsets of microtubules including those found in the centrioles, mitotic spindle, and cilia. However, whether this modification alters intrinsic microtubule dynamics or affects extrinsic associations with specific interacting partners remains to be determined. Here we identify the microtubule-binding protein centriole and spindle–associated protein (CSAP), which colocalizes with polyglutamylated tubulin to centrioles, spindle microtubules, and cilia in human tissue culture cells. Reducing tubulin polyglutamylation prevents CSAP localization to both spindle and cilia microtubules. In zebrafish, CSAP is required for normal brain development and proper left–right asymmetry, defects that are qualitatively similar to those reported previously for depletion of polyglutamylation-conjugating enzymes. We also find that CSAP is required for proper cilia beating. Our work supports a model in which polyglutamylation can target selected microtubule-associated proteins, such as CSAP, to microtubule subpopulations, providing specific functional capabilities to these populations. PMID:22493317

Backer, Chelsea B.; Gutzman, Jennifer H.; Pearson, Chad G.; Cheeseman, Iain M.

2012-01-01

390

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

E-print Network

microtubules and the instability of PAR-6 and PAR-2 domains during maintenance phase. Our data support design, data collection and analysis, decision to publish, or preparation of the manuscript. CompetingLong Astral Microtubules and RACK-1 Stabilize Polarity Domains during Maintenance Phase

Skop, Ahna

391

Kinesin's Neck-Linker Determines its Ability to Navigate Obstacles on the Microtubule Surface  

E-print Network

-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence

Hancock, William O.

392

A Kinesin Switch I Arginine to Lysine Mutation Rescues Microtubule Function  

Microsoft Academic Search

Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster ki- nesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved ar- ginine

Lisa M. Klumpp; Andrew T. Mackey; Christopher M. Farrell; John M. Rosenberg; Susan P. Gilbert

2003-01-01

393

Microtubule-targeting agents in oncology and therapeutic potential in hepatocellular carcinoma  

PubMed Central

In mammalian cells, microtubules are present both in interphase and dividing cells. In the latter, microtubules forming the mitotic spindle are highly dynamic and exquisitely sensitive to therapeutic inhibitors. Developed to alter microtubule function, microtubule-binding agents have been proven to be highly active as an anticancer treatment. Significant development of microtubule-binding agents has taken place in recent years, with newer anti-tubulin agents now showing novel properties of enhanced tumor specificity, reduced neurotoxicity, and insensitivity to chemoresistance mechanisms. Hepatocellular carcinoma remains one of the most difficult cancers to treat, with chemotherapies being relatively ineffective. There is now evidence to suggest that microtubule-binding agents may be effective in the treatment of hepatocellular carcinoma, especially when used in combination with mammalian target of rapamycin inhibitors. Preclinical models have suggested that the latter may be able to overcome resistance to microtubule binding agents. In this review article, recent developments of novel microtubule binding agents and their relevance to the treatment of hepatocellular carcinoma will be discussed. PMID:24790457

Loong, Herbert H; Yeo, Winnie

2014-01-01

394

Identification of a Novel Protein Regulating Microtubule Stability through a Chemical Approach  

Microsoft Academic Search

To identify novel proteins regulating the microtubule cytoskeleton, we screened a library of purine derivatives using mitotic spindle assembly in Xenopus egg extracts as an assay. Out of a collection of 1561 compounds, we identified 15 that destabilized microtubules without targeting tubulin directly, resulting in small spindles. Affinity chromatography with one compound, named diminutol, revealed a potential target as NQO1,

Sarah M Wignall; Nathanael S Gray; Young-Tae Chang; Lolita Juarez; Richard Jacob; Al Burlingame; Peter G Schultz; Rebecca Heald

2004-01-01

395

A model of cytoplasmically driven microtubule-based motion in the single-celled  

E-print Network

A model of cytoplasmically driven microtubule- based motion in the single-celled Caenorhabditis of cytoplasmically driven microtubule-based pronuclear motion in the single-celled Caenorhabditis elegans em- bryo Institute of Mathematical Sciences, b Center for Genomics and Systems Biology, c Sackler Institute

Shelley, Michael

396

Consistent handedness of microtubule helical arrays in maize and Arabidopsis primary roots  

Microsoft Academic Search

Summary The orientation of cortical microtubules in plant cells has been extensively studied, in part because of their influence on the expansion of most plant cell types. Cortical microtubules are often arranged in helical arrays, which are well known to occur with a specific pitch as a function of development or experimental treatment; however, it is not known if the

B. M. Liang; A. M. Dennings; R. E. Sharp; T. I. Baskin

1996-01-01

397

The spindle assembly function of Caenorhabditis elegans katanin does not require microtubule-severing activity  

PubMed Central

Katanin is a heterodimeric microtubule-severing protein that is conserved among eukaryotes. Loss-of-function mutations in the Caenorhabditis elegans katanin catalytic subunit, MEI-1, cause specific defects in female meiotic spindles. To determine the relationship between katanin’s microtubule-severing activity and its role in meiotic spindle formation, we analyzed the MEI-1(A338S) mutant. Unlike wild-type MEI-1, which mediated disassembly of microtubule arrays in Xenopus fibroblasts, MEI-1(A338S) had no effect on fibroblast microtubules, indicating a lack of microtubule-severing activity. In C. elegans, MEI-1(A338S) mediated assembly of extremely long bipolar meiotic spindles. In contrast, a nonsense mutation in MEI-1 caused assembly of meiotic spindles without any poles as assayed by localization of the spindle-pole protein, ASPM-1. These results indicated that katanin protein, but not katanin’s microtubule-severing activity, is required for assembly of acentriolar meiotic spindle poles. To understand the nonsevering activities of katanin, we characterized the N-terminal domain of the katanin catalytic subunit. The N-terminal domain was necessary and sufficient for binding to the katanin regulatory subunit. The katanin regulatory subunit in turn caused a dramatic change in the microtubule-binding properties of the N-terminal domain of the catalytic subunit. This unique bipartite microtubule-binding structure may mediate the spindle-pole assembly activity of katanin during female meiosis. PMID:21372175

McNally, Karen Perry; McNally, Francis J.

2011-01-01

398

Disrupting microtubule network immobilizes amoeboid chemotactic receptor in the plasma membrane  

E-print Network

Disrupting microtubule network immobilizes amoeboid chemotactic receptor in the plasma membrane S of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21218, USA b Department receptor Directed diffusion Microtubules Actin FRAP Signaling cascades are initiated in the plasma membrane

Devreotes, Peter

399

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce ( Picea glauca )  

Microsoft Academic Search

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing

L. C. Fowke; S. M. Attree; H. Wang; D. I. Dunstan

1990-01-01

400

Pushing forces drive the comet-like motility of microtubule arrays in Dictyostelium.  

PubMed

Overexpression of dynein fragments in Dictyostelium induces the movement of the entire interphase microtubule array. Centrosomes in these cells circulate through the cytoplasm at rates between 0.4 and 2.5 microm/s and are trailed by a comet-tail like arrangement of the microtubule array. Previous work suggested that these cells use a dynein-mediated pulling mechanism to generate this dramatic movement and that similar forces are at work to maintain the interphase MTOC position in wild-type cells. In the present study, we address the nature of the forces used to produce microtubule movement. We have used a laser microbeam to sever the connection between the motile centrosomes and trailing microtubules, demonstrating that the major force for such motility results from a pushing on the microtubules. We eliminate the possibility that microtubule assembly/disassembly reactions are significant contributors to this motility and suggest that the cell cortex figures prominently in locating force-producing molecules. Our findings indicate that interphase microtubules in Dictyostelium are subject to both dynein- and kinesin-like forces and that these act in concert to maintain centrosome position in the cell and to support the radial character of the microtubule network. PMID:15857957

Brito, Daniela A; Strauss, Joshua; Magidson, Valentin; Tikhonenko, Irina; Khodjakov, Alexey; Koonce, Michael P

2005-07-01

401

A tethered delivery mechanism explains the catalytic action of a microtubule polymerase  

PubMed Central

Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple ??-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast ??-tubulin. Like TOG1, TOG2 binds selectively to a fully ‘curved’ conformation of ??-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two ??-tubulins. Preferential interactions between TOGs and fully curved ??-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized ??-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end. DOI: http://dx.doi.org/10.7554/eLife.03069.001 PMID:25097237

Ayaz, Pelin; Munyoki, Sarah; Geyer, Elisabeth A; Piedra, Felipe-Andrés; Vu, Emily S; Bromberg, Raquel; Otwinowski, Zbyszek; Grishin, Nick V; Brautigam, Chad A; Rice, Luke M

2014-01-01

402

Microtubule remodelling is required for the front–rear polarity switch during contact inhibition of locomotion  

PubMed Central

When migrating mesenchymal cells collide, they exhibit a ‘contact inhibition of locomotion’ response that results in reversal of their front–rear polarity by extension of a new leading edge, which enables their migration away from the opposing contacted cell. The critical cytoskeletal rearrangements underpinning these mutual repulsion events are currently unknown. We found that during fibroblast cell–cell collisions, microtubules at the region of contact increase their frequency of catastrophe, their rates of shrinkage and growth, and concomitantly, a new microtubule array is established at a new leading edge. We show that Rho and ROCK activity is necessary for this repulsion response, and we observed increased microtubule stabilisation as a consequence of ROCK inhibition. Importantly, partial destabilisation of microtubules, by co-treatment with a low dose of nocodazole, restored microtubule dynamics to that of untreated cells and rescued contact inhibition of locomotion in ROCK-inhibited cells. Although there was an increase in microtubule growth or shrinkage rates in Y27632 cell–cell collisions, these failed to reach the same level of dynamicity compared with untreated collisions. Our data suggest that microtubule dynamics at contact sites must increase beyond a threshold for a cell to switch its front–rear polarity, and that microtubule stabilisation can lead to a failure of contact inhibition of locomotion. PMID:21750190

Kadir, Shereen; Astin, Jonathan W.; Tahtamouni, Lubna; Martin, Paul; Nobes, Catherine D.

2011-01-01

403

Microtubule remodelling is required for the front-rear polarity switch during contact inhibition of locomotion.  

PubMed

When migrating mesenchymal cells collide, they exhibit a 'contact inhibition of locomotion' response that results in reversal of their front-rear polarity by extension of a new leading edge, which enables their migration away from the opposing contacted cell. The critical cytoskeletal rearrangements underpinning these mutual repulsion events are currently unknown. We found that during fibroblast cell-cell collisions, microtubules at the region of contact increase their frequency of catastrophe, their rates of shrinkage and growth, and concomitantly, a new microtubule array is established at a new leading edge. We show that Rho and ROCK activity is necessary for this repulsion response, and we observed increased microtubule stabilisation as a consequence of ROCK inhibition. Importantly, partial destabilisation of microtubules, by co-treatment with a low dose of nocodazole, restored microtubule dynamics to that of untreated cells and rescued contact inhibition of locomotion in ROCK-inhibited cells. Although there was an increase in microtubule growth or shrinkage rates in Y27632 cell-cell collisions, these failed to reach the same level of dynamicity compared with untreated collisions. Our data suggest that microtubule dynamics at contact sites must increase beyond a threshold for a cell to switch its front-rear polarity, and that microtubule stabilisation can lead to a failure of contact inhibition of locomotion. PMID:21750190

Kadir, Shereen; Astin, Jonathan W; Tahtamouni, Lubna; Martin, Paul; Nobes, Catherine D

2011-08-01

404

Nucleation of microtubules in vitro by isolated spindle pole bodies of the yeast Saccharomyces cerevisiae  

PubMed Central

Spindle pole bodies (SPBs) were isolated from the yeast Saccharomyces cerevisiae by an adaptation of the Kleinschmidt monolayer technique. Spheroplasts prepared from the cells were lysed on an air-water interface. Spread preparations were picked up on grids, transferred to experimental test solutions, and prepared for whole-mount electron microscopy. Using purified exogenous tubulin from porcine brain tissue, the isolated SPBs were shown to nucleate the assembly of microtubules in vitro. Microtubule growth was directional and primarily onto the intranuclear face of the SPB. Neither the morphology nor the microtubule-initiating capacity of the SPB was affected by treatment with the enzymes DNase, RNase, or phospholipase although both properties were sensitive to trypsin. Analysis of SPBs at various stages of the cell cycle showed that newly replicated SPBs had the capacity to nucleate microtubules. SPBs isolated from exponentially growing cells initiated a subset of the yeast spindle microtubules equivalent to the number of pole-to-pole microtubules seen in vivo. However, SPBs isolated from cells in stationary phase and therefore arrested in G1 nucleated a number of microtubules equal to the total chromosomal and pole-to-pole tubules in the yeast spindle. This may mean that in G1-arrested cells, the SPB is associated with microtubule attachment sites of the yeast chromatin. PMID:357437

1978-01-01

405

DEFORMABLE TRELLISES ON FACTOR GRAPHS FOR ROBUST MICROTUBULE TRACKING IN CLUTTER  

E-print Network

DEFORMABLE TRELLISES ON FACTOR GRAPHS FOR ROBUST MICROTUBULE TRACKING IN CLUTTER Rahul Kidambi in microtubule tracking is due to clutter, or the presence of many similar intersecting structures. This paper images, (b) considerable clutter due to frequent intersections with other MTs. The focus of this paper

California at Santa Barbara, University of

406

The organization of cortical microtubule arrays in the radish root hair  

Microsoft Academic Search

Summary Cortical microtubule arrays in the radish root hair were analyzed from reconstructions of serial ultra-thin sections in order to test extant hypotheses concerning the role of microtubules in the deposition of oriented microfibrils of cellulose. Passing away from the tip, root hairs exhibit a transition from random to oriented deposition of microfibrils at approximately 25 µm. Along the root

R. W. Seagull; I. B. Heath

1980-01-01

407

Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins.  

PubMed

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles--including their nucleotide-free states--at ? 7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface. PMID:25209998

Atherton, Joseph; Farabella, Irene; Yu, I-Mei; Rosenfeld, Steven S; Houdusse, Anne; Topf, Maya; Moores, Carolyn A

2014-01-01

408

The Multipurpose 15-protofilament Microtubules in C. elegans Have Specific Roles in Mechanosensation  

PubMed Central

Summary Because microtubules perform many essential functions in neurons, delineating unique roles attributable to these organelles presents a formidable challenge. Microtubules endow neurons with shape and structure and are required for developmental processes including neurite outgrowth [1], intracellular transport [2], and synaptic formation and plasticity [3, 4]; microtubules in sensory neurons may be required for the above processes in addition to a specific sensory function. In Caenorhabditis elegans six touch receptor neurons (TRNs) sense gentle touch [5] and uniquely contain 15-protofilament microtubules [6]. Disruption of these microtubules by loss of either the MEC-7 ?-tubulin [7] or MEC-12 a-tubulin [8] or by growth in 1 mM colchicine causes touch insensitivity [5, 6], altered distribution of the touch transduction channel, and a general reduction in protein levels. We show that the effect on touch sensitivity can be separated from the others; microtubule depolymerization in mature TRNs causes touch insensitivity but does not result in protein distribution and production defects. In addition, the mec-12(e1605) mutation selectively causes touch insensitivity without affecting microtubule formation and other cellular processes. Touching e1605 animals produces a reduced mechanoreceptor current that inactivates more rapidly than in wild type, suggesting a specific role of the microtubules in mechanotransduction. PMID:19615905

Bounoutas, Alexander; O’Hagan, Robert; Chalfie, Martin

2009-01-01

409

The synthesis of organic charge transfer hetero-microtubules by crack welding.  

PubMed

The strain-induced cracks in organic microtubules composed of an organic charge transfer (CT) complex of 1,2,4,5-tetracyanobenzene (TCNB) and naphthalene were selectively welded via the formation of secondary CT complexes; this process, in turn, led to the formation of organic hetero-microtubules consisting of multiple segments of two organic CT complexes. PMID:25054622

Kim, J; Chung, J; Hyon, J; Kwon, T; Seo, C; Nam, J; Kang, Y

2014-09-14

410

Model based dynamics analysis in live cell microtubule images Alphan Altinok1  

E-print Network

Department of Molecular, Cellular, and Developmental Biology2 , University of California ­ Santa Barbara and querying microtubule image databases, are introduced to quantify and analyze microtubule dynamic behavior of various anti-cancer drugs, such as Taxol, [3]. For these and a host of basic biology issues

California at Santa Barbara, University of

411

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

SciTech Connect

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}.

Martinez, Homero [CEA, Saclay, DSM-IRFU-SPP (France); Melfo, Alejandra [ICTP, Trieste (Italy); Universidad de Los Andes, Merida (Venezuela, Bolivarian Republic of); Nesti, Fabrizio [Universita di Ferrara, Ferrara (Italy); Senjanovic, Goran [ICTP, Trieste (Italy)

2011-05-13

412

CP-conserving two-Higgs-doublet model: The approach to the decoupling limit  

Microsoft Academic Search

A CP-even neutral Higgs boson with Standard-Model-like couplings may be the\\u000alightest scalar of a two-Higgs-doublet model. We study the decoupling limit of\\u000athe most general CP-conserving two-Higgs-doublet model, where the mass of the\\u000alightest Higgs scalar is significantly smaller than the masses of the other\\u000aHiggs bosons of the model. In this case, the properties of the lightest Higgs

John F. Gunion; Howard E. Haber

2003-01-01

413

Microscopic study of doublet bands in odd-odd A ? 100 nuclei  

NASA Astrophysics Data System (ADS)

A systematic study of the doublet bands observed in odd-odd mass ?100 is performed using the microscopic triaxial projected shell model approach. This mass region has depicted some novel features which are not observed in other mass regions, for instance, it has been observed that doublet bands cross diabatically in 106Ag. It is demonstrated that this unique feature is due to crossing of the two 2-quasiparticle configurations having different intrinsic structures. Further, we provide a complete set of transition probabilities for all the six-isotopes studied in this work and it is shown that the predicted transitions are in good agreement with the available experimental data.

Dar, W. A.; Sheikh, J. A.; Bhat, G. H.; Palit, R.; Ali, R. N.; Frauendorf, S.

2015-01-01

414

Three extra mirror or sequential families: case for a heavy Higgs boson and inert doublet.  

PubMed

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(Z). PMID:21668143

Martínez, Homero; Melfo, Alejandra; Nesti, Fabrizio; Senjanovi?, Goran

2011-05-13

415

Microtubule assembly in cold-adapted organisms: Functional properties and structural adaptations of tubulins from Antarctic fishes  

Microsoft Academic Search

Fishes native to the coastal waters of the Antarctic have adapted to habitat and body temperatures in the range ?1.8 to +2°C. Their cytoplasmic microtubules, unlike those of mammals and temperate poikilotherms, have evolved to assemble efficiently at these low temperatures. To learn about the underlying molecular adaptations, my laboratory is studying microtubule proteins [tubulin ?? dimers and microtubule-associated proteins

H. William Detrich

1997-01-01

416

A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity  

Microsoft Academic Search

Background: In eukaryotes, assembly of the mitotic spindle requires the interaction of chromosomes with microtubules. During this process, several motor proteins that move along microtubules promote formation of a bipolar microtubule array, but the precise mechanism is unclear. In order to examine the roles of different motor proteins in building a bipolar spindle, we have used a simplified system in

Claire E. Walczak; Isabelle Vernos; Timothy J. Mitchison; Eric Karsenti; Rebecca Heald

1998-01-01

417

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

E-print Network

Microtubules are dynamic structures that present the peculiar characteristic to be ice- cold-labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6 and shrinking phases ended by catastrophes and rescues, respectively. In vitro, microtubule dynamics are under

Paris-Sud XI, Université de

418

Cellulose microfibril orientation and cell shaping in developing guard cells of Allium : The role of microtubules and ion accumulation  

Microsoft Academic Search

The role of microtubules and ions in cell shaping was investigated in differentiating guard cells of Allium using light and electron microscopy and cytochemistry. Microtubules appear soon after cytokinesis in a discrete zone close to the plasmalemma adjacent to the common wall between guard cells. The microtubules fan out from this zone, which corresponds to the future pore site, towards

B. A. Palevitz; P. K. Hepler

1976-01-01

419

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

NASA Astrophysics Data System (ADS)

Microtubules associate to form bundles in vivo in a wide variety of contexts including the mitotic spindle, neuronal processes, and the cortical array in plant cells. These supramolecular assemblies differ in size and shape, and in their internal structure, but the principles that determine this variation in morphology are not understood. To help elucidate such principals we constructed microtubule bundles in vitro using a variety of bundling agents. We have characterized the structure of these supramolecular assemblies of microtubules from the nanoscale to the mesoscale using synchrotron x-ray scattering and diffraction, video enhanced DIC and fluorescence microscopy, and electron microscopy. In the presence of inert polymers, an osmotic pressure imbalance between the inside and the outside of the microtubules may cause them to buckle to a non-circular cross-section. Depletion effects cause these distorted microtubules to bundle into a lattice with rectangular symmetry. The critical buckling pressure provides a measure of the stiffness of the inter-protofilament bond, and we determined that microtubule associated proteins enhance the strength of this bond, while the chemotherapeutic drug taxol has no effect. Multivalent ions cause microtubules to associate into bundles whose morphology depends on the condensing ion. Tightly packed hexagonal bundles with controllable diameters are observed for large tri-, tetra-, and pentavalent counterions. Unexpectedly, in the presence of small divalent cations, we have discovered a living necklace bundle phase, comprised of dynamical assemblies of MT nematic membranes with linear, branched, and loop topologies. Cations may also cause tubulin to assemble into non-microtubule structures. For example, in the presence of spermine, over time the microtubule bundles transform into a columnar phase of inverted tubules, such that the surface which was facing outside of the microtubules switches to the inside. This rearrangement between two arrays of hierarchically structured nanotubules occurs through a novel phase transition driven by a discrete conformational change in the constituent tubulin subunit. The microtubule associated protein tau may cause microtubules to bundle and the binding of tau seems to affect the microtubule structure. The interaction of tau with microtubules is strongly dependent on the particular tau isoform.

Needleman, Daniel Joseph

420

Proteomics of cancer cell lines resistant to microtubule stabilizing agents  

PubMed Central

In spite of the clinical success of microtubule interacting agents (MIAs), a significant challenge for oncologists is the inability to predict the response of individual cancer patients to these drugs. In the present study, six cell lines were compared by 2D DIGE proteomics to investigate cellular resistance to the class of MIAs known as microtubule stabilizing agents (MSAs). The human lung cancer cell line A549 was compared to two drug-resistant daughter cell lines, a Taxol resistant cell line (AT12) and an epothilone B (EpoB) resistant cell line (EpoB40). The ovarian cancer cell line Hey was compared to two drug-resistant daughter cell lines, an EpoB resistant cell line (EpoB8) and an ixabepilone resistant cell line (Ixab80). All 2D DIGE results were validated by Western blot analyses. A variety of cytoskeletal and cytoskeleton-associated proteins were differentially expressed in drug resistant cells. Differential abundance of 14-3-3?, galectin-1 and phosphorylation of stathmin are worthy of further studies as candidate predictive biomarkers for MSAs. This is especially true for galectin-1, a ?-galactose-binding lectin that mediates tumor invasion and metastasis. Galectin-1 was greatly increased in EpoB- and ixabepilone-resistant cells and its suppression caused an increase in drug sensitivity in both drug-sensitive and -resistant Hey cells. Furthermore, the growth medium from resistant Hey cells contained higher levels of galectin-1, suggesting that galectin-1 could play a role in resistance to microtubule stabilizing agents. PMID:24252851

Albrethsen, Jakob; Angeletti, Ruth H.; Horwitz, Susan Band; Yang, Chia-Ping Huang

2013-01-01

421

Gadkin: A novel link between endosomal vesicles and microtubule tracks.  

PubMed

Different types of endosomal vesicles show distinct distribution patterns within cells. While early endosomes can be found throughout the cell, recycling endosomal vesicles and tubules tend to cluster near the microtubule organizing center in the perinuclear region in most cell types. The molecular mechanisms underlying the steady-state distribution and dynamics of various types of endosomal vesicles has long remained enigmatic. However, during the past decade it has become evident that microtubule-based motor proteins of the kinesin family play a pivotal role in the positioning of endosomes. Early endosomes were shown to cluster in the perinuclear area in the absence of KIF16B,1 KIF3A is required for the steady-state distribution of late endosomes/lysosomes,2 and KIF13A directs M6PR-containing vesicles from the TGN to the plasma membrane3 to name only a few examples. In the case of Tf-containing recycling endosomes antibody-injection experiments implicated kinesin-1, a heteromer comprised of KIF5 heavy and KLC light chains, as a motor for their transport towards the cell periphery.4 Indeed, KIF5B knockdown experiments confirmed that kinesin-1 is necessary to maintain the peripheral pool of recycling endosomes.5 But how is kinesin-1 linked to endosomal vesicles? Work from our own laboratory has identified the AP-1-binding protein Gadkin as a molecular link between AP-1-mediated traffic and kinesin-1-based transport along microtubules.5 This work as well as hypothetical models for kinesin-dependent endosomal membrane traffic will be discussed here. PMID:20798811

Maritzen, Tanja; Haucke, Volker

2010-07-01

422

Kinesin superfamily proteins and the regulation of microtubule dynamics in morphogenesis.  

PubMed

Kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motors that serve as sources of force for intracellular transport and cell division. Recent studies have revealed new roles of KIFs as microtubule stabilizers and depolymerizers, and these activities are fundamental to cellular morphogenesis and mammalian development. KIF2A and KIF19A have microtubule-depolymerizing activities and regulate the neuronal morphology and cilia length, respectively. KIF21A and KIF26A work as microtubule stabilizers that regulate axonal morphology. Morphological defects that are similar to human diseases are observed in mice in which these KIF genes have been deleted. Actually, KIF2A and KIF21A have been identified as causes of human neuronal diseases. In this review, the functions of these atypical KIFs that regulate microtubule dynamics are discussed. Moreover, some interesting unanswered questions and hypothetical answers to them are discussed. PMID:25347970

Niwa, Shinsuke

2015-01-01

423

CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content  

PubMed Central

Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule–membrane links there. Ultrastructural analysis revealed defects in these microtubule–membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule–membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition. PMID:20819941

Craige, Branch; Tsao, Che-Chia; Diener, Dennis R.; Hou, Yuqing; Lechtreck, Karl-Ferdinand; Rosenbaum, Joel L.

2010-01-01

424

Genetic Evidence That Cellulose Synthase Activity Influences Microtubule Cortical Array Organization1[W][OA  

PubMed Central

To identify factors that influence cytoskeletal organization we screened for Arabidopsis (Arabidopsis thaliana) mutants that show hypersensitivity to the microtubule destabilizing drug oryzalin. We cloned the genes corresponding to two of the 131 mutant lines obtained. The genes encoded mutant alleles of PROCUSTE1 and KORRIGAN, which both encode proteins that have previously been implicated in cellulose synthesis. Analysis of microtubules in the mutants revealed that both mutants have altered orientation of root cortical microtubules. Similarly, isoxaben, an inhibitor of cellulose synthesis, also altered the orientation of cortical microt