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1

Molecular architecture of axonemal microtubule doublets revealed by cryo-electron tomography  

Microsoft Academic Search

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 that contain a number of proteins besides tubulin, and are usually found in arrays of nine doublets arranged around two singlet microtubules. Coordinated sliding

Haixin Sui; Kenneth H. Downing

2006-01-01

2

SLIDING VELOCITY BETWEEN OUTER DOUBLET MICROTUBULES OF SEA-URCHIN SPERM AXONEMES  

Microsoft Academic Search

SUMMARY Using a dark-field microscope equipped with a high-efficiency TV camera including a video tape-recorder, we recorded the sliding movement between outer doublet microtubules of the demembranated axonemes of sea-urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella by adding ATP and trypsin at 25 °C. The time and length of the sliding doublet microtubules from axonemes were measured directly from

Y. YANO; T. MIKI-NOUMURA

1980-01-01

3

Cyclical Interactions between Two Outer Doublet Microtubules in Split Flagellar Axonemes  

PubMed Central

The beating of cilia and flagella is based on the localized sliding between adjacent outer doublet microtubules; however, the mechanism that produces oscillatory bending is unclear. To elucidate this mechanism, we examined the behavior of frayed axonemes of Chlamydomonas by using high-speed video recording. A pair of doublet microtubules frequently displayed association and dissociation cycles in the presence of ATP. In many instances, the dissociation of two microtubules was not accompanied by noticeable bending, suggesting that the dynein-microtubule interaction is not necessarily regulated by the microtubule curvature. On rare occasions, association and dissociation occurred simultaneously in the same interacting pair, resulting in a tip-directed movement of a stretch of gap between the pair. Based on these observations, we propose a model for cyclical bend propagation in the axoneme.

Aoyama, Susumu; Kamiya, Ritsu

2005-01-01

4

Analysis of microtubule sliding patterns in Chlamydomonas flagellar axonemes reveals dynein activity on specific doublet microtubules  

Microsoft Academic Search

Generating the complex waveforms characteristic of beating eukaryotic cilia and flagella requires spatial regulation of dynein-driven microtubule sliding. To generate bending, one prediction is that dynein arms alternate between active and inactive forms on specific subsets of doublet microtubules. Using an in vitro microtubule sliding assay combined with a structural approach, we determined that ATP induces sliding between specific subsets

Matthew J. Wargo; Mark A. McPeek; Elizabeth F. Smith

2004-01-01

5

Inner arm dynein ATPase fraction of sea urchin sperm flagella causes active sliding of axonemal outer doublet microtubule.  

PubMed

In order to clarify the role of the inner arms of the axoneme in sperm flagellar movement, we prepared an ATPase fraction (12S) from the outer arm-depleted axonemes of sea urchin sperm flagella. When both arm-depleted axonemes were incubated with the 12S ATPase, they exhibited the sliding disintegration of outer doublet microtubules. Electron microscopy revealed that the ATPase rebound to the original inner arm sites of the axoneme. Therefore, it is quite likely that the 12S ATPase is one of the components of the inner arms. We referred to it as "inner arm dynein". PMID:1825599

Wada, S; Okuno, M; Mohri, H

1991-02-28

6

Motile flagellar axonemes with a 9 + 1 microtubule configuration  

Microsoft Academic Search

Electron microscope (EM) studies of the eukaryotic flagellum reveal that the organelle contains a 9 + 2 arrangement of microtubules, the axoneme, with nine doublets surrounding two singlets enveloped by a membrane which is continuous with that of the cell; various linkages and projections are associated with the microtubules1. Strong experimental evidence supports the idea that the forces required for

S. Marchese-Ragona; M. E. J. Holwill

1980-01-01

7

The Parkin co-regulated gene product, PACRG, is an evolutionarily conserved axonemal protein that functions in outer-doublet microtubule morphogenesis  

Microsoft Academic Search

Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Comparative genomics of flagellated and non- flagellated eukaryotes provides one way to identify new putative flagellar proteins. We identified the Parkin co- regulated gene, or PACRG, from such a screen. Male mice deficient in PACRG are sterile, but its function has been little explored. The

Helen R. Dawe; Helen Farr; Neil Portman; Michael K. Shaw; Keith Gull

2005-01-01

8

Microtubule sliding in mutant Chlamydomonas axonemes devoid of outer or inner dynein arms  

Microsoft Academic Search

To clarify the functional differentiation be- tween the outer and inner dynein arms in eukaryotic flagella, their mechanochemical properties were as- sessed by measuring the sliding velocities of outer- doublet microtubules in disintegrating axonemes of Chlamydomonas, using wild-type and mutant strains that lack either of the arms. A special procedure was developed to induce sliding disintegration in Chlamydomonas axonemes which

Tsuyoshi Okagaki; Ritsu Kamiya

1986-01-01

9

One of the Nine Doublet Microtubules of Eukaryotic Flagella Exhibits Unique and Partially Conserved Structures  

PubMed Central

The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1–2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella.

Lin, Jianfeng; Heuser, Thomas; Song, Kangkang; Fu, Xiaofeng; Nicastro, Daniela

2012-01-01

10

One of the nine doublet microtubules of eukaryotic flagella exhibits unique and partially conserved structures.  

PubMed

The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1-2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella. PMID:23071579

Lin, Jianfeng; Heuser, Thomas; Song, Kangkang; Fu, Xiaofeng; Nicastro, Daniela

2012-10-10

11

Direct measurements of sliding between outer doublet microtubules in swimming sperm flagella.  

PubMed

The relative motion of 40-nanometer gold beads bound to the exposed outer doublet microtubules of demembranated sea urchin sperm flagella has been observed and photographed during adenosine triphosphate (ATP)-reactivated swimming. This direct demonstration and measure of sliding displacements between outer doublet microtubules in actively bending flagella verifies the original sliding microtubule model for ciliary bending that was established by electron microscopy of fixed cilia and provides a new, functional measure for the diameter of the flagellar axoneme of 132 +/- 8 nanometers. PMID:2928796

Brokaw, C J

1989-03-24

12

The A and B tubules of the outer doublets of sea urchin sperm axonemes are composed of different tubulin variants.  

PubMed

The alpha beta-tubulin heterodimer, the structural unit of microtubules, comes in many variants. There are different alpha and beta isotypes encoded by multigene families. Additional heterogeneity is generated by a set of posttranslational modifications. Detyrosination of alpha-tubulin, removal of the carboxy-terminal Glu-Tyr dipeptide of alpha-tubulin, phosphorylation of some tubulins, polyglutamylation, and polyglycylation of alpha- and beta-tubulins all involve the acidic carboxy-terminal region. We have investigated the distribution of tubulin variants in the axonemal microtubules of sea urchin sperm flagella by immunological procedures and by direct sequence and mass spectrometric analysis of the carboxy-terminal peptides. The A and B tubules that comprise the nine outer doublets differ strongly in tubulin variants. A tubules contain over 95% unmodified, tyrosinated alpha beta-tubulin. In B tubules, alpha-tubulin is approximately 65% detyrosinated and both alpha- and beta-tubulin are 40-45% polyglycylated. These results show a segregation of tubulin variants between two different axonemal structures and raise the possibility that posttranslational modifications of tubulins reflect or specify structurally and functionally distinct microtubules. PMID:8718878

Multigner, L; Pignot-Paintrand, I; Saoudi, Y; Job, D; Plessmann, U; Rüdiger, M; Weber, K

1996-08-20

13

Effects of trypsin-digested outer-arm dynein fragments on the velocity of microtubule sliding in elastase-digested flagellar axonemes.  

PubMed

Flagellar movement is caused by the coordinated activity of outer and inner dynein arms, which induces sliding between doublet microtubules. In trypsin-treated flagellar axonemes, microtubule sliding induced by ATP is faster in the presence than in the absence of the outer arms. To elucidate the mechanism by which the outer arms regulate microtubule sliding, we studied the effect of trypsin-digested outer-arm fragments on the velocity of microtubule sliding in elastase-treated axonemes of sea urchin sperm flagella. We found that microtubule sliding was significantly slower in elastase-treated axonemes than in trypsin-treated axonemes, and that this difference disappeared after the complete removal of the outer arms. After about 95% of the outer arms were removed, however, the velocity of sliding induced by elastase and ATP increased significantly by adding outer arms that had been treated with trypsin in the presence of ATP. The increase in sliding velocity did not occur in the elastase-treated axonemes from which the outer arms had been completely removed. Among the outer arm fragments obtained by trypsin treatment, a polypeptide of about 350 kDa was found to be possibly involved in the regulation of sliding velocity. These results suggest that the velocity of sliding in the axonemes with only inner arms is similar to that in the axonemes with both inner and outer arms, and that the 350 kDa fragment, probably of the alpha heavy chains, increases the sliding activity of the intact outer and inner arms on the doublet microtubules. PMID:12655153

Imai, Hiroshi; Shingyoji, Chikako

2003-02-01

14

Incorporation of Paramecium axonemal Tubulin into Higher Plant Cells Reveals Functional Sites of Microtubule Assembly  

Microsoft Academic Search

Incorporation of Paramecium axonemal tubulin into lysed endosperm cells of the higher plant Haemanthus enabled us to identify sites of microtubule assembly. This exogenous Paramecium tubulin could be traced by specific antibodies that do not stain endogenous plant microtubules. Intracellular copolymerization of protozoan and higher plant tubulins gave rise to hybrid polymers that were visualized by immunofluorescence and by immunoelectron

Marylin Vantard; Nicolette Levilliers; Anne-Marie Hill; Andre Adoutte; Anne-Marie Lambert

1990-01-01

15

Cryo-electron tomography reveals conserved features of doublet microtubules in flagella.  

PubMed

The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction. PMID:21930914

Nicastro, Daniela; Fu, Xiaofeng; Heuser, Thomas; Tso, Alan; Porter, Mary E; Linck, Richard W

2011-09-19

16

The molecular architecture of axonemes revealed by cryoelectron tomography.  

PubMed

Eukaryotic flagella and cilia are built on a 9 + 2 array of microtubules plus >250 accessory proteins, forming a biological machine called the axoneme. Here we describe the three-dimensional structure of rapidly frozen axonemes from Chlamydomonas and sea urchin sperm, using cryoelectron tomography and image processing to focus on the motor enzyme dynein. Our images suggest a model for the way dynein generates force to slide microtubules. They also reveal two dynein linkers that may provide "hard-wiring" to coordinate motor enzyme action, both circumferentially and along the axoneme. Periodic densities were also observed inside doublet microtubules; these may contribute to doublet stability. PMID:16917055

Nicastro, Daniela; Schwartz, Cindi; Pierson, Jason; Gaudette, Richard; Porter, Mary E; McIntosh, J Richard

2006-08-18

17

Regulation of dynein-driven microtubule sliding by the axonemal protein kinase CK1 in Chlamydomonas flagella  

PubMed Central

Experimental analysis of isolated ciliary/flagellar axonemes has implicated the protein kinase casein kinase I (CK1) in regulation of dynein. To test this hypothesis, we developed a novel in vitro reconstitution approach using purified recombinant Chlamydomonas reinhardtii CK1, together with CK1-depleted axonemes from the paralyzed flagellar mutant pf17, which is defective in radial spokes and impaired in dynein-driven microtubule sliding. The CK1 inhibitors (DRB and CK1-7) and solubilization of CK1 restored microtubule sliding in pf17 axonemes, which is consistent with an inhibitory role for CK1. The phosphatase inhibitor microcystin-LR blocked rescue of microtubule sliding, indicating that the axonemal phosphatases, required for rescue, were retained in the CK1-depleted axonemes. Reconstitution of depleted axonemes with purified, recombinant CK1 restored inhibition of microtubule sliding in a DRB– and CK1-7–sensitive manner. In contrast, a purified “kinase-dead” CK1 failed to restore inhibition. These results firmly establish that an axonemal CK1 regulates dynein activity and flagellar motility.

Gokhale, Avanti; Wirschell, Maureen

2009-01-01

18

Two proteins isolated from sea urchin sperm flagella: structural components common to the stable microtubules of axonemes and centrioles.  

PubMed

Biochemical fractionation of axonemal microtubules yields the protofilament ribbon (pf-ribbon), an insoluble structure of 3-4 longitudinal protofilaments composed primarily of alpha/beta tubulin, tektins A, B and C, and two previously uncharacterized polypeptides of 77 kDa and 83 kDa. We have isolated the 77/83 kDa polypeptides (termed Sp77 and Sp83) from sperm flagella of the sea urchin Stronglyocentrotus purpuratus and raised polyclonal antibodies against them. Sp77 and Sp83 copurify exclusively with the pf-ribbon. Both the anti-Sp77 and anti-Sp83 antibodies detected the nine outer doublets and the basal bodies of sea urchin sperm by immunofluorescence microscopy. In addition, the anti-Sp83 antibody, but not the anti-Sp77 antibody, detected a single 83 kDa polypeptide on immunoblots of unfertilized sea urchin egg cytoplasm, and a single polypeptide of 80 kDa on blots of isolated mitotic spindles from Chinese hamster ovary (CHO) cells. Previous studies have shown that tektins are present in the basal bodies and centrosomes/centrioles of cells ranging from clam to human. We found that anti-Sp83 decorates the spindle poles in sea urchin zygotes, and the interphase centrosome and spindle poles in CHO cells. In CHO cells arrested in S phase with aphidicolin, anti-Sp83 detects multiple centrosomes. The staining of the centrosome was not disrupted by prolonged nocodazole treatment, suggesting that the 80 kDa polypeptide is associated with the centrioles themselves. Our observations demonstrate that, like tektins, Sp77 and Sp83 are structural proteins associated with stable doublet microtubules, and may be components of basal bodies and centrioles of sea urchins and mammalian cells. PMID:9454732

Hinchcliffe, E H; Linck, R W

1998-03-01

19

Displacement-weighted velocity analysis of gliding assays reveals that chlamydomonas axonemal dynein preferentially moves conspecific microtubules.  

PubMed

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

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

2013-05-01

20

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

PubMed Central

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

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

2001-01-01

21

Turnover of tubulin in ciliary outer doublet microtubules.  

PubMed

Previous pulse-chase labeling studies have shown that structural proteins incorporate into fully assembled sea urchin embryonic cilia at rates approaching those of full regeneration. When all background ciliogenesis was suppressed by taxol, the turnover of most proteins, including tubulin, continued. The present study utilized chemical dissection to explore the route of tubulin incorporation in the presence of taxol and also in steady-state cilia from prism stage embryos. Surprisingly, in cilia from untreated embryos, the most heavily labeled tubulin was found in the most stable portion of the doublet microtubles, the junctional protofilaments. With taxol, this preferential incorporation was suppressed, although control-level turnover still took place in the remainder of the doublet. This paradoxical result was confirmed by pulse-chase labeling and immediately isolating steady-state cilia, then isolating two additional crops of cilia regenerated, respectively, from pools of high and then decreased label. In each case, the level of label occurring in the tubulin from the junctional protofilaments, compared with that from the remainder of the doublet, correlated with the level of pool label from which it must exchange or assemble. These data indicate that ciliary outer doublet microtubules are dynamic structures and that the junctional region is not inert. Plausible mechanisms of incorporation and turnover of tubulin in fully-assembled, fully-motile cilia can now be assessed with regared to recent discoveries, particularly intraflagellar transport, distal tip incorporation, and treadmilling. PMID:15216899

Stephens, R E

1999-10-01

22

Structures attached to doublet microtubules of cilia: computer modeling of thin-section and negative-stain stereo images.  

PubMed Central

With a single set of positional coordinates for longitudinal and transverse attachment of the inner and outer rows of dynein arms with respect to the doublet microtubules of Tetrahymena ciliary axonemes, a computer model has been constructed at 4-nm resolution that reconciles negative-stain en face stereo images of arm and spoke positions to traditional images of tannic acid/glutaraldehyde-fixed sections. In this model, inner and outer arms correspond in substructure; both repeat with a 24-nm periodicity without stagger between rows, and a pair of arms is in exact alignment with the first spoke (S1) in each doublet spoke group. The model and the supporting micrographs suggest that each arm cycles in three dimensions and that, during cycling, the inner and outer arms move in opposite directions with respect to the center of subfiber A of the doublet (N). Attachment is off-center with respect to subfiber B of the adjacent doublet (N + 1), causing the sliding doublets to skew with respect to one another. Images

Avolio, J; Glazzard, A N; Holwill, M E; Satir, P

1986-01-01

23

Asymmetry of inner dynein arms and inter-doublet links in Chlamydomonas flagella  

PubMed Central

Although the widely shared “9 + 2” structure of axonemes is thought to be highly symmetrical, axonemes show asymmetrical bending during planar and conical motion. In this study, using electron cryotomography and single particle averaging, we demonstrate an asymmetrical molecular arrangement of proteins binding to the nine microtubule doublets in Chlamydomonas reinhardtii flagella. The eight inner arm dynein heavy chains regulate and determine flagellar waveform. Among these, one heavy chain (dynein c) is missing on one microtubule doublet (this doublet also lacks the outer dynein arm), and another dynein heavy chain (dynein b or g) is missing on the adjacent doublet. Some dynein heavy chains either show an abnormal conformation or were replaced by other proteins, possibly minor dyneins. In addition to nexin, there are two additional linkages between specific pairs of doublets. Interestingly, all these exceptional arrangements take place on doublets on opposite sides of the axoneme, suggesting that the transverse functional asymmetry of the axoneme causes an in-plane bending motion.

Bui, Khanh Huy; Sakakibara, Hitoshi; Movassagh, Tandis; Oiwa, Kazuhiro

2009-01-01

24

The regulation of dynein-driven microtubule sliding in Chlamydomonas flagella by axonemal kinases and phosphatases.  

PubMed

The purpose of this chapter is to review the methodology and advances that have revealed conserved signaling proteins that are localized in the 9+2 ciliary axoneme for regulating motility. Diverse experimental systems have revealed that ciliary and eukaryotic flagellar motility is regulated by second messengers including calcium, pH, and cyclic nucleotides. In addition, recent advances in in vitro functional studies, taking advantage of isolated axonemes, pharmacological approaches, and biochemical analysis of axonemes have demonstrated that otherwise ubiquitous, conserved protein kinases and phosphatases are transported to and anchored in the axoneme. Here, we focus on the functional/pharmacological, genetic, and biochemical approaches in the model genetic system Chlamydomonas that have revealed highly conserved kinases, anchoring proteins (e.g., A-kinase anchoring proteins), and phosphatases that are physically located in the axoneme where they play a direct role in control of motility. PMID:20409803

Elam, Candice A; Sale, Winfield S; Wirschell, Maureen

2009-11-21

25

Splayed Tetrahymena cilia. A system for analyzing sliding and axonemal spoke arrangements.  

PubMed

This study makes use of a procedure designed to illustrate, without serial section analysis, the three-dimensional changes in the ciliary axoneme produced by microtubule sliding, and to confirm essential features of the sliding microtubule hypothesis of ciliary movement. Cilia, isolated from Tetrahymena pyriformis by the dibucaine procedure, are attached to polylysine substratum, and treated with Triton X-100. Critical point drying maintains three-dimensional structure without embedding. The detergent removes the membrane and many axonemes unroll, always in an organized fashion so that doublets follow one another in sequence, according to the enantiomorphic form of the cilium. The central pair of microtubules fall to the side as a unit. The parallel doublet microtubules retain relative longitudinal positions in part by interdoublet or nexin links. Spoke organization and tip patterns are preserved in the opened axonemes. We generalize the work of Warner and Satir (Warner, F. D., and P. Satir, 1976. J. Cell Biol. 63:35-63) to show that spoke group arrangements are maintained for all doublets in straight regions, while systematic displacements occur in bent regions. The conclusion that local contraction of microtubles is absent in the axoneme is strengthened, and direct graphic demonstrations of sliding at the ciliary tip are shown. A morphogenetic numbering scheme is presented which results in a quantitative fit of the tip images to the images predicated by the equation for doublet sliding, and which makes possible new comparisons of structural parameters between axonemes and with cilia of other organisms. PMID:825521

Sale, W S; Satir, P

1976-11-01

26

The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules  

Microsoft Academic Search

BACKGROUND: Impairment of cilia and flagella function underlies a growing number of human genetic diseases. Mutations in hydin in hy3 mice cause lethal communicating hydrocephalus with early onset. Hydin was recently identified as an axonemal protein; however, its function is as yet unknown. RESULTS: Here we use RNAi in Trypanosoma brucei to address this issue and demonstrate that loss of

Helen R Dawe; Michael K Shaw; Helen Farr; Keith Gull

2007-01-01

27

Geometry drives the "deviated-bending" of the bi-tubular structures of the 9 + 2 axoneme in the flagellum.  

PubMed

The axoneme "9 + 2" is basically a system constituted of a cylinder of 9 microtubule doublets surrounding a central pair of microtubules. These bi-tubular structures are considered as the support system of the active molecular complexes that generate and regulate the axonemal movement. Schoutens has calculated their moments of inertia [Schoutens, 1994: Journal of Theoretical Biology 171:163-177]. The results obtained allowed us to assume that these bi-tubular systems are endowed with dynamic properties that could be involved in the regulation of the axonemal machinery. For the first time, using the finite elements methods and the resistance of material principles, we have now calculated that the curvature of the axoneme induces the deviated-bending of the bi-tubular structures of the axoneme, because of their geometry only; they behave as beams in a framework. This approach is similar to the one used to measure the deflection of a single microtubule [Kasas et al., 2004: Chem Phys Chem 5:252-257]. These behaviors induce internal movement or constraints of either couples or triplets of doublets within the axonemal cylinder that could be directly involved in a constrained or a spontaneous "convergence/divergence" equilibrium of the cylindrical generatrices that they draw along the axonemal cylinder, which could apparently regulate the activity of the axonemal motors (the dynein arms). These results are discussed here, taking into consideration the dynamic propagation of the wave train along the flagellar axoneme, and the regulated balance between the activities of the two opposite sides of the axoneme during the beat. This study raises a few questions about the architecture-activity duo of the axonemal doublets. PMID:15368611

Cibert, Christian; Heck, Jean-Vivien

2004-11-01

28

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

Microsoft Academic Search

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

T. Miki-Noumura; R. KAMIYA

1979-01-01

29

A Tektin Homologue Is Decreased in Chlamydomonas Mutants Lacking an Axonemal Inner-Arm Dynein  

PubMed Central

In ciliary and flagellar axonemes, various discrete structures such as inner and outer dynein arms are regularly arranged on the outer doublet microtubules. Little is known about the basis for their regular arrangement. In this study, proteins involved in the attachment of inner-arm dyneins were searched by a microtubule overlay assay on Chlamydomonas mutant axonemes. A 58-kDa protein (p58) was found ?80% diminished in the mutants ida6 and pf3, both lacking one (species e) of the seven inner-arm species (a–g). Analysis of its cDNA indicated that p58 is homologous to tektin, a protein that was originally found in sea urchin and thought to be crucial for the longitudinal periodicity of the doublet microtubule. Unlike sea urchin tektin, which is a component of protofilament ribbons that occur after Sarkosyl treatment of axonemes, p58 was not contained in similar Sarkosyl-resistant ribbons from Chlamydomonas axonemes. Immunofluorescence microscopy showed that p58 was localized uniformly along the axoneme and on the basal body. The p58 signal was reduced in ida6 and pf3. These results suggest that a reduced amount of p58 is sufficient for the production of outer doublets, whereas an additional amount of it is involved in inner-arm dynein attachment.

Yanagisawa, Haru-aki; Kamiya, Ritsu

2004-01-01

30

Integrated Control of Axonemal Dynein AAA+ Motors  

PubMed Central

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.

King, Stephen M.

2012-01-01

31

Direction of active sliding of microtubules in Tetrahymena cilia.  

PubMed

Axonemes of protozoan (Tetrahymena thermophila BIII) cilia, isolated by the dibucaine method, were treated briefly with trypsin after removal of the ciliary membranes by treatment with Triton X-100. After attachment to polylysine-coated surfaces, the partially digested axonemes remained mainly intact cylinders. Such attached axonemes can be treated with ATP, which induces microtubles sliding. ATP-treated preparations showed disrupted axonemes in which doublets had telescoped out of the original cylinders. These could be captured in place for electron microscopy after critical point drying. Images of this type were used to determine relative movement between adjacent doublet microtubules. Each doublet actively slid relative to its neighbors in a single direction, in which the polarity of force generation of the dynein arms was from base to tip. PMID:266725

Sale, W S; Satir, P

1977-05-01

32

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

PubMed Central

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

1989-01-01

33

Purealin blocks the sliding movement of sea urchin flagellar axonemes by selective inhibition of half the ATPase activity of axonemal dyneins.  

PubMed

Ciliary and flagellar movements are explained by active sliding between the outer doublet microtubules of an axoneme via their inner and outer dynein arms. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, blocked the motility of Triton-demembranated sea urchin sperm flagella within 5 min at concentrations above 20 microM. In a similar concentration range, purealin blocked the sliding movement of the flagellar axonemes in vitro within a few minutes judging from the turbidity measurements. The ATPase activity of axonemes was partially inhibited by purealin in a concentration-dependent manner. The maximum inhibition reached approximately 50% at concentrations above 20 microM, indicating that half the axonemal ATPase activity is sensitive to purealin. Similar results were observed on the ATPase activity of outer-arm-depleted axonemes and that of a mixture of 21S dynein and salt-extracted axonemes. On the other hand, ATPase activity of isolated 21S dynein was not inhibited by purealin. The inhibitory action of purealin on the axonemal ATPases was reversed by dilution of purealin. The effect of purealin on the double-reciprocal plot of the ATPase activity as a function of ATP concentrations showed that the inhibition was not a competitive type. In accord with this finding, purealin did not affect the vanadate-mediated UV photocleavage of axonemal dyneins. These results suggest that purealin binds reversibly to a site other than the catalytic ATP-binding site and inhibits half the ATPase activity of axonemes. Taken together, our results suggest that purealin-sensitive ATPase activity of the dynein arms plays an essential role in generating the sliding movement of flagellar axonemes. PMID:9398284

Fang, Y I; Yokota, E; Mabuchi, I; Nakamura, H; Ohizumi, Y

1997-12-16

34

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

Microsoft Academic Search

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

W. S. Sale

1986-01-01

35

cAMP-stimulated phosphorylation of an axonemal polypeptide that copurifies with the 22S dynein arm regulates microtubule translocation velocity and swimming speed in Paramecium.  

PubMed Central

In Paramecium tetraurelia, cyclic nucleotides are important physiological second messengers that could regulate dynein mechanochemistry by phosphorylation. A 29-kDa polypeptide that is phosphorylated in a cAMP- and Ca(2+)-sensitive manner in permeabilized cells and isolated axonemes is the only significant phosphorylated moiety that consistently copurifies with 22S dynein from paramecium cilia. It is not a component of 14S dynein. This polypeptide can be thiophosphorylated in a cAMP-sensitive manner, and this form of 22S dynein is stable when stored at -70 degrees C. cAMP-mediated thiophosphorylation of the 29-kDa polypeptide significantly increases the velocity with which 22S dynein causes microtubules to glide in vitro. The increase is abolished, together with the thiophosphorylation of the 29-kDa polypeptide, by preincubation with high Ca2+. Pretreatment with high Ca2+ does not alter the thiophosphorylation pattern of, or the velocity of microtubule translocation by, 14S dynein. The same preincubation conditions that permit or abolish the increase in velocity of microtubule translocation by 22S dynein permit or fail to permit swimming speed of permeabilized cells to increase on reactivation even after cAMP is removed. The effect of cAMP on swimming speed can therefore be accounted for by changes in the mechanically coupled 22S dynein activity via phosphorylation or thiophosphorylation of the 29-kDa polypeptide, which could act as a regulatory dynein light chain. Images

Hamasaki, T; Barkalow, K; Richmond, J; Satir, P

1991-01-01

36

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

PubMed

The "9+2" axoneme is a highly specific cylindrical machine whose periodic bending is due to the cumulative shear of its 9 outer doublets of microtubules. Because of the discrete architecture of the tubulin monomers and the active appendices that the outer doublets carry (dynein arms, nexin links and radial spokes), this movement corresponds to the relative shear of these topological verniers, whose characteristics depend on the geometry of the wave train. When an axonemal segment bends, this induces the compressed and dilated conformations of the tubulin monomers and, consequently, the modification of the spatial frequencies of the appendages that the outer doublets carry. From a dynamic point of view, the adjustments of the spatial frequencies of the elements of the two facing verniers that must interact create different longitudinal periodic patterns of distribution of the joint probability of the molecular interaction as a function of the location of the doublet pairs around the axonemal cylinder and their spatial orientation within the axonemal cylinder. During the shear, these patterns move along the outer doublet intervals at a speed that ranges from one to more than a thousand times that of sliding, in two opposite directions along the two opposite halves of the axoneme separated by the bending plane, respecting the polarity of the dynein arms within the axoneme. Consequently, these waves might be involved in the regulation of the alternating activity of the dynein arms along the flagellum, because they induce the necessary intermolecular dialog along the axoneme since they could be an element of the local dynamic stability/instability equilibrium of the axoneme. This complements the geometric clutch model [Lindemann, C., 1994. A "geometric clutch" hypothesis to explain oscillations of the axoneme of cilia and flagella. J. Theor. Biol. 168, 175-189]. PMID:18405921

Cibert, Christian

2008-02-08

37

Effects of imposed bending on microtubule sliding in sperm flagella.  

PubMed

The movement of eukaryotic flagella is characterized by its oscillatory nature. In sea urchin sperm, for example, planar bends are formed in alternating directions at the base of the flagellum and travel toward the tip as continuous waves. The bending is caused by the orchestrated activity of dynein arms to induce patterned sliding between doublet microtubules of the flagellar axoneme. Although the mechanism regulating the dynein activity is unknown, previous studies have suggested that the flagellar bending itself is important in the feedback mechanism responsible for the oscillatory bending. If so, experimentally bending the microtubules would be expected to affect the sliding activity of dynein. Here we report on experiments with bundles of doublets obtained by inducing sliding in elastase-treated axonemes. Our results show that bending not only "switches" the dynein activity on and off but also affects the microtubule sliding velocity, thus supporting the idea that bending is involved in the self-regulatory mechanism underlying flagellar oscillation. PMID:15589153

Morita, Yutaka; Shingyoji, Chikako

2004-12-14

38

Purification of axonemal dyneins and dynein-associated components from Chlamydomonas.  

PubMed

Axonemal dyneins are responsible for generating the force required to power ciliary and flagellar motility. These highly complex enzymes form the inner and outer arms associated with the outer doublet microtubules. They are built around one or more ~520kD heavy chains that exhibit motor activity and also include additional components that are required for assembly within the axonemal superstructure and/or regulation of motor function in response to a broad range of signaling inputs. The dyneins from flagella of Chlamydomonas have been extensively studied as this organism is amenable to genetic, biochemical, and physiological approaches. In this chapter, I describe methods that have been devised by a number of laboratories to extract and purify individual dyneins from Chlamydomonas. When combined with the wide range of available mutants, these methods allow for the analysis of dyneins lacking individual components or motor units. PMID:20409797

King, Stephen M

2009-11-21

39

Microtubule sliding in cilia of the rabbit trachea and oviduct.  

PubMed

Evidence for active sliding of microtubules during ciliary activity has been demonstrated in a number of organisms: sea urchin sperm flagella, protozoan cilia, and mollusc gill cilia. Although there is evidence that active sliding also occurs in mammalian sperm flagella, there is little or no information on whether active sliding of microtubules also occurs in the short (5-micron) cilia of the mammalian trachea or oviduct. Since these cilia are important in tracheobronchial clearance and ovum transport, respectively, it has been important to demonstrate that microtubule sliding is also involved in the activity of somatic cilia. Ciliated apical portions (cortices) and cilia were isolated from rabbit trachea and oviduct, using Triton X-100 to demembranate the cilia. Most of the ciliated cortices reactivated upon addition of ATP, whereas isolated cilia reactivated to a lesser extent. When preparations of cilia were digested with trypsin before or after ATP addition, disintegration of axonemal doublets occurred with about the same frequency as reactivation. These events were recorded using Nomarski optics and dark-field microscopy. When isolated cilia which had been digested by trypsin and exposed to ATP were also prepared for electron microscopy by negative staining, telescoping of doublet microtubules from axonemes could be shown. These results demonstrate that mammalian somatic ciliary doublet microtubules actively slide in a manner similar to that described for invertebrate cilia. PMID:7348603

Dirksen, E R; Zeira, M

1981-01-01

40

Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase  

Microsoft Academic Search

Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axone- mal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First,

David R. Howard; Geoffrey Habermacher; David B. Glass; Elizabeth E Smith; Winfield S. Sale

1994-01-01

41

The Molecular Architecture of Axonemes Revealed by Cryoelectron Tomography  

Microsoft Academic Search

Eukaryotic flagella and cilia are built on a 9 + 2 array of microtubules plus >250 accessory proteins, forming a biological machine called the axoneme. Here we describe the three-dimensional structure of rapidly frozen axonemes from Chlamydomonas and sea urchin sperm, using cryoelectron tomography and image processing to focus on the motor enzyme dynein. Our images suggest a model for

Daniela Nicastro; Cindi Schwartz; Jason Pierson; Richard Gaudette; Mary E. Porter; J. Richard McIntosh

2006-01-01

42

Microtubule sliding in reactivated flagella.  

PubMed

Recent experimental studies of microtubule sliding in demembranated sea urchin sperm flagella are described. A local iontophoretic application of ATP to a Triton-extracted flagellum elicits a local bending response whose form is in exact conformity with the predictions of the sliding microtubule model. Cinematographic analysis of the microtubule sliding initiated by treating fragments of demembranated flagella with trypsin in the presence of ATP reveals that the speed of sliding is almost constant. This implies that the speed does not depend on the number of dynein arms participating in the generation of sliding force. The distribution of apparent sliding velocities indicates that there is no difference in sliding velocity among the doublets. The sliding velocity depends on MgATP concentration in a manner consistent with Michaelis-Menten kinetics. The sliding velocity of doublets in trypsin-treated axonemes is close to the maximum velocity of relative sliding taking place between adjacent doublets in beating flagella reactivated at the same MgATP concentration. PMID:6764040

Takahashi, K; Shingyoji, C; Kamimura, S

1982-01-01

43

cAMP-Stimulated Phosphorylation of an Axonemal Polypeptide That Copurifies with the 22S Dynein Arm Regulates Microtubule Translocation Velocity and Swimming Speed in Paramecium  

Microsoft Academic Search

In Paramecium tetraurelia, cyclic nucleotides are important physiological second messengers that could regulate dynein mechanochemistry by phosphorylation. A 29-kDa polypeptide that is phosphorylated in a cAMP- and Ca2+-sensitive manner in permeabilized cells and isolated axonemes is the only significant phosphorylated moiety that consistently copurifies with 22S dynein from paramecium cilia. It is not a component of 14S dynein. This polypeptide

Toshikazu Hamasaki; Kurt Barkalow; Jeffrey Richmond; Peter Satir

1991-01-01

44

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

PubMed

The axoneme is the skeleton and motor axis of flagella and cilia in eukaryotic organisms. Basically it consists of a series of longitudinal fibers (outer doublets of microtubules) that design a cylinder and whose sliding, due to the coordinated activities of dedicated molecular motors (the dynein arms), is converted into a bending because outer doublets pairs are stabilized by elastic links (the nexine molecules). In spite of these interesting mechanical properties, mechanical and robotics engineers have never considered this amazing molecular machinery as a model. The aim of this paper is to propose the robotic design and the kinematic modeling of the '3 + 0' axoneme that makes motile the flagellum of Diplauxis hatti, the simplest that exists. The model that we propose bends and twists and combines the two movements. It is able to propagate wave trains that could be involved in the development of biomimetic actuators of various mechanisms such as (sub)aquatic robotic propellers as well as robotic trunks. PMID:23579109

Cibert, Christian

2013-04-12

45

Gamma-tubulin functions in the nucleation of a discrete subset of microtubules in the eukaryotic flagellum.  

PubMed

gamma-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of gamma-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of gamma-tubulin in the formation of the 9 + 2 flagellum axoneme. T. brucei possesses a single flagellum and forms a new flagellum during each cell cycle. We have used an inducible RNA interference (RNAi) approach to ablate expression of gamma-tubulin, and, after induction, we observe that the new flagellum is still formed but is paralyzed, while the old flagellum is unaffected. Electron microscopy reveals that the paralyzed flagellum lacks central pair microtubules but that the outer doublet microtubules are formed correctly. These differences in microtubule nucleation mechanisms during flagellum growth provide insights into spatial and temporal regulation of gamma-tubulin-dependent processes within cells and explanations for the organization and evolution of axonemal structures such as the 9 + 0 axonemes of sensory cells and primary cilia. PMID:12676092

McKean, Paul G; Baines, Andrea; Vaughan, Sue; Gull, Keith

2003-04-01

46

Mechanism of flagellar oscillation-bending-induced switching of dynein activity in elastase-treated axonemes of sea urchin sperm.  

PubMed

Oscillatory movement of eukaryotic flagella is caused by dynein-driven microtubule sliding in the axoneme. The mechanical feedback from the bending itself is involved in the regulation of dynein activity, the main mechanism of which is thought to be switching of the activity of dynein between the two sides of the central pair microtubules. To test this, we developed an experimental system using elastase-treated axonemes of sperm flagella, which have a large Ca(2+)-induced principal bend (P-bend) at the base. On photoreleasing ATP from caged ATP, they slid apart into two bundles of doublets. When the distal overlap region of the slid bundles was bent in the direction opposite to the basal P-bend, backward sliding of the thinner bundle was induced along the flagellum including the bent region. The velocity of the backward sliding was significantly lower than that of the forward sliding, supporting the idea that the dynein activity alternated between the two sides of the central pair on bending. Our results show that the combination of the direction of bending and the conformational state of dynein-microtubule interaction induce the switching of the dynein activity in flagella, thus providing the basis for flagellar oscillation. PMID:18682495

Hayashi, Shuichi; Shingyoji, Chikako

2008-08-05

47

Conserved axoneme symmetry altered by a component ?-tubulin  

Microsoft Academic Search

Ninefold microtubule symmetry of the eukaryotic basal body and motile axoneme has been long established [1–3]. In Drosophila, these organelles contain distinct but similar ?-tubulin isoforms [4–10]: basal bodies contain only ?1-tubulin, and only ?2-tubulin is used for assembly of sperm axonemes. A single ?-tubulin functions throughout spermatogenesis [11,12]. Thus, differences in organelle assembly reside in ?-tubulin. We tested the

Elizabeth C. Raff; Jeffrey A. Hutchens; Henry D. Hoyle; Mark G. Nielsen; F. Rudolf Turner

2000-01-01

48

Bending-induced switching of dynein activity in elastase-treated axonemes of sea urchin sperm--roles of Ca2+ and ADP.  

PubMed

Flagellar beating is caused by microtubule sliding, driven by the activity of dynein, between adjacent two of the nine doublet microtubules. An essential process in the regulation of dynein is to alternate its activity (switching) between the two sides of the central pair microtubules. The switching of dynein activity can be detected, in an in vitro system using elastase-treated axonemes of sea urchin sperm flagella, as a reversal of the relative direction of ATP-induced sliding between the two bundles of doublets at high Ca(2+) (10(-4) M) at pH 7.8-8.0. The reversal is triggered by externally applied bending of the doublet bundle. However, the mechanism of this bending-induced reversal (or backward sliding) remains unclear. To understand how the switching of dynein activity in flagella can be induced by bending, we studied the roles of ADP, which is an important factor for the dynein motile activity, and of Ca(2+) in the bending-induced reversal of microtubule sliding between two bundles of doublets at pH 7.5 and 7.2. We found that the reversal of sliding direction was induced regardless of the concentrations of Ca(2+) at low pH, but occurred more frequently at low Ca(2+) (<10(-9) M) than at high Ca(2+). At pH 7.5, an application of ADP increased the frequency of occurrence of backward sliding at high as well as low concentrations of Ca(2+). The results indicate that ADP-dependent activation of dynein, probably resulting from ADP-binding to dynein, is involved in the regulation of the bending-induced switching of dynein activity in flagella. PMID:19343792

Hayashi, Shuichi; Shingyoji, Chikako

2009-05-01

49

Structural Analysis of Mutations in the Drosophila ?2-Tubulin Isoform Reveals Regions in the ?-Tubulin Molecule Required for General and for Tissue-Specific Microtubule Functions  

PubMed Central

We have determined the lesions in a number of mutant alleles of ?Tub85D, the gene that encodes the testis-specific ?2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the ?2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all ?-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t(6) contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate ?-tubulins. Correspondingly, B2t(6) disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that ?3, a developmentally regulated Drosophila ?-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type ?2-tubulin. We show here by complementation analysis that ?3 and the B2t(6) product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the ?2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the ?2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the ?2 variant lacking the carboxy terminus and the B2t(6) variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate ?-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type ?-tubulins. We propose that the integrity of this structure in the Drosophila testis ?2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.

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

1995-01-01

50

Direction of Active Sliding of Microtubules in Tetrahymena Cilia  

Microsoft Academic Search

Axonemes of protozoan (Tetrahymena thermophila BIII) cilia, isolated by the dibucaine method, were treated briefly with trypsin after removal of the ciliary membranes by treatment with Triton X-100. After attachment to polylysine-coated surfaces, the partially digested axonemes remained mainly intact cylinders. Such attached axonemes can be treated with ATP, which induces microtubule sliding. ATP-treated preparations showed disrupted axonemes in which

Winfield S. Sale; Peter Satir

1977-01-01

51

Microtubule dynamics in a cytosolic extract of fetal rat brain  

Microsoft Academic Search

Brain microtubule dynamics were studied by video-enhanced differential interference contrast microscopy in a cytosolic extract from fetal rat brain, prepared under conditions designed to produce minimal alterations in microtubule stability. With urchin sperm axoneme fragments as nucleation seeds, the extract was shown to support cellular-like microtubule dynamics. Microtubules elongated from one end of the axonemes, and did not spontaneously self-assemble

J. R Simon; R. D Graff; P. F Maness

1998-01-01

52

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

2012-06-25

53

Sensing the mechanical state of the axoneme and integration of Ca2+ signaling by outer arm dynein.  

PubMed

Axonemal dyneins have been demonstrated to monitor the mechanical state of the axoneme and must also alter activity in response to various signaling pathways. The central pair/radial spoke systems are clearly involved in controlling inner dynein arm function; however, the mechanisms by which the outer dynein arm transduces regulatory signals appear quite distinct at the molecular level. In Chlamydomonas, these regulatory components include thioredoxins involved in response to redox changes, molecules that tether the gamma heavy-chain motor unit to the A-tubule of the outer doublet and a Ca(2+)-binding protein that controls the structure of the gamma heavy-chain N-terminal domain. Together, these studies now suggest that the gamma heavy chain acts as a key regulatory node for controlling outer arm function in response to alterations in curvature and ligand binding. Furthermore, they allow us to propose a testable molecular mechanism by which altered Ca(2+) levels might lead to a change in ciliary waveform by controlling whether one heavy chain of outer arm dynein acts as a microtubule translocase or as an ATP-dependent brake that limits the amount of interdoublet sliding. PMID:20186692

King, Stephen M

2010-04-01

54

The velocity of microtubule sliding: its stability and load dependency.  

PubMed

It is now well understood that ATP-driven active sliding between the doublet microtubules in the sperm axoneme generates flagellar movement. However, much remains to be learned about how this movement is controlled. Detailed analyses of the flagellar beating of the mammalian spermatozoa revealed that there were two beating modes at a constant rate of microtubule sliding: that is, a nearly constant-curvature beating in nonhyperactivated spermatozoa and a nearly constant-frequency beating in hyperactivated spermatozoa. The constant rate of microtubule sliding suggests that the beat frequency and waveform of the flagellar beating are dependently regulated. Comparison of the sliding velocity of several mammalian and sea urchin sperm flagella with their mechanical property clarified that the sliding velocity of the microtubule was determined by the stiffness of the flagellum at its base, and that its relationship was expressed by a logarithmic equation that is similar to the classical force-velocity equation of the muscle contraction. Data from sea urchin spermatozoa also satisfied the equation, suggesting that the same microtubule sliding system functions in both the mammalian and echinoderm spermatozoa. PMID:17685439

Ishijima, Sumio

2007-11-01

55

Second harmonic microscopy of axonemes.  

PubMed

We performed Second Harmonic Microscopy of axonemes obtained from sea urchin sperm. Using polarization analysis and a trade-off between signal and photodamage, we were able to determine, for the first time to our knowledge, the nonlinear susceptibility chizxx/chixzx = 1.1+/-0.2 and chizzz/chixzx = 4+/-0.5 of axonemes. PMID:19466174

Odin, Christophe; Heichette, Claire; Chretien, Denis; Le Grand, Yann

2009-05-25

56

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

PubMed

We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 ?m, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-?m segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, ~2 ?m) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20°, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general. PMID:22503702

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

2012-04-05

57

Diameter oscillation of axonemes in sea-urchin sperm flagella.  

PubMed

The 9 + 2 configuration of axonemes is one of the most conserved structures of eukaryotic organelles. Evidence so far has confirmed that bending of cilia and flagella is the result of active sliding of microtubules induced by dynein arms. If the conformational change of dynein motors, which would be a key step of force generation, is occurring in a three-dimensional manner, we can easily expect that the microtubule sliding should contain some transverse component, i.e., a motion in a direction at a right angle to the longitudinal axis of axonemes. Using a modified technique of atomic force microscopy, we found such transverse motion is actually occurring in an oscillatory manner when the axonemes of sea-urchin sperm flagella were adhered onto glass substrates. The motion was adenosine triphosphate-dependent and the observed frequency of oscillation was similar to that of oscillatory sliding of microtubules that had been shown to reflect the physiological activity of dynein arms (S. Kamimura and R. Kamiya. 1989. Nature: 340:476-478; 1992. J. Cell Biol. 116:1443-1454). Maximal amplitude of the diameter oscillation was around 10 nm, which was within a range of morphological change observed with electron microscopy (F. D. Warner. 1978. J. Cell Biol. 77:R19-R26; N. C. Zanetti, D. R. Mitchell, and F. D. Warner. 1979. J. Cell Biol. 80:573-588). PMID:14695276

Sakakibara, Hajime M; Kunioka, Yuki; Yamada, Takenori; Kamimura, Shinji

2004-01-01

58

Diameter Oscillation of Axonemes in Sea-Urchin Sperm Flagella  

PubMed Central

The 9 + 2 configuration of axonemes is one of the most conserved structures of eukaryotic organelles. Evidence so far has confirmed that bending of cilia and flagella is the result of active sliding of microtubules induced by dynein arms. If the conformational change of dynein motors, which would be a key step of force generation, is occurring in a three-dimensional manner, we can easily expect that the microtubule sliding should contain some transverse component, i.e., a motion in a direction at a right angle to the longitudinal axis of axonemes. Using a modified technique of atomic force microscopy, we found such transverse motion is actually occurring in an oscillatory manner when the axonemes of sea-urchin sperm flagella were adhered onto glass substrates. The motion was adenosine triphosphate-dependent and the observed frequency of oscillation was similar to that of oscillatory sliding of microtubules that had been shown to reflect the physiological activity of dynein arms (S. Kamimura and R. Kamiya. 1989. Nature. 340:476–478; 1992. J. Cell Biol. 116:1443–1454). Maximal amplitude of the diameter oscillation was around 10 nm, which was within a range of morphological change observed with electron microscopy (F. D. Warner. 1978. J. Cell Biol. 77:R19–R26; N. C. Zanetti, D. R. Mitchell, and F. D. Warner. 1979. J. Cell Biol. 80:573–588).

Sakakibara, Hajime M.; Kunioka, Yuki; Yamada, Takenori; Kamimura, Shinji

2004-01-01

59

Effects of the central pair apparatus on microtubule sliding velocity in sea urchin sperm flagella.  

PubMed

To produce oscillatory bending movement in cilia and flagella, the activity of dynein arms must be regulated. The central-pair microtubules, located at the centre of the axoneme, are often thought to be involved in the regulation, but this has not been demonstrated definitively. In order to determine whether the central-pair apparatus are directly involved in the regulation of the dynein arm activity, we analyzed the movement of singlet microtubules that were brought into contact with dynein arms on bundles of doublets obtained by sliding disintegration of elastase-treated flagellar axonemes. An advantage of this new assay system was that we could distinguish the bundles that contained the central pair apparatus from those that did not, the former being clearly thicker than the latter. We found that microtubule sliding occurred along both the thinner and the thicker bundles, but its velocity differed between the two kinds of bundles in an ATP concentration dependent manner. At high ATP concentrations, such as 0.1 and 1 mM, the sliding velocity on the thinner bundles was significantly higher than that on the thicker bundles, while at lower ATP concentrations the sliding velocity did not change between the thinner and the thicker bundles. We observed similar bundle width-related differences in sliding velocity after removal of the outer arms. These results provide first evidence suggesting that the central pair and its associated structures may directly regulate the activity of the inner (and probably also the outer) arm dynein. PMID:10355878

Yoshimura, M; Shingyoji, C

1999-02-01

60

Birefringence of single and bundled microtubules.  

PubMed Central

We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07 nm. The peak retardance of a small bundle increased linearly with the number of MTs in the bundle. Axonemes (prepared from sea urchin sperm) had a peak retardance 20 times higher than that of single MTs, in accordance with the nine doublets and two singlets arrangement of parallel MTs in the axoneme. Measured filament retardance decreased when the filament was defocused or the numerical aperture of the imaging system was decreased. However, the retardance "area," which we defined as the image retardance integrated along a line perpendicular to the filament axis, proved to be independent of focus and of numerical aperture. These results are in good agreement with a theory that we developed for measuring retardances with imaging optics. Our theoretical concept is based on Wiener's theory of mixed dielectrics, which is well established for nonimaging applications. We extend its use to imaging systems by considering the coherence region defined by the optical set-up. Light scattered from within that region interferes coherently in the image point. The presence of a filament in the coherence region leads to a polarization dependent scattering cross section and to a finite retardance measured in the image point. Similar to resolution measurements, the linear dimension of the coherence region for retardance measurements is on the order lambda/(2 NA), where lambda is the wavelength of light and NA is the numerical aperture of the illumination and imaging lenses.

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

1998-01-01

61

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

PubMed

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

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

2000-04-01

62

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

PubMed

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

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

2004-07-01

63

Regulation of Flagellar Dynein by Calcium and a Role for an Axonemal Calmodulin and Calmodulin-dependent Kinase  

PubMed Central

Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonas mutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at pCa4, dynein activity in pf18 and pf15 axonemes was restored to wild-type level. The calcium-induced increase in dynein activity in pf18 axonemes was inhibited by antagonists of calmodulin and calmodulin-dependent kinase II. Axonemes lacking the C1 central tubule (pf16) or lacking radial spoke components (pf14 and pf17) do not exhibit calcium-induced increase in dynein activity in pCa4 buffer. We conclude that calcium regulation of flagellar motility involves regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase II may mediate the calcium signal, and that the central apparatus and radial spokes are key components of the calcium signaling pathway.

Smith, Elizabeth F.

2002-01-01

64

Assembly of Chick Brain Tubulin onto Flagellar Microtubules from Chlamydomonas and Sea Urchin Sperm  

Microsoft Academic Search

Flagellar microtubules from Chlamydomonas and sea urchin sperm were used as in vitro assembly sites for chick brain tubulin. Brain microtubules assembled onto the A-tubules, central tubules, and, to a limited extent, onto the B-tubules of flagellar axonemes. Assembly occurred onto the distal ends of the axonemes at low tubulin concentrations and onto distal and proximal ends at high tubulin

Lester I. Binder; William L. Dentler; Joel L. Rosenbaum

1975-01-01

65

Calcium regulation of ciliary motility analysis of axonemal calcium-binding proteins.  

PubMed

Substantial data have contributed to a model in which the axonemal microtubules act as a scaffold for the assembly of molecules that form a signal transduction pathway that ultimately regulates dynein. We have also known for some time that for virtually all motile cilia and flagella, the second messenger, calcium, impacts upon these signaling pathways to modulate beating in response to extracellular cues. Yet we are only beginning to identify the axonemal proteins that bind this second messenger and determine their role in regulating dynein-driven microtubule sliding to alter the size and shape of ciliary bends. Here, we review our current understanding of calcium regulation of motility, emphasizing recent advances in the detection and characterization of calcium-binding proteins anchored to the axoneme. PMID:20409805

DiPetrillo, Christen; Smith, Elizabeth

2009-11-21

66

Regulations of microtubule sliding by Ca2+ and cAMP and their roles in forming flagellar waveforms.  

PubMed

The function of Ca(2+) and cAMP in extruding doublet microtubules from sea urchin sperm axoneme and generating flagellar waves was investigated in order to clarify the regulatory mechanism of microtubule sliding and the formation mechanism of beating patterns of cilia and flagella. Almost all potentially asymmetric spermatozoa that were demembranated with Triton in the absence of Ca(2+) and reactivated with MgATP(2-) (Gibbons, B.H. and Gibbons, I.R. (1980). J. Cell Biol., 84: 13-27), beat with planar waves closely resembling those of the intact spermatozoa, whereas potentially symmetric spermatozoa, in which axonemal calmodulin was removed by detergent extraction in the presence of millimolar Ca(2+) (Brokaw, C.J. and Nagayama, S.M. (1985). J. Cell Biol., 100: 1875-1883), beat with three-dimensional waves if they were reactivated with low MgATP(2-). At a high MgATP(2-), almost all demembranated spermatozoa beat with planar waves. cAMP enhanced the three-dimensionality of the flagellar waves at a low Ca(2+). These changes in the flagellar waves were caused by different regulations of the microtubule sliding by calcium, cAMP, and MgATP(2-). PMID:23546177

Ishijima, Sumio

2013-04-02

67

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

PubMed

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

Linck, R W; Amos, L A; Amos, W B

1985-01-01

68

CEP162 is an axoneme-recognition protein promoting ciliary transition zone assembly at the cilia base.  

PubMed

The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly. CEP162 interacts with core transition zone components, and mediates their association with microtubules. Loss of CEP162 arrests ciliogenesis at the stage of transition zone assembly. Abolishing its centriolar tethering, however, allows CEP162 to stay on the growing end of the axoneme and ectopically assemble transition zone components at cilia tips. This generates extra-long cilia with strikingly swollen tips that actively release ciliary contents into the extracellular environment. CEP162 is thus an axoneme-recognition protein pre-tethered at centriole distal ends before ciliogenesis to promote and restrict transition zone formation specifically at the cilia base. PMID:23644468

Wang, Won-Jing; Tay, Hwee Goon; Soni, Rajesh; Perumal, Geoffrey S; Goll, Mary G; Macaluso, Frank P; Asara, John M; Amack, Jeffrey D; Tsou, Meng-Fu Bryan

2013-05-05

69

Visualization of the GDP-dependent switching in the growth polarity of microtubules.  

PubMed

Microtubules are filamentous polar polymers with plus and minus ends. This polarity plays a crucial role in a variety of cellular functions such as chromosome movement and organelle transport. To examine the relationship between the growth polarity of microtubules and guanine nucleotide dependence, we polymerized microtubules from axonemes of sea urchin sperm flagella either with GTP or with GTP and GDP, and observed individual microtubules by dark-field microscopy. Tubulin concentrations were adjusted in each case to grow microtubules from only one end of each axoneme. The growth polarity of microtubules was determined using N-ethylmaleimide-modified tubulin (NEM-tubulin). In the presence of GTP only and at low tubulin concentrations, microtubules grew from the plus ends of axonemes. Surprisingly, in the presence of GTP and GDP, microtubules grew from the minus ends, even at high tubulin concentrations. To confirm these results, we used a perfusion chamber to monitor the growth polarity of microtubules from the same axoneme under different conditions. Exchanging a solution containing only GTP for one containing GTP and GDP elicited a switch in the growth polarity of microtubules from the plus ends to the minus ends. These results suggest that GDP directly affects microtubule polymerization and inverts microtubule growth polarity, probably by inhibiting microtubule growth at the plus ends. PMID:9665843

Tanaka-Takiguchi, Y; Itoh, T J; Hotani, H

1998-07-17

70

Axonemal activity relative to the 2D/3D-waveform conversion of the flagellum.  

PubMed

The waveform of the flagellum of the sea urchin spermatozoon is mainly planar, but its 3D-properties were evoked for dynamic reasons and described as helical. In 1975, the apparent twisting pattern of the sea urchin axoneme was described [Gibbons I. 1975. The molecular basis of flagellar motility in sea urchin spermatozoa. In: Inoué S, Stephens R, editors. Molecular and cellular movement. New York: Raven Press, p. 207-232.] and was considered to be one of the main elements involved in axonemal behaviour. Recently, planar, quasi-planar, and helical waveforms were observed when the flagellum of sea urchin sperm cells was submitted to an increase in viscosity. The quasi-planar conformation seemed to be due to the alternating torsion of the inter-bend segments [Woolley D, Vernon G. 2001. A study of helical and planar waves on sea urchin sperm flagella, with a theory of how they are generated. J. Exp. Biol. 204:1333-1345]. These three waveforms, which are due to a change in axonemal activity, are possibly used by the sperm cells to adapt their movement to variations in the physico-chemical characteristics of the medium (seawater) in which the cells normally swim. We constructed a simple model to describe qualitatively the central shear (between the axonemal doublets and the central pair) and the tangential shear (between the doublets themselves). In this model, the 3D-bending is resolved into components in two perpendicular planes and each of the nine planes of inter-doublet interaction defines a potential bending plane that is independently regulated. These shears were calculated for the three waveforms and their inter-conversion. This allowed us to propose that axoneme is resolved in successive modules delineated by abscissas where the sliding is always nil. We discuss these data concerning the axonemal machinery, and especially the alternating activity of opposite sides of (two) neutral surface(s) that seem(s) to be responsible for this inter-conversion, and for the possible twist of the axoneme during the beating. PMID:11921166

Cibert, C

2002-02-01

71

Target molecules of calmodulin on microtubules of Tetrahymena cilia  

SciTech Connect

In the course of an attempt to isolate the calmodulin-binding proteins (CaMBPs) from cilia of Tetrahymena, it was found that some CaMBPs tend to interact with axonemal microtubules. The present study demonstrates this interaction by cosedimentation experiments using in vitro polymerized Tetrahymena axonemal microtubules and Tetrahymena CaMBPs purified from axonemes by calmodulin affinity column chromatography. Analysis by the ({sup 125}I)calmodulin overlay method showed that at least three CaMBPs (M{sub r} 69, 45, and 37 kDa) cosediment with microtubules. Furthermore, without any addition of exogenous CaMBPs, microtubules purified after three cycles of temperature-dependent polymerization and depolymerization included the above CaMBPs and additional CaMBPs which could not cosediment with microtubules. From the results, the authors have classified these microtubule-associated CaMBPs into two groups: (i) CaMBPs which interact with microtubules only during polymerization, and (ii) CaMBPs which interact not only with microtubules during polymerization, but also with polymerized microtubules. These results suggest that the microtubule-associated CaMBPs, especially those of the latter group, are located on the surface of ciliary microtubules, and may become the target molecules of calmodulin at Ca{sup 2+}-triggered ciliary reversal.

Hirano-Ohnishi, Junko; Watanabe, Yoshio (Univ. of Tsukuba, Ibaraki (Japan))

1988-09-01

72

The 78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein  

PubMed Central

A previous study (King et al., 1991. J. Biol. Chem. 266:8401-8407) showed that the 78,000-M(r) intermediate chain (IC78) from the Chlamydomonas outer arm dynein is in direct contact with alpha-tubulin in situ, suggesting that this protein may be involved in binding the dynein to the doublet microtubules. Molecular genetic analysis of this chain recently demonstrated that it is a WD repeat protein essential for outer arm assembly (Wilkerson et al., 1995.J. Cell Biol. 129:169- 178). We have now transcribed and translated IC78 in vitro, and demonstrate that this molecule binds axonemes and microtubules, whereas a homologous protein (the 69,000-M(r) intermediate chain [IC69] of Chlamydomonas outer arm dynein) does not. Thus, IC78 is a bona fide microtubule-binding protein. Taken together with the previous results, these findings indicate that IC78 is likely to provide at least some of the adhesive force that holds the dynein to the doublet microtubule, and support the general hypothesis that the dynein intermediate chains are involved in targeting different dyneins to the specific cell organelles with which they associate. Analysis of the binding activities of various IC78 deletion constructs translated in vitro identified discrete regions of IC78 that affected the binding to microtubules; two of these regions are specifically missing in IC69. Previous studies also showed that IC78 is in direct contact with IC69; the current work indicates that the region of IC78 that mediates this interaction is coincident with two of IC78's WD repeats. This supports the hypothesis that these repeats are involved in protein-protein interactions within the dynein complex.

1995-01-01

73

Stabilization of sea urchin flagellar microtubules by histone H1  

Microsoft Academic Search

Complex microtubule assemblies are essential components of eukaryotic cilia and flagella. They are extremely stable and are not affected by agents that normally induce polymer disassembly. The molecular basis of this microtubular stability is unknown, and it is not related to any feature of the constitutive tubulin. In sea urchin sperm flagella, axonemal microtubules are found to be stabilized by

Luc Multigner; Jean Gagnon; Alain van Dorsselaer; Didier Job

1992-01-01

74

Polarity of dynein-microtubule interactions in vitro: cross-bridging between parallel and antiparallel microtubules  

PubMed Central

Ciliary doublet microtubules produced by sliding disintegration in 20 muM MgATP2-reassociate in the presence of exogenous 30S dynein and 6 mM MgSO4. The doublets form overlapping arrays, held together by dynein cross-bridges. Dynein arms on both A and B subfibers serve as unambiguous markers of microtubule polarity within the arrays. Doublets reassociate via dynein cross-bridges in both parallel and antiparallel modes, although parallel interactions are favored 2:1. When 20 muM ATP is added to the arrays, the doublets undergo both vanadate-sensitive and insensitive forms of secondary disintegration to reproduce the original population of doublets. The results demonstrate that both parallel and antiparallel doublet cross-bridging is sensitive to dissociation by ATP even though normal ciliary motion depends strictly on dynein interactions between parallel microtubules.

1981-01-01

75

Time-dependent measure of a nanoscale force-pulse driven by the axonemal dynein motors in individual live sperm cells  

Microsoft Academic Search

Nanoscale mechanical forces generated by motor proteins are crucial to normal cellular and organismal functioning. The ability to measure and exploit such forces is important to developing motile biomimetic nanodevices powered by biological motors for nanomedicine. Axonemal dynein motors positioned inside the sperm flagellum drive microtubule sliding and give rise to rhythmic beating. This force-generating action pushes the sperm cell

Michael J. Allen; Robert E. Rudd; Mike W. McElfresh; Rod Balhorn

2010-01-01

76

Analysis of the role of nucleotides in axonemal dynein function.  

PubMed

Axonemal dynein in flagella and cilia is a motor molecule that produces microtubule sliding, powered by the energy of ATP hydrolysis. Our goal is to understand how dynein motile activity is controlled to produce the characteristic oscillatory movement of flagella. ATP, the energy source for dynein, is also important as a regulator of dynein activity. Among the four nucleotide-binding sites of a dynein heavy chain, one is the primary ATP hydrolyzing site while the others are noncatalytic sites and thought to perform regulatory functions. Stable binding of both ATP and ADP to these regulatory sites is probably essential for the chemomechanical energy transduction in dynein. Although the ATP concentration in beating flagella is physiologically high and constant, at any moment in the oscillatory cycle some dynein molecules are active while others are not, and the motile activity of dynein oscillates temporally and spatially in the axoneme. It is likely that the basic mechanism underlying the highly dynamic control of dynein activity involves the ATP-dependent inhibition and ADP-dependent activation (or release of inhibition) of dynein. How the inhibition and activation can be induced in beating flagella is still unknown. It seems, however, that the mechanical force of bending is involved in the activation of dynein, probably through the control of noncatalytic nucleotide binding to dynein. This chapter provides an overview of several approaches, using sea urchin sperm flagella, to studying the roles of ATP and ADP in the regulation of dynein activity with or without the mechanical signal of bending. PMID:20409802

Shingyoji, Chikako

2009-11-21

77

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

PubMed Central

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

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

2012-01-01

78

Biochemical and physiological analysis of axonemal dyneins.  

PubMed

Axonemal dyneins are highly complex molecular motors that power the beating of cilia/flagella. In addition to the motor subunits, these enzymes contain components that allow for assembly at the correct axonemal location and also enable the motor to respond to a broad array of signals including phosphorylation, Ca(2+), redox changes, and mechanical activation. The green alga Chlamydomonas reinhardtii has become the premier system in which to analyze these motors, as it allows for classical/molecular genetic approaches to be combined with biochemical fractionation, and physiological measurements to gain an integrated view of dynein function. Furthermore, Chlamydomonas provides the opportunity to study axonemal dyneins in the cytoplasm prior to their transport into the cilium/flagellum, thus allowing the nature of the assembly process to be defined. In this chapter, I describe methods used in my laboratory to prepare and fractionate cytoplasmic extracts and to localize axonemal dynein components within the flagellum at both the light microscope level and by biochemical and genetic approaches. Finally, I also detail how to assess dynein-driven flagella motility by measuring beat frequency and propulsive force of both intact cells and reactivated cell models. PMID:23498738

King, Stephen M

2013-01-01

79

ATP hydrolysis coupled to microtubule sliding in sea-urchin sperm flagella.  

PubMed

Using sea urchin (Hemicentrotus pulcherimus) sperm flagella, ATP hydrolysis coupled to sliding movement of microtubules was investigated. Flagellar axonemes were pretreated with trypsin and the microtubules induced to slide by addition of ATP (50-1,000 microM) at 0-20 degrees C. Motion-dependent hydrolysis of ATP was observed immediately after the addition of ATP, the rate of which was higher than that of steady state hydrolysis in axonemes without trypsin-treatment, or after complete disintegration. The rate of hydrolysis of ATP divided by the sliding velocity of microtubules reflects the ATP consumption necessary per unit distance of microtubule sliding. This parameter varied according to the experimental conditions in that it increased when the ATP concentration or temperature was decreased. Our results suggest that there is not a strict stoichiometric relationship between ATP hydrolysis and sliding distance in the dynein-tubulin system, indicating that the mechanochemical coupling is different from that in beating axonemes. PMID:4030735

Kamimura, S; Yano, M; Shimizu, H

1985-05-01

80

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

PubMed

Dynein transduces the chemical energy of ATP hydrolysis into mechanical work through conformational changes. To identify the factors governing the coupling between the ATPase activity and the motile activity of the dynein molecule, we examined the effects of potassium iodide, which can unfold protein tertiary structures, on dynein activity in reactivated sea urchin sperm flagella. The presence of low concentrations of KI (0.05-0.1 M) in the reactivating solution did not influence the stable beating of demembranated flagella at 0.02-1 mM ATP, when the total concentration of potassium was kept at 0.15 M by adding K-acetate. However, double-reciprocal plots of ATP concentration and beat frequency showed a mixed type of inhibition by KI, indicating the possibility that KI inhibits the ATP hydrolysis and decreases the maximum sliding velocity. The ATPase activity of 21S dynein with or without microtubules did not decrease with the KI concentration. In the elastase-treated axonemes, KI decreased the velocity of sliding disintegration, while it increased the frequency of occurrence of axonemes showing no sliding. This may be related to some defect in the coordination of dynein activities. On 21S dynein adsorbed on a glass surface, however, the velocity of microtubule sliding was increased by KI, while KI lowered the dynein-microtubule affinity. The velocity further increased under lower salt conditions enhancing the dynein-microtubule interactions. The results suggest the importance of organized regulation of the dynamic states of dynein-microtubule interactions through the stalk for the coupling between the ATPase activity and the motile activity of dynein in beating flagella. PMID:21520430

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

2011-05-03

81

Isolated flagellar outer arm dynein translocates brain microtubules in vitro  

Microsoft Academic Search

The inner and outer arms of the flagellar axoneme generate the forces needed for flagellar movement; these arms contain ATPases called dyneins. To date, there has been no method for studying the mechanochemical transducing activity of isolated dyneins. Recently, it was found that the brain microtubule-associated protein (MAP) 1C is a microtubule-activated ATPase with the structural and force-producing properties of

Bryce Mark Paschal; Stephen M. King; Anthony G. Moss; Christine A. Collins; Richard B. Vallee; George B. Witman

1987-01-01

82

Analysis of cytoplasmic microtubules and flagellar roots in the zoospores of Allomyces macrogynus  

Microsoft Academic Search

Summary Cytoskeletal and flagellar microtubules in the zoospores of the aquatic fungusAllomyces macrogynus are resistant to microtubule depolymerizing drugs. Consequently, we have analyzed the partial composition and organization of microtubules (Mts) in the cytoplasm and flagellar apparatus in the zoospores ofA. macrogynus. Evidence from two-dimensional gel electrophoresis demonstrated the presence of two a-tubulin isoforms in axonemal and cytoplasmic Mts. In

G. R. Aliaga; J. C. Pommerville

1990-01-01

83

Axonemal radial spokes: 3D structure, function and assembly.  

PubMed

The radial spoke (RS) is a complex of at least 23 proteins that works as a mechanochemical transducer between the central-pair apparatus and the peripheral microtubule doublets in eukaryotic flagella and motile cilia. The RS contributes to the regulation of the activity of dynein motors, and thus to flagellar motility. Despite numerous biochemical, physiological and structural studies, the mechanism of the function of the radial spoke remains unclear. Detailed knowledge of the 3D structure of the RS protein complex is needed in order to understand how RS regulates dynein activity. Here we review the most important findings on the structure of the RS, including results of our recent cryo-electron tomographic analysis of the RS protein complex. PMID:22754630

Pigino, Gaia; Ishikawa, Takashi

2012-02-01

84

Calcium control of waveform in isolated flagellar axonemes of chlamydomonas  

PubMed Central

The effect of Ca(++) on the waveform of reactivated, isolated axonemes of chlamydomonas flagella was investigated. Flagella were detached and isolated by the dibucaine procedure and demembranated by treatment with the detergent Nonidet; the resulting axomenes lack the flagellar membrane and basal bodies. In Ca(++)-buffered reactivation solutions containing 10(-6) M or less free Ca(++), the axonemes beat with a highly asymmetrical, predominantly planar waveform that closely resembled that of in situ flagella of forward swimming cells. In solutions containing 10(-4) M Ca(++), the axonemes beat with a symmetrical waveform that was very similar to that of in situ flagella during backward swimming. In 10(-5) M Ca(++), the axonemes were predominantly quiescent, a state that appears to be closely associated with changes in axomenal waveform or direction of beat in many organisms. Experiments in which the concentrations of free Ca(++), not CaATP(--) complex were independently varied suggested that free Ca(++), not CaATP(--), was responsible for the observed changes. Analysis of the flagellar ATPases associated with the isolated axonemes and the nonidet- soluble membrane-matrix fraction obtained during preparation of the axonemes showed that the axonemes lacked the 3.0S Ca(++)-activated ATPase, almost all of which was recovered in the membrane-matrix fraction. These results indicate that free Ca(++) binds directly to an axonemal component to alter flagellar waveform, and that neither the 3.0S CaATPase nor the basal bodies are directly involved in this change.

Bessen, M; Fay, RB; Witman, GB

1980-01-01

85

Septins 2, 7 and 9 and MAP4 colocalize along the axoneme in the primary cilium and control ciliary length.  

PubMed

Septins are a large, evolutionarily conserved family of GTPases that form hetero-oligomers and interact with the actin-based cytoskeleton and microtubules. They are involved in scaffolding functions, and form diffusion barriers in budding yeast, the sperm flagellum and the base of primary cilia of kidney epithelial cells. We investigated the role of septins in the primary cilium of retinal pigmented epithelial (RPE) cells, and found that SEPT2 forms a 1:1:1 complex with SEPT7 and SEPT9 and that the three members of this complex colocalize along the length of the axoneme. Similar to observations in kidney epithelial cells, depletion of cilium-localized septins by siRNA-based approaches inhibited ciliogenesis. MAP4, which is a binding partner of SEPT2 and controls the accessibility of septins to microtubules, was also localized to the axoneme where it appeared to negatively regulate ciliary length. Taken together, our data provide new insights into the functions and regulation of septins and MAP4 in the organization of the primary cilium and microtubule-based activities in cells. PMID:23572511

Ghossoub, Rania; Hu, Qicong; Failler, Marion; Rouyez, Marie-Christine; Spitzbarth, Benjamin; Mostowy, Serge; Wolfrum, Uwe; Saunier, Sophie; Cossart, Pascale; Jamesnelson, W; Benmerah, Alexandre

2013-04-09

86

Direct interaction of Gas11 with microtubules: implications for the dynein regulatory complex.  

PubMed

We previously described the Trypanin family of cytoskeleton-associated proteins that have been implicated in dynein regulation [Hill et al., J Biol Chem2000; 275(50):39369-39378; Hutchings et al., J Cell Biol2002;156(5):867-877; Rupp and Porter, J Cell Biol2003;162(1):47-57]. Trypanin from T. brucei is part of an evolutionarily conserved dynein regulatory system that is required for regulation of flagellar beat. In C. reinhardtii, the trypanin homologue (PF2) is part of an axonemal 'dynein regulatory complex' (DRC) that functions as a reversible inhibitor of axonemal dynein [Piperno et al., J Cell Biol1992;118(6):1455-1463; Gardner et al., J Cell Biol1994;127(5):1311-1325]. The DRC consists of an estimated seven polypeptides that are tightly associated with axonemal microtubules. Association with the axoneme is critical for DRC function, but the mechanism by which it attaches to the microtubule lattice is completely unknown. We demonstrate that Gas11, the mammalian trypanin/PF2 homologue, associates with microtubules in vitro and in vivo. Deletion analyses identified a novel microtubule-binding domain (GMAD) and a distinct region (IMAD) that attenuates Gas11-microtubule interactions. Using single-particle binding assays, we demonstrate that Gas11 directly binds microtubules and that the IMAD attenuates the interaction between GMAD and the microtubule. IMAD is able to function in either a cis- or trans-orientation with GMAD. The discovery that Gas11 provides a direct linkage to microtubules provides new mechanistic insight into the structural features of the dynein-regulatory complex. PMID:17366626

Bekker, Janine M; Colantonio, Jessica R; Stephens, Andrew D; Clarke, W Thomas; King, Stephen J; Hill, Kent L; Crosbie, Rachelle H

2007-06-01

87

Stabilization of sea urchin flagellar microtubules by histone H1.  

PubMed

Complex microtubule assemblies are essential components of eukaryotic cilia and flagella. They are extremely stable and are not affected by agents that normally induce polymer disassembly. The molecular basis of this microtubular stability is unknown, and it is not related to any feature of the constitutive tubulin. In sea urchin sperm flagella, axonemal microtubules are found to be stabilized by a protein identical to histone H1, a result that defines a new role for this histone and provides evidence for a concerted evolution of chromatin and microtubular structures. PMID:1436071

Multigner, L; Gagnon, J; Van Dorsselaer, A; Job, D

1992-11-01

88

Direct measurement of the force of microtubule sliding in flagella  

Microsoft Academic Search

The movement of eukaryotic cilia and flagella is caused by ATP-driven active sliding between the doublet microtubules1-3, and the force for the sliding is believed to be generated by the dynein arms of the A-tubule interacting with the B-tubule of the adjoining doublet. To understand the mechanochemical basis of this force-generating reaction and to correlate it with the overt motile

Shinji Kamimura; Keiichi Takahashi

1981-01-01

89

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

PubMed

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

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

2005-12-01

90

Functionally significant central-pair rotation in a primitive eukaryotic flagellum  

Microsoft Academic Search

There is now considerable evidence that the basis for ciliary and flagellar movement is an active sliding between peripheral doublet microtubules which, when resisted by structures within the axoneme, leads to axonemal bend formation. In contrast, relatively little is known about the control mechanisms which coordinate the interdoublet sliding and axonemal binding to produce the effective motion observed in various

Charlotte K. Omoto; George B. Witman

1981-01-01

91

Calcium regulation of microtubule sliding in reactivated sea urchin sperm flagella.  

PubMed

The changes in the bending pattern of flagella induced by an increased intracellular Ca(2+) concentration are caused by changes in the pattern and velocity of microtubule sliding. However, the mechanism by which Ca(2+) regulates microtubule sliding in flagella has been unclear. To elucidate it, we studied the effects of Ca(2+) on microtubule sliding in reactivated sea urchin sperm flagella that were beating under imposed head vibration. We found that the maximum microtubule sliding velocity obtainable by imposed vibration, which was about 170-180 rad/second in the presence of 250 microM MgATP and <10(-9) M Ca(2+), was decreased by 10(-6)-10(-5) M Ca(2+) by about 15-20%. Similar decrease of the sliding velocity was observed at 54 and 27 microM MgATP. The Ca(2+)-induced decrease of the sliding velocity was due mainly to a decrease in the reverse bend angle. When the plane of beat was artificially rotated by rotating the plane of vibration of the pipette that held the sperm head, the asymmetric bending pattern also rotated at 10(-5) M Ca(2+) as well as at <10(-9) M Ca(2+). The rotation of the bending pattern was observed at MgATP higher than 54 microM ( approximately 100 microM ATP). These results indicate that the Ca(2+)-induced decrease of the sliding velocity is mediated by a rotatable component or components (probably the central pair) at high MgATP, but is not due to specific dynein arms on particular doublets. We further investigated the effects of a mild trypsin treatment and of trifluoperazine on the Ca(2+)-induced decrease in sliding velocity. Axonemes treated for 3 minutes with a low concentration (0.1 microgram/ml) of trypsin beat with a more symmetrical waveform than before the treatment. Also, their microtubule sliding velocity and reverse bend angle were not affected by high Ca(2+) concentrations. Trifluoperazine (25-50 microM) had no effect on the decrease of the sliding velocity in beating flagella at 10(-5) M Ca(2+). However, the flagella that had been 'quiescent' at 10(-4) M Ca(2+) resumed asymmetrical beating following an application of 10-50 microM trifluoperazine. In such beating flagella, both the sliding velocity and the reverse bend angle were close to their respective values at 10(-5) M Ca(2+). Trypsin treatment induced a similar recovery of beating in quiescent flagella at 10(-)(4) M Ca(2+), albeit with a more symmetrical waveform. These results provide first evidence that, at least at ATP concentrations higher than approximately 100 microM, 10(-6)-10(-5) M Ca(2+) decreases the maximum sliding velocity of microtubules in beating flagella through a trypsin-sensitive regulatory mechanism which possibly involves the central pair apparatus. They also suggest that calmodulin may be associated with the mechanism underlying flagellar quiescence induced by 10(-4) M Ca(2+). PMID:10671372

Bannai, H; Yoshimura, M; Takahashi, K; Shingyoji, C

2000-03-01

92

Effects of Imposed Bending on Microtubule Sliding in Sperm Flagella  

Microsoft Academic Search

The movement of eukaryotic flagella is characterized by its oscillatory nature [1]. In sea urchin sperm, for example, planar bends are formed in alternating directions at the base of the flagellum and travel toward the tip as continuous waves. The bending is caused by the orchestrated activity of dynein arms to induce patterned sliding between doublet microtubules of the flagellar

Yutaka Morita; Chikako Shingyoji

2004-01-01

93

MICROTUBULE SURFACE LATTICE AND SUBUNIT STRUCTURE AND OBSERVATIONS ON REASSEMBLY  

PubMed Central

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

Erickson, Harold P.

1974-01-01

94

Simulation of Cyclic Dynein-Driven Sliding, Splitting, and Reassociation in an Outer Doublet Pair  

PubMed Central

Abstract A regular cycle of dynein-driven sliding, doublet separation, doublet reassociation, and resumption of sliding was previously observed by Aoyama and Kamiya in outer doublet pairs obtained after partial dissociation of Chlamydomonas flagella. In the work presented here, computer programming based on previous simulations of oscillatory bending of microtubules was extended to simulate the cycle of events observed with doublet pairs. These simulations confirm the straightforward explanation of this oscillation by inactivation of dynein when doublets separate and resumption of dynein activity after reassociation. Reassociation is augmented by a dynein-dependent “adhesive force” between the doublets. The simulations used a simple mathematical model to generate velocity-dependent shear force, and an independent elastic model for adhesive force. Realistic results were obtained with a maximum adhesive force that was 36% of the maximum shear force. Separation between a pair of doublets is the result of a buckling instability that also initiates a period of uniform sliding that enlarges the separation. A similar instability may trigger sliding initiation events in flagellar bending cycles.

Brokaw, Charles J.

2009-01-01

95

Functional and molecular diversity of dynein heavy chains  

Microsoft Academic Search

The directed translocation of dynein along microtubules is the basis for a wide variety of essential cellular movements. In eukaryotic flagella and cilia, dynein produces the active sliding of outer doublet microtubules that underlies axonemal bending. Several dynein heavy chain isoforms are precisely located in the axoneme in order to initiate and propagate bends, and it is believed that these

David J. Asai

1996-01-01

96

Katanin Knockdown Supports a Role for Microtubule Severing in Release of Basal Bodies before Mitosis in Chlamydomonas  

Microsoft Academic Search

Katanin is a microtubule-severing protein that participates in the regulation of cell cycle progression and in ciliary disassembly, but its precise role is not known for either activity. Our data suggest that in Chlamydomonas, katanin severs doublet microtubules at the proximal end of the flagellar transition zone, allowing disengagement of the basal body from the flagellum before mitosis. Using an

M. Qasim Rasi; Jeremy D. K. Parker; Jessica L. Feldman; Wallace F. Marshall; Lynne M. Quarmby

2009-01-01

97

EXTRACELLULAR MICROTUBULES  

PubMed Central

Mastigonemes (Flimmer) from the sperm of Ascophyllum and Fucus were found to consist of a tripartite structure—a ca. 2000-A tapered basal region, a closed microtubular shaft, and a group of terminal filaments. Each of these regions appears to be constructed of globular subunits with a center-to-center distance of about 45 A. The mastigoneme microtubule is of smaller diameter (170–190 A) than cytoplasmic microtubules in these or other plant cells. During the initial stages of flagellar ontogeny, structures similar to mastigonemes (presumptive mastigonemes) are found within membrane-limited sacs in the cytoplasm or within the perinuclear space. Mastigonemes at this time are generally not found on the flagellar surface. Later, when the anterior flagellum acquires mastigonemes, the presumptive mastigonemes are absent from the cytoplasm. The regularity of attachment of mastigonemes to the flagellar surface suggests that specific attachment sites are constructed on the plasma membrane during flagellar ontogeny. No evidence for penetration of the mastigoneme through the plasma membrane was obtained. The origin and structure of mastigonemes are discussed in relation to reports of the origin and structure of other microtubular systems.

Bouck, G. Benjamin

1969-01-01

98

Absence of axonemal arms in nasal mucosa cilia in Kartagener's syndrome  

Microsoft Academic Search

A TOTAL lack of axonemal arms in the spermatozoa of infertile men has been described1-3 and those results included those from four men, three of whom had situs inversus of the thoracic organs while the fourth was a brother to one of the others. The total absence of axonemal arms (dynein arms) was deemed to be responsible for the complete

Henning Pedersen; Niels Mygind

1976-01-01

99

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

100

Titanium gettering in Doublet III  

SciTech Connect

The application of mild titanium gettering in the Doublet III tokamak has led to a significant improvement in the obtainable operating regimes and discharge parameters for all of the many plasma cross-sectional shapes studied. With gettering, low-Z impurities and radiated power are greatly reduced. The maximum line averaged electron density has increased 50% (anti n/sub e max/ approx. 1 x 10/sup 20//m/sup 3/), corresponding to a Murakami coefficient of nearly 6.

de Grassie, J.S.; Callis, R.; Campbell, G.

1980-08-01

101

Inner and outer arm axonemal dyneins from the Antarctic rockcod Notothenia coriiceps.  

PubMed

Adaptive compensation of enzymatic activities is common among cold-living poikilotherms. Their enzymes often demonstrate higher activities at low temperatures than do homologs from temperate or thermophilic species. To understand the molecular features necessary for cold adaptation of microtubule motor proteins, we have initiated studies of the flagellar dynein ATPases of Antarctic fishes (body temperature range = -1.8 to +2 degrees C). Dyneins were isolated by high-salt extraction of demembranated sperm axonemes from the Antarctic yellowbelly rockcod, Notothenia coriiceps. Although solubilization of inner arms was incomplete, an inner arm dynein was recognized as a discrete complex containing one major dynein heavy chain (DHC) and sedimenting through sucrose gradients at approximately 12 S. Like inner arm dyneins from Chlamydomonas, the fish complex contained an actin-immunoreactive protein of 43 kDa and a 30-kDa protein. One isoform of the inner arm DHC gene family of N. coriiceps was detected by the polymerase chain reaction, and Southern analysis established that this DHC gene is present at one copy per haploid genome. Outer arm dynein was extracted quantitatively by high-salt treatment, contained two DHCs (one major, one minor), and sedimented through sucrose gradients as a polydisperse, aggregating system. Associated with the outer arm DHCs were five presumptive intermediate chains (ICs) of 66-91 kDa, immunologically defined by their cross-reactivity to four monoclonal antibodies specific for ICs from other organisms. The basal (non-microtubule-stimulated) specific ATPase activities of the N. coriiceps inner and outer arm dyneins were approximately 0.07 and approximately 0.04 micromol of P(i) min(-1) mg(-1), respectively, at 0 degrees C, attained their maxima (approximately 0.1 micromol of P(i) min(-1) mg(-1)) at 9 and 19 degrees C, respectively, and at higher temperatures declined substantially. Furthermore, the activities of the fish dyneins at temperatures < or = 15 degrees C were significantly larger than that of outer arm dynein from the mesophile Tetrahymena. These results suggest that the greater catalytic efficiencies of N. coriiceps inner and outer arm dyneins at low temperatures are due to enhanced polypeptide flexibility in the active sites of their protein subunits. We conclude that temperature adaptation of flagellar dyneins from Antarctic fishes is compatible with substantial conservation of primary and quaternary structure. PMID:9063878

King, S M; Marchese-Ragona, S P; Parker, S K; Detrich, H W

1997-02-11

102

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

PubMed Central

Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000- dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two- dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin.

1980-01-01

103

Structural-Functional Relationships of the Dynein, Spokes, and Central-Pair Projections Predicted from an Analysis of the Forces Acting within a Flagellum  

Microsoft Academic Search

In the axoneme of eukaryotic flagella the dynein motor proteins form crossbridges between the outer doublet microtubules. These motor proteins generate force that accumulates as linear tension, or compression, on the doublets. When tension or compression is present on a curved microtubule, a force per unit length develops in the plane of bending and is transverse to the long axis

Charles B. Lindemann

2003-01-01

104

Coordination of Dynein Motor Enzymes for Generation of 3-dimensional Flagellar Bending Waves  

Microsoft Academic Search

Eukaryotic flagella contain tens of thousands of dynein motor enzymes, arranged in regular arrays along the outer doublet microtubules of the axoneme -- the internal cytoskeleton of a flagellum or cilium. These dyneins produce longitudinal sliding between the doublet microtubules. They must interact cooperatively to generate the bending waves that are required for efficient propulsion of spermatozoa, or other situations.

Charles J. Brokaw

105

Power spectral density of Transmit Reference Doublet trains and Reference Sharing Doublet trains in ultra wideband systems  

Microsoft Academic Search

The power spectral densities of the Transmit Reference doublet (TR-doublet) train and the Reference Sharing doublets (RS-doublet) train are computed with the mathematics presented. It is shown that the PSD of the RS-doublet train has similar characteristics to the TR-doublet train and it can be shown that time dithering on the doublets can improve the overall PSD pattern. However, only

Chun Yi Lee; Chris Toumazou

2004-01-01

106

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.

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

107

Splice-site mutations in the axonemal outer dynein arm docking complex gene CCDC114 cause primary ciliary dyskinesia.  

PubMed

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

2012-12-20

108

Threshold Anomaly in Doublet nd-Scattering.  

National Technical Information Service (NTIS)

Calculations of the doublet nd-scattering phase shift which prove to be the evidence of its irregular behaviour near the three-particle threshold are carried out. The case when the doublet nd-scattering length a/sub 2/ is close to experimental value is sh...

D. V. Shapoval I. V. Simenog A. I. Sitnichenko

1987-01-01

109

Microtubule Self Assembly  

Microsoft Academic Search

Microtubules are important structural elements for neurons. Microtubles are cylindrical pipes that are self-assembled from tubulin dimers, These structures are intimately related to the neuron transport system. Abnormal microtubule disintegration contributes to neuro-disease. For several decades, experimentalists investigated the structure of the microtubules using TEM and Cryo-EM. However, the detailed structure at a molecular level remain incompletely understood. . In

Yongseok Jho; M. C. Choi; O. Farago; Mahnwon Kim; P. A. Pincus

2008-01-01

110

Nanomechanics of microtubules.  

PubMed

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 2 orders of magnitude lower than the Young's, giving rise to a length-dependent flexural rigidity of microtubules. The temperature dependence of the microtubule's bending stiffness in the (5-40) degrees C range shows a strong variation upon cooling coming from the increasing interaction between the protofilaments. PMID:12484982

Kis, A; Kasas, S; Babi?, B; Kulik, A J; Benoît, W; Briggs, G A D; Schönenberger, C; Catsicas, S; Forró, L

2002-11-21

111

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

112

THE MECHANISM OF ACTION OF COLCHICINE: Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin  

Microsoft Academic Search

The thermal depolymerization procedure of Stephens (1970 . J.Mol. Biol .47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay

LESLIE WILSON; ISAURA MEZA

1973-01-01

113

Meiosis: organizing microtubule organizers.  

PubMed

During meiosis in fission yeast, the zygote nucleus undergoes microtubule-driven oscillatory movements that ultimately serve to promote genetic recombination. An essential component of this is a meiosis-specific consolidation of microtubule-organizing activity to the the spindle pole body, driven by the novel coiled-coil protein mcp6/hrs1p. PMID:16111936

Sawin, Kenneth E

2005-08-23

114

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

PubMed

Cilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled by a central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. We characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. We show that assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and that the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135's role in assembling highly stable MT-based structures, such as motile axonemes and centrioles. PMID:22898782

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

2012-08-14

115

Structural analysis of flagellar axonemes from inner arm dynein knockdown strains of Trypanosoma brucei.  

PubMed

Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Flagellar function in this organism is critical for life cycle progression and pathogenesis, however the regulation of flagellar motility is not well understood. The flagellar axoneme produces a complex beat through the precisely coordinated firing of many proteins, including multiple dynein motors. These motors are found in the inner arm and outer arm complexes. We are studying one of the inner arm dynein motors in the T. brucei flagellum: dynein-f. RNAi knockdown of genes for two components of dynein-f: DNAH10, the alpha heavy chain, and IC138, an intermediate chain, cause severe motility defects including immotility. To determine if motility defects result from structural disruption of the axoneme, we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy (TEM). Our analysis showed that inner arm dynein size, axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures. These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme. PMID:23682429

Zukas, Randi; Chang, Alex J; Rice, Marian; Springer, Amy L

2012-12-01

116

Threshold anomaly in doublet nd-scattering.  

National Technical Information Service (NTIS)

The doublet nd-scattering amplitude behaviour near the three-particle threshold is studied analytically and numerically. The scattering amplitude possesses an anomaly caused by the small energy of singlet deuteron virtual level. The anomaly manifests itse...

D. V. Shapoval I. V. Simenog

1988-01-01

117

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

118

Oscillation modes of microtubules.  

PubMed

Microtubules are long, filamentous protein complexes which play a central role in several cellular physiological processes, such as cell division transport and locomotion. Their mechanical properties are extremely important since they determine the biological function. In a recently published experiment [Phys. Rev. Lett. 89 (2002) 248101], microtubule's Young's and shear moduli were simultaneously measured, proving that they are highly anisotropic. Together with the known structure, this finding opens the way to better understand and predict their mechanical behavior under a particular set of conditions. In the present study, we modeled microtubules by using the finite elements method and analyzed their oscillation modes. The analysis revealed that oscillation modes involving a change in the diameter of the microtubules strongly depend on the shear modulus. In these modes, the correlation times of the movements are just slightly shorter than diffusion times of free molecules surrounding the microtubule. It could be therefore speculated that the matching of the two timescales could play a role in facilitating the interactions between microtubules and MT associated proteins, and between microtubules and tubulins themselves. PMID:15567524

Kasas, S; Cibert, C; Kis, A; De Los Rios, P; Riederer, B M; Forró, L; Dietler, G; Catsicas, S

2004-12-01

119

Cyclic AMP induces maturation of trout sperm axoneme to initiate motility  

NASA Astrophysics Data System (ADS)

Cyclic AMP has long been implicated as an activator of sperm motility1-5. From more recent experiments using demembranated mammalian and sea urchin spermatozoa6,7, it was concluded that cyclic AMP only increases the motility of the axoneme after it has been initiated by MgATP2-. We have now carried out similar experiments using spermatozoa collected from the rainbow trout and demembranated by treatment with the detergent Triton X-100. Our results suggest that in this species, cyclic AMP is required before MgATP2- to trigger maturation of the nonmotile axoneme. Subsequent addition of an energy source then induces motility.

Morisawa, Masaaki

1982-02-01

120

Microtubule Self- Assembly  

NASA Astrophysics Data System (ADS)

Microtubules are important structural elements for neurons. Microtubles are cylindrical pipes that are self-assembled from tubulin dimers, These structures are intimately related to the neuron transport system. Abnormal microtubule disintegration contributes to neuro-disease. For several decades, experimentalists investigated the structure of the microtubules using TEM and Cryo-EM. However, the detailed structure at a molecular level remain incompletely understood. . In this presentation, we report numerically studies of the self-assembly process using a toy model for tubulin dimers. We investigate the nature of the interactions which are essential to stabilize such the cylindrical assembly of protofilaments. We use Monte Carlo simulations to suggest the pathways for assembly and disassembly of the microtubules.

Jho, Yongseok; Choi, M. C.; Farago, O.; Kim, Mahnwon; Pincus, P. A.

2008-03-01

121

The dynein microtubule motor  

Microsoft Academic Search

Dyneins are large multi-component microtubule-based molecular motors involved in many fundamental cellular processes including vesicular transport, mitosis and ciliary\\/flagellar beating. In order to achieve useful work, these enzymes must contain motor, cargo-binding and regulatory components. The ATPase and microtubule motor domains are located within the very large dynein heavy chains that form the globular heads and stems of the complex.

Stephen M King

2000-01-01

122

Luminal particles within cellular microtubules  

PubMed Central

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.

Garvalov, Boyan K.; Zuber, Benoit; Bouchet-Marquis, Cedric; Kudryashev, Mikhail; Gruska, Manuela; Beck, Martin; Leis, Andrew; Frischknecht, Friedrich; Bradke, Frank; Baumeister, Wolfgang; Dubochet, Jacques; Cyrklaff, Marek

2006-01-01

123

Functional state of the axonemal dyneins during flagellar bend propagation.  

PubMed Central

When mouse spermatozoa swim in media of high viscosity, additional waves of bending are superimposed on the primary traveling wave. The additional (secondary) waves are relatively small in scale and high in frequency. They originate in the proximal part of the interbend regions. The initiation of secondary bending happens only in distal parts of the flagellum. The secondary waves propagate along the interbends and then tend to die out as they encounter the next-most-distal bend of the primary wave, if that bend exceeds a certain angle. The principal bends of the primary wave, being of greater angle than the reverse bends, strongly resist invasion by the secondary waves; when a principal bend of the primary wave propagates off the flagellar tip, the secondary wave behind it suddenly increases in amplitude. We claim that the functional state of the dynein motors in relation to the primary wave can be deduced from their availability for recruitment into secondary wave activity. Therefore, only the dyneins in bends are committed functionally to the maintenance and propagation of the flagellar wave; dyneins in interbend regions are not functionally committed in this way. We equate functional commitment with tension-generating activity, although we argue that the regions of dynein thus engaged nevertheless permit sliding displacements between the doublets.

Woolley, D M; Vernon, G G

2002-01-01

124

Characterization of an A-Kinase Anchoring Protein in Human Ciliary Axonemes  

PubMed Central

Although protein kinase A (PKA) activation is known to increase ciliary beat frequency in humans the molecular mechanisms involved are unknown. We demonstrate that PKA is associated with ciliary axonemes where it specifically phosphorylates a 23-kDa protein. Because PKA is often localized to subcellular compartments in proximity to its substrate(s) via interactions with A-kinase–anchoring proteins (AKAPs), we investigated whether an AKAP was also associated with ciliary axonemes. This study has identified a novel 28 kDa AKAP (AKAP28)that is highly enriched in airway axonemes. The mRNA for AKAP28 is up-regulated as primary airway cells differentiate and is specifically expressed in tissues containing cilia and/or flagella. Additionally, both Western blot and immunostaining data show that AKAP28 is enriched in airway cilia. These data demonstrate that we have identified the first human axonemal AKAP, a protein that likely plays a role in the signaling necessary for efficient modulation of ciliary beat frequency.

Kultgen, Patricia L.; Byrd, Sherell K.; Ostrowski, Lawrence E.; Milgram, Sharon L.

2002-01-01

125

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.

1992-01-01

126

Lattice defects in microtubules: protofilament numbers vary within individual microtubules  

Microsoft Academic Search

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

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

1992-01-01

127

Neutral density measurements in Doublet 3  

NASA Astrophysics Data System (ADS)

In the Doublet III Tokamak a variety of cross sectionally shaped plasmas were investigated. Simple magnetically shielded ionization gauges monitoring the working gas pressure show a striking dependence upon plasma shape. Average neutral density varies over two orders of magnitude. In the extreme of the simplified poloidal divertor shapes the pressure exceeds 0.001 torr.

Degrassie, J. S.; Deboo, J. C.; Mahdavi, M. A.; Ohyabu, N.; Shimada, M.

1981-08-01

128

Neutral density measurements in doublet III  

Microsoft Academic Search

In the Doublet III tokamak a variety of cross-sectionally-shaped plasmas has been investigated. Simple magnetically-shielded ionization gauges monitoring the working gas pressure show a striking dependence upon plasma shape. Average neutral density varies over two orders of magnitude. In the exteme cases of the simplified poloidal divertor shapes, the pressure exceeds 10⁻³ Torr.

J. S. deGrassie; J. C. DeBoo; M. A. Mahdavi; N. Ohyabu; M. Shimada

1982-01-01

129

Doublet effects in the verbal maze during acquisition and relearning  

Microsoft Academic Search

144 Ss divided into 3 doublet conditions (0, 2, and 4 doublets) under each of three retention intervals (|14, 1, and 24 hr.) were used. Each S learned and relearned a verbal maze under the correction procedure. The results demonstrate that the effect of the doublet upon the serial-position error curve is reversed from acquisition to relearning. Further, the results

Charles P. Thompson

1966-01-01

130

Bidirectional Transport along Microtubules  

Microsoft Academic Search

Active transport by microtubule motors has a plethora of crucial roles in eukaryotic cells. Organelles often move bidirectionally, employing both plus-end and minus-end directed motors. Bidirectional motion is widespread and may allow dynamic regulation, error correction and the establishment of polarized organelle distributions. Emerging evidence suggests that motors for both directions are simultaneously present on cellular ‘cargo’, but that their

Michael A. Welte

2004-01-01

131

Microtubule circumferential vibrations in cytosol.  

PubMed

Microtubules are key components of the cytoskeleton and perform a variety of functions, including chromosome movement during cell division, intracellular transport of materials, movement of organelles and intracellular tracking. A combination of essential and up-to-date methods is needed for investigating the biology of microtubules and understanding the mechanisms of microtubule-drug interaction. Coupled cytosol-microtubule mechanical vibrations of microtubules are studied in this article. Such investigations provide helpful insights on the functional mechanisms of microtubules and their interactions with other proteins and drugs. The viscous cytosol and the microtubule are coupled through the continuity condition across the microtubule-cytosol interface. The stress field in the cytosol induced by vibrating microtubule is analytically determined and the coupled circumferential vibrations of the cytosol-microtubule system are investigated by developing a coupled polynomial eigenvalue problem. Finally, the variations of vibration frequencies of a coupled system with cytosol dynamic viscosity, and microtubule circumferential Young's modulus are examined. Furthermore, the validity of the present analysis is confirmed by comparing the results with those obtained from the literature. PMID:23057232

Daneshmand, Farhang

2012-08-01

132

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

SciTech Connect

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

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

2009-04-23

133

Mice Deficient in the Axonemal Protein Tektin-t Exhibit Male Infertility and Immotile-Cilium Syndrome Due to Impaired Inner Arm Dynein Function  

PubMed Central

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.

Tanaka, Hiromitsu; Iguchi, Naoko; Toyama, Yoshiro; Kitamura, Kouichi; Takahashi, Tohru; Kaseda, Kazuhiro; Maekawa, Mamiko; Nishimune, Yoshitake

2004-01-01

134

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

PubMed

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

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

2013-02-11

135

SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing.  

PubMed

One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtubule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1-6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7-10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pavement cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mechanism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs. PMID:24055158

Wightman, Raymond; Chomicki, Guillaume; Kumar, Manoj; Carr, Paul; Turner, Simon R

2013-09-19

136

SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing  

PubMed Central

Summary One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtubule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1–6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7–10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pavement cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mechanism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs.

Wightman, Raymond; Chomicki, Guillaume; Kumar, Manoj; Carr, Paul; Turner, Simon R.

2013-01-01

137

Orbifold GUT model with nine Higgs doublets  

NASA Astrophysics Data System (ADS)

We describe a non-supersymmetric orbifold GUT based on SU(5) symmetry. It is a modification of Kawamura's 5-D orbifold GUT model. The difference lies in the choice of Higgs scalars as we have allowed only 5-plets of SU(5) in the GUT scale. This variant was originally proposed by Brahmachari and Raychoudhuri. Proton decay problem and the doublet triplet splitting problems are solved by extra dimensional mechanism. The unification scale is around 5.0×1013 GeVs. In low energy there are nine Higgs doublets. One at the 100 GeV region and eight others degenerate at around 1.4 TeV. It is an attractive non-supersymmetric extension of standard model with very rich collider physics phenomenology.

Brahmachari, Biswajoy

2008-05-01

138

Ofd1 controls dorso-ventral patterning and axoneme elongation during embryonic brain development.  

PubMed

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

D'Angelo, Anna; De Angelis, Amalia; Avallone, Bice; Piscopo, Immacolata; Tammaro, Roberta; Studer, Michèle; Franco, Brunella

2012-12-27

139

Trilepton signals in the inert doublet model  

SciTech Connect

In this work, we investigate the prospects for detecting the Inert Doublet Model via the trilepton channel at the LHC. We present a set of representative benchmark scenarios in which all applicable constraints are satisfied, and show that in some of these scenarios, it is possible to obtain a signal at the 5{sigma} significance level or better with integrated luminosity of 300 fb{sup -1}.

Miao, Xinyu; Su, Shufang [Department of Physics, University of Arizona, Tucson, Arizona 85721 (United States); Thomas, Brooks [Department of Physics, University of Arizona, Tucson, Arizona 85721 (United States); Department of Physics, University of Maryland, College Park, Maryland 20742 (United States)

2010-08-01

140

A supersymmetric one Higgs doublet model  

Microsoft Academic Search

We present a supersymmetric extension of the Standard Model in which only one electroweak doublet acquires a vacuum expectation\\u000a value and gives mass to Standard Model fermions. As well as the novel accommodation of a Standard Model Higgs within a supersymmetric\\u000a framework, this leads to a very predictive model, with some advantages over the MSSM. In particular, problems with proton

Rhys Davies; John March-Russell; Matthew McCullough

2011-01-01

141

Doublet III neutral beam cryopumping system  

Microsoft Academic Search

Two neutral beamlines, each capable of injectingroughly-equal 3.6 MW H° beam of 80 keV, are employed for the upcoming neutral beam injection heating experiments in Doublet III tokamak. The large influx of hydrogen gas in a beamline is pumped by a sophisticated system of cryopanels that are cooled by a closed-loop forced flow of liquid helium. Two cryopanels per beamline

M. A. Otavka; A. P. Colleraine; M. M. Holland; J. Kim

1981-01-01

142

Higgs properties in a softly broken Inert Doublet Model  

NASA Astrophysics Data System (ADS)

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

Enberg, Rikard; Rathsman, Johan; Wouda, Glenn

2013-08-01

143

Rho Proteins and Microtubules  

Microsoft Academic Search

Rho GTPases have increasingly become recognized as prominent regulators of the microtubule (MT) cytoskeleton. Whereas Rho\\u000a GTPases regulate the de novo formation of distinct actin arrays (stress fibers, lamellipodia, and filopodia), with MTs, which\\u000a are present as extensive and dynamic arrays in the absence of Rho GTPase signaling, Rho GTPases principally modify the behavior\\u000a and dynamics of individual MTs within

Christina H. Eng; Gregg G. Gundersen

144

Generation of microtubule stability subclasses by microtubule- associated proteins: implications for the microtubule "dynamic instability" model  

PubMed Central

We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.

1985-01-01

145

Microtubule Organization in the Phragmoplast  

Microsoft Academic Search

\\u000a The phragmoplast harnesses the actions of microtubules and actin microfilaments to deliver Golgi-derived vesicles for the\\u000a assembly of the cell plate which divides the cytoplasm of the mother cell. This review emphasizes on how microtubules are\\u000a organized in the phragmoplast to allow cytokinesis to take place in a spatially and temporally regulated fashion. The phragmoplast\\u000a microtubule array consists of two

Bo Liu; Takashi Hotta; Chin-Min Kimmy Ho; Yuh-Ru Julie Lee

146

Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase  

SciTech Connect

Photochemical cross-linking of both tetrahymena and aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge-membrane complex and the dynein-like membrane-associated ATPase. Photochemical cross-linking of ciliary membrane proteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement.

Dentler, W.L.; Pratt, M.M.; Stephens, R.E.

1980-02-01

147

An earthquake doublet in Ometepec, Guerrero, Mexico  

NASA Astrophysics Data System (ADS)

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 hours of each other. We determine the source parameters of these events by inverting surface-wave data at a period of 256 s. The results are for the first event, strike = 116°, dip = 77°, slip = 88° and seismic moment of 2.8 × 1026 dyne . cm, and for the second event strike = 116°, dip = 78°, slip = 89° and seismic moment of 2.8 × 1026 dyne . cm. Modeling of long-period P waves suggests that the first event has a depth of 20 km and is represented by a single trapezoidal source time function, with an effective duration of 6 s. The second event is best modeled by two sources at depths of 15 and 10 km. The combined effective source duration time for the two sources is about 10 s. The ratio of the seismic moment, obtained from body waves to that from surface waves, is ~ 0.5 for the first event and 1 for the second. Adding the seismic moment of the two events and considering the first week aftershock area, 3200 km2, the stress drop is ~4 bars. These results suggest that the first event, that involved a deeper asperity, caused an incremental stress change large enough to trigger the second event. If the two events of the doublet broke distinct areas of the subduction zone, the coseismic slip is 0.58 m, and accounts for about 25% of the total plate motion between the Cocos Plate and the North America Plate, accumulated since the last large earthquake in the region. Other doublets similar to the Ometepec doublet have occurred along the Middle America Trench during the past 70 years. A regional distribution of comparable-size asperities may be responsible for this relatively frequent occurrence of doublets and for the simplicity of earthquakes in the region. The high convergence rate, which produces rapid strain accumulation and short recurrence intervals for large earthquakes, and the smooth sea-floor subducted along the middle America Trench, may contribute to the homogeneous distribution of comparable size asperities. We found a relation, log T ~ 1/3log M0 (T is the average recurrence time and M0 is the average seismic moment) for large earthquakes along the subduction zone in the Guerrero-Oaxaca region, where the convergence rate and the properties of the subducted plate are considered relatively uniform. A simple asperity model predicts this relation.

Astiz, Luciana; Kanamori, Hiroo

1984-04-01

148

Doublet III neutral beam cryopumping system  

SciTech Connect

Two neutral beamlines, each capable of injectingroughly-equal 3.6 MW H/sup 0/ beam of 80 keV, are employed for the upcoming neutral beam injection heating experiments in Doublet III tokamak. The large influx of hydrogen gas in a beamline is pumped by a sophisticated system of cryopanels that are cooled by a closed-loop forced flow of liquid helium. Two cryopanels per beamline at sub-cooled liquid helium temperatures provide a combined pumping speed of over 10/sup 6/ l/s. The system of cryopanels, transfer lines, and a liquifier is described, together with preliminary test results in the actual beamline.

Otavka, M.A.; Colleraine, A.P.; Holland, M.M.; Kim, J.

1981-04-01

149

Microrheology of microtubule solutions and actin-microtubule composite networks.  

PubMed

We perform local or microrheological measurements on microtubule solutions, as well as composite networks. The viscoelastic properties of microtubules as reported from two-point microrheology agree with the macroscopic measurement at high frequencies, but appear to show a discrepancy at low frequencies, at time scales on the order of a second. A composite of filamentous actin (F-actin) and microtubules has viscoelastic behavior between that of F-actin and pure microtubules. We further show that the Poisson ratio of the composite, measured by the length-scale dependent two-point microrheology, is robustly smaller than that of the F-actin network at time scales tau>1 s, suggesting that a local compressibility is conferred by the addition of microtubules to the F-actin network. PMID:19518917

Pelletier, Vincent; Gal, Naama; Fournier, Paul; Kilfoil, Maria L

2009-05-07

150

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

151

Microtubules, MAPs and Xylem Formation  

Microsoft Academic Search

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

Edouard Pesquet; Clive Lloyd

2011-01-01

152

Anomalous flexural behaviors of microtubules.  

PubMed

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

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

2012-04-18

153

Template synthesis of organic microtubules  

Microsoft Academic Search

Organic microtubules have recently caused a great deal of excitement in the physics, chemistry, and materials science communities. Such tubules are inherently intriguing chemical systems and have myriad proposed technolgical applications; they may also be useful as mimics of biological microtubules. The only known synthetic route for preparing organic microtubles involves extremely expensive reagents and produces tubules with a broad

Charles R. Martin; Leon S. Van Dyke; Zhihua Cai; Wenbin Liang

1990-01-01

154

Synchronous Oscillations in Microtubule Polymerization  

Microsoft Academic Search

Under conditions where microtubule nucleation and growth are fast (i.e., high magnesium ion and tubulin concentrations and absence of glycerol), microtubule assembly in vitro exhibits an oscillatory regime preceding the establishment of steady state. The amplitude of the oscillations can represent >50% of the maximum turbidity change and oscillations persist for up to 20 periods of 80 s each. Oscillations

M. F. Carlier; R. Melki; D. Pantaloni; T. L. Hill; Y. Chen

1987-01-01

155

Tempered two-Higgs-doublet model  

NASA Astrophysics Data System (ADS)

We discuss the phenomenological consequences of requiring the cancellation of quadratic divergences up to the leading two-loop order within the two-Higgs-doublet model. Taking into account existing experimental constraints, allowed regions in the parameter space, permitting the cancellation, are determined. A degeneracy between masses of scalar bosons is observed for tan???40. The possibility for CP violation in the scalar potential is discussed and regions of tan??-MH± with a substantial amount of CP violation are determined. In order to provide a source for dark matter in a minimal manner, a scalar gauge singlet is introduced and discussed. The model allows to ameliorate the little hierarchy problem by lifting the minimal scalar Higgs-boson mass and by suppressing the quadratic corrections to scalar masses. The cutoff originating from the naturality arguments is therefore lifted from ˜0.6TeV in the standard model to ?2.5TeV in two-Higgs-doublet model depending on the mass of the lightest scalar.

Grzadkowski, B.; Osland, P.

2010-12-01

156

Applications research studies on microtubules  

NASA Astrophysics Data System (ADS)

This final report describes progress completed on two of three phases of work aimed at defining new applications areas for microtubules formed as self-organizing microstructures from lipids with diacetylenic lecithin structures. The emphasis in the first phase was on the suspension, orientation, and electro-optical properties of metalized microtubule dispersions in fluids, liquid crystals, and polymers, and on techniques for controlling the orientation and attachment of microtubules to surfaces. The emphasis in the second phase was on phase shift effects in the millimeter wave region with field effects on metalized microtubule dispersions, in regard to potential applications such as scanning radar antennas and radar lenses. In these studies it was discovered that composite fluids consisting of metalized microtubules dispersed in liquid crystals have the highest birefringence values which have been reported in the millimeter wave region and that these composite fluids are readily aligned by applied electrical and magnetic fields.

Margerum, J. David; Lim, K. C.; Lackner, Anna M.; Miller, Leroy J.; Sherman, Elena; Smith, Willis H., Jr.; Vanast, Camille I.

1990-09-01

157

Compressed microtubules: Splitting or buckling  

NASA Astrophysics Data System (ADS)

Microtubule (MT) is the mechanically strongest cytoskeletal element in eukaryotic cells and plays a key role in maintaining cell's geometrical shape by bearing compressive forces. MTs are highly dynamic, and ``dynamic instability'' is referred to the switch between polymerization and depolymerization phases (the latter is characterized by splitting of protofilaments at the plus end). A micromechanics model is proposed here to study whether an axially compressed microtubule, protected by a ``cap'' consisted of a few layers of strongly bonded GTP dimers at the plus end, can split prior to overall buckling. Our basic conclusion is that compression-driven splitting of a capped microtubule can happen prior to overall buckling when the microtubule is very short (typically shorter than few hundreds of nanometers). For example, compression-driven splitting from middle of a capped microtubule can happen prior to buckling when the length of microtubule is shorter than a few hundreds of nanometers. In addition, for capped microtubules shorter than 125-180 nm (depending on specific values of axial Young's modulus and adhesion energy between protofilaments), mechanical compression will cause splitting of the microtubule at its plus end prior to overall buckling. On the other hand, however, for microtubules of length longer than 0.3-0.75 micron (depending on specific values of axial Young's modulus and adhesion energy between protofilaments), the present model shows that a cap composed of even one single layer of GTP dimers is sufficient to prevent compression-driven splitting prior to buckling, in agreement with the known observations that dynamic instability or splitting of moderately long microtubules could happen only when the cap is completely lost at the plus end.

Jin, M. Z.; Ru, C. Q.

2012-03-01

158

Evolution of microtubule organizing centers across the tree of eukaryotes.  

PubMed

The architecture of eukaryotic cells is underpinned by complex arrrays of microtubules that stem from an organizing center, referred to as the MTOC. With few exceptions, MTOCs consist of two basal bodies that anchor flagellar axonemes and different configurations of microtubular roots. Variations in the structure of this cytoskeletal system, also referred to as the 'flagellar apparatus', reflect phylogenetic relationships and provide compelling evidence for inferring the overall tree of eukaryotes. However, reconstructions and subsequent comparisons of the flagellar apparatus are challenging, because these studies require sophisticated microscopy, spatial reasoning and detailed terminology. In an attempt to understand the unifying features of MTOCs and broad patterns of cytoskeletal homology across the tree of eukaryotes, we present a comprehensive overview of the eukaryotic flagellar apparatus within a modern molecular phylogenetic context. Specifically, we used the known cytoskeletal diversity within major groups of eukaryotes to infer the unifying features (ancestral states) for the flagellar apparatus in the Plantae, Opisthokonta, Amoebozoa, Stramenopiles, Alveolata, Rhizaria, Excavata, Cryptophyta, Haptophyta, Apusozoa, Breviata and Collodictyonidae. We then mapped these data onto the tree of eukaryotes in order to trace broad patterns of trait changes during the evolutionary history of the flagellar apparatus. This synthesis suggests that: (i) the most recent ancestor of all eukaryotes already had a complex flagellar apparatus, (ii) homologous traits associated with the flagellar apparatus have a punctate distribution across the tree of eukaryotes, and (iii) streamlining (trait losses) of the ancestral flagellar apparatus occurred several times independently in eukaryotes. PMID:23398214

Yubuki, Naoji; Leander, Brian S

2013-03-22

159

The 78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein  

Microsoft Academic Search

A previous study (King et al., 1991. J. Biol. Chem. 266:8401-8407) showed that the 78,000-M(r) intermediate chain (IC78) from the Chlamydomonas outer arm dynein is in direct contact with alpha-tubulin in situ, suggesting that this protein may be involved in binding the dynein to the doublet microtubules. Molecular genetic analysis of this chain recently demonstrated that it is a WD

Stephen M. King; Ramila S. Patel-King; Curtis G. Wilkerson; George B. Witman

1995-01-01

160

Model independence of the nd system in the doublet state  

SciTech Connect

We have obtained a model-independent solution of the problem of nd scattering in the doublet state. The solution reveals qualitative characteristics of a three-nucleon system at low energies and correlations between the parameters of the doublet scattering amplitude.

Simenog, I.V.; Shapoval, D.V.

1988-04-01

161

On composite two Higgs doublet models  

NASA Astrophysics Data System (ADS)

We investigate the issue of anomalous contribution to the T parameter and to Flavor Changing Neutral Currents in models with two Higgs doublets arising as composite pseudo Nambu-Goldstone modes. The non linear Lagrangians of several models are explicitly derived and the anomalous contributions to T are identified. The breaking patterns SU(5) ? SU(4) × U(1) and SU(5) ? SU(4), are analyzed first and we show how anomalous contributions to T arise in both models. Apart from that, the embedding of the Standard Model fermions in a 10 of SU(5) avoids at the same time large corrections to the Zboverline{b} coupling and Flavor Changing Neutral Current transitions. Finally, we propose a model based on the breaking SO(9) /SO(8) that is free from anomalous contributions to T and in which the problems of the Zboverline{b} coupling and of Flavor Changing Neutral Currents can be simultaneously solved.

Bertuzzo, Enrico; Ray, Tirtha Sankar; de Sandes, Hiroshi; Savoy, Carlos A.

2013-05-01

162

Engineering oscillating microtubule bundles.  

PubMed

From motility of simple protists to determining the handedness of complex vertebrates, highly conserved eukaryotic cilia and flagella are essential for the reproduction and survival of many biological organisms. Despite extensive studies, the exact mechanism by which individual components coordinate their activity to produce ciliary beating patterns remains unknown. We describe a novel approach toward studying ciliary beating. Instead of deconstructing a fully functional organelle from the top-down, we describe a process by which synthetic cilia-like structures are assembled from the bottom-up and we present methods for engineering such structures. We demonstrate how simple mixtures of microtubules, kinesin clusters, and a bundling agent assemble into structures that produce spontaneous oscillations, suggesting that self-organized beating may be a generic feature of internally driven bundles. Synthetic cilia-like structures can be assembled at high density, leading to synchronization and metachronal traveling waves, reminiscent of the waves seen in biological ciliary fields. PMID:23498742

Sanchez, Timothy; Dogic, Zvonimir

2013-01-01

163

Microtubule-depolymerizing kinesins.  

PubMed

The microtubule (MT) cytoskeleton supports a broad range of cellular functions, from providing tracks for intracellular transport, to supporting movement of cilia and flagella, to segregating chromosomes in mitosis. These functions are facilitated by the organizational and dynamic plasticity of MT networks. An important class of enzymes that alters MT dynamics is the depolymerizing kinesin-like proteins, which use their catalytic activities to regulate MT end dynamics. In this review, we discuss four topics surrounding these MT-depolymerizing kinesins. We provide a historical overview of studies focused on these motors and discuss their phylogeny. In the second half, we discuss their enzymology and biophysics and give an overview of their known cellular functions. This discussion highlights the fact that MT-depolymerizing kinesins exhibit a diverse range of design principles, which in turn increases their functional versatility in cells. PMID:23875646

Walczak, Claire E; Gayek, Sophia; Ohi, Ryoma

2013-07-17

164

Possible multiple chiral doublet bands in 107Ag  

NASA Astrophysics Data System (ADS)

Two pairs of nearly degenerate doublet bands in 107Ag are studied via the relativistic mean-field (RMF) theory and the multiparticle plus rotor model (PRM), which suggests these bands as two distinct sets of chiral doublet bands. For the suggested ?g9/2-1??h11/22 and ?g9/2-1??h11/2d5/2 configurations, the favorable triaxial deformation ? for nuclear chirality can be obtained from the configuration-fixed constrained triaxial RMF calculations. Adopting the PRM, the data available are reproduced very well for the two pairs of doublet bands. Chiral geometry is further conformed by analyzing the angular momentum components. We suggest that two pairs of doublet bands in 107Ag would be another example of multiple chiral doublet bands.

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

2013-08-01

165

Ap58: A novel in situ outer dynein arm-binding protein  

Microsoft Academic Search

Outer arm dynein is a molecular motor that is positioned at 24nm intervals on outer doublet microtubules in cilia and flagella. In the present paper, we report identification of a 58kDa novel protein with a tetratricopeptide repeat (TPR), referred to as ap58 (for 58kDa axonemal protein) in sea urchin sperm axonemes. Ap58 is extracted along with the outer arm dynein

Kazuo Ogawa; Kazuo Inaba

2006-01-01

166

Doublet Ridge Formation on Europa: Evidence from Topographic Data  

NASA Astrophysics Data System (ADS)

Galileo images show that in many places the surface of Europa is dominated by plains of densely criss-crossing doublet ridges. In most doublet ridges a central V-shaped trough divides the two opposing ridge flanks, which are roughly bilaterally symmetric. Several formational mechanisms for doublet ridges have been proposed. Some models involve new material ejected or squeezed through cracks to the surface, forming twin debris piles on either side of the crack. These models generally require liquid water very near or at the surface at the time of doublet ridge formation. Other models attribute doublet ridge relief to the upwarping of pre-exisiting terrain on either side of a crack. These models require solid-state deformation of the surface and vertical movement of solid-state material in the near-subsurface, but do not require the presence of liquid water as close to the surface as in the first group of models. Thus doublet ridge formation has important implications for whether substantial liquid water influenced the formation of surface features on Europa. So far, this question remains controversial and more evidence is required. Additional evidence was provided by Galileo stereo observations (56m/pxl, 29m/pxl) of a doublet ridge located at (195W, 16S). The stereo images allowed to reconstruct the doublet ridge topography at a resolution of about 200m. Based on the topographic data, there was clear evidence that this doublet ridge was formed by upwarping of terrain rather than piling up of debris. Specifically, we found preexisting terrain features on one of the doublet ridge flanks and a pronounced asymmetry in slopes between the trough and the flanks. The upwarping angle is about 20deg.

Giese, B.; Wagner, R.; Neukum, G.; Sullivan, R.; Galileo SSI Team

1999-09-01

167

Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules.  

PubMed

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48-50 kDa in isolated spindles and centrosomes from CHO cells. PMID:7983171

Steffen, W; Fajer, E A; Linck, R W

1994-08-01

168

STRUCTURE OF CORTICAL MICROTUBULE ARRAYS  

Microsoft Academic Search

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.

A. R. HARDHAM; B. E. S. GUNNING

169

Microtubule and Cell Shape Determination  

Microsoft Academic Search

\\u000a The ordered organization of cortical microtubules promotes directional expansion of plant cells, and thus is a critical determinant\\u000a of cell shape. Changes in cell shape in turn lead to the positional differences of the mechanical stress, which may align\\u000a cortical microtubules. A large fraction of morphological mutants with altered cell shapes are caused by defects in organization\\u000a of cortical arrays,

Takashi Hashimoto

170

Unconventional functions of microtubule motors.  

PubMed

With the functional characterization of proteins advancing at fast pace, the notion that one protein performs different functions - often with no relation to each other - emerges as a novel principle of how cells work. Molecular motors are no exception to this new development. Here, we provide an account on recent findings revealing that microtubule motors are multifunctional proteins that regulate many cellular processes, in addition to their main function in transport. Some of these functions rely on their motor activity, but others are independent of it. Of the first category, we focus on the role of microtubule motors in organelle biogenesis, and in the remodeling of the cytoskeleton, especially through the regulation of microtubule dynamics. Of the second category, we discuss the function of microtubule motors as static anchors of the cargo at the destination, and their participation in regulating signaling cascades by modulating interactions between signaling proteins, including transcription factors. We also review atypical forms of transport, such as the cytoplasmic streaming in the oocyte, and the movement of cargo by microtubule fluctuations. Our goal is to provide an overview of these unexpected functions of microtubule motors, and to incite future research in this expanding field. PMID:22306515

Muresan, Virgil; Muresan, Zoia

2012-01-28

171

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

172

Reconstitution of flagellar sliding.  

PubMed

The motile structure within eukaryotic cilia and flagella is the axoneme. This structure typically consists of nine doublet microtubules arranged around a pair of singlet microtubules. The axoneme contains more than 650 different proteins that have structural, force-generating, and regulatory functions. Early studies on sea urchin sperm identified the force-generating components, the dynein motors. It was shown that dynein can slide adjacent doublet microtubules in the presence of ATP. How this sliding gives rise to the beating of the axoneme is still unknown. Reconstitution assays provide a clean system, free from cellular effects, to elucidate the underlying beating mechanisms. These assays can be used to identify the components that are both necessary and sufficient for the generation of flagellar beating. PMID:23498749

Alper, Joshua; Geyer, Veikko; Mukundan, Vikram; Howard, Jonathon

2013-01-01

173

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

PubMed Central

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

1996-01-01

174

Signals of inert doublet dark matter in neutrino telescopes  

SciTech Connect

One of the simplest extensions of the standard model that explains the observed abundance of dark matter is the inert doublet model. In this theory a discrete symmetry ensures that the neutral component of an additional electroweak doublet scalar is stable and constitutes a dark matter candidate. As massive bodies such as the Sun and Earth move through the dark matter halo, dark matter particles can become gravitationally trapped in their cores. Annihilations of these particles result in neutrinos, which can potentially be observed with neutrino telescopes. We calculate the neutrino detection rate at these experiments from inert doublet dark matter annihilations in the cores of the Sun and the Earth.

Agrawal, Prateek [Department of Physics, University of Maryland, College Park, Maryland 20742 (United States); Dolle, Ethan M. [Department of Physics, University of Arizona, Tucson, Arizona 85721 (United States); Krenke, Christopher A. [Department of Physics, University of Maryland, College Park, Maryland 20742 (United States); Department of Physics, University of Arizona, Tucson, Arizona 85721 (United States)

2009-01-01

175

Surface Engineering for Microtubule Manipulation  

NASA Astrophysics Data System (ADS)

Microtubule filaments act as dynamic structures inside cells for cargo transport and cell motility. We have used self-assembled monolayers and lithographic techniques to control surface interactions between microtubules and synthetic substrates. Switchable protein adsorption has been achieved using an electrode coated with a non-fouling polyethylene glycol self-assembled silane monolayer (SAM). Novel integration of the SAM into current electron-beam lithography techniques has allowed for the underlying electrode to be patterned with much freedom of geometry while preventing permanent adsorption of the protein. In this configuration, microtubules assemble on top of the patterned electrode but are blocked from the surrounding regions. The reversible adsorption permits study of microtubules under spatially controlled electric fields. Furthermore, such active test surfaces can be used to study microtubule assembly and to simulate kinesin motor transport in neurons. This method is also compatible with DNA and other biomolecules and, unlike soft lithography, can be scaled down to tens of nanometers in a straightforward manner.

Noel, John; Teizer, Winfried; Hwang, Wonmuk

2007-10-01

176

Constraints on the two-Higgs-doublet model  

NASA Astrophysics Data System (ADS)

The two-Higgs-doublet model provides a simple, yet interesting, generalization of the SM Higgs sector. We study the CP-conserving version of this model with general, flavor-diagonal, Yukawa couplings. Indirect constraints are obtained from flavor physics on the charged Higgs boson mass and couplings. The relation of these bounds to those for the more specialized two-Higgs-doublet model types with a Z2 symmetry is discussed.

Sta?L, Oscar

2010-02-01

177

Constraints on the two-Higgs-doublet model  

SciTech Connect

The two-Higgs-doublet model provides a simple, yet interesting, generalization of the SM Higgs sector. We study the CP-conserving version of this model with general, flavor-diagonal, Yukawa couplings. Indirect constraints are obtained from flavor physics on the charged Higgs boson mass and couplings. The relation of these bounds to those for the more specialized two-Higgs-doublet model types with a Z{sub 2} symmetry is discussed.

Staal, Oscar [Department of Physics and Astronomy, Uppsala University, Box 516, SE-751 20 Uppsala (Sweden)

2010-02-10

178

Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro  

Microsoft Academic Search

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biologi- cal, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug

Eric J. Aamodt; Joseph G. Culotti

1986-01-01

179

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

180

Structure of kinetochore fibers: Microtubule continuity and inter-microtubule bridges  

Microsoft Academic Search

To understand how microtubules interact in forming the mitotic apparatus and orienting and moving chromosomes, the precise arrangement of microtubules in kinetochore fibers in Chinese hamster ovary cells was examined. Individual microtubules were traced, using high voltage electron microscopy of serial 0.25 µm sections, from the kinetochore toward the pole. Microtubule arrangement in kinetochore fibers in untreated mitotic cells and

Patricia L. Witt; Hans Ris; Gary G. Borisy

1981-01-01

181

Regulation of Microtubule Stability in Breast Cancer.  

National Technical Information Service (NTIS)

The dynamic turnover of microtubules within the cell is essential for a number of cellular processes including progression through the cell cycle and intracellular transport. A number of chemotherapeutic drugs target microtubules and disrupt their turnove...

S. M. Hanash

1999-01-01

182

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

PubMed Central

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

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

2009-01-01

183

Optimization of isopolar microtubule arrays.  

PubMed

Isopolar arrays of aligned cytoskeletal filaments are components in a number of designs of hybrid nanodevices incorporating biomolecular motors. For example, a combination of filament arrays and motor arrays can form an actuator or a molecular engine resembling an artificial muscle. Here, isopolar arrays of microtubules are fabricated by flow alignment, and their quality is characterized by their degree of alignment. We find, in agreement with our analytical models, that the degree of alignment is ultimately limited by thermal forces, while the kinetics of the alignment process are influenced by the flow strength, the microtubule stiffness, the gliding velocity, and the tip length. Strong flows remove microtubules from the surface and reduce the filament density, suggesting that there is an optimal flow strength for the fabrication of ordered arrays. PMID:23330965

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

2013-02-06

184

Template synthesis of organic microtubules  

SciTech Connect

Organic microtubules have recently caused a great deal of excitement in the physics, chemistry, and materials science communities. Such tubules are inherently intriguing chemical systems and have myriad proposed technolgical applications; they may also be useful as mimics of biological microtubules. The only known synthetic route for preparing organic microtubles involves extremely expensive reagents and produces tubules with a broad range of diameters and lengths. The authors have recently discovered a new method for synthesizing organic microtubules. This method uses a microporous membrane as a template during tubule synthesis. The most significant advantage of this template method is that it yields tubules with monodisperse diameters and lengths. They describe this template synthetic method in this paper.

Martin, C.R.; Van Dyke, L.S.; Cai, Z.; Liang, W. (Colorado State Univ., Fort Collins (USA))

1990-11-21

185

Making more microtubules by severing: a common theme of noncentrosomal microtubule arrays?  

PubMed Central

Two related enzymes, katanin and spastin, use the energy from ATP hydrolysis to sever microtubules. Two new studies (one in this issue; see McNally et al., p. 881) show that microtubule severing by katanin provides a means for increasing microtubule density in meiotic spindles. Interestingly, loss of spastin leads to a sparser microtubule array in axons and synaptic boutons. Together, these studies hint at a wider role for microtubule-severing enzymes in the formation and organization of noncentrosomal microtubule arrays by generating new seeds for microtubule growth.

Roll-Mecak, Antonina; Vale, Ronald D.

2006-01-01

186

The chirality of ciliary beats  

Microsoft Academic Search

Many eukaryotic cells possess cilia which are motile, whip-like appendages that can oscillate and thereby induce motion and fluid flows. These organelles contain a highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of nine doublets of microtubules. Complex bending waves emerge from the interplay of active internal forces generated by dynein motor proteins

A. Hilfinger; F. Jülicher

2008-01-01

187

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.

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

2012-01-01

188

The dynamic kinetochore-microtubule interface.  

PubMed

The kinetochore is a control module that both powers and regulates chromosome segregation in mitosis and meiosis. The kinetochore-microtubule interface is remarkably fluid, with the microtubules growing and shrinking at their point of attachment to the kinetochore. Furthermore, the kinetochore itself is highly dynamic, its makeup changing as cells enter mitosis and as it encounters microtubules. Active kinetochores have yet to be isolated or reconstituted, and so the structure remains enigmatic. Nonetheless, recent advances in genetic, bioinformatic and imaging technology mean we are now beginning to understand how kinetochores assemble, bind to microtubules and release them when the connections made are inappropriate, and also how they influence microtubule behaviour. Recent work has begun to elucidate a pathway of kinetochore assembly in animal cells; the work has revealed that many kinetochore components are highly dynamic and that some cycle between kinetochores and spindle poles along microtubules. Further studies of the kinetochore-microtubule interface are illuminating: (1) the role of the Ndc80 complex and components of the Ran-GTPase system in microtubule attachment, force generation and microtubule-dependent inactivation of kinetochore spindle checkpoint activity; (2) the role of chromosomal passenger proteins in the correction of kinetochore attachment errors; and (3) the function of microtubule plus-end tracking proteins, motor depolymerases and other proteins in kinetochore movement on microtubules and movement coupled to microtubule poleward flux. PMID:15509863

Maiato, Helder; DeLuca, Jennifer; Salmon, E D; Earnshaw, William C

2004-11-01

189

Microtubule particle dispersion in liquid crystal hosts  

Microsoft Academic Search

Microtubule particles and metal-coated microtubules were dispersed in various host liquid crystal mixtures. Dispersion effects were evaluated as a function of liquid crystal type, viscosity, dielectric anisotropy and surface interaction. Experimental results indicated that all the types of liquid crystals studied were aligned perpendicular to the microtubule surfaces, regardless of liquid crystal composition or various surface coatings used on the

A. M. Lackner; K. C. Lim; J. D. Margerum; E. Sherman

1993-01-01

190

Centrosome composition and microtubule anchoring mechanisms  

Microsoft Academic Search

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

Michel Bornens

2002-01-01

191

Microtubule assembly nucleated by isolated centrosomes  

Microsoft Academic Search

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

Tim Mitchison; Marc Kirschner

1984-01-01

192

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

193

Axonemal Beta Heavy Chain Dynein DNAH9: cDNA Sequence, Genomic Structure, and Investigation of Its Role in Primary Ciliary Dyskinesia  

Microsoft Academic Search

Dyneins are multisubunit protein complexes that couple ATPase activity with conformational changes. They are involved in the cytoplasmatic movement of organelles (cytoplasmic dyneins) and the bending of cilia and flagella (axonemal dyneins). Here we present the first complete cDNA and genomic sequences of a human axonemal dynein beta heavy chain gene, DNAH9, which maps to 17p12. The 14-kb-long cDNA is

Lucia Bartoloni; Jean-Louis Blouin; Amit K. Maiti; Amanda Sainsbury; Colette Rossier; Corinne Gehrig; Jin-Xiong She; Michele P. Marron; Eric S. Lander; Maggie Meeks; Eddie Chung; Miquel Armengot; Mark Jorissen; Hamish S. Scott; Celia D. Delozier-Blanchet; R. Marc Gardiner; Stylianos E. Antonarakis

2001-01-01

194

Immunological dissimilarity in protein component (dynein 1) between outer and inner arms within sea urchin sperm axonemes  

Microsoft Academic Search

The 0.5 M KCI-treatment solubilizes the outer arms from sea urchin sperm axo- nemes. -30% of A-polypeptide, corresponding to dynein 1 in SDS-polyacrylamide gel, was solubilized by this treatment (as SEA-dynein 1) . Electron microscopic observation indicated that the extracted axonemes lacked the outer arms in various degrees. The SEA-dynein 1 was purified and an antiserum against it was prepared

KAZUO OGAWA; SUMIKO NEGISHI; MASATAKA OBIKA

1982-01-01

195

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

PubMed

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 with 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

2012-05-22

196

Dynein-ADP as a force-generating intermediate revealed by a rapid reactivation of flagellar axoneme.  

PubMed Central

Fragmented flagellar axonemes of sand dollar spermatozoa were reactivated by rapid photolysis of caged ATP. After a time lag of 10 ms, axonemes treated with protease started sliding disintegration. Axonemes without protease digestion started nanometer-scale high-frequency oscillation after a similar time lag. Force development in the sliding disintegration was measured with a flexible glass needle and its time course was corresponded well to that of the dynein-ADP intermediate production estimated using kinetic rates previously reported. However, with a high concentration ( approximately 80 microM) of vanadate, which binds to the dynein-ADP intermediate and forms a stable complex of dynein-ADP-vanadate, the time course of force development in sliding disintegration was not affected at all. In the case of high frequency oscillation, the time lag to start the oscillation, the initial amplitude, and the initial frequency were not affected by vanadate, though the oscillation once started was damped more quickly at higher concentrations of vanadate. These results suggest that during the initial turnover of ATP hydrolysis, force generation of dynein is not blocked by vanadate. A vanadate-insensitive dynein-ADP is postulated as a force-generating intermediate.

Tani, T; Kamimura, S

1999-01-01

197

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.

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

2012-01-01

198

Posttranslational modifications regulate microtubule function  

Microsoft Academic Search

The ??-tubulin heterodimer, the building block of microtubules, is subject to a large number of post-translational modifications, comparable in diversity to the intensively studied histone modifications. Although these unusual modifications are conserved throughout evolution, their functions have remained almost completely elusive. Recently, however, important advances in the understanding of how tubulin modifications regulate function and organization have been made.

Klaus Weber; Stefan Westermann

2003-01-01

199

Information processing in brain microtubules  

Microsoft Academic Search

Models of the mind are based on the idea that neuron microtubules can perform computation. From this point of view, information processing is the fundamental issue for understanding the brain mechanisms that produce consciousness. The cytoskeleton polymers could store and process information through their dynamic coupling mediated by mechanical energy. We analyze the problem of information transfer and storage in

Jean Faber; Renato Portugal; Luiz Pinguelli Rosa

2006-01-01

200

Template Synthesis of Metal Microtubules.  

National Technical Information Service (NTIS)

We have recently described a template method for the synthesis of organic microtubules. This method entails the use of the pores in a microporous membrane as templates for tubule formation. The key to the tubule-formation process in the presence of molecu...

C. J. Brumlik C. R. Martin

1991-01-01

201

Microtubule dynamics regulation contributes to endothelial morphogenesis  

PubMed Central

Because little is known how microtubules contribute to cell migration in a physiological three-dimensional environment, we analyzed microtubule function and dynamics during in vitro angiogenesis in which endothelial cells form networks on a reconstituted basement membrane. Endothelial network formation resulted from distinct cell behaviors: matrix reorganization by myosin-mediated contractile forces, and active cell migration along reorganized, bundled matrix fibers. Inhibition of microtubule dynamics inhibited persistent cell migration, but not matrix reorganization. In addition, microtubule polymerization dynamics and CLASP2-binding to microtubules were spatially regulated to promote microtubule growth into endothelial cell protrusions along matrix tension tracks. We propose that microtubules counter-act contractile forces of the cortical actin cytoskeleton and are required to stabilize endothelial cell protrusions in a soft three-dimensional environment.

Lyle, Karen S.; Corleto, Jose A.; Wittmann, Torsten

2012-01-01

202

Microtubule nucleation: gamma-tubulin and beyond.  

PubMed

Centrosomes and their fungal equivalents, spindle pole bodies (SPBs), are the main microtubule (MT)-organizing centers in eukaryotic cells. Several proteins have been implicated in microtubule formation by centrosomes and SPBs, including microtubule-minus-end-binding proteins and proteins that bind along the length or stabilize the plus ends of microtubules. Recent work has improved our understanding of the molecular mechanisms of MT formation. In particular, it has shown that gamma-tubulin and its associated proteins play key roles in microtubule nucleation and spindle assembly in evolutionarily distant species ranging from fungi to mammals. Other work indicates that gamma-tubulin-mediated microtubule nucleation, although necessary, is not sufficient for mitotic spindle assembly but requires additional proteins that regulate microtubule nucleation independently of centrosomes. PMID:17038541

Wiese, Christiane; Zheng, Yixian

2006-10-15

203

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

204

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

PubMed Central

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

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

2012-01-01

205

The CSC connects three major axonemal complexes involved in dynein regulation  

PubMed Central

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

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

2012-01-01

206

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

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

1994-01-01

207

DYX1C1 is required for axonemal dynein assembly and ciliary motility.  

PubMed

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2-4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4). PMID:23872636

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

2013-07-21

208

MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS  

PubMed Central

The role of microtubules and microtubule nucleating sites in the unicell, Ochromonas has been examined through the use of two mitotic inhibitors, isopropyl N-phenylcarbamate (IPC) and isopropyl N-3-chlorophenyl carbamate (CIPC). Although IPC and CIPC have little or no effect on intact microtubules, the assembly of three separate sets of microtubules in Ochromonas has been found to be differentially affected by IPC and CIPC. The assembly of flagellar microtubules after mechanical deflagellation is partially inhibited; the reassembly of rhizoplast microtubules after pressure depolymerization is totally inhibited (however, macrotubules may form at the sites of microtubule initiation or elsewhere); and, the reassembly of the beak set of microtubules after pressure depolymerization may be unaffected although similar concentrations of IPC and CICP completely inhibit microtubule regeneration on the rhizoplast. These effects on microtubule assembly, either inhibitory or macrotubule inducing, are fully reversible. The kinetics of inhibition and reversal are found to be generally similar for both flagellar and cell shape regeneration. Incorporation data suggest that neither IPC nor CIPC has significant effects on protein synthesis in short term experiments. Conversely, inhibiting protein synthesis with cycloheximide has little effect on microtubule regeneration when IPC or CIPC is removed. Although the exact target for IPC and CIPC action remains uncertain, the available evidence suggests that the microtubule protein pool or the microtubule nucleating sites are specifically and reversibly affected. Comparative experiments using the mitotic inhibitor colchicine indicate some similarities and differences in its mode of action with respect to that of IPC and CIPC on assembly and disassembly of microtubules in these cells.

Brown, David L.; Bouck, G. Benjamin

1974-01-01

209

Two-Higgs-doublet models with Minimal Flavour Violation  

SciTech Connect

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

Carlucci, Maria Valentina [Physik-Department, Technische Universitaet Muenchen, James-Franck-Strasse, D-85748 Garching (Germany)

2010-12-22

210

SUSY GUT with automatic doublet-triplet hierarchy  

NASA Astrophysics Data System (ADS)

A mechanism is suggested to resolve the problem of doublet-triplet hierarchy in the SU(5) SUSY GUT. In this mechanism, which we call ``GIFT'' (``Goldstones instead of fine tuning''), the doublets are pseudo-Goldstone bosons of a certain broken global symmetry of the superpotential. With supergravity as the source of SUSY breaking we show that the SU(2)L×U(1)Y-->U(1)em breaking as well as the mass of the lightest Higgs boson are due to the radiative corrections.

Anselm, A. A.; Johansen, A. A.

1988-01-01

211

Magnetic quadrupole doublet focusing system for high energy ions  

SciTech Connect

A high energy focused ion beam microprobe using a doublet arrangement of short magnetic quadrupole lenses was used to focus 1-3 MeV protons to spot sizes of 1x1 {mu}m{sup 2} and 1-4.5 MeV carbon and silicon ion beams to spot sizes of 1.5x1.5 {mu}m{sup 2}. The results presented clearly demonstrate that this simple doublet configuration can provide high energy microbeams for microananalysis and microfabrication applications.

Glass, Gary A.; Dymnikov, Alexander D.; Dias, Johnny F.; Houston, Louis M.; LeBlanc, Jared [Louisiana Accelerator Center/Physics Department, The University of Louisiana at Lafayette, P.O. Box 44210, Lafayette, Louisiana 70504-4210 (United States); Rout, Bibhudutta [Department of Physics, University of North Texas, P.O. Box 311427, Denton, Texas 76203 (United States)

2008-03-15

212

Chapter 14 Monitoring Temporal Variations of Physical Properties in the Crust by Cross?Correlating the Waveforms of Seismic Doublets  

Microsoft Academic Search

Doublets or multiplets are earthquakes with nearly identical waveforms. First observed on volcanoes, doublets are found in tectonic environments. Doublets can be relocated relatively with a precision of a few meters. Very good doublets separated by a large time lapse are essential for detecting slow temporal variations of crustal properties. We present basic techniques for selecting and processing doublets. In

Georges Poupinet; Florent Brenguier

2008-01-01

213

Glycolytic enzymes and assembly of microtubule networks.  

PubMed

The ability of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase (PK), and lactate dehydrogenase muscle-type (LDH(M)), to generate interactive microtubule networks was investigated. Bundles have previously been defined as the parallel alignment of several microtubules and are one form of microtubule networks. Utilizing transmission electron microscopy, interactive networks of microtubules as well as bundles were readily observed in the presence of GAPDH, aldolase, or PK. These networks appear morphologically as cross-linked microtubules, oriented in many different ways. Light scattering indicated that the muscle forms of GAPDH, aldolase, PK and LDH(m) caused formation of the microtubule networks. Triose phosphate isomerase (TPI) and lactate dehydrogenase heart-type (LDH(H)), glycolytic enzymes which do not interact with tubulin or microtubules, did not produce bundles, or interactive networks. Sedimentation experiments confirmed that the enzymes that cross-link also co-pellet with the microtubules. Such cross-linking of microtubules indicate that the enzymes are multivalent with the capability of simultaneous binding to more than one microtubule. PMID:8529027

Volker, K W; Reinitz, C A; Knull, H R

1995-11-01

214

Microtubule-associated proteins from Antarctic fishes.  

PubMed

Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization. PMID:1980093

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

1990-01-01

215

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

PubMed

Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert on microtubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process. PMID:23674690

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

2013-05-14

216

Multiscale Trend Analysis of Microtubule Transport in Melanophores  

Microsoft Academic Search

Microtubule-based transport is critical for trafficking of organelles, organization of endomembranes, and mitosis. The driving force for microtubule-based transport is provided by microtubule motors, which move organelles specifically to the plus or minus ends of the microtubules. Motor proteins of opposite polarities are bound to the surface of the same cargo organelle. Transport of organelles along microtubules is discontinuous and

Ilya Zaliapin; Irina Semenova; Anna Kashina; Vladimir Rodionov

2005-01-01

217

+TIPs: SxIPping along microtubule ends  

PubMed Central

+TIPs are a heterogeneous class of proteins that specifically bind to growing microtubule ends. Because dynamic microtubules are essential for many intracellular processes, +TIPs likely play important roles in regulating microtubule dynamics and microtubule interactions with other intracellular structures. End-binding proteins (EBs) recognize a structural cap at growing microtubule ends, and have emerged as central adaptors that mediate microtubule plus-end-tracking of potentially all other +TIPs. The majority of these +TIPs bind EBs through a short hydrophobic SxIP sequence motif and surrounding electrostatic interactions. These recent discoveries have resulted in a rapid expansion of the number of possible +TIPs. In this review, we outline our current understanding of the molecular mechanism of plus-end-tracking and provide an overview of SxIP-recruited +TIPs.

Kumar, Praveen; Wittmann, Torsten

2012-01-01

218

Microtubule targeting agents: from biophysics to proteomics.  

PubMed

This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin polymerization and microtubule targeting agent binding, we then highlight how the three-dimensional structures of drug-tubulin complexes obtained on stabilized tubulin are seeded by precise biological and biophysical data. New avenues opened by thermodynamics analysis, high throughput screening, and proteomics for the molecular pharmacology of these drugs are presented. The amount of data generated by biophysical, proteomic and cellular techniques shed more light onto the microtubule-tubulin equilibrium and tubulin-drug interaction. Combining these approaches provides new insight into the mechanism of action of known microtubule interacting agents and rapid in-depth characterization of next generation molecules targeting the interaction between microtubules and associated modulators of their dynamics. This will facilitate the design of improved and/or alternative chemotherapies targeting the microtubule cytoskeleton. PMID:20107862

Calligaris, D; Verdier-Pinard, P; Devred, F; Villard, C; Braguer, D; Lafitte, Daniel

2010-01-28

219

Beta and Current Limits in the Doublet III Tokamak.  

National Technical Information Service (NTIS)

Neutral-beam heated discharges in Doublet III exhibit an operational beta limit, beta /sub T/(%) less than or equal to 3.5 I(MA)/a(m)B(T), in good agreement with several theoretical predictions for ideal external kink or ballooning modes. These theories p...

E. J. Strait M. S. Chu G. L. Jahns J. S. Kim A. G. Kellman

1986-01-01

220

Weak magnetic dipole moments in two-Higgs-doublet models  

Microsoft Academic Search

We investigate the effects of the new scalars in a two-Higgs-doublet model on the weak magnetic dipole moments of the fermions at the Z peak. The proportionality of the Yukawa couplings to the fermion masses, and to tanbeta, makes such effects more important for the third family, and potentially relevant. For the tau lepton, the new diagrams are suppressed by

J. Bernabéu; D. Comelli; L. Lavoura; João P. Silva

1996-01-01

221

Weak magnetic dipole moments in two-Higgs-doublet models  

Microsoft Academic Search

We investigate the effects of the new scalars in a two-Higgs-doublet model on the weak magnetic dipole moments of the fermions at the {ital Z} peak. The proportionality of the Yukawa couplings to the fermion masses, and to tan, makes such effects more important for the third family, and potentially relevant. For the lepton, the new diagrams are suppressed by

J. Bernabeu; D. Comelli; L. Lavoura; Joao P. Silva

1996-01-01

222

The 2008 May 29 earthquake doublet in SW Iceland  

Microsoft Academic Search

On 2008 May 29 an earthquake doublet shook the southwestern part of Iceland. The first main shock originated beneath Mt Ingólfsfjall, located near the western margin of the South Iceland Seismic Zone (SISZ) approximately 40 km east of the capital Reykjavík. Immediate aftershock activity was recorded by the SIL seismic network, operated by the Icelandic Meteorological Office (IMO), with both

J. Decriem; T. Árnadóttir; A. Hooper; H. Geirsson; F. Sigmundsson; M. Keiding; B. G. Ófeigsson; S. Hreinsdóttir; P. Einarsson; P. Lafemina; R. A. Bennett

2010-01-01

223

Performance of Doublet III neutral beam injector cryopumping system  

Microsoft Academic Search

The Doublet III neutral beam injector system is based on three beamlines; each beamline employs two 80 kV\\/80 A hydrogen ion sources. Two liquid helium (LHe) cooled cryopanel arrays were designed as an integral part of the beamline in order to provide high differential pumping of hydrogen gas along the beamline. The cryopanel arrays consist of a front (nearer to

A. R. Langhorn; J. Kim; M. L. Tupper; J. P. Williams; J. Fasolo

1984-01-01

224

The Doublet III Neutral Beam Source Cryopanel System  

Microsoft Academic Search

A cryogenic pumping system is designed to provide 1.4 ?? 106 l\\/sec. of hydrogen pumping speed for the Doublet III neutral beam ion source. The cryopump is made up of two elements; a cylindrical unit which uses a \\

Jack Tanabe; Robert Yamamoto; Peter Vander Arend

1979-01-01

225

Thermal Contact Conductance Measurements on Doublet III Armor Tile Graphite.  

National Technical Information Service (NTIS)

Several tests were performed on the Doublet III wall armor tiles to determine the cool-down rate and to evaluate improvements made by changing the conditions at the interface between the graphite tile and the stainless steel backing plate. Thermal diffusi...

D. W. Doll E. Reis

1983-01-01

226

Microtubules of guard cells are light sensitive.  

PubMed

Guard cells of stomata are characterized by ordered bundles of microtubules radiating from the ventral side toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial arrangement of the cellulose micro-fibrils of guard cells. However, the role of microtubules in daily cycles of opening and closing of stomata is not clear. The organization of microtubules in guard cells of Commelina communis leaves was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were organized in parallel, straight and dense bundles, in the dark they were less straight and oriented randomly near the stomatal pore. The effect of blue and red light on the organization of guard cell microtubules resembled the effects of white light and dark respectively. When stomata were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore, when incubated in the light, guard cell microtubules were more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein-tubulin alpha 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The dynamics of microtubule rearrangement upon transfer of intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light. Our data suggest that microtubule organization in guard cells is responsive to light signals. PMID:15169939

Lahav, Maoz; Abu-Abied, Mohamad; Belausov, Eduard; Schwartz, Amnon; Sadot, Einat

2004-05-01

227

Molecular Mechanisms of Microtubule Acting Cancer Drugs  

Microsoft Academic Search

Here the molecular mechanism of antimitotic drugs, biological compounds that bind to tubulin and microtubules and suppress\\u000a microtubule dynamics are reviewed. A common feature of tubulin-interacting compounds is that binding to tubulin is linked\\u000a to assembly, either the stabilization of a microtubule lattice by compounds like the taxanes and epothilones, or the induction\\u000a of alternate, nonmicrotubule polymer forms. The nonmicrotubule

John J. Correia; Sharon Lobert

228

Microtubule Interaction Site of the Kinesin Motor  

Microsoft Academic Search

Kinesin and myosin are motor proteins that share a common structural core and bind to microtubules and actin filaments, respectively. While the actomyosin interface has been well studied, the location of the microtubule-binding site on kinesin has not been identified. Using alanine-scanning mutagenesis, we have found that microtubule-interacting kinesin residues are located in three loops that cluster in a patch

Günther Woehlke; Aaron K Ruby; Cynthia L Hart; Bernice Ly; Nora Hom-Booher; Ronald D Vale

1997-01-01

229

Microtubule-associated proteins of neurons  

Microsoft Academic Search

Microtubule-associated proteins (MAP) have been identified in cultures of rat sympathetic neurons. In all of the experiments performed here, the cultures consisted of >97% neurons. 26 proteins were identified in these neuronal cultures that (a) remained associated with cytoskeletons prepared with a Triton X-100-containing microtubule-stabilizing buffer, (b) were released from such cytoskeletons by incubation in microtubule-depolymerizing buffers, (c) were not

MARK M. BLACK; JEFREY T. KURDYLA

1983-01-01

230

Role of nucleotide hydrolysis in microtubule assembly  

Microsoft Academic Search

MICROTUBULE assembly in vitro requires (in normal conditions) that GTP be bound to the exchangeable nucleotide-binding site of tubulin1-3. The bound GTP is hydrolysed during polymerisation and the resulting GDP remains bound to the microtubule while the phosphate is released into the medium. Although hydrolysis normally occurs during polymerisation, microtubules will form in a non-hydrolysable analogue of GTP, guanylyl imidodiphosphate

R. C. Weisenberg; W. J. Deery

1976-01-01

231

Novel Response to Microtubule Perturbation in Meiosis  

Microsoft Academic Search

During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G1 or G2, instead of metaphase. Cells arrest in G1 if microtubule perturbation

Andreas Hochwagen; Gunnar Wrobel; Marie Cartron; Philippe Demougin; Christa Niederhauser-Wiederkehr; Monica G. Boselli; Michael Primig; Angelika Amon

2005-01-01

232

Microtubule-targeting-dependent reorganization of filopodia.  

PubMed

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

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

2007-03-13

233

Temperature-dependent elasticity of microtubules.  

PubMed

Central to the biological function of microtubules is their ability to modify their length which occurs by addition and removal of subunits at the ends of the polymer, both in vivo and in vitro. This dynamic behavior is strongly influenced by temperature. Here, we show that the lateral interaction between tubulin subunits forming microtubule is strongly temperature dependent. Microtubules deposited on prefabricated substrates were deformed in an atomic force microscope during imaging, in two different experimental geometries. Microtubules were modeled as anisotropic, with the Young's modulus corresponding to the resistance of protofilaments to stretching and the shear modulus describing the weak interaction between the protofilaments. Measurements involving radial compression of microtubules deposited on flat mica confirm that microtubule elasticity depends on the temperature. Bending measurements performed on microtubules deposited on lithographically fabricated substrates show that this temperature dependence is due to changing shear modulus, implying that the lateral interaction between the protofilaments is strongly determined by the temperature. These measurements are in good agreement with previously reported measurements of the disassembly rate of microtubules, demonstrating that the mechanical and dynamic properties of microtubules are closely related. PMID:18494514

Kis, A; Kasas, S; Kulik, A J; Catsicas, S; Forró, L

2008-05-22

234

Extending the Microtubule/Microfibril Paradigm1  

PubMed Central

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.

Fisher, Deborah D.; Cyr, Richard J.

1998-01-01

235

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms.  

PubMed

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo. PMID:2415535

Piperno, G; Fuller, M T

1985-12-01

236

Precise source location of AE doublets by spectral matrix analysis of triaxial hodogram  

Microsoft Academic Search

We have developed a precise relative source loca- tion technique using acoustic emission doublets (AE doublets) in the triaxial hodogram method to evaluate the direction and distance of subsurface extension cracks. An AE doublet is a pair of acoustic emissions with similar waveforms and adjacent locations on the same crack but which occur at different times. The relative source location

Hirokazu Moriya; Koji Nagano; Hiroaki Niitsuma

1994-01-01

237

Taxanes, microtubules and chemoresistant breast cancer  

Microsoft Academic Search

The taxanes, paclitaxel and docetaxel are microtubule-stabilizing agents that function primarily by interfering with spindle microtubule dynamics causing cell cycle arrest and apoptosis. However, the mechanisms underlying their action have yet to be fully elucidated. These agents have become widely recognized as active chemotherapeutic agents in the treatment of metastatic breast cancer and early-stage breast cancer with benefits gained in

Barbara T. McGrogan; Breege Gilmartin; Desmond N. Carney; Amanda McCann

2008-01-01

238

GTPase activity at ends of microtubules  

Microsoft Academic Search

MICROTUBULES are involved in many important biological processes, including cell motility, cell division, morphogenesis and axonal transport, and it is of fundamental interest to understand the mechanism and regulation of tubulin polymerisation in order to clarify their function. Previously we presented evidence that brain tubulin, devoid of microtubule associated proteins (MAPs) after fractionation by phosphocellulose chromatography, exhibits a characteristic GTPase

Therese David-Pfeuty; Jean Laporte; Dominique Pantaloni

1978-01-01

239

Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces: Teleostei).  

PubMed

The mature spermatozoon of Aponurus laguncula, a parasite of the unicorn leatherjacket Aluterus monoceros, was studied by transmission electron microscopy. The spermatozoon possesses 2 axonemes of the 9+"1" trepaxonematan pattern, attachment zones, a nucleus, a mitochondrion, external ornamentation of the plasma membrane and cortical microtubules. The major features are the presence of: 1) external ornamentation in the anterior part of the spermatozoon not associated with cortical microtubules; 2) one mitochondrion; and 3) cortical microtubules arranged as a single field in the ventral side. The maximum number of microtubules is in the nuclear region. The extremities of the axonemes are characterized by the disappearance of the central core and the presence of microtubule doublets or singlets. This study is the first undertaken with a member of the Lecithasteridae and exemplifies the sperm ultrastructure for the superfamily Hemiuroidea. PMID:19559102

Quilichini, Y; Foata, J; Justine, J-L; Bray, R A; Marchand, B

2009-06-24

240

Ciliary and flagellar structure and function--their regulations by posttranslational modifications of axonemal tubulin.  

PubMed

Eukaryotic cilia and flagella are evolutionarily conserved microtubule-based organelles protruding from the cell surface. They perform dynein-driven beating which contributes to cell locomotion or flow generation. They also play important roles in sensing as cellular antennae, which allows cells to respond to various external stimuli. The main components of cilia and flagella, ?- and ?-tubulins, are known to undergo various posttranslational modifications (PTMs), including phosphorylation, palmitoylation, tyrosination/detyrosination, ?2 modification, acetylation, glutamylation, and glycylation. Recent identification of tubulin-modifying enzymes, especially tubulin tyrosine ligase-like proteins which perform tubulin glutamylation and glycylation, has demonstrated the importance of tubulin modifications for the assembly and functions of cilia and flagella. In this chapter, we review recent work on PTMs of ciliary and flagellar tubulins in conjunction with discussing the basic knowledge. PMID:22364873

Konno, Alu; Setou, Mitsutoshi; Ikegami, Koji

2012-01-01

241

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

PubMed

Congenital hydrocephalus, the accumulation of excess cerebrospinal fluid (CSF) in the ventricles of the brain, affects one of every 1000 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 (Jhy(lacZ)) 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 Jhy(lacZ/lacZ) brains, and ultrastructural analysis revealed disorganization of the expected 9+2 microtubule pattern. Rather, the majority of Jhy(lacZ/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 Jhy(lacZ/lacZ) mice, and the Jhy transcript and protein are decreased in Jhy(lacZ/lacZ) mice. Partial phenotypic rescue was achieved in Jhy(lacZ/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

2013-07-29

242

Isolation and characterization of a novel dynein that contains C and A heavy chains from sea urchin sperm flagellar axonemes.  

PubMed

A novel dynein (C/A dynein), which is composed of C and A heavy chains, two intermediate chains and several light chains, was isolated from sea urchin sperm flagella. The C/A dynein was released by the treatment with 0.7 M NaCl plus 5 mM ATP from the axonemes depleted of outer arm 21 S dynein. Sedimentation coefficient of this dynein was estimated by sucrose density gradient centrifugation to be 22-23 S. The C/A dynein particle appeared to be composed of three distinct domains; two globular head domains and one rod domain as seen by negative staining electron microscopy. The mobility of 'A' heavy chain of C/A dynein on SDS-gel electrophoresis was similar to that of A heavy chains (A alpha and A beta) of 21 S dynein. However, UV-cleavage patterns of C and A heavy chains of C/A dynein were different from those of A heavy chains of 21 S dynein. Furthermore, an antiserum raised against A heavy chain of C/A dynein did not crossreact with A heavy chains of 21 S dynein. Under the conditions in which the C/A dynein was released, some of inner arms were removed concomitantly from axonemes as observed by electron microscopy. These results suggested that C/A dynein is a component of the inner arms. PMID:8207066

Yokota, E; Mabuchi, I

1994-02-01

243

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

244

Visualizing individual microtubules by bright field microscopy  

NASA Astrophysics Data System (ADS)

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

Gutiérrez-Medina, Braulio; Block, Steven M.

2010-11-01

245

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

246

New description of the doublet bands in doubly odd nuclei  

NASA Astrophysics Data System (ADS)

The experimentally observed ?I=1 doublet bands in some odd-odd nuclei are analyzed within the orthosymplectic extension of the interacting vector boson model (IVBM). A new, purely collective interpretation of these bands is given on the basis of the obtained boson-fermion dynamical symmetry of the model. It is illustrated by its application to three odd-odd nuclei from the A~130 region, namely Pr126, Pr134, and La132. The theoretical predictions for the energy levels of the doublet bands as well as E2 and M1 transition probabilities between the states of the yrast band in the last two nuclei are compared with experiment and the results of other theoretical approaches. The obtained results reveal the applicability of the orthosymplectic extension of the IVBM.

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

2009-04-01

247

Improved electroweak phase transition with subdominant inert doublet dark matter  

NASA Astrophysics Data System (ADS)

The inert doublet dark matter model has recently gained attention as a possible means of facilitating a strongly first-order electroweak phase transition (EWPT), as needed for baryogenesis. We extend previous results by considering the regime where the DM is heavier than half the Higgs mass, and its relic density is determined by annihilation into W, Z and Higgs bosons. We find a large natural region of parameter space where the EWPT is strongly first order, while the lightest inert doublet state typically contributes only 0.1%-3% of the total dark matter. Despite this small density, its interactions with nucleons are strong enough to be directly detectable given a factor of 5 improvement over the current sensitivity of XENON100. A 10% increase in the branching ratio for Higgs decays to two photons is predicted.

Cline, James M.; Kainulainen, Kimmo

2013-04-01

248

A doublet microlens array for imaging micron-sized objects.  

PubMed

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

Tripathi, A; Chronis, N

2011-09-21

249

Evidence for Multiple Chiral Doublet Bands in Ce133  

NASA Astrophysics Data System (ADS)

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

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

2013-04-01

250

Screening doublet law and X-ray satellite spectra  

Microsoft Academic Search

1. A critical examination of the existing data on X-ray satellite spectra has led to a fuller information regarding the occurrence of pairs of satellite lines in the K, L and M regions, showing more or less constant value of Delta&surd;nu\\/R, similar to the screening doublets in the X-ray energy levels of singly ionised atoms. 2. In the K region

G. B. Deodhar; B. D. Padalia

1963-01-01

251

Pseudospin doublet aligned structure in doubly odd 186Ir  

NASA Astrophysics Data System (ADS)

186Ir has been restudied through the 180Hf(11B,5n) reaction at 65 MeV using in-beam ?-ray and conversion-electron spectroscopy. The unfavored component of the doubly decoupled band was established and shown to be consistent with a description in terms of the ?h9/2??[411~1/2,3/2] structure, i.e., the coupling of an aligned proton and a neutron pseudospin doublet.

Cardona, M. A.; Debray, M. E.; García Bermúdez, G.; Hojman, D.; Kreiner, A. J.; Somacal, H.; Burlon, A.; Davidson, J.; Davidson, M.; Levinton, G.; Ozafrán, M.; Vázquez, M.; Napoli, D. R.; Rico, J.; Bazzacco, D.; Burch, R.; de Acuña, D.; Lenzi, S. M.; Medina, N.; Rossi Alvarez, C.; Blasi, N.; Lo Bianco, G.; de Boer, J.; Frischke, D.; Maier, H. J.

1997-01-01

252

Artificial apposition compound eye using aspherical cylindrical micro-doublets  

NASA Astrophysics Data System (ADS)

An ultrathin objective with the configuration of an artificial apposition compound eye was designed in order to obtain a miniaturized camera. The optical design was based in a nonconventional multi aperture configuration of a diurnal insect eye that uses ommatidia as individual units. An aspherical cylindrical micro doublet (CMD), obtained by ALSIE, is used in this optical design to improve the image quality in more than 200 percent in comparison with the first design with the spherical CMD.

Garza-Rivera, Anel; Renero-Carrillo, Francisco J.

2011-08-01

253

Reconstruction of inert doublet scalars at the international linear collider  

NASA Astrophysics Data System (ADS)

We study collider signatures for extra scalar bosons in the inert doublet model at the international linear collider (ILC). The inert doublet model is a simple extension of the standard model by introducing an additional isospin-doublet scalar field which is odd under an unbroken Z2 symmetry. The model predicts four kinds of Z2-odd scalar bosons, and the lightest of them becomes stable and a candidate of the dark matter as long as it is electrically neutral. Taking into account the constraints from various theoretical and phenomenological conditions, we perform a simulation study for the distinctive signatures of the extra scalars over the standard-model background contributions at the ILC with the center-of-mass energy of s=250 GeV and 500 GeV. We further discuss observables for determination of the mass of the scalars. We find that the parameter regions which cannot be detected at the large hadron collider can be probed at the ILC.

Aoki, Mayumi; Kanemura, Shinya; Yokoya, Hiroshi

2013-10-01

254

SHS Observations of the 3727Å O+ Doublet  

NASA Astrophysics Data System (ADS)

We present ground-based observations of the terrestrial O+ doublet (2D -- 4S) emission at 3726 and 3729 Å. These O+ emission lines were detected as part of a Galactic O+ interstellar medium program underway at the University of Wisconsin's Pine Bluff Observatory using a Field-Widened Spatial Heterodyne Spectrometer (FW-SHS). The FW-SHS produces Fizeau fringes by replacing the return mirrors in a Michelson interferometer with diffraction gratings. The FW-SHS combines interferometric and field-widening gains to achieve sensitivities much larger than conventional grating instruments of similar size and resolving power. We present the initial O+ FW-SHS observations, believed to be originating in the F region of the ionosphere. The emission intensities are estimated to be in the range of 0.5-1.5 R. Results from several observation periods ranging from December 2003 through October 2005 are discussed. We include the doublet ratio results for all observation periods as well as an implied seasonal variability in intensity. As this terrestrial O+ emission is a relatively unstudied phenomenon, we outline the methodology we are using to study this doublet and discuss its importance to the F region of the ionosphere.

Briczinski, S. J.; Mierkiewicz, E.; Roesler, F.; Nossal, S.

2009-05-01

255

Microtubule organization by kinesin motors and microtubule crosslinking protein MAP65.  

PubMed

Microtubules are rigid, proteinaceous filaments required to organize and rearrange the interior of cells. They organize space by two mechanisms, including acting as the tracks for long-distance cargo transporters, such as kinesin-1, and by forming a network that supports the shape of the cell. The microtubule network is composed of microtubules and a bevy of associated proteins and enzymes that self-organize using non-equilibrium dynamic processes. In order to address the effects of self-organization of microtubules, we have utilized the filament-gliding assay with kinesin-1 motors driving microtubule motion. To further enhance the complexity of the system and determine if new patterns are formed, we added the microtubule crosslinking protein MAP65-1. MAP65-1 is a microtubule-associated protein from plants that crosslinks antiparallel microtubules, similar to mammalian PRC1 and fission yeast Ase1. We find that MAP65 can slow and halt the velocity of microtubules in gliding assays, but when pre-formed microtubule bundles are added to gliding assays, kinesin-1 motors can pull apart the bundles and reconstitute cell-like protrusions. PMID:23945219

Pringle, Joshua; Muthukumar, Amutha; Tan, Amanda; Crankshaw, Laura; Conway, Leslie; Ross, Jennifer L

2013-08-15

256

Microtubule organization by kinesin motors and microtubule crosslinking protein MAP65  

NASA Astrophysics Data System (ADS)

Microtubules are rigid, proteinaceous filaments required to organize and rearrange the interior of cells. They organize space by two mechanisms, including acting as the tracks for long-distance cargo transporters, such as kinesin-1, and by forming a network that supports the shape of the cell. The microtubule network is composed of microtubules and a bevy of associated proteins and enzymes that self-organize using non-equilibrium dynamic processes. In order to address the effects of self-organization of microtubules, we have utilized the filament-gliding assay with kinesin-1 motors driving microtubule motion. To further enhance the complexity of the system and determine if new patterns are formed, we added the microtubule crosslinking protein MAP65-1. MAP65-1 is a microtubule-associated protein from plants that crosslinks antiparallel microtubules, similar to mammalian PRC1 and fission yeast Ase1. We find that MAP65 can slow and halt the velocity of microtubules in gliding assays, but when pre-formed microtubule bundles are added to gliding assays, kinesin-1 motors can pull apart the bundles and reconstitute cell-like protrusions.

Pringle, Joshua; Muthukumar, Amutha; Tan, Amanda; Crankshaw, Laura; Conway, Leslie; Ross, Jennifer L.

2013-09-01

257

Observations of microtubules and microtubule-microfilament associations in osmotically treated cells of Micrasterias denticulata Bréb.  

PubMed

As an extension of the observation and interpretation regarding the different microtubule systems of Micrasterias denticulata [12, 19], the existence of intertubular structures, such as microfilaments, which are strongly marked in osmotically treated cells, is especially interesting. The complex of microtubules and microfilaments occurs during post-telophase nuclear migration, probably engaged in the mechanism of movement. The arrangement of microtubules either parallel or perpendicular to the nuclear membrane is characteristic for the stage of nuclear migration. Another microtubule system, the microtubule band in the cortical protoplasm of the isthmus region [12], is described during morphogenesis of the new half cell. Osmotically treated cells in the stage of septum formation demonstrate the presence of cross-linked microtubules near the plasmalemma and microtubule bundles, situated in the protoplasm between the secondary wall and the chloroplast, probably representing the microtubule system in the cortical protoplasm of the old half cell described by Kiermayer [12, 16]. The frequent appearance of microtubules and intertubular structures in differentiating cells of Micrasterias denticulata after osmotic treatment is discussed along with implication for stabilization of microtubules, cross bridges, and microfilaments. PMID:6889505

Neuhaus-Url, G; Kiermayer, O

1982-06-01

258

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

PubMed Central

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

Laan, Liedewij; Roth, Sophie; Dogterom, Marileen

2012-01-01

259

Kinetochores distinguish GTP from GDP forms of the microtubule lattice  

Microsoft Academic Search

During prometaphase in mitotic cell division, chromosomes attach to the walls of microtubules and subsequently move to microtubule ends, where they stay throughout mitosis,. This end-attachment seems to be essential for correct chromosome segregating. However, the mechanism by which kinetochores, the multiprotein complexes that link chromosomes to the microtubules of the mitotic spindle,, recognize and stay attached to microtubule ends

Fedor F. Severin; Peter K. Sorger; Anthony A. Hyman

1997-01-01

260

Theoretical Description of Microtubule Dynamics in Fission Yeast During Interphase  

Microsoft Academic Search

Fission yeast (S. pombe) is a unicellular organism with a characteristic cylindrical shape. Cell growth during interphase is strongly influenced by microtubule self-organization - a process that has been experimentally well characterised. The microtubules are organized in 3 to 4 bundles, called ``interphase microtubule assemblies'' (IMAs). Each IMA is composed of several microtubules, arranged with their dynamic ``plus'' ends facing

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

2009-01-01

261

Origin of kinetochore microtubules in Chinese hamster ovary cells  

Microsoft Academic Search

We have attempted to determine whether chromosomal microtubules arise by kinetochore nucleation or by attachment of pre-existing microtubules. The appearance of new microtubules was investigated in vivo on kinetochores to which microtubules had not previously been attached. The mitotic apparatus of Chinese hamster ovary cells was reconstructed in three dimensions from 0.25 µm thick serial sections, and the location of

Patricia L. Witt; Hans Ris; Gary G. Borisy

1980-01-01

262

Axonal microtubules: a computer-linked quantitative analysis  

Microsoft Academic Search

Employing current computer-aided morphometric techniques, axonal microtubule density was determined for the rat sural nerve. Analysis of extensive data showed that while microtubule number increases with axon size, the increase is not directly proportional. Thus the relationship between microtubule density and axonal size is inversely related, so that microtubule density is greater in smaller axons than in larger axons. When

A. M. B. Malbouisson; M. N. Ghabriel; G. Allt

1985-01-01

263

Microtubules in viral replication and transport.  

PubMed

Viruses use and subvert host cell mechanisms to support their replication and spread between cells, tissues and organisms. Microtubules and associated motor proteins play important roles in these processes in animal systems, and may also play a role in plants. Although transport processes in plants are mostly actin based, studies, in particular with Tobacco mosaic virus (TMV) and its movement protein (MP), indicate direct or indirect roles of microtubules in the cell-to-cell spread of infection. Detailed observations suggest that microtubules participate in the cortical anchorage of viral replication complexes, in guiding their trafficking along the endoplasmic reticulum (ER)/actin network, and also in developing the complexes into virus factories. Microtubules also play a role in the plant-to-plant transmission of Cauliflower mosaic virus (CaMV) by assisting in the development of specific virus-induced inclusions that facilitate viral uptake by aphids. The involvement of microtubules in the formation of virus factories and of other virus-induced inclusions suggests the existence of aggresomal pathways by which plant cells recruit membranes and proteins into localized macromolecular assemblies. Although studies related to the involvement of microtubules in the interaction of viruses with plants focus on specific virus models, a number of observations with other virus species suggest that microtubules may have a widespread role in viral pathogenesis. PMID:23379770

Niehl, Annette; Peña, Eduardo J; Amari, Khalid; Heinlein, Manfred

2013-03-22

264

Signaling function of ?-catenin in microtubule regulation  

PubMed Central

Centrosomes control microtubule dynamics in many cell types, and their removal from the cytoplasm leads to a shift from dynamic instability to treadmilling behavior and to a dramatic decrease of microtubule mass (Rodionov et al., 1999; PNAS 96:115). In cadherin-expressing cells, these effects can be reversed: non-centrosomal cytoplasts that form cadherin-mediated adherens junctions display dense arrays of microtubules (Chausovsky et al., 2000; Nature Cell Biol 2:797). In adherens junctions, cadherin’s cytoplasmic domain binds p120 catenin and ?-catenin, which in turn binds ?-catenin. To elucidate the roles of the cadherin-associated proteins in regulating microtubule dynamics, we prepared GFP-tagged, plasma membrane targeted or untargeted p120 catenin, ?-catenin and ?-catenin and tested their ability to rescue the loss of microtubule mass caused by centrosomal removal in the poorly adhesive cell line CHO-K1. Only membrane targeting of ?-catenin led to a significant increase in microtubule length and density in centrosome-free cytoplasts. Expression of non-membrane-targeted ?-catenin produced only a slight effect, while both membrane-targeted and non-targeted p120 and ?-catenin were ineffective in this assay. Together, these findings suggest that ?-catenin is able to regulate microtubule dynamics in a centrosome-independent manner.

Shtutman, Michael; Chausovsky, Alexander; Prager-Khoutorsky, Masha; Schiefermeier, Natalia; Boguslavsky, Shlomit; Kam, Zvi; Fuchs, Elaine; Geiger, Benjamin; Borisy, Gary G.; Bershadsky, Alexander D.

2009-01-01

265

Microtubule dynamics alter the interphase nucleus.  

PubMed

Microtubules are known to drive chromosome movements and to induce nuclear envelope breakdown during mitosis and meiosis. Here we show that microtubules can enforce nuclear envelope folding and alter the levels of nuclear envelope-associated heterochromatin during interphase, when the nuclear envelope is intact. Microtubule reassembly, after chemically induced depolymerization led to folding of the nuclear envelope and to a transient accumulation of condensed chromatin at the site nearest the microtubule organizing center (MTOC). This microtubule-dependent chromatin accumulation next to the MTOC is dependent on the composition of the nuclear lamina and the activity of the dynein motor protein. We suggest that forces originating from simultaneous polymerization of microtubule fibers deform the nuclear membrane and the underlying lamina. Whereas dynein motor complexes localized to the nuclear envelope that slide along the microtubules transfer forces and/or signals into the nucleus to induce chromatin reorganization and accumulation at the nuclear membrane folds. Thus, our study identified a molecular mechanism by which mechanical forces generated in the cytoplasm reshape the nuclear envelope, alter the intranuclear organization of chromatin, and affect the architecture of the interphase nucleus. PMID:23117601

Gerlitz, Gabi; Reiner, Orly; Bustin, Michael

2012-11-02

266

Microtubules regulate disassembly of epithelial apical junctions  

PubMed Central

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

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

2006-01-01

267

Molecular Motors Control Length of Antiparallel Microtubule Overlaps  

NASA Astrophysics Data System (ADS)

Using Monte Carlo simulation, we studied the controlling length of antiparallel microtubule overlaps by motors in the presence of PRC1. Two models for the inhibition mechanism of microtubule dynamics are developed. The comparison of the simulation results and the experimental data shows that the inhibition of microtubule dynamics is probably not due to a direct inhibition of polymerization and depolymerization of microtubule by the motor at plus end of microtubule but rather caused by global structural changes in the microtubule due to the presence of bound motor on the microtubule.

Wang, Ziqing; Zhang, Caihua; Wang, Guodong

268

Imaging individual spindle microtubule dynamics in fission yeast.  

PubMed

Microtubules exhibit dynamic instability, stochastically switching between infrequent phases of growth and shrinkage. In the cell, microtubule dynamic instability is further modulated by microtubule-associated proteins and motors, which are specifically tuned to cell cycle stages. For example, mitotic microtubules are more dynamic than interphase microtubules. The different parameters of microtubule dynamics can be measured from length versus time data, which are generally obtained from time-lapse acquisition using the optical microscope. The typical maximum resolution of the optical microscope is ~?/2 or ~300nm. This scale represents a challenge for imaging fission yeast microtubule dynamics specifically during early mitosis, where the bipolar mitotic spindle contains many short dynamic microtubules of ~1-?m scale. Here, we present a novel method to image short fission yeast mitotic microtubules. The method uses the thermosensitive reversible kinesin-5 cut7.24(ts) to create monopolar spindles, where asters of individual mitotic microtubules are presented for imaging and subsequent analysis. PMID:23973085

Costa, Judite; Fu, Chuanhai; Syrovatkina, Viktoriya; Tran, Phong T

2013-01-01

269

MOVING IN FOR THE KILL: MOTILE MICROTUBULE REGULATORS  

PubMed Central

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

Su, Xiaolei; Ohi, Ryoma; Pellman, David

2012-01-01

270

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

Microsoft Academic Search

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

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

2002-01-01

271

Electron Tomography Reveals Novel Microtubule Lattice and Microtubule Organizing Centre Defects in +TIP Mutants  

PubMed Central

Mal3p and Tip1p are the fission yeast (Schizosaccharomyces pombe) homologues of EB1 and CLIP-170, two conserved microtubule plus end tracking proteins (+TIPs). These proteins are crucial regulators of microtubule dynamics. Using electron tomography, we carried out a high-resolution analysis of the phenotypes caused by mal3 and tip1 deletions. We describe the 3-dimensional microtubule organization, quantify microtubule end structures and uncover novel defects of the microtubule lattices. We also reveal unexpected structural modifications of the spindle pole bodies (SPBs), the yeast microtubule organizing centers. In both mutants we observe an increased SPB volume and a reduced number of MT/SPB attachments. The discovered defects alter previous interpretations of the mutant phenotypes and provide new insights into the molecular functions of the two protein families.

Brunner, Damian; Antony, Claude

2013-01-01

272

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

273

Electron tomography reveals novel microtubule lattice and microtubule organizing centre defects in +TIP mutants.  

PubMed

Mal3p and Tip1p are the fission yeast (Schizosaccharomyces pombe) homologues of EB1 and CLIP-170, two conserved microtubule plus end tracking proteins (+TIPs). These proteins are crucial regulators of microtubule dynamics. Using electron tomography, we carried out a high-resolution analysis of the phenotypes caused by mal3 and tip1 deletions. We describe the 3-dimensional microtubule organization, quantify microtubule end structures and uncover novel defects of the microtubule lattices. We also reveal unexpected structural modifications of the spindle pole bodies (SPBs), the yeast microtubule organizing centers. In both mutants we observe an increased SPB volume and a reduced number of MT/SPB attachments. The discovered defects alter previous interpretations of the mutant phenotypes and provide new insights into the molecular functions of the two protein families. PMID:23613905

Höög, Johanna L; Huisman, Stephen M; Brunner, Damian; Antony, Claude

2013-04-16

274

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

Microsoft Academic Search

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

Vaart van der B

2011-01-01

275

Actomyosin Transports Microtubules and Microtubules Control Actomyosin Recruitment during Xenopus Oocyte Wound Healing  

Microsoft Academic Search

Background: Interactions between microtubules and actin filaments (F-actin) are critical for cellular motility processes ranging from directed cell locomotion to cytokinesis. However, the cellular bases of these interactions remain poorly understood. We have analyzed the role of microtubules in generation of a contractile array comprised of F-actin and myosin-2 that forms around wounds made in Xenopus oocytes.Results: After wounding, microtubules

Craig A. Mandato; William M. Bement

2003-01-01

276

Tubulin requires tau for growth onto microtubule initiating sites.  

PubMed Central

Tubulin purified by phosphocellulose chromatography and free of accessory proteins will not form microtubules in the absence or presence of microtubule initiating sites (flagellar microtubules). The capacity for growth onto pre-existing "seeds" can be restored by the addition of small quantities of partially purified tau protein. Larger quantities restore the capacity for spontaneous assembly. These results suggest that tubulin requires tau for both initiation and growth of microtubules and that tau is incorporated into the microtubule throughout its length. Images

Witman, G B; Cleveland, D W; Weingarten, M D; Kirschner, M W

1976-01-01

277

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

2013-07-03

278

How calcium controls microtubule anisotropic phase formation in the presence of microtubule-associated proteins in vitro  

Microsoft Academic Search

Here we show a new effect of Ca2+ on microtubule morphology: Ca2+ can cause smooth curving of microtubules in the presence of microtubule-associated proteins (MAPs). In vitro, microtubules self-organize, forming complex dissipative structures. Such structures may be strongly affected by relatively weak external factors. A factor such as Ca2+ potentially influences spatiotemporal patterns of microtubule assembly, but the dynamics are

Vlado Buljan; Elena P. Ivanova; Karen M Cullen

2009-01-01

279

Microtubules Coordinate VEGFR2 Signaling and Sorting  

PubMed Central

VEGF signaling is a key regulator of vessel formation and function. In vascular endothelial cells, this signaling is mediated through its cognate receptor VEGFR2, which is dynamically sorted in response to ligand. Little is known about the underlying mechanism of this intracellular sorting. Here we examined the role of different components of the cytoskeleton in this process. We found that VEGFR2 mainly associates with microtubule fibers and to a lesser extent with intermediate filaments and actin. Microtubule disruption leads to accumulation of VEGFR2 protein in the membrane and cytoplasm leading to defects in VEGF signaling. In contrast, inhibition of actin filaments results in no accumulation of VEGFR2 total protein or apparent changes in microtubule association. Instead, actin inhibition leads to a more global signaling disruption of the ERK1/2 pathway. This is the first report demonstrating that VEGFR2 associates closely with microtubules in modulating the subcellular sorting and signaling of VEGFR2.

Czeisler, Catherine; Mikawa, Takashi

2013-01-01

280

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

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

1975-01-01

281

Novel Response to Microtubule Perturbation in Meiosis†  

PubMed Central

During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G1 or G2, instead of metaphase. Cells arrest in G1 if microtubule perturbation occurs as they enter the meiotic cell cycle and in G2 if cells are already undergoing premeiotic S phase. Concomitantly, cells down-regulate genes required for cell cycle progression, meiotic differentiation, and spore formation in a highly coordinated manner. Decreased expression of these genes is likely to be responsible for halting both cell cycle progression and meiotic development. Our results point towards the existence of a novel surveillance mechanism of microtubule integrity that may be particularly important during specialized cell cycles when coordination of cell cycle progression with a developmental program is necessary.

Hochwagen, Andreas; Wrobel, Gunnar; Cartron, Marie; Demougin, Philippe; Niederhauser-Wiederkehr, Christa; Boselli, Monica G.; Primig, Michael; Amon, Angelika

2005-01-01

282

Lower hybrid wave electron heating experiments in Doublet IIA  

SciTech Connect

Experiments designed to heat electrons by Landau damping of waves at approximately twice the lower hybrid frequency have been carried out on Doublet IIA. This objective is in contrast to other lower hybrid experiments which are designed to heat ions using frequencies corresponding to the lower hybrid resonance frequency. Up to 500 kW of rf power was applied to discharge with approximately 100 kW ohmic input using parallel wavelengths chosen to optimize the spatial distribution of the power deposition based on linear or quasi-linear Landau damping.

Freeman, R.L.; Luxon, J.L.; Chan, V.S.

1980-07-01

283

B ? D(*)??? decays in two-Higgs-doublet models  

NASA Astrophysics Data System (ADS)

A sizable excess with respect to the SM expectation has been reported recently by the BaBar collaboration in the decay rates B ? D(*)??, normalized by the corresponding light lepton modes. A violation of lepton flavor universality as suggested by this excess could be due to a charged Higgs mediating these processes at tree level. In this talk we analyze the implications of the observed excess within the framework of two-Higgs-doublet models, considering also the bounds from other semileptonic and leptonic decays of B and D(s) mesons. Prospects for B ? D(*)?? decays at future Super-Flavor Factories are also discussed.

Celis, Alejandro; Jung, Martin; Li, Xin-Qiang; Pich, Antonio

2013-07-01

284

Effective-range expansion for doublet nd scattering  

SciTech Connect

We derived an expression for the effective range r of doublet nd scattering and perform systematic calculations of r for a broad class of separable interaction profiles. We predict the value (1/2)..cap alpha../sup 3/a/sup 2/r = 1.25 for the slope parameter of the amplitude and establish the quadratic correlation between this parameter and the scattering length a. This correlation and the well-known relation between the triton binding energy and a allow the model-independent explanation of the low-energy behavior of the nd scattering amplitude.

Simenog, I.V.; Sitnichenko, A.I.; Shapoval, D.V.

1987-01-01

285

Pseudospin doublet aligned structure in doubly odd {sup 186}Ir  

SciTech Connect

{sup 186}Ir has been restudied through the {sup 180}Hf({sup 11}B,5n) reaction at 65 MeV using in-beam {gamma}-ray and conversion-electron spectroscopy. The unfavored component of the doubly decoupled band was established and shown to be consistent with a description in terms of the {pi}h{sub 9/2}{circle_times}{nu}[{number_sign}411{number_sign}{number_sign}&dbigwig;1/2,3/2] structure, i.e., the coupling of an aligned proton and a neutron pseudospin doublet. {copyright} {ital 1997} {ital The American Physical Society}

Cardona, M.A.; Debray, M.E.; Garcia Bermudez, G.; Hojman, D.; Kreiner, A.J.; Somacal, H.; Burlon, A.; Davidson, J.; Davidson, M.; Levinton, G.; Ozafran, M.; Vazquez, M.; Napoli, D.R.; Rico, J.; Bazzacco, D.; Burch, R.; De Acuna, D.; Lenzi, S.M.; Medina, N.; Rossi Alvarez, C.; Blasi, N.; Lo Bianco, G.; de Boer, J.; Frischke, D.; Maier, H.J. [Departamento de Fisica, Comision Nacional de Energia Atomica, 1429 Buenos Aires (Argentina)

1997-01-01

286

AMPK attenuates microtubule proliferation in cardiac hypertrophy.  

PubMed

Cell hypertrophy requires increased protein synthesis and expansion of the cytoskeletal networks that support cell enlargement. AMPK limits anabolic processes, such as protein synthesis, when energy supply is insufficient, but its role in cytoskeletal remodeling is not known. Here, we examined the influence of AMPK in cytoskeletal remodeling during cardiomyocyte hypertrophy, a clinically relevant condition in which cardiomyocytes enlarge but do not divide. In neonatal cardiomyocytes, activation of AMPK with 5-aminoimidazole carboxamide ribonucleotide (AICAR) or expression of constitutively active AMPK (CA-AMPK) attenuated cell area increase by hypertrophic stimuli (phenylephrine). AMPK activation had little effect on intermediate filaments or myofilaments but dramatically reduced microtubule stability, as measured by detyrosinated tubulin levels and cytoskeletal tubulin accumulation. Importantly, low-level AMPK activation limited cell expansion and microtubule growth independent of mTORC1 or protein synthesis repression, identifying a new mechanism by which AMPK regulates cell growth. Mechanistically, AICAR treatment increased Ser-915 phosphorylation of microtubule-associated protein 4 (MAP4), which reduces affinity for tubulin and prevents stabilization of microtubules (MTs). RNAi knockdown of MAP4 confirmed its critical role in cardiomyocyte MT stabilization. In support of a pathophysiological role for AMPK regulation of cardiac microtubules, AMPK ?2 KO mice exposed to pressure overload (transverse aortic constriction; TAC) demonstrated reduced MAP4 phosphorylation and increased microtubule accumulation that correlated with the severity of contractile dysfunction. Together, our data identify the microtubule cytoskeleton as a sensitive target of AMPK activity, and the data suggest a novel role for AMPK in limiting accumulation and densification of microtubules that occurs in response to hypertrophic stress. PMID:23316058

Fassett, John T; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie; Bache, Robert J

2013-01-11

287

Resistance to Microtubule-Targeting Drugs  

Microsoft Academic Search

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

Paraskevi Giannakakou; James P. Snyder

288

The Rib43a Protein Is Associated with Forming the Specialized Protofilament Ribbons of Flagellar Microtubules in Chlamydomonas  

PubMed Central

Ciliary and flagellar microtubules contain a specialized set of three protofilaments, termed ribbons, that are composed of tubulin and several associated proteins. Previous studies of sea urchin sperm flagella identified three of the ribbon proteins as tektins, which form coiled-coil filaments in doublet microtubules and which are associated with basal bodies and centrioles. To study the function of tektins and other ribbon proteins in the assembly of flagella and basal bodies, we have begun an analysis of ribbons from the unicellular biflagellate, Chlamydomonas reinhardtii, and report here the molecular characterization of the ribbon protein rib43a. Using antibodies against rib43a to screen an expression library, we recovered a full-length cDNA clone that encodes a 42,657-Da polypeptide. On Northern blots, the rib43a cDNA hybridized to a 1.7-kb transcript, which was up-regulated upon deflagellation, consistent with a role for rib43a in flagellar assembly. The cDNA was used to isolate RIB43a, an ?4.6-kb genomic clone containing the complete rib43a coding region, and restriction fragment length polymorphism analysis placed the RIB43a gene on linkage group III. Sequence analysis of the RIB43a gene indicates that the substantially coiled-coil rib43a protein shares a high degree of sequence identity with clones from Trypanosoma cruzi and Homo sapiens (genomic, normal fetal kidney, and endometrial and germ cell tumors) but little sequence similarity to other proteins including tektins. Affinity-purified antibodies against native and bacterially expressed rib43a stained both flagella and basal bodies by immunofluorescence microscopy and stained isolated flagellar ribbons by immuno-electron microscopy. The structure of rib43a and its association with the specialized protofilament ribbons and with basal bodies is relevant to the proposed role of ribbons in forming and stabilizing doublet and triplet microtubules and in organizing their three-dimensional structure.

Norrander, Jan M.; deCathelineau, Aimee M.; Brown, Jennifer A.; Porter, Mary E.; Linck, Richard W.

2000-01-01

289

The Rib43a protein is associated with forming the specialized protofilament ribbons of flagellar microtubules in Chlamydomonas.  

PubMed

Ciliary and flagellar microtubules contain a specialized set of three protofilaments, termed ribbons, that are composed of tubulin and several associated proteins. Previous studies of sea urchin sperm flagella identified three of the ribbon proteins as tektins, which form coiled-coil filaments in doublet microtubules and which are associated with basal bodies and centrioles. To study the function of tektins and other ribbon proteins in the assembly of flagella and basal bodies, we have begun an analysis of ribbons from the unicellular biflagellate, Chlamydomonas reinhardtii, and report here the molecular characterization of the ribbon protein rib43a. Using antibodies against rib43a to screen an expression library, we recovered a full-length cDNA clone that encodes a 42,657-Da polypeptide. On Northern blots, the rib43a cDNA hybridized to a 1. 7-kb transcript, which was up-regulated upon deflagellation, consistent with a role for rib43a in flagellar assembly. The cDNA was used to isolate RIB43a, an approximately 4.6-kb genomic clone containing the complete rib43a coding region, and restriction fragment length polymorphism analysis placed the RIB43a gene on linkage group III. Sequence analysis of the RIB43a gene indicates that the substantially coiled-coil rib43a protein shares a high degree of sequence identity with clones from Trypanosoma cruzi and Homo sapiens (genomic, normal fetal kidney, and endometrial and germ cell tumors) but little sequence similarity to other proteins including tektins. Affinity-purified antibodies against native and bacterially expressed rib43a stained both flagella and basal bodies by immunofluorescence microscopy and stained isolated flagellar ribbons by immuno-electron microscopy. The structure of rib43a and its association with the specialized protofilament ribbons and with basal bodies is relevant to the proposed role of ribbons in forming and stabilizing doublet and triplet microtubules and in organizing their three-dimensional structure. PMID:10637302

Norrander, J M; deCathelineau, A M; Brown, J A; Porter, M E; Linck, R W

2000-01-01

290

Using Microtubules to Illustrate Polymer Properties  

NSDL National Science Digital Library

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

Hess, Henry; Jeune, Yoli

2009-10-07

291

Glycolytic enzyme interactions with tubulin and microtubules.  

PubMed

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice. PMID:2553125

Walsh, J L; Keith, T J; Knull, H R

1989-11-01

292

Harnessing microtubule dynamic instability for nanostructure assembly  

NASA Astrophysics Data System (ADS)

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

Bouchard, Ann M.; Warrender, Christina E.; Osbourn, Gordon C.

2006-10-01

293

Microtubule motor Ncd induces sliding of microtubules in vivo.  

PubMed

The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, we developed an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts. We found that green fluorescent protein (GFP)-Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic "looping" behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 microm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data, for the first time, demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis. PMID:17596520

Oladipo, Abiola; Cowan, Ann; Rodionov, Vladimir

2007-06-27

294

Organization of neuronal microtubules in the nematode Caenorhabditis elegans.  

PubMed

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

Chalfie, M; Thomson, J N

1979-07-01

295

Array of doublets: A branch of cellular solutions in directional solidification  

SciTech Connect

In directional solidification of alloys, the interface pattern assumes a cellular structure, with a periodic array of cells, when the velocity is increased beyond the threshold of planar interface instability. A detailed experimental study in the succinonitrile-acetone system has revealed a branch of cellular structure in which the interface pattern consists of a periodic array of coupled cells or doublets. This doublet interface evolves with two characteristic length scales at the advancing front: a small intraspacing between the cells in a doublet, whose selection is sharp, and a larger interspacing corresponding to the distance between cells in adjoining doublets, whose selection is weak. The dynamics of the time-dependent evolution of a doublet interface is investigated by statistical analysis of tip spacings, by using a Fourier transform of the interface shape and through the study of the variation of shape parameters with time. These dynamical studies have confirmed the selection of doublet interface as a stable solution of the cellular pattern formation. A range of stable regime for an array with doublets is determined and the key factor that controls the formation of regular cells or doublets in an array is discussed. When doublets are unstable, time dependency may follow due to a source mechanism at grain boundaries, which induces strong spatiotemporal chaos.

Jamgotchian, H. (Ames Laboratory and Department of Materials Science and Engineering, Iowa State University, Ames, Iowa 50011 (United States)); Trivedi, R. (Ames Laboratory and Department of Materials Science and Engineering, Iowa State University, Ames, Iowa 50011 (United States)); Billia, B.

1993-06-01

296

Chiral doublets and ?-softness in the A ~ 130 region  

NASA Astrophysics Data System (ADS)

Systematic properties of doublet bands related to chiral symmetry restoration in odd-odd A ~130 nuclei were recently established by experimental studies [1]. In the case of ^134Pr, the doublet ?h_11/2?h_11/2 bands become nearly degenerate starting at spin ~ 14hbar, while in other N=75 and N=73 isotones the bands are separated by ~ 300 keV. The degenerate bands are predicted for this region from the coupling of the valence h_11/2 proton and neutron to a triaxial rotor core with ?=30^circ. Current work explores effects of ?-softness on the properties of these bands. The calculations were conducted using a core-particle-hole coupling model. The Hamiltonian consists of the collective core, the single-particle valence nucleons, and qQ interactions. The Kerman-Klein method was used to find eigenstates, which provided a convenient way for exploring core effects. Calculations were made for triaxial cores with various ?-softness using the General Collective Model keeping < ? > =30^circ. The degeneracy in the ?h_11/2?h_11/2 bands calculated for the triaxial rotor of fixed ?=30^circ is indeed lifted for ?-soft cores, but consistent with the values observed experimentally. The effects of ?-softness on transition rates and other observables will be discussed. [1mm] [1] K. Starosta et al (to be published) [1mm] [Supported by NSF-PHY9870280; DOE-DE-FG02-91ER-40609

Starosta, K.; Chiara, C. J.; Fossan, D. B.; Koike, T.; Caprio, M. A.; Beausang, C. W.; Krücken, R.

2000-10-01

297

Kinetochore microtubules in PTK cells  

PubMed Central

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

1992-01-01

298

Microtubules and axoplasmic transport. Inhibition of transport by podophyllotoxin: an interaction with microtubule protein.  

PubMed

Pharmacological evidence is presented for the involvement of microtubules in the process of fast axoplasmic transport. A quantitative measure of the inhibition of axoplasmic transport in an in vitro preparation of rat sciatic nerve is described. The alkaloids colchicine, podophyllotoxin, and vinblastine, which are known both to disrupt microtubules and to bind to the protein subunit of microtubules, are inhibitors of axoplasmic transport. Lumicolchine and picropodophyllin, unlike their respective isomers colchicine and podophyllotoxin, are poor inhibitors of axoplasmic transport. The dissociation constants for the binding of colchicine, lumicolchicine, podophyllotoxin, and picropodophyllin to purified microtubule protein from rat brain have been measured. Inhibition of axoplasmic transport by these drugs correlates favorably with their affinities of microtubule protein. PMID:53233

Paulson, J C; McClure, W O

1975-11-01

299

EMAP, an echinoderm microtubule-associated protein found in microtubule-ribosome complexes.  

PubMed

The major non-tubulin polypeptide found associated with microtubules purified from unfertilized sea urchin eggs by cycles of pH-dependent assembly has a Mr of 77,000. The 77,000 Mr polypeptide is heat- and acid-labile, and is antigenically distinct from the mammalian brain MAPs, MAP-2 and tau. Affinity-purified antiserum against the 77,000 Mr polypeptide was used to survey a variety of cells and tissues for the presence of antigenically related polypeptides. A cross-reacting polypeptide, ranging in Mr from 72,000 to 80,000, was found in microtubule preparations from a wide variety of echinoderms, including sea urchins, starfish and sand dollars. Indirect immunofluorescence showed that the polypetide was found in interphase as well as mitotic microtubule arrays. No cross-reacting material was detected in microtubules isolated from marine molluscs, mammalian brain or mouse B16 cultured cells. Because the 77,000 Mr MAP is abundant in echinoderms, we have called it EMAP for echinoderm microtubule-associated protein. Although the precise function of the EMAP is not known, our data suggest that the EMAP is involved in the attachment of ribosomes to microtubules. Large numbers of ribosomes are attached to the walls of EMAP-containing microtubules, but not EMAP-deficient microtubules. Removal of the EMAP from the microtubule by salt-extraction results in the release of ribosomes from the microtubule, indicating that the EMAP may form part or all of the long tapered stalk that connects these two organelles. PMID:9867489

Suprenant, K A; Dean, K; McKee, J; Hake, S

1993-02-01

300

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

301

Extragenic bypass suppressors of mutations in the essential gene BLD2 promote assembly of basal bodies with abnormal microtubules in Chlamydomonas reinhardtii.  

PubMed Central

bld2-1 mutant Chlamydomonas reinhardtii strains assemble basal bodies with singlet microtubules; bld2-1 cells display flagellar assembly defects as well as positioning defects of the mitotic spindle and cleavage furrow. To further understand the role of the BLD2 gene, we have isolated three new bld2 alleles and three partially dominant extragenic suppressors, rgn1-1, rgn1-2, and rgn1-3. bld2 rgn1-1 strains have phenotypes intermediate between those of bld2 and wild-type strains with respect to flagellar number, microtubule rootlet organization, cleavage furrow positioning, and basal body structural phenotypes. Instead of the triplet microtubules of wild-type cells, bld2 rgn1-1 basal bodies have mixtures of no, singlet, doublet, and triplet microtubules. The bld2-4 allele was made by insertional mutagenesis and identified in a noncomplementation screen in a diploid strain. The bld2-4 allele has a lethal phenotype based on mitotic segregation in diploid strains and in haploid strains generated by meiotic recombination. The lethal phenotype in haploid strains is suppressed by rgn1-1; these suppressed strains have similar phenotypes to other bld2 rgn1-1 double mutants. It is likely that BLD2 is an essential gene that is needed for basal body assembly and function.

Preble, A M; Giddings, T H; Dutcher, S K

2001-01-01

302

Model Independence of a Nd-System in a Doublet State.  

National Technical Information Service (NTIS)

A model-independent solution of the nd-scattering problem in a doublet state, that explains the qualitative characteristics of a three-nucleon system at low energies and the correlations between the parameters of a doublet scattering amplitude is obtained...

I. V. Simenog D. V. Shapoval

1987-01-01

303

Doublet III US\\/Japan cooperation upgrade program, final technical report, May 1979September 1984  

Microsoft Academic Search

The US\\/Japan agreement provided for sharing of Doublet III experimental time between scientific teams from GA and the Japan Atomic Energy Research Institute (JAERI), and also provided for upgrading the experimental capabilities of Doublet III with additional power systems, plasma heating capability, and plasma diagnostic systems.

C. H. Fox; L. G. Davis; C. R. Harder; K. C. Shoolbred

1984-01-01

304

Tree Level Vacuum Stability in Multi Higgs Doublet Models: A Cumbersome Analysis?  

Microsoft Academic Search

We introduce a simple approach to obtain the constraint for multi Higgs doublet potentials to be stable, i.e. to have tree-level\\u000a vacuum stability. We use the methodology for some special cases with three doublets.

S. Zarrinkamar; H. Hassanabadi; A. A. Rajabi

2010-01-01

305

Unusual doublet structure in proton magnetic-resonance spectra of yttrium and lutetium trihydrides  

Microsoft Academic Search

The proton magnetic resonance spectra of yttrium and lutetium trihydride, Y H3 and Lu H3 , respectively, show an unusual doublet structure qualitatively similar to the Pake doublet that results from the mutual dipolar interaction of closely spaced, isolated proton pairs. However, both the magnitude of the splittings, roughly 70 kHz , and the second moment of the spectra, roughly

G. Majer; A. Telfah; F. Grinberg; R. G. Barnes

2004-01-01

306

Highly Segmented SPL as a Mixture of Doublet and FoDo  

Microsoft Academic Search

A new structure is designed for the CERN Superconducting Proton LINAC as a mixture of Doublet and FoDo focusing schemes using room temperature quadrupoles. This architecture is compared to the baseline (doublet) and FoDo architectures of SPL on the basis of its beam dynamics performance and the required investment.

M Eshraqi; A M Lombardi; P A Posocco

2010-01-01

307

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

308

Thermodynamics of dense hadronic matter in a parity doublet model  

SciTech Connect

We study thermodynamics of nuclear matter in a two-flavored parity doublet model within the mean-field approximation. Parameters of the model are chosen to reproduce correctly the properties of the nuclear ground state. The model predicts two phase transitions in nuclear matter, a liquid-gas phase transition at normal nuclear density and a chiral transition at higher density. At finite temperature the pion decay constant exhibits a considerable reduction at intermediate values of chemical potential, which is traced back to the presence of the liquid-gas transition, and approaches zero at higher chemical potential associated with the chiral symmetry restoration. A 'transition' from meson-rich to baryon-rich matter is also discussed.

Sasaki, Chihiro [Frankfurt Institute for Advanced Studies, D-60438 Frankfurt am Main (Germany); Mishustin, Igor [Frankfurt Institute for Advanced Studies, D-60438 Frankfurt am Main (Germany); Kurchatov Institute, Russian Research Center, Moscow 123182 (Russian Federation)

2010-09-15

309

Infrared monitoring of the Doublet III beam armor plate  

SciTech Connect

An 80 keV, 3.6 MW neutral beam injection system has recently been installed on Doublet III, and the installation of a second system is scheduled within several months. Armor plate consisting of /approximately equals/100 graphite tiles (10 cm x 10 cm) coated with TiC has been plated over portions of the inner vacuum wall lying in the line of sight of the ion sources. In order to monitor the condition of the armor plate an infrared camera and a set of optical pyrometers have been installed alongside the beamline and view the armor plate through a CaF/sub 2/ window. The pyrometers measure the temperature of the armor plate associated with the maximum of the intensity distribution of each ion source.

McMahon, T.; Kamperschroer, J.; Colleraine, A.; Kim, J.; McColl, D.; Tooker, J.; Treglio, J.

1981-01-01

310

Search for multiple chiral doublets in rhodium isotopes  

SciTech Connect

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

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

2008-02-15

311

Microtubule motors: a new hope for kinesin-5 inhibitors?  

PubMed

A new study demonstrates that two microtubule plus end-directed kinesins can oppose each other. The cause of this apparent contradiction is the specific orientation of microtubules on which each motor exerts its force. PMID:23885879

Groen, Aaron

2013-07-22

312

Two Microtubule-associated Proteins of Arabidopsis MAP65s Promote Antiparallel Microtubule Bundling  

PubMed Central

The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that “zipper up” antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter.

Gaillard, Jeremie; Neumann, Emmanuelle; Van Damme, Daniel; Stoppin-Mellet, Virginie; Ebel, Christine; Barbier, Elodie; Geelen, Danny

2008-01-01

313

2HDMC - two-Higgs-doublet model calculator  

NASA Astrophysics Data System (ADS)

We describe the public C++ code 2HDMC which can be used to perform calculations in a general, CP-conserving, two-Higgs-doublet model (2HDM). The program features simple conversion between different parametrizations of the 2HDM potential, a flexible Yukawa sector specification with choices of different Z-symmetries or more general couplings, a decay library including all two-body - and some three-body - decay modes for the Higgs bosons, and the possibility to calculate observables of interest for constraining the 2HDM parameter space, as well as theoretical constraints from positivity and unitarity. The latest version of the 2HDMC code and full documentation is available from: http://www.isv.uu.se/thep/MC/2HDMC. Catalogue identifier: AEFI_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEFI_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU GPL No. of lines in distributed program, including test data, etc.: 12?032 No. of bytes in distributed program, including test data, etc.: 90?699 Distribution format: tar.gz Programming language: C++ Computer: Any computer running Linux Operating system: Linux RAM: 5 Mb Classification: 11.1 External routines: GNU Scientific Library (http://www.gnu.org/software/gsl/) Nature of problem: Determining properties of the potential, calculation of mass spectrum, couplings, decay widths, oblique parameters, muon g-2, and collider constraints in a general two-Higgs-doublet model. Solution method: From arbitrary potential and Yukawa sector, tree-level relations are used to determine Higgs masses and couplings. Decay widths are calculated at leading order, including FCNC decays when applicable. Decays to off-shell vector bosons are obtained by numerical integration. Observables are computed (analytically or numerically) as function of the input parameters. Restrictions: CP-violation is not treated. Running time: Less than 0.1 s on a standard PC

Eriksson, David; Rathsman, Johan; Stål, Oscar

2010-01-01

314

Theory and phenomenology of two-Higgs-doublet models  

NASA Astrophysics Data System (ADS)

We discuss theoretical and phenomenological aspects of two-Higgs-doublet extensions of the Standard Model. In general, these extensions have scalar mediated flavour changing neutral currents which are strongly constrained by experiment. Various strategies are discussed to control these flavour changing scalar currents and their phenomenological consequences are analysed. In particular, scenarios with natural flavour conservation are investigated, including the so-called type I and type II models as well as lepton-specific and inert models. Type III models are then discussed, where scalar flavour changing neutral currents are present at tree level, but are suppressed by either a specific ansatz for the Yukawa couplings or by the introduction of family symmetries leading to a natural suppression mechanism. We also consider the phenomenology of charged scalars in these models. Next we turn to the role of symmetries in the scalar sector. We discuss the six symmetry-constrained scalar potentials and their extension into the fermion sector. The vacuum structure of the scalar potential is analysed, including a study of the vacuum stability conditions on the potential and the renormalization-group improvement of these conditions is also presented. The stability of the tree level minimum of the scalar potential in connection with electric charge conservation and its behaviour under CP is analysed. The question of CP violation is addressed in detail, including the cases of explicit CP violation and spontaneous CP violation. We present a detailed study of weak basis invariants which are odd under CP. These invariants allow for the possibility of studying the CP properties of any two-Higgs-doublet model in an arbitrary Higgs basis. A careful study of spontaneous CP violation is presented, including an analysis of the conditions which have to be satisfied in order for a vacuum to violate CP. We present minimal models of CP violation where the vacuum phase is sufficient to generate a complex CKM matrix, which is at present a requirement for any realistic model of spontaneous CP violation.

Branco, G. C.; Ferreira, P. M.; Lavoura, L.; Rebelo, M. N.; Sher, Marc; Silva, João P.

2012-07-01

315

Spag16, an Axonemal Central Apparatus Gene, Encodes a Male Germ Cell Nuclear Speckle Protein that Regulates SPAG16 mRNA Expression  

PubMed Central

Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the “9+2” axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L and SPAG16S have a profound defect in spermatogenesis. We show here that transduction of SPAG16S into cultured dispersed mouse male germ cells and BEAS-2B human bronchial epithelial cells increases SPAG16L expression, but has no effect on the expression of several other axoneme components. We also demonstrate that the Spag16L promoter shows increased activity in the presence of SPAG16S. The distinct nuclear localization of SPAG16S and its ability to modulate Spag16L mRNA expression suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells. This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions.

Nagarkatti-Gude, David R.; Jaimez, Ruth; Henderson, Scott C.; Teves, Maria E.; Zhang, Zhibing; Strauss, Jerome F.

2011-01-01

316

Microtubules viewed as molecular ant colonies.  

PubMed

Populations of ants and other social insects self-organize and develop 'emergent' properties through stigmergy in which individual ants communicate with one another via chemical trails of pheromones that attract or repulse other ants. In this way, sophisticated properties and functions develop. Under appropriate conditions, in vitro microtubule preparations, initially comprised of only tubulin and GTP, behave in a similar manner. They self-organize and develop other higher-level emergent phenomena by a process where individual microtubules are coupled together by the chemical trails they produce by their own reactive growing and shrinking. This behaviour is described and compared with the behaviour of ant colonies. Viewing microtubules as populations of molecular ants may provide new insights as to how the cytoskeleton may spontaneously develop high-level functions. It is plausible that such processes occur during the early stages of embryogenesis and in cells. PMID:16968217

Tabony, James

2006-10-01

317

Modern methods to interrogate microtubule dynamics.  

PubMed

Microtubules are essential protein filaments required to organize and rearrange the interior of the cell. They must be stiff with mechanical integrity to support the structure of the cell. Yet, they must also be dynamic to enable rearrangements of the cell during cell division and development. This dynamic nature is inherent to microtubules and comes about through the hydrolysis of chemical energy stored in guanosine triphosphate (GTP). Dynamic instability has been studied with a number of microscopy techniques both in cells and in reconstituted systems. In this article, we review the techniques used to examine microtubule dynamic instability and highlight future avenues and still open questions about this vital and fascinating activity. PMID:24061278

Bailey, Megan; Conway, Leslie; Gramlich, Michael W; Hawkins, Taviare L; Ross, Jennifer L

2013-09-24

318

[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.

1975-01-01

319

Assays for the detection of microtubule depolymerization inhibitors  

US Patent & Trademark Office Database

This invention provides assays for agents that modulate (e.g. upregulate, downregulate or completely inhibit) microtubule depolymerizing or microtubule severing proteins. Such agents will have profound effects on progression of the cell cycle and act as potent anti-mitotic agents. The microtubule severing protein or microtubule depolymerizing protein is preferably a katanin, a p60 subunit of a katanin, an XKCM 1, or an OP18 polypeptide.

Vale; Ronald D. (San Francisco, CA); Hartman; James J. (San Francisco, CA)

2004-03-02

320

Microtubule-binding agents: a dynamic field of cancer therapeutics  

Microsoft Academic Search

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

Mary Ann Jordan; Charles Dumontet

2010-01-01

321

Organization of organelles and membrane traffic by microtubules  

Microsoft Academic Search

Organelles of the central membrane system of higher eukaryotes have been shown to utilize microtubules both for maintenance of their characteristic spatial distributions and for efficient transport of their protein and lipid to diverse sites within the cell. Recent work addressing the mechanisms that underlie this organization provides new insights regarding the roles of microtubules and microtubule motors in influencing

Nelson B. Cole; Jennifer Lippincott-Schwartz

1995-01-01

322

Ethylene-induced microtubule reorientations: mediation by helical arrays  

Microsoft Academic Search

Entire microtubule arrays, within outer cortical and epidermal cells of pea epicotyl and mung-bean hypocotyl, have been visualized by indirect immunofluorescence. In all cells the microtubule arrangement can be interpreted as being a single multistart helix of variable pitch. In control cells the predominant pattern is a tightly compressed helix with the microtubules consequently in a net transverse direction with

I. N. Roberts; C. W. Lloyd; K. Roberts

1985-01-01

323

Microtubules and Neuronal Polarity: Review Lessons from Mitosis  

Microsoft Academic Search

Several lines of evidence suggest that the cytoskeletal minally postmitotic neurons abandon the mechanisms elements known as microtubules may be central to all that mitotic cells use to organize their microtubules and of these issues. Microtubules are dynamic polymers develop entirely novel strategies. This assumption, based made up of tubulin subunits. They provide architectural on the fact that the neuronal

Peter W. Baas

1999-01-01

324

Unidirectional motion of microtubules and microspheres by Dynein motor protein  

Microsoft Academic Search

This paper reports a nanotransport system with the directional movement control. The system consists of motor proteins, dynein and kinesin, and their tracks, microtubules. Dynein molecules are specifically immobilized on a microchannel glass surface and fluorescently labeled microtubules were transported by dynein motion. Moving directions of gliding microtubules were controlled by a pressure-driven flow through the microchannel, and their polarities

Tetsutaro Murakami; Takeshi Sugie; Takahide Kon; Ryuji Yokokawa

2007-01-01

325

Microtubule polarity in the nutritive tubes of insect ovarioles  

Microsoft Academic Search

The enormous numbers of microtubules within the nutritive tubes of hemipteran ovarioles are amenable to the hook-decoration technique for determining microtubule structural polarity, as they can be microdissected from ovarioles intact. This has allowed the correlation between the polarity of this continuously elongating complex of microtubules within a nutritive tube and the direction of transport along its length; and has

Howard Stebbings; Cherryl Hunt

1983-01-01

326

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

327

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

328

Phosphorylation of sperm axoneme central apparatus protein SPAG16L by a testis specific kinase, TSSK2*  

PubMed Central

Mammalian SPAG16L, the orthologue of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites and the protein was confirmed to be phosphorylated in vivo. A yeast-two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by co-immunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their co-localization was also noted by confocal microscopy in CHO cells where they were co-expressed. TSSK2 associates with SPAG16L via its C terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.

Zhang, Zhibing; Shen, Xuening; Jones, Brian H.; Xu, Bingfang; Herr, John C; Strauss, Jerome F

2009-01-01

329

An Integrative Computational Model of Multiciliary Beating  

Microsoft Academic Search

The coordinated beating of motile cilia is responsible for ovum transport in the oviduct, transport of mucus in the respiratory\\u000a tract, and is the basis of motility in many single-celled organisms. The beating of a single motile cilium is achieved by\\u000a the ATP-driven activation cycles of thousands of dynein molecular motors that cause neighboring microtubule doublets within\\u000a the ciliary axoneme

Xingzhou Yang; Robert H. Dillon; Lisa J. Fauci

2008-01-01

330

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring  

Microsoft Academic Search

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether inter- mediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140)

Pinfen Yang; Winfield S. Sale

1998-01-01

331

Microtubule actin cross-linking factor 1 regulates cardiomyocyte microtubule distribution and adaptation to hemodynamic overload.  

PubMed

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r(2)?=?0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKC? and ?1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload. PMID:24086300

Fassett, John T; Xu, Xin; Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J; Chen, Yingjie

2013-09-26

332

Microtubule nucleating ?TuSC assembles structures with 13-fold microtubule-like symmetry  

PubMed Central

Microtubules are nucleated in vivo by ?-tubulin complexes. The 300 kDa ?-tubulin small complex (?TuSC), consisting of two molecules of ?-tubulin and one copy each of the accessory proteins Spc97p and Spc98p, is the conserved, essential core of the microtubule nucleating machinery1,2. In metazoa multiple ?TuSCs assemble with other proteins into ?-tubulin ring complexes (?TuRCs). The structure of ?TuRC suggested that it functions as a microtubule template2–5. Because each ?TuSC contains two molecules of ?-tubulin, it was assumed that the ?TuRC-specific proteins are required to organize ?TuSCs to match thirteen-fold microtubule symmetry. Here, we show that ?TuSC forms rings even in the absence of other ?TuRC components. The yeast adaptor protein Spc110p stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8Å cryo-EM reconstruction of the filament reveals thirteen ?-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97p and Spc98p suggest functions for conserved sequence motifs, with implications for the ?TuRC-specific proteins. The ?TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

Kollman, Justin M.; Polka, Jessica K.; Zelter, Alex; Davis, Trisha N.; Agard, David A.

2010-01-01

333

MAP 4: a microtubule-associated protein specific for a subset of tissue microtubules  

Microsoft Academic Search

The cytological distribution of microtubule-associated protein 4 (MAP 4) (L. M. Parysek, C. F. Asnes, J. B. Olmsted, 1984, J. Cell Biol., 99:1309-1315) in mouse tissues has been examined. Adjacent 0.5-0.9-um sections of polyethylene glycol-embedded tissues were incubated with affinity-purified MAP 4 or tubulin antibodies, and the immunofluorescent images were compared. Tubulin antibody labeling showed distinct microtubules in all tissues

LINDA M. PARYSEK; JOHN J. WOLOSEWICK; J. B. OLMSTED

1984-01-01

334

Microtubule Actin Cross-Linking Factor 1 Regulates Cardiomyocyte Microtubule Distribution and Adaptation to Hemodynamic Overload  

PubMed Central

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r2?=?0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKC? and ?1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.

Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J.; Chen, Yingjie

2013-01-01

335

Intracytoplasmic ciliary elements in epidermal cells of Syndesmis echinorum and Paravortex cardii (Platyhelminthes, Dalyellioida).  

PubMed

Epidermal cells of Syndesmis echinorum and Paravortex cardii contain many intracytoplasmic ciliary components: clusters of centrioles disorganized and incomplete short axonemes composed of loosely organized microtubules of irregular lengths, fully formed axonemes though some with fewer than nine doublets, and ciliary rootlets. Furthermore, conspicuous dense granules are found in solitary groups in the cytoplasm. Clusters of dense granules are also closely associated with Golgi complexes and developing axonemal microtubules. Since the dense granules decrease in number as the axonemes increase, it is likely that the granules are involved in the formation of axonemal microtubules. Ciliary elements are especially abundant in epidermal cells of Paravortex cardii embryos, some of them resembling those previously described by several authors in differentiating ciliated cells engaged in centriologenesis and ciliogenesis. Attention has been focused on the relative proportion and position of these elements, as well as the different morphology and several assembling states that they exhibit in epidermal cells of adult S. echinorum and adults and embryos of P. cardii. A functional interpretation of some of the findings is given, which allows us to suggest a sequence of ciliogenetic events that occur in epidermal cells of both species. PMID:1518068

Cifrian, B; García-Corrales, P; Martínez-Alos, S

1992-08-01

336

Reconstituting the kinetochore-microtubule interface: what, why, and how  

PubMed Central

The kinetochore is the proteinaceous complex that governs the movement of duplicated chromosomes by interacting with spindle microtubules during mitosis and meiosis. Faithful chromosome segregation requires that kinetochores form robust load-bearing attachments to the tips of dynamic spindle microtubules, correct microtubule attachment errors, and delay the onset of anaphase until all chromosomes have made proper attachments. To understand how this macromolecular machine operates to segregate duplicated chromosomes with exquisite accuracy, it is critical to reconstitute and study kinetochore-microtubule interactions in vitro using defined components. Here, we review the current status of reconstitution as well as recent progress in understanding the microtubule binding functions of kinetochores in vivo.

Akiyoshi, Bungo; Biggins, Sue

2012-01-01

337

Taxanes, microtubules and chemoresistant breast cancer.  

PubMed

The taxanes, paclitaxel and docetaxel are microtubule-stabilizing agents that function primarily by interfering with spindle microtubule dynamics causing cell cycle arrest and apoptosis. However, the mechanisms underlying their action have yet to be fully elucidated. These agents have become widely recognized as active chemotherapeutic agents in the treatment of metastatic breast cancer and early-stage breast cancer with benefits gained in terms of overall survival (OS) and disease-free survival (DFS). However, even with response to taxane treatment the time to progression (TTP) is relatively short, prolonging life for a matter of months, with studies showing that patients treated with taxanes eventually relapse. This review focuses on chemoresistance to taxane treatment particularly in relation to the spindle assembly checkpoint (SAC) and dysfunctional regulation of apoptotic signaling. Since spindle microtubules are the primary drug targets for taxanes, important SAC proteins such as MAD2, BUBR1, Synuclein-gamma and Aurora A have emerged as potentially important predictive markers of taxane resistance, as have specific checkpoint proteins such as BRCA1. Moreover, overexpression of the drug efflux pump MDR-1/P-gp, altered expression of microtubule-associated proteins (MAPs) including tau, stathmin and MAP4 may help to identify those patients who are most at risk of recurrence and those patients most likely to benefit from taxane treatment. PMID:18068131

McGrogan, Barbara T; Gilmartin, Breege; Carney, Desmond N; McCann, Amanda

2007-11-12

338

Vesicle deformation by microtubules: A phase diagram  

NASA Astrophysics Data System (ADS)

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

Emsellem, Virginie; Cardoso, Olivier; Tabeling, Patrick

1998-10-01

339

Acentrosomal microtubule nucleation in higher plants  

Microsoft Academic Search

Higher plants have developed a unique pathway to control their cytoskeleton assembly and dynamics. In most other eukaryotes, microtubules are nucleated in vivo at the nucleation and organizing centers and are involved in the establishment of polarity. Although the major cytoskeletal components are common to plant and animal cells, which suggests conserved regulation mechanisms, plants do not possess centrosome-like organelles.

Anne-Catherine Schmit

2002-01-01

340

Automatic tip selection for microtubule dynamics quantification  

Microsoft Academic Search

Microtubule (MT) dynamics quantification includes modeling of elongation, rapid shortening, and pauses. It indicates the effect of the cancer treatment drug paclitaxel because the drug causes MTs to bundle, which will in turn inhibit successful mitosis of cancerous cells. Thus, automatic MT dynamics analysis has been researched intensely because it allows for faster evaluation of potential cancer treatments and better

Mario O. Malavé; Xuran Zhao; Koon Yin Kong; A. I. Marcus; M. D. Wang

2010-01-01

341

Track Switching and Crossing by Microtubule Motors.  

NASA Astrophysics Data System (ADS)

Cytoskeletal filaments in cells form a network of crossing tracks for motor proteins carrying vesicular and protein cargoes. The ability to pass through, switch, or dissociate at such intersections is relevant to the motor's ability to effectively navigate in the cell and deliver goods to the appropriate location. We have formed an in vitro system of crossed microtubules to study the outcome of kinesin motors and dynein-dynactin complexes when they encounter an intersection. Microtubules were flowed into the sample chamber from two orthogonal directions and aligned with the flow direction when they attached to glass cover slips via biotin-streptavidin. The first flow direction defined the microtubules closest to the glass surface. Using total internal reflection fluorescence (TIRF) microscopy, we visualized single GFP-kinesin motors and dynein-dynactin-GFP complexes during processive motility at 1 mM ATP. Both dynein and kinesin can switch microtubules, pass by an intersection, or dissociate. Using optical trapping, we placed 1 ?m polymer beads decorated with multiple motors to simulate a large cargo encountering an intersection at 1 mM ATP. Beads are more likely to pause at the intersection at high motor number and can pass and switch as the motor concentration is titrated down. The differences between kinesin and dynein could inform of the ability of these motors to navigate the cell, both separately and in coordination. Supported by NIH grant AR51174.

Ross, Jennifer; Wallace, Karen; Shuman, Henry; Holzbaur, Erika; Goldman, Yale

2006-03-01

342

Microtubule motors: doin' it without dynactin.  

PubMed

The minus-end directed microtubule motor protein cytoplasmic dynein contributes to many aspects of cell division and it is generally believed that these mitotic functions require the dynein activator and processivity factor, dynactin. New research now shows that dynein accomplishes many of its mitotic functions without dynactin. PMID:23845243

Wadsworth, Patricia; Lee, Wei-Lih

2013-07-01

343

Immunoperoxidase visualisation of microtubules and microtubular proteins  

Microsoft Academic Search

MICROTUBULES are ubiquitous eukaryotic organelles, formed by polymerised tubulin and presumably some additional proteins, which are involved in a wide variety of cell functions (for review see refs 1 and 2). Evidence has been presented for the existence of a dynamic equilibrium between polymerised and non-polymerised microtubular proteins3. Recently immunofluorescence of cultured cells demonstrated a fine cytoplasmic network that was

J. de Mey; J. Hoebeke; M. DE BRABANDER; G. GEUENS; M. JONIAU

1976-01-01

344

Plant cytoskeleton: DELLA connects gibberellins to microtubules.  

PubMed

A new study reveals that DELLA proteins directly interact with the prefoldin complex, thus regulating tubulin subunit availability in a gibberellin-dependent manner. This finding provides a mechanistic link between the growth-promoting plant hormone gibberellin and cortical microtubule organization. PMID:23743413

Dixit, Ram

2013-06-01

345

Coordination of opposite-polarity microtubule motors  

Microsoft Academic Search

any cargoes move bidirectionally, frequently re- versing course between plus- and minus-end microtubule travel. For such cargoes, the extent and importance of interactions between the opposite-polarity motors is unknown. In this paper we test whether opposite- polarity motors on lipid droplets in Drosophila embryos are coordinated and avoid interfering with each other's activity, or whether they engage in a tug

Steven P. Gross; Michael A. Welte; Steven M. Block; Eric F. Wieschaus

2002-01-01

346

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

347

2HDMC — two-Higgs-doublet model calculator  

NASA Astrophysics Data System (ADS)

We describe version 1.0.6 of the public C++ code 2HDMC, which can be used to perform calculations in a general, CP-conserving, two-Higgs-doublet model (2HDM). The program features simple conversion between different parametrizations of the 2HDM potential, a flexible Yukawa sector specification with choices of different Z-symmetries or more general couplings, a decay library including all two-body — and some three-body — decay modes for the Higgs bosons, and the possibility to calculate observables of interest for constraining the 2HDM parameter space, as well as theoretical constraints from positivity and unitarity. The latest version of the 2HDMC code and full documentation is available from: http://www.isv.uu.se/thep/MC/2HDMC.New version program summaryProgram title: 2HDMC Catalogue identifier: AEFI_v1_1 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEFI_v1_1.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU GPL No. of lines in distributed program, including test data, etc.: 12?110 No. of bytes in distributed program, including test data, etc.: 92?731 Distribution format: tar.gz Programming language: C++ Computer: Any computer running Linux Operating system: Linux RAM: 5 Mb Catalogue identifier of previous version: AEFI_v1_0 Journal reference of previous version: Comput. Phys. Comm. 180 (2010) 189 Classification: 11.1 External routines: GNU Scientific Library (http://www.gnu.org/software/gsl/) Does the new version supersede the previous version?: Yes Nature of problem: Determining properties of the potential, calculation of mass spectrum, couplings, decay widths, oblique parameters, muon g-2, and collider constraints in a general two-Higgs-doublet model. Solution method: From arbitrary potential and Yukawa sector, tree-level relations are used to determine Higgs masses and couplings. Decay widths are calculated at leading order, including FCNC decays when applicable. Decays to off-shell vector bosons are obtained by numerical integration. Observables are computed (analytically or numerically) as function of the input parameters. Reasons for new version: Improved calculation of the oblique parameters. Summary of revisions: The computation of the oblique parameters has been improved to give reliable results in the case of degenerate masses for the Higgs bosons. Another issue in the oblique parameter calculation, affecting the numerical values of S, U, V, and X (independently of the Higgs boson masses), has been corrected. Restrictions: CP-violation is not treated. Running time: Less than 0.1 s on a standard PC.

Eriksson, David; Rathsman, Johan; Stål, Oscar

2010-04-01

348

Three-dimensional structure of cytoplasmic dynein bound to microtubules  

PubMed Central

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

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

2007-01-01

349

Mechanism of microtubule array expansion in the cytokinetic phragmoplast  

PubMed Central

In land plants, the cell plate partitions the daughter cells at cytokinesis. The cell plate initially forms between daughter nuclei and expands centrifugally until reaching the plasma membrane. The centrifugal development of the cell plate is driven by the centrifugal expansion of the phragmoplast microtubule array, but the molecular mechanism underlying this expansion is unknown. Here, we show that the phragmoplast array comprises stable microtubule bundles and dynamic microtubules. We find that the dynamic microtubules are nucleated by ?-tubulin on stable bundles. The dynamic microtubules elongate at the plus ends and form new bundles preferentially at the leading edge of the phragmoplast. At the same time, they are moved away from the cell plate, maintaining a restricted distribution of minus ends. We propose that cycles of attachment of ?-tubulin complexes onto the microtubule bundles, microtubule nucleation and bundling, accompanied by minus-end-directed motility, drive the centrifugal development of the phragmoplast.

Murata, Takashi; Sano, Toshio; Sasabe, Michiko; Nonaka, Shigenori; Higashiyama, Tetsuya; Hasezawa, Seiichiro; Machida, Yasunori; Hasebe, Mitsuyasu

2013-01-01

350

Measuring the Dynamic Parameters of MCF7 Cell Microtubules  

NASA Astrophysics Data System (ADS)

Microtubules are the key component of the cytoskeleton. They are intrinsically dynamic displaying dynamic instability in which they randomly switch between a phase of growing and shrinking, both in vitro and in vivo. This dynamic is specified by the following parameters: growing rate, shrinking rate, frequency of catastrophe, and frequency of rescue. In this work, we will present our primary results in which we measured the dynamic parameters of a single microtubule polymerized from MCF7 tubulin in vitro. The results are significant since the MCF7 microtubules are non-neural mammalian consisting of different beta tubulin isotypes in their structures as compared to neural mammalian microtubules, such as bovine brain. The unique dynamic parameters of individual MCF7 microtubules in vitro, which are reported for the first time, indicate that non-neural microtubules can be fundamentally different from neural microtubules.

Winton, Carly; Shojania Feizabadi, Mitra

2013-03-01

351

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

352

Partial Depletion of Gamma-Actin Suppresses Microtubule Dynamics  

PubMed Central

Actin and microtubule interactions are important for many cellular events, however these interactions are poorly described. Alterations in ?-actin are associated with diseases such as hearing loss and cancer. Functional investigations demonstrated that partial depletion of ?-actin affects cell polarity and induces resistance to microtubule-targeted agents. To determine whether ?-actin alterations directly affect microtubule dynamics, microtubule dynamic instability was analyzed in living cells following partial siRNA depletion of ?-actin. Partial depletion of ?-actin suppresses interphase microtubule dynamics by 17.5% due to a decrease in microtubule shortening rates and an increase in microtubule attenuation. ?-Actin partial depletion also increased distance-based microtubule catastrophe and rescue frequencies. In addition, knockdown of ?-actin delayed mitotic progression, partially blocking metaphase–anaphase transition and inhibiting cell proliferation. Interestingly, in the presence of paclitaxel, interphase microtubule dynamics were further suppressed by 24.4% in the ?-actin knockdown cells, which is comparable to 28.8% suppression observed in the control siRNA treated cells. Paclitaxel blocked metaphase–anaphase transition in both the ?-actin knockdown cells and the control siRNA cells. However, the extent of mitotic arrest was much higher in the control cells (28.4%), compared to the ?-actin depleted cells (8.5%). Therefore, suppression of microtubule dynamics by partial depletion of ?-actin is associated with marked delays in metaphase-anaphase transition and not mitotic arrest. This is the first demonstration that ?-actin can modulate microtubule dynamics by reducing the microtubule shortening rate, promoting paused/attenuated microtubules, and increasing transition frequencies suggesting a mechanistic link between ?-actin and microtubules. © 2013 Wiley Periodicals, Inc

Po'uha, Sela T; Honore, Stephane; Braguer, Diane; Kavallaris, Maria

2013-01-01

353

Characterizing and engineering microtubule properties for use in hybrid nanodevices  

NASA Astrophysics Data System (ADS)

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

Jeune-Smith, Yolaine

354

Gallagher-Moszkowski (GM) doublet bands in {sup 162}Ho  

SciTech Connect

High-spin states in {sup 162}Ho have been populated in the reaction {sup 160}Gd({sup 7}Li, 5n) at a beam energy of 49 MeV. The K {sup {pi}}=1{sup -} band, the low-K Gallagher-Moszkowski (GM) partner band of known high-K(K {sup {pi}}=6{sup -}) band, based on the configuration {pi}7/2{sup -}[523]{center_dot}{nu}5/2{sup +}[642], and the K {sup {pi}}=6{sup +} band, the high-K GM partner band of known low-K(K{sup {pi}}=1{sup +}) band, based on the configuration {pi}7/2{sup -}[523]{center_dot}{nu}5/2{sup -}[523], have been identified. GM splitting energies, defined as {delta}E{sub GM}=E{sub int}{sup K-}E{sub int}{sup K,} 80 keV and -135 keV were extracted from these two sets of GM doublet bands, respectively. They are comparable with 65 keV and -145 keV, reported recently by Hojman et al. for the corresponding configurations {pi}7/2{sup -}[523]{center_dot}{nu}5/2{sup +}[642] and {pi}7/2{sup -}[523]{center_dot}{nu}5/2{sup -}[523] in {sup 164}Ho, respectively.

Liang Guodong; Wang Shouyu; Liu Yunzuo; Ma Yingjun; Lu Jingbin; Li Mingfei; Cui Xingzhu; Zhao Guangyi; Li Xianfeng; Wu Xiaoguang; Zhu Lihua; Li Guangshen; Wen Shuxian; Yang Chunxiang [Department of Physics, Jilin University, Changchun 130023 (China); China Institute of Atomic Energy, P.O. Box 275, Beijing 102413 (China)

2005-12-15

355

Gallagher-Moszkowski (GM) doublet bands in 162Ho  

NASA Astrophysics Data System (ADS)

High-spin states in 162Ho have been populated in the reaction 160Gd(7Li, 5n) at a beam energy of 49 MeV. The K ?=1- band, the low-K Gallagher-Moszkowski (GM) partner band of known high-K(K ?=6-) band, based on the configuration ?7/2-[523]??5/2+[642], and the K ?=6+ band, the high-K GM partner band of known low-K(K?=1+) band, based on the configuration ?7/2-[523]??5/2-[523], have been identified. GM splitting energies, defined as ?EGM=EintK<-EintK>, 80 keV and -135 keV were extracted from these two sets of GM doublet bands, respectively. They are comparable with 65 keV and -145 keV, reported recently by Hojman et al. for the corresponding configurations ?7/2-[523]??5/2+[642] and ?7/2-[523]??5/2-[523] in 164Ho, respectively.

Liang, Guodong; Wang, Shouyu; Liu, Yunzuo; Ma, Yingjun; Lu, Jingbin; Li, Mingfei; Cui, Xingzhu; Zhao, Guangyi; Li, Xianfeng; Wu, Xiaoguang; Zhu, Lihua; Li, Guangshen; Wen, Shuxian; Yang, Chunxiang

2005-12-01

356

GTP?S microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs)  

PubMed Central

Microtubule plus-end-tracking proteins (+TIPs) localize to growing microtubule plus ends to regulate a multitude of essential microtubule functions. End-binding proteins (EBs) form the core of this network by recognizing a distinct structural feature transiently existing in an extended region at growing microtubule ends and by recruiting other +TIPs to this region. The nature of the conformational difference allowing EBs to discriminate between tubulins in this region and other potential tubulin binding sites farther away from the microtubule end is unknown. By combining in vitro reconstitution, multicolor total internal reflection fluorescence microscopy, and electron microscopy, we demonstrate here that a closed microtubule B lattice with incorporated GTP?S, a slowly hydrolyzable GTP analog, can mimic the natural EB protein binding site. Our findings indicate that the guanine nucleotide ?-phosphate binding site is crucial for determining the affinity of EBs for lattice-incorporated tubulin. This defines the molecular mechanism by which EBs recognize growing microtubule ends.

Maurer, Sebastian P.; Bieling, Peter; Cope, Julia; Hoenger, Andreas; Surrey, Thomas

2011-01-01

357

Dynein arms are oscillating force generators  

NASA Astrophysics Data System (ADS)

Eukaryotic flagella beat rhythmically. Dynein is a protein that powers flagellar motion, and oscillation may be inherent to this protein. Here we determine whether oscillation is a property of dynein arms themselves or whether oscillation requires an intact axoneme, which is the central core of the flagellum and consists ofa regular array of microtubules. Using optical trapping nanometry,, we measured the force generated by a few dynein arms on an isolated doublet microtubule. When the dynein arms on the doublet microtubule contact a singlet microtubule and are activated by photolysis of caged ATP, they generate a peak force of ~6pN and move the singlet microtubule over the doublet microtubule in a processive manner. The force and displacement oscillate with a peak-to-peak force and amplitude of ~2pN and ~30nm, respectively. The geometry of the interaction indicates that very few (possibly one) dynein arms are needed to generate the oscillation. The maximum frequency of the oscillation at 0.75mM ATP is ~70Hz this frequency decreases as the ATP concentration decreases. A similar oscillatory force is also generated by inner dynein arms alone on doublet microtubules that are depleted of outer dynein arms. The oscillation of the dynein arm may be a basic mechanism underlying flagellar beating.

Shingyoji, Chikako; Higuchi, Hideo; Yoshimura, Misako; Katayama, Eisaku; Yanagida, Toshio

1998-06-01

358

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

PubMed Central

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

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

2013-01-01

359

The Sodium Doublets as Youth Indicators for Low-Mass Stars  

NASA Astrophysics Data System (ADS)

We investigate the use of the Na I doublets at 5890 and 5896 Å (the Fraunhofer D lines) and 8183 and 8195 Å as gravity indicators for stars of late K and M spectral type. As is well known, the equivalent widths (EWs) of these doublets increase with photospheric log(g). We show that the EWs of members of the ? Pictoris moving group (BPMG) (age 10-20 Myr) lie between the EWs of giants and main sequence stars based on the analysis of approx. 200 spectra collected with the MDM 1.3-meter McGraw-Hill telescope and the SMARTS 1.5-meter telescope. We find the Na D lines are useful age indicators for low mass BPMG candidates earlier than M2 and the 8200 Å doublet becomes useful for stars later than M4. The EWs of the Na doublets may therefore be used to establish low gravity, hence youth, among low mass stars in general.

Schlieder, J. E.; Fielding, D.; Lepine, S.; Rice, E.; Tomasino, R.; Simon, M.; Shara, M. M.

2011-12-01

360

Self-organization of interphase microtubule arrays in fission yeast.  

PubMed

Microtubule organization is key to eukaryotic cell structure and function. In most animal cells, interphase microtubules organize around the centrosome, the major microtubule organizing centre (MTOC). Interphase microtubules can also become organized independently of a centrosome, but how acentrosomal microtubules arrays form and whether they are functionally equivalent to centrosomal arrays remains poorly understood. Here, we show that the interphase microtubule arrays of fission yeast cells can persist independently of nuclear-associated MTOCs, including the spindle pole body (SPB)--the centrosomal equivalent. By artificially enucleating cells, we show that arrays can form de novo (self-organize) without nuclear-associated MTOCs, but require the microtubule nucleator mod20-mbo1-mto1 (refs 3-5), the bundling factor ase1 (refs 6,7), and the kinesin klp2 (refs 8,9). Microtubule arrays in enucleated and nucleated cells are morphologically indistinguishable and similarly locate to the cellular axis and centre. By simultaneously tracking nuclear-independent and SPB-associated microtubule arrays within individual nucleated cells, we show that both define the cell centre with comparable precision. We propose that in fission yeast, nuclear-independent, self-organized, acentrosomal microtubule arrays are structurally and functionally equivalent to centrosomal arrays. PMID:16998477

Carazo-Salas, Rafael E; Nurse, Paul

2006-09-24

361

Doublet splitting due to intercell tunneling in three-Josephson-junction flux qubit  

Microsoft Academic Search

It has been shown that for a three-Josephson-junction flux qubit with non-negligible loop inductance, the intercell tunneling in the three-dimensional potential of the system can cause doublet splitting in the ground state and the first excited state. This paper presents the calculation results of the magnitude of doublet splitting for various values of parameters of the system. For realistic values

Y. Shimazu; K. Ochiai; E. Shinozaki

2010-01-01

362

Low-Temperature Elastic Properties of Non-Kramers Doublet Compound PrMg3  

Microsoft Academic Search

We have investigated low-temperature elastic properties of PrMg3 with a non-Kramers Gamma3 doublet ground state by using ultrasonic measurements under high field up to 18 T down to 20 mK. A softening of the elastic constant (C11-C12)\\/2 has been observed below 8 K due to the non-magnetic Gamma3 doublet possessing electric quadrupole Ou and Ov with Gamma3 symmetry and magnetic

K. Araki; K. Mitsumoto; M. Akatsu; Y. Nemoto; T. Goto; H. S. Suzuki; H. Tanida; S. Takagi; S. Yasin; S. Zherlitsyn; J. Wosnitza

2011-01-01

363

Doublet frequencies and codon weighting in the DNA of Escherichia coli and its phages  

Microsoft Academic Search

Summary A compilation of nucleic acid sequences fromE.coli and its phages has been analysed for the frequency of occurrence of nearest neighbour base doublets and codons. Several statistically significant deviations from random are found in both doublet and codon frequencies. The deviations inE.coli also appear to occur in ? and in the coat protein gene of MS2, whereas T4 and

R. A. Elton; G. J. Russell; J. H. Subak-Sharpe

1976-01-01

364

Subpellicular Microtubules in Apicomplexa and Trypanosomatids  

Microsoft Academic Search

\\u000a The cytoskeleton plays a fundamental role in various processes such as the establishment of cell shape, cell locomotion, and\\u000a the intracellular motility of various structures found in eukaryotic cells. Microtubules are among the most conspicuous structures\\u000a in the cytoskeleton. They can be found free in the cytoplasm, forming the mitotic spindle or assembled in various structures.\\u000a A special type of

Wanderley de Souza; Marcia Attias

365

Rapid Movement of Microtubules in Axons  

Microsoft Academic Search

Cytoskeletal and cytosolic proteins are transported along axons in the slow components of axonal transport at average rates of about 0.002–0.1 ?m\\/s. This movement is essential for axonal growth and survival, yet the mechanism is poorly understood. Many studies on slow axonal transport have focused on tubulin, the subunit protein of microtubules, but attempts to observe the movement of this

Lei Wang; Anthony Brown

2002-01-01

366

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

PubMed Central

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

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

2004-01-01

367

STUDIES ON THE MICROTUBULES IN HELIOZOA  

PubMed Central

Electron microscope preparations were made of specimens of Actinosphaerium nucleofilum fixed in glutaraldehyde before, during, and after exposure to high pressures (4,000 to 8,000 psi). A study of this material showed that, although other organelles were relatively stable, the microtubular elements of the axopodia and cytosome became unstable under pressure. Their rapid disintegration under pressure was correlated with beading and retraction of the axopodia. Moreover, after the release of pressure, microtubules reappeared as soon as, or sooner than the reextension of the axopodia. The rate of disintegration increased as the pressure was raised. At 4,000 psi, few if any tubules remained after 10 min, whereas at 6,000 and 8,000 psi the disintegration was much more rapid. Some adaptational reorganization of the microtubules and axopodia occurred while relatively low pressures were maintained. This was accompanied by an actual elongation of the axopodia in specimens maintained for 20 min at 4,000 psi, but was confined to knoblike axopodial remnants in animals kept at 6,000 psi. No regeneration of tubules or axopodia occurred at 8,000 psi. The presence of fibers and a finely fibrillar material in pressurized animals suggests that these may be derivatives of microtubular disintegration. This evidence, though purely morphological, is consistent with the hypothesis that microtubules play an important role not only in maintaining the formstability of the axopodia, but also in the active process by which the axopodia reextend themselves after retraction.

Tilney, Lewis G.; Hiramoto, Yukio; Marsland, Douglas

1966-01-01

368

Molecular Communication: Simulation of Microtubule Topology  

NASA Astrophysics Data System (ADS)

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

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

369

CLASP-mediated cortical microtubule organization guides PIN polarization axis.  

PubMed

Recent evidence indicates a correlation between orientation of the plant cortical microtubule cytoskeleton and localization of polar cargoes. However, the molecules and mechanisms that create this correlation have remained unknown. Here we show that, in Arabidopsis thaliana, the microtubule orientation regulators CLASP and MAP65 (refs 3, 4) control the abundance of polarity regulator PINOID kinase at the plasma membrane. By localized upregulation of clathrin-dependent endocytosis at cortical microtubule- and clathrin-rich domains orthogonal to the axis of polarity, PINOID accelerates the removal of auxin transporter PIN proteins from those sites. This mechanism links directional microtubule organization to the polar localization of auxin transporter PIN proteins, and clarifies how microtubule-enriched cell sides are kept distinct from polar delivery domains. Our results identify the molecular machinery that connects microtubule organization to the regulation of the axis of PIN polarization. PMID:23515161

Kakar, Klementina; Zhang, Hongtao; Scheres, Ben; Dhonukshe, Pankaj

2013-03-20

370

Properties of microtubule bundles induced by Glyceraldehyde3-phosphate dehydrogenase  

Microsoft Academic Search

The binding of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) to microtubules causes the microtubules to assemble into large bundles. This bundling can be considered as a further step in the assembly of supramolecular structures. The rate of bundle formation, after addition of GAPDH to preformed microtubules, is not dependent on the GAPDH concentration and reflects bundling kinetics. Bundle disassembly can be

Marijke Somers; Yves Engelborghs

1991-01-01

371

EB1 identifies sites of microtubule polymerisation during neurite development  

Microsoft Academic Search

EB1 is a microtubule associated protein which interacts with the APC tumour suppressor protein and components of the cytoplasmic dynein\\/dynactin complex. EB1 is also a specific marker of growing microtubule tips. Here we demonstrate that EB1 protein levels are increased during axon but not dendrite formation in differentiated N2A neuroblastoma cells, and that EB1 localises to microtubule tips throughout extending

E. E Morrison; P. M Moncur; J. M Askham

2002-01-01

372

Regulation of microtubule assembly by human EB1 family proteins  

Microsoft Academic Search

The EB1 family proteins are highly conserved microtubule-associated proteins. The EB1 protein in yeast has been shown to play an important role in regulating microtubule dynamics and chromosome segregation. Human EB1 family proteins include EB1, RP1 and EBF3. Although EB1 and RP1 have been shown to associate with microtubules, the subcellular localization of endogenous EBF3 had not been characterized. The

Wen Bu; Li-Kuo Su

2001-01-01

373

Microtubule organization by the budding yeast spindle pole body  

Microsoft Academic Search

In budding yeast microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the ?-tubulin Tub4p, and two additional proteins, the SPB components

Michael Knop; Gislene Pereira; Elmar Schiebel

1999-01-01

374

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

PubMed

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

2013-01-08

375

Sperm ultrastructure of the European hornet Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae).  

PubMed

This study represents the first sperm description of a Vespinae species (Vespa crabro). The acrosome consists of an acrosomal vesicle and a perforatorium. The nucleus has compact chromatin and shows lenticular structures on the nuclear envelope. These structures, which have never been observed in a hymenopteran sperm, could be clusters of nuclear pores. The centriolar adjunct has an asymmetric pattern and shows a structured periphery. The centriole consists of 9 accessory microtubules and 9 doublet microtubules devoid of arms and spokes. The axoneme has a 9+9+2 microtubule pattern and the accessory microtubules have 16 protofilaments. The mitochondrial derivatives differ in length and diameter. The larger one is adjacent to the nuclear base, while the smaller one begins below the centriolar adjunct. They possess three distinct areas and a large paracrystalline region, which occurs only in the large one. The large mitochondrial derivative ends first, followed by the small one. The axoneme gradually disorganizes: first the central microtubules disappear, then the doublets, which show opened B-tubules, and finally the accessory microtubules. The sperm morphology of V. crabro is very similar to that of the polistine wasp, Agelaia vicina. This can indicate that, in Vespidae, sperm morphology is maintained without important variations among subfamilies and/or that this similarity indicates close phylogenetic relationship between these two subfamilies. Although Vespidae phylogenetically related to Formicidae, these data suggest that the former more closely related to Apoidea than to Formicidae. PMID:18675936

Mancini, Karina; Lino-Neto, José; Dolder, Heidi; Dallai, Romano

2008-09-09

376

Theoretical Description of Microtubule Dynamics in Fission Yeast During Interphase  

NASA Astrophysics Data System (ADS)

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

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

2009-03-01

377

Force-generation and dynamic instability of microtubule bundles  

PubMed Central

Individual dynamic microtubules can generate pushing or pulling forces when their growing or shrinking ends are in contact with cellular objects such as the cortex or chromosomes. These microtubules can operate in parallel bundles, for example when interacting with mitotic chromosomes. Here, we investigate the force-generating capabilities of a bundle of growing microtubules and study the effect that force has on the cooperative dynamics of such a bundle. We used an optical tweezers setup to study microtubule bundles growing against a microfabricated rigid barrier in vitro. We show that multiple microtubules can generate a pushing force that increases linearly with the number of microtubules present. In addition, the bundle can cooperatively switch to a shrinking state, due to a force-induced coupling of the dynamic instability of single microtubules. In the presence of GMPCPP, bundle catastrophes no longer occur, and high bundle forces are reached more effectively. We reproduce the observed behavior with a simple simulation of microtubule bundle dynamics that takes into account previously measured force effects on single microtubules. Using this simulation, we also show that a constant compressive force on a growing bundle leads to oscillations in bundle length that are of potential relevance for chromosome oscillations observed in living cells.

Laan, Liedewij; Husson, Julien; Munteanu, E. Laura; Kerssemakers, Jacob W. J.; Dogterom, Marileen

2008-01-01

378

Microtubule-binding agents: a dynamic field of cancer therapeutics  

PubMed Central

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

Dumontet, Charles; Jordan, Mary Ann

2010-01-01

379

Anti-vascular actions of microtubule-binding drugs  

PubMed Central

Microtubule-binding drugs (MBDs) are widely used in cancer chemotherapy and also have clinically relevant anti-angiogenic and vascular-disrupting properties. These anti-vascular actions are due in part to direct effects on endothelial cells, and all MBDs (i.e. both microtubule-stabilizing and -destabilizing) inhibit endothelial cell proliferation, migration, and tube formation in vitro, actions which are thought to correspond to therapeutic anti-angiogenic 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 sub-cellular 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 anti-vascular actions of the microtubule-binding drugs, information which could be useful in the identification or design of agents whose actions more selectively target the tumor vasculature.

Schwartz, Edward L.

2009-01-01

380

Surface landing of microtubule nanotracks influenced by lithographically patterned channels  

NASA Astrophysics Data System (ADS)

Microtubules, which serve as cellular structural components in nature, can be placed within a lithographically patterned channel as engineered nanoscale tracks for bionanotechnology applications. We study the landing behavior of microtubules upon their diffusion onto a kinesin-coated glass surface in the presence of the channel. The influence of channel geometry on the landing rate of microtubules is experimentally characterized using channels with varying width. Additionally, we develop a theoretical model to quantitatively analyze our data by accounting for geometrical constraints due to both the width and height of the channels against the diffusion of the landing microtubules.

Lin, Chih-Tin; Kao, Ming-Tse; Meyhofer, Edgar; Kurabayashi, Katsuo

2009-09-01

381

A divergent canonical WNT-signaling pathway regulates microtubule dynamics  

PubMed Central

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

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

2004-01-01

382

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

383

Nonlinear dynamics of cilia and flagella  

NASA Astrophysics Data System (ADS)

Cilia and flagella are hairlike extensions of eukaryotic cells which generate oscillatory beat patterns that can propel micro-organisms and create fluid flows near cellular surfaces. The evolutionary highly conserved core of cilia and flagella consists of a cylindrical arrangement of nine microtubule doublets, called the axoneme. The axoneme is an actively bending structure whose motility results from the action of dynein motor proteins cross-linking microtubule doublets and generating stresses that induce bending deformations. The periodic beat patterns are the result of a mechanical feedback that leads to self-organized bending waves along the axoneme. Using a theoretical framework to describe planar beating motion, we derive a nonlinear wave equation that describes the fundamental Fourier mode of the axonemal beat. We study the role of nonlinearities and investigate how the amplitude of oscillations increases in the vicinity of an oscillatory instability. We furthermore present numerical solutions of the nonlinear wave equation for different boundary conditions. We find that the nonlinear waves are well approximated by the linearly unstable modes for amplitudes of beat patterns similar to those observed experimentally.

Hilfinger, Andreas; Chattopadhyay, Amit K.; Jülicher, Frank

2009-05-01

384

The 65-kDa Carrot Microtubule-Associated Protein Forms Regularly Arranged Filamentous Cross-Bridges between Microtubules  

Microsoft Academic Search

In plants, cortical microtubules (MTs) occur in characteristically parallel groups maintained up to one microtubule diameter apart by fine filamentous cross-bridges. However, none of the plant microtubule-associated proteins (MAPs) so far purified accounts for the observed separation between MTs in cells. We previously isolated from carrot cytoskeletons a MAP fraction including 120-and 65-kDa MAPs and have now separated the 65-kDa

Jordi Chan; Cynthia G. Jensen; Lawrence C. W. Jensen; Max Bush; Clive W. Lloyd

1999-01-01

385

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

Microsoft Academic Search

A subset of mRNAs, polyribosomes, and poly(A)-binding proteins copurify with microtubules from sea urchin embryos. Several lines of evidence in- dicate that the interaction of microtubules with ribo- somes is specific: a distinct stalk-like structure ap- pears to mediate their association; ribosomes bind to microtubules with a constant stoichiometry through several purification cycles; and the presence of ribo- somes in

Danielle Hamill; Jill Davis; Julie Drawbridge; Kathy A. Suprenant

1994-01-01

386

Roles of Microtubule Bias and Joining in the Self-Organization of Microtubule Driven by Dynein C - A Modeling Study  

Microsoft Academic Search

Microtubules driven by dynein protein motors may form self-organized circular patterns, understanding which could be of importance to the development of potential nano-bio-machines. It was expected that the microtubule bias and joining could play crucial roles in self-organized movement of microtubules. A Monte Carlo modeling study was conducted to confirm and evaluate contributions of these two parameters to self-organization of

Q. Chen; D. Y. Li; K. Oiwa

2009-01-01

387

Laser-transected microtubules exhibit individuality of regrowth, however most free new ends of the microtubules are stable  

Microsoft Academic Search

To study the possible mechanism of microtubule turnover in interphase cells, we have used the 266-nm wavelength of a short-pulsed Nd\\/YAG la- ser to transect microtubules in situ in PtK2 cells at predefined regions. The regrowth and shrinkage of the transected microtubules have been examined by stain- ing the treated cells with antitubulin mAb at various time points after laser

Wen Tao; Robert J. Walter; Michael W. Berns

1988-01-01

388

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

Microsoft Academic Search

During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation.\\u000a Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These\\u000a kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they\\u000a can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the

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

2010-01-01

389

Genetic variation in SPAG16 regions encoding the WD40 repeats is not associated with reduced sperm motility and axonemal defects in a population of infertile males  

PubMed Central

Background SPAG16 is a critical structural component of motile cilia and flagella. In the eukaryotic unicellular algae Chlamydomonas, loss of gene function causes flagellar paralysis and prevents assembly of the “9?+?2” axoneme central pair. In mice, we have previously shown that loss of Spag16 gene function causes male infertility and severe sperm motility defects. We have also reported that a heterozygous mutation of the human SPAG16 gene reduces stability of the sperm axonemal central apparatus. Methods In the present study, we analyzed DNA samples from 60 infertile male volunteers of Western European (Italian) origin, to search for novel SPAG16 gene mutations, and to determine whether increased prevalence of SPAG16 single nucleotide polymorphisms (SNPs) was associated with infertility phenotypes. Semen parameters were evaluated by light microscopy and sperm morphology was comprehensively analyzed by transmission electron microscopy (TEM). Results For gene analysis, sequences were generated covering exons encoding the conserved WD40 repeat region of the SPAG16 protein and the flanking splice junctions. No novel mutations were found, and the four SNPs in the assessed gene region were present at expected frequencies. The minor alleles were not associated with any assessed sperm parameter in the sample population. Conclusions Analysis of the SPAG16 regions encoding the conserved WD repeats revealed no evidence for association of mutations or genetic variation with sperm motility and ultrastructural sperm characteristics in a cohort of Italian infertile males.

2012-01-01

390

Estimation of the diffusion-limited rate of microtubule assembly.  

PubMed Central

Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth. Images FIGURE 1

Odde, D J

1997-01-01

391

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

392

What is the Nature of the Doublets in the E-Methanol Lamb-Dip Spectra?  

NASA Astrophysics Data System (ADS)

A large number of methanol lines have been measured with a Lamb-dip technique in the frequency range 75-510 GHz. A few series of doublets for the transitions with selection rules ? J = 0, ? K = ± 1, ± 3, ± 5, E_1 leftrightarrow E_2 and ? J = ± 1, ? K = ± 1 between the torsional-rotational levels of E-methanol in the ?_t = 0 state have been observed. These doublets were not predicted and were not observed earlier. In a traditional approach to the methanol molecule (as a nearly prolate asymmetric top with an internal rotation) these doublets may only originate from the magnetic hyperfine structure of the E-methanol torsional-rotational levels. However, there are some signs in spectra indicating that the doublets are sensitive to the parity selections rules. If so, the origin of the doublets is an inversion splitting of the E(+) and E(-) energy levels. This exciting interpretation seems to be feasible. The results of the experimental measurements will be presented and the possibility of a new type of the inversion motion in the CH_3OH molecule due to the proton tunneling in the H-O-C-H plane will be discussed .

Belov, S. P.; Burenin, A. V.; Golubiatnikov, G. Yu.; Lapinov, A. V.

2013-06-01

393

Energy separation of the 1+/1- parity doublet in 20Ne  

NASA Astrophysics Data System (ADS)

The parity doublet of 1+/1- states of Ne-20 at 11.26 MeV excitation energy is one of the best known test cases to study the weak part of the nuclear Hamiltonian. The feasibility of parity violation experiments depend on the effective nuclear enhancement factor (RN/|E(1+) - E(l-)|) which amplifies the impact of the matrix element of the weak interaction on observables indicating parity mixing. An extreme large value of Rn/|E(1+) - E(l-)| = (670 ± 7000) MeV-1 was reported for the doublet in 20Ne. The large uncertainty depends amongst others on the large uncertainty of |E(1+) - E(l-)| = 7.7±5.5 keV of the parity doublet. Nuclear resonance fluorescence (NRF) experiments with linearly and circularly polarized photon beams were performed at the High Intensity Gamma-Ray Source at Duke University, Durham, NC, USA, to determine the energy difference of the parity doublet with higher precision. The different angular distributions for 0+ ? 1- ? 0+ and 0+ ? 1+ ? 0+ NRF cascades in polarized ?-ray beams were used to determine the energy difference of the parity doublet to 2.9(13) keV.

Beller, J.; Wagner, J.; Ahmed, M.; Deleanu, D.; Filipescu, D. M.; Glodariu, T.; Isaak, J.; Kelley, J. H.; Kwan, E.; Pietralla, N.; Raut, R.; Romig, C.; Rusev, G.; Scheck, M.; Stave, S. C.; Tonchev, A.; Tornow, W.; Weller, H. R.; Zamfir, N.-V.; Zweidinger, M.

2012-05-01

394

Molecular tools for studying the radial spoke.  

PubMed

Studies of cilia and flagella often entail biochemical analysis of axonemal complexes that either associate with the nine outer doublet microtubules or the two singlet microtubules in the 9+2 axoneme. Each complex contains multiple subunits, a few of which are ubiquitous vital proteins, while many are novel with prevalent domains that remain to be characterized. Investigation of axoneme biochemistry will continue providing insights into flagellar biology as well as molecular complexes in general. Yet, the complicated contents and extensive molecular interactions pose significant challenges in experimentation. As such, most biochemical studies remain limited to dynein motors and often require extensive training and expensive equipment. The rapid accumulation of high-throughput database and versatile research tools has now lessened the obstacles significantly. Here, we describe the strategies and methods that were used to circumvent some of the common difficulties to characterize the radial spoke in Chlamydomonas axoneme, some of which were tailored to students with little research experience. They could be adapted for the study of many other axonemal complexes and for classroom settings as well. PMID:23498732

Zhu, Xiaoyan; Liu, Yi; Sivadas, Priyanka; Gupta, Anjali; Yang, Pinfen

2013-01-01

395

The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella  

PubMed Central

Cilia and flagella are highly conserved microtubule (MT)-based organelles with motile and sensory functions, and ciliary defects have been linked to several human diseases. The 9 + 2 structure of motile axonemes contains nine MT doublets interconnected by nexin links, which surround a central pair of singlet MTs. Motility is generated by the orchestrated activity of thousands of dynein motors, which drive interdoublet sliding. A key regulator of motor activity is the dynein regulatory complex (DRC), but detailed structural information is lacking. Using cryoelectron tomography of wild-type and mutant axonemes from Chlamydomonas reinhardtii, we visualized the DRC in situ at molecular resolution. We present the three-dimensional structure of the DRC, including a model for its subunit organization and intermolecular connections that establish the DRC as a major regulatory node. We further demonstrate that the DRC is the nexin link, which is thought to be critical for the generation of axonemal bending.

Heuser, Thomas; Raytchev, Milen; Krell, Jeremy; Porter, Mary E.

2009-01-01

396

The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella.  

PubMed

Cilia and flagella are highly conserved microtubule (MT)-based organelles with motile and sensory functions, and ciliary defects have been linked to several human diseases. The 9 + 2 structure of motile axonemes contains nine MT doublets interconnected by nexin links, which surround a central pair of singlet MTs. Motility is generated by the orchestrated activity of thousands of dynein motors, which drive interdoublet sliding. A key regulator of motor activity is the dynein regulatory complex (DRC), but detailed structural information is lacking. Using cryoelectron tomography of wild-type and mutant axonemes from Chlamydomonas reinhardtii, we visualized the DRC in situ at molecular resolution. We present the three-dimensional structure of the DRC, including a model for its subunit organization and intermolecular connections that establish the DRC as a major regulatory node. We further demonstrate that the DRC is the nexin link, which is thought to be critical for the generation of axonemal bending. PMID:20008568

Heuser, Thomas; Raytchev, Milen; Krell, Jeremy; Porter, Mary E; Nicastro, Daniela

2009-12-14

397

Ultrastructural study on the spermiogenesis and spermatozoon of the metacercariae of Microphallus primas (Digenea), a parasite of Carcinus maenas.  

PubMed

The thread-like spermatozoon of the crab parasite Microphallus primas was studied by electron microscopy. A survey of the head region of the spermatozoon reveals three features hitherto unknown in Platyhelminthes spermatozoa. The first is the aberrant inclusion of the nucleus within one of the two axonemes, limited to the head end region. The second is the coexistence, in the same axoneme, of two patterns, 9 + 0 (doublets without dynein arms) and 9 + "1". The third is the presence of a layer of cortical microtubules running longitudinally from the zone where the nucleus goes from axoneme to the tail region (where the two flagella start). The sequence of events in spermatogenesis is similar to that described for most of the Digenea trematodes, and the spermiogenesis process conforms to a common plan in nearly the whole group. PMID:2310565

Castilho, F; Barandela, T

1990-02-01

398

Microtubules in Dendritic Spine Development and Plasticity  

PubMed Central

Recent studies indicate that microtubules (MTs) may play an important role in spine development and dynamics. Several imaging studies have now documented the exploration of dendritic spines by dynamic MTs in an activity-dependent manner. Furthermore, it was found that alterations of MT dynamics by pharmacological and molecular approaches exert profound influence on the development and plasticity of spines associated with neuronal activity. It is reasonable to speculate that dynamic MTs may be responsible for targeted delivery of specific cargos to a selected number of spines and/or for interacting with the actin cytoskeleton to generate the structural changes of spines associated with synaptic modifications.

Gu, Jiaping; Zheng, James Q.

2010-01-01

399

Microtubule Length Regulation by Molecular Motors  

NASA Astrophysics Data System (ADS)

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

Melbinger, Anna; Reese, Louis; Frey, Erwin

2012-06-01

400

Microtubule-Mediated and Microtubule-Independent Transport of Adenovirus Type 5 in HEK293 Cells?  

PubMed Central

Adenovirus serotypes 2 and 5 are taken into cells by receptor-mediated endocytosis, and following release from endosomes, destabilized virions travel along microtubules to accumulate around the nucleus. The entry process culminates in delivery of the viral genome through nuclear pores. This model is based on studies with conventional cell lines, such as HeLa and HEp-2, but in HEK293 cells, which are routinely used in this laboratory because they are permissive for replication of multiple adenovirus serotypes, a different trafficking pattern has been observed. Nuclei of 293 cells have an irregular shape, with an indented region, and virions directly labeled with carboxyfluorescein accumulate in a cluster within that indented region. The clusters, which form in close proximity to the microtubule organizing center (MTOC) and to the Golgi apparatus, are remarkably stable; a fluorescent signal can be seen in the MTOC region up to 16 h postinfection. Furthermore, if cells are infected and then undergo mitosis after the cluster is formed, the signal is found at each spindle pole. Despite the sequestration of virions near the MTOC, 293 cells are no less sensitive than other cells to productive infection with adenovirus. Even though cluster formation depends on intact microtubules, infectivity is not compromised by disruption of microtubules with either nocodazole or colchicine, as determined by expression of an enhanced green fluorescent protein reporter gene inserted in the viral genome. These results indicate that virion clusters do not represent the infectious pathway and suggest an alternative route to the nucleus that does not depend on nocodazole-sensitive microtubules.

Yea, Carmen; Dembowy, Joanna; Pacione, Laura; Brown, Martha

2007-01-01

401

Microtubule-mediated and microtubule-independent transport of adenovirus type 5 in HEK293 cells.  

PubMed

Adenovirus serotypes 2 and 5 are taken into cells by receptor-mediated endocytosis, and following release from endosomes, destabilized virions travel along microtubules to accumulate around the nucleus. The entry process culminates in delivery of the viral genome through nuclear pores. This model is based on studies with conventional cell lines, such as HeLa and HEp-2, but in HEK293 cells, which are routinely used in this laboratory because they are permissive for replication of multiple adenovirus serotypes, a different trafficking pattern has been observed. Nuclei of 293 cells have an irregular shape, with an indented region, and virions directly labeled with carboxyfluorescein accumulate in a cluster within that indented region. The clusters, which form in close proximity to the microtubule organizing center (MTOC) and to the Golgi apparatus, are remarkably stable; a fluorescent signal can be seen in the MTOC region up to 16 h postinfection. Furthermore, if cells are infected and then undergo mitosis after the cluster is formed, the signal is found at each spindle pole. Despite the sequestration of virions near the MTOC, 293 cells are no less sensitive than other cells to productive infection with adenovirus. Even though cluster formation depends on intact microtubules, infectivity is not compromised by disruption of microtubules with either nocodazole or colchicine, as determined by expression of an enhanced green fluorescent protein reporter gene inserted in the viral genome. These results indicate that virion clusters do not represent the infectious pathway and suggest an alternative route to the nucleus that does not depend on nocodazole-sensitive microtubules. PMID:17442712

Yea, Carmen; Dembowy, Joanna; Pacione, Laura; Brown, Martha

2007-04-18

402

Tubulin folding cofactor D is a microtubule destabilizing protein  

Microsoft Academic Search

A rapid switch between growth and shrinkage at microtubule ends is fundamental for many cellular processes. The main structural components of microtubules, the ??-tubulin heterodimers, are generated through a complex folding process where GTP hydrolysis [Fontalba et al. (1993) J. Cell Sci. 106, 627–632] and a series of molecular chaperones are required [Sternlicht et al. (1993) Proc. Natl. Acad. Sci.

Lara Mart??n; Mónica L Fanarraga; Kerman Aloria; Juan C Zabala

2000-01-01

403

Targeting the Microtubules in Breast Cancer Beyond Taxanes: The Epothilones  

Microsoft Academic Search

Microtubule-targeting agents such as the taxanes are highly active against breast cancer and have become a cornerstone in the treatment of patients with early and advanced breast cancer. The natural epothilones and their analogs are a novel class of microtubule- stabilizing agents that bind tubulin and result in ap- optotic cell death. Among this family of compounds, patupilone, ixabepilone, BMS-310705,

JAVIER CORTES; J OSE BASELGA

2007-01-01

404

?-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

405

Molecular control of kinetochore-microtubule dynamics and chromosome oscillations  

PubMed Central

Summary Chromosome segregation in metazoans requires the alignment of sister-kinetochores onto the metaphase plate. During chromosome alignment, bioriented kinetochores move chromosomes by regulating the plus-end dynamics of the attached microtubules. The bundles of kinetochore-bound microtubules alternate between growth and shrinkage, leading to regular oscillations along the spindle axis. However, the molecular mechanisms that coordinate microtubule plus-end dynamics remain unknown. Here we show that CENP-H, a subunit of the CENP-A NAC/CAD kinetochore complex, is essential for this coordination, as kinetochores lacking CENP-H establish bioriented attachments, but fail to generate regular oscillations, due to an uncontrolled rate of microtubule plus-end turnover. These alterations lead to rapid erratic movements that disrupt metaphase plate organization. Moreover, we show that the abundance of the CENP-A NAC/CAD subunits CENP-H and CENP-I dynamically change on individual sister-kinetochores in vivo, as they preferentially bind the sister-kinetochore attached to growing microtubules, and that one other subunit, CENP-Q, binds microtubules in vitro. Thus, we propose that CENP-A NAC/CAD is a direct regulator of kinetochore-microtubule dynamics, which physically links centromeric DNA to microtubule plus-ends.

Amaro, Ana C.; Samora, Catarina P.; Holtackers, Rene; Wang, Enxiu; Kingston, Isabel J.; Alonso, Maria; Lampson, Michael; McAinsh, Andrew D.; Meraldi, Patrick

2010-01-01

406

Cdt1 throws kinetochore-microtubule attachments for a loop  

PubMed Central

The Ndc80 complex links spindle microtubules to the kinetochore to ensure the proper segregation of chromosomes during mitosis. Analysis of the replication licensing factor Cdt1 during mitosis now reveals a cooperative role with the Ndc80 complex in establishing stable microtubule attachments to the spindle.

Matson, Daniel R.; Stukenberg, P. Todd

2013-01-01

407

Conserved microtubule–actin interactions in cell movement and morphogenesis  

Microsoft Academic Search

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

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

2003-01-01

408

Microtubule-severing enzymes at the cutting edge  

PubMed Central

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.

Sharp, David J.; Ross, Jennifer L.

2012-01-01

409

EB1 Targets to Kinetochores with Attached, Polymerizing Microtubules  

PubMed Central

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end–kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.

Tirnauer, Jennifer S.; Canman, Julie C.; Salmon, E.D.; Mitchison, Timothy J.

2002-01-01

410

Leading at the Front: How EB Proteins Regulate Microtubule Dynamics  

NASA Astrophysics Data System (ADS)

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

Hawkins, Taviare

2012-02-01

411

Microtubule distribution in gravitropic protonemata of the moss Ceratodon.  

PubMed

Tip cells of dark-grown protonemata of the moss Ceratodon purpureus are negatively gravitropic (grow upward). They possess a unique longitudinal zonation: (1) a tip group of amylochloroplasts in the apical dome, (2) a plastid-free zone, (3) a zone of significant plastid sedimentation, and (4) a zone of mostly non-sedimenting plastids. Immunofluorescence of vertical cells showed microtubules distributed throughout the cytoplasm in a mostly axial orientation extending through all zones. Optical sectioning revealed a close spatial association between microtubules and plastids. A majority (two thirds) of protonemata gravistimulated for > 20 min had a higher density of microtubules near the lower flank compared to the upper flank in the plastid-free zone. This apparent enrichment of microtubules occurred just proximal to sedimented plastids and near the part of the tip that presumably elongates more to produce curvature. Fewer than 5% of gravistimulated protonemata had an enrichment in microtubules near the upper flank, whereas 14% of vertical protonemata were enriched near one of the side walls. Oryzalin and amiprophos-methyl (APM) disrupted microtubules, gravitropism, and normal tip growth and zonation, but did not prevent plastid sedimentation. We hypothesize that a microtubule redistribution plays a role in gravitropism in this protonema. This appears to be the first report of an effect of gravity on microtubule distribution in plants. PMID:11537091

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

1990-01-01

412

Calmodulin is required for paraflagellar rod assembly and flagellum-cell body attachment in trypanosomes.  

PubMed

In the flagellum of the African sleeping sickness parasite Trypanosoma brucei calmodulin (CaM) is found within the paraflagellar rod (PFR), an elaborate extra-axonemal structure, and the axoneme. In dissecting mechanisms of motility regulation we analysed CaM function using RNAi. Unexpectedly CaM depletion resulted in total and catastrophic failure in PFR assembly; even connections linking axoneme to PFR failed to form following CaM depletion. This provides an intriguing parallel with the role in the green alga Chlamydomonas of a CaM-related protein in docking outer-dynein arms to axoneme outer-doublet microtubules. Absence of CaM had no discernible effect on axoneme assembly, but the failure in PFR assembly was further compounded by loss of the normal linkage between PFR and axoneme to the flagellum attachment zone of the cell body. Thus, flagellum detachment was a secondary, time-dependent consequence of CaM RNAi, and coincided with the loss of normal trypomastigote morphology, thereby linking the presence of PFR architecture with maintenance of cell form, as well as cell motility. Finally, wider comparison between the flagellum detachment phenotypes of RNAi mutants for CaM and the FLA1 glycoprotein potentially provides new perspective into the function of the latter into establishing and maintaining flagellum-cell body attachment. PMID:23787017

Ginger, Michael L; Collingridge, Peter W; Brown, Robert W B; Sproat, Rhona; Shaw, Michael K; Gull, Keith

2013-06-19

413

Comparative analysis of doublets versus single-layer diffractive optical elements in eyepiece or magnifier design.  

PubMed

We quantify the impact of eye clearance requirement on the performance of eyepieces utilizing doublets versus diffractive optical elements on aspheric substrates. In this study, the doublets were designed to be cemented on-axis elements. Specifically, four different values of eye clearance were implemented: 17, 20, 23, and 26 mm. For each value, axial and lateral color, spherical aberration, coma, astigmatism, field curvature, and distortion were compared. Each system under comparison was optimized for the same focal length, a 9 mm exit pupil, photopic wavelengths (513-608 nm), and a 40 degrees full field of view. We demonstrate that the single-layer diffractive optical element supports an eye clearance value of approximately 80% of the effective focal length, while the doublet drops below desired specifications at approximately 65% of the effective focal length. PMID:18026553

Cakmakci, Ozan; Rolland, Jannick

2007-11-20

414

Classification of finite reparametrization symmetry groups in the three-Higgs-doublet model  

NASA Astrophysics Data System (ADS)

Symmetries play a crucial role in electroweak symmetry breaking models with non-minimal Higgs content. Within each class of these models, it is desirable to know which symmetry groups can be implemented via the scalar sector. In N-Higgs-doublet models, this classification problem was solved only for N=2 doublets. Very recently, we suggested a method to classify all realizable finite symmetry groups of Higgs-family transformations in the three-Higgs-doublet model (3HDM). Here, we present this classification in all detail together with an introduction to the theory of solvable groups, which play the key role in our derivation. We also consider generalized- CP symmetries, and discuss the interplay between Higgs-family symmetries and CP-conservation. In particular, we prove that presence of the ?4 symmetry guarantees the explicit CP-conservation of the potential. This work completes classification of finite reparametrization symmetry groups in 3HDM.

Ivanov, Igor P.; Vdovin, E.

2013-02-01

415

Two Higgs doublets with fourth-generation fermions: Models for TeV-scale compositeness  

NASA Astrophysics Data System (ADS)

We construct a class of two Higgs doublets models with a 4th sequential generation of fermions that may effectively accommodate the low-energy characteristics and phenomenology of a dynamical electroweak symmetry breaking scenario which is triggered by the condensates of the 4th family fermions. In particular, we single out the heavy quarks by coupling the heavier Higgs doublet (?h) which possesses a much larger VEV only to them while the lighter doublet (??) couples only to the light fermions. We study the constraints on these models from precision electroweak data as well as from flavor data. We also discuss some distinct new features that have direct consequences on the production and decays of the 4th family quarks and leptons in high-energy colliders;, in particular, the conventional search strategies for t' and b' may need to be significantly revised.

Bar-Shalom, Shaouly; Nandi, Soumitra; Soni, Amarjit

2011-09-01

416

Structure of the doublet bands in doubly odd nuclei: The case of Cs128  

NASA Astrophysics Data System (ADS)

The structure of the ?J=1 doublet bands in Cs128 is investigated within the framework of the interacting vector boson-fermion model. A new, purely collective interpretation of these bands is given on the basis of the used boson-fermion dynamical symmetry of the model. The energy levels of the doublet bands as well as the absolute B(E2) and B(M1) transition probabilities between the states of both yrast and yrare bands are described quite well. The observed odd-even staggering of both B(M1) and B(E2) values is reproduced by the introduction of an appropriate interaction term of quadrupole type, which produces such a staggering effect in the transition strengths. The calculations show that the appearance of doublet bands in certain odd-odd nuclei could be a consequence of the realization of a larger dynamical symmetry based on the noncompact supersymmetry group OSp(2?/12,R).

Ganev, H. G.; Brant, S.

2010-09-01

417

A model for quasi-parity-doublet spectra in odd-mass nuclei  

NASA Astrophysics Data System (ADS)

A model of a quadrupole-octupole vibrating and rotating core plus a particle is proposed to describe and classify the quasi-parity-doublet spectra in odd-mass nuclei. The yrast levels are described as low-energy rotation-vibration modes built on the ground state. The non-yrast split parity-doublet sequences are considered as higher-energy rotation-vibration modes coupled to one-quasi-particle states. The even-even core is considered within the model of a coherent quadrupole-octupole motion, while the odd nucleon is described within the reflection-asymmetric deformed shell model with pairing interaction. The Coriolis decoupling and K-mixing interactions are calculated microscopically through a parity projection of the single-particle wave function. The unified model scheme was tested on the yrast and non-yrast quasi-parity-doublet spectra in the nuclei 223Ra and 221Fr.

Minkov, Nikolay

2013-05-01

418

Significance of microtubule catastrophes at focal adhesion sites  

PubMed Central

Directional cell migration requires cell polarization and asymmetric distribution of cell signaling. Focal adhesions and microtubules are two systems which are essential for these. It was shown that these two systems closely interact with each other. It is known that microtubule targeting stimulates focal adhesion dissociation. Our recent study shows that focal adhesions, in turn, specifically induce microtubule catastrophe via a biochemical mechanism. We were able to track down one of the focal adhesion proteins paxillin which is involved in this process. Paxillin phosphorylation was previously shown to be the key component in the regulation of focal adhesion assembly or disassembly. Since microtubule catastrophe dynamic differs at the leading edge and cell rear, similar to paxillin phosphorylation levels, we suggest a model connecting asymmetric distribution of focal adhesions and asymmetric distribution of microtubule catastrophes at adhesion sites as a feedback loop.

Kaverina, I

2009-01-01

419

The Ndc80 kinetochore complex forms oligomeric arrays along microtubules  

PubMed Central

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

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

2010-01-01

420

Developmental neurotoxicity of methylmercury: the role of microtubules  

SciTech Connect

The purpose of this research was to investigate the interaction of methylmercury with microtubules as a possible mechanism for methylmercury-caused developmental neurotoxicity. Methylmercury effects on developing cerebellar cortex, an area of rapid proliferation, were examined. This model was used to test the hypothesis that microtubules of the mitotic spindle are sensitive to methylmercury in vivo as well as in cultured cells. The effect of methylmercury on non-spindle microtubules was studied in cultured cells. Cellular levels of methylmercury were determined and were used to construct a dose-response relationship. The direct effects of methylmercury on microtubule assembly in vitro were also documented. The data from these three systems have been integrated to form a hypothesis for the role of microtubules in developmental neurotoxicity caused by methylmercury. 159 references, 23 figures, 16 tables.

Sager, P.R.

1982-01-01

421

Simulation of Second Harmonic Generation from Heterogeneous Microtubule Structures  

NASA Astrophysics Data System (ADS)

Second harmonic generation imaging is a coherent nonlinear microscopy with contrast arising from certain asymmetric endogenous structures in cells, including spindle microtubules. As a second-order nonlinear optical process, SHG requires a noncentrosymmetric macromolecular organization to generate signal, so it can be used as a measure of microtubule polarity within spindles or other microtubule structures. We developed a simulation of SHG microscopy accounting for 3-dimensional orientation and circularly polarized excitation in order to quantify the dependence of SHG signal on microtubule density, spacing, polarity, and rotational order. SHG can be used to assess spindle polarity in living cells using simultaneous ratio imaging with two-photon excited fluorescence from labeled tubulin. The results from simulation are used to quantify microtubule polarity from SHG and TPEF images of spindles in the one-cell C. elegans embryo and Xenopus oocyte extract.

Langowitz, Noah; Yu, Che-Hang; Needleman, Daniel

2012-02-01

422

Selective vulnerability of dopaminergic neurons to microtubule depolymerization.  

PubMed

Parkinson disease (PD) is characterized by the specific degeneration of dopaminergic (DA) neurons in substantia nigra and has been linked to a variety of environmental and genetic factors. Rotenone, an environmental PD toxin, exhibited much greater toxicity to DA neurons in midbrain neuronal cultures than to non-DA neurons. The effect was significantly decreased by the microtubule-stabilizing drug taxol and mimicked by microtubule-depolymerizing agents such as colchicine or nocodazole. Microtubule depolymerization disrupted vesicular transport along microtubules and caused the accumulation of dopamine vesicles in the soma. This led to increased oxidative stress due to oxidation of cytosolic dopamine leaked from vesicles. Inhibition of dopamine metabolism significantly reduced rotenone toxicity. Thus, our results suggest that microtubule depolymerization induced by PD toxins such as rotenone plays a key role in the selective death of dopaminergic neurons. PMID:16091364

Ren, Yong; Liu, Wenhua; Jiang, Houbo; Jiang, Qian; Feng, Jian

2005-08-09

423

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 tomograp