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1

The axonemal microtubules of the Chlamydomonas flagellum differ in tubulin isoform content.  

PubMed

Little is known of the molecular basis for the diversity of microtubule structure and function found within the eukaryotic flagellum. Antibodies that discriminate between tyrosinated alpha tubulin and post-translationally detyrosinated alpha tubulin were used to localize these complementary tubulin isoforms in flagella of the single-celled green alga Chlamydomonas reinhardtii. Immunofluorescence analysis of intact axonemes detected both isoforms along most of the lengths of flagella; however, each had a short distal zone rich in tyrosinated tubulin. Localizations on splayed axonemes revealed that the microtubules of the central-pair apparatus were rich in tyrosinated tubulin, while outer doublets contained a mixture of both isoforms. Immunoelectron analysis of individual outer doublets revealed that while tyrosinated tubulin was present in both A and B tubules, detyrosinated tubulin was largely confined to the wall of the B hemi-tubules. The absence of detyrosinated tubulin from the A tubules of the outer doublets and the microtubules of the central pair, both of which extend past the B hemi-tubules of the outer doublets in the flagellar tip, explained the appearance of a tyrosinated tubulin-rich distal zone on intact axonemes. Localizations performed on cells regenerating flagella revealed that flagellar assembly used tyrosinated tubulin; detyrosination of the B tubule occurred during later stages of regeneration, well after microtubule polymerization. The developmental timing of detyrosination, which occurs over a period during which the regrowing flagella begin to beat more effectively, suggests that post-translational modification of the B tubule surface may enhance dynein/B tubule interactions that power flagellar beating. PMID:9427680

Johnson, K A

1998-02-01

2

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

PubMed

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

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

2000-06-01

3

The doublet microtubules of rods of the rabbit retina  

Microsoft Academic Search

The connecting cilium of the rabbit photoreceptor rod is composed of nine outer doublets, lacking dynein side arms. The central singlet microtubules are absent. In cross section, there is an inner dense ring situated between the doublets and the center core of the cilium. As the doublet microtubules progress from the connecting region into outer segments, the cylindrical array of

G. Y. Wen; D. Soifer; H. M. Wisniewski

1982-01-01

4

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

5

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

PubMed Central

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.

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

2013-01-01

6

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

7

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

PubMed

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

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

2014-05-01

8

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

PubMed Central

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

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

2014-01-01

9

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

PubMed Central

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 Hydin causes slow growth and a loss of cell motility. We show that two separate defects in newly-formed flagellar central pair microtubules underlie the loss of cell motility. At early time-points after RNAi induction, the central pair becomes mispositioned, while at later time points the central pair is lost. While the basal body is unaffected, both defects originate at the basal plate, reflecting a role for TbHydin throughout the length of the central pair. Conclusion Our data provide the first evidence of Hydin's role within the trypanosome axoneme, and reveal central pair anomalies and thus impairment of ependymal ciliary motility as the likely cause of the hydrocephalus observed in the hy3 mouse.

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

2007-01-01

10

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

PubMed Central

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

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

2014-01-01

11

Biochemical characterization of tektins from sperm flagellar doublet microtubules  

PubMed Central

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

1987-01-01

12

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

13

Asymmetry of the central apparatus defines the location of active microtubule sliding in Chlamydomonas flagella  

PubMed Central

Regulation of ciliary and flagellar motility requires spatial control of dynein-driven microtubule sliding. However, the mechanism for regulating the location and symmetry of dynein activity is not understood. One hypothesis is that the asymmetrically organized central apparatus, through interactions with the radial spokes, transmits a signal to regulate dynein-driven microtubule sliding between subsets of doublet microtubules. Based on this model, we hypothesized that the orientation of the central apparatus defines positions of active microtubule sliding required to control bending in the axoneme. To test this, we induced microtubule sliding in axonemes isolated from wild-type and mutant Chlamydomonas cells, and then used electron microscopy to determine the orientation of the central apparatus. Transverse sections of wild-type axonemes revealed that the C1 microtubule is predominantly oriented toward the position of active microtubule sliding. In contrast, the central apparatus is randomly oriented in axonemes isolated from radial spoke deficient mutants. For outer arm dynein mutants, the C1 microtubule is oriented toward the position of active microtubule sliding in low calcium buffer, but is randomly oriented in high calcium buffer. These results provide evidence that the central apparatus defines the position of active microtubule sliding, and may regulate the size and shape of axonemal bends through interactions with the radial spokes. In addition, our results indicate that in high calcium conditions required to generate symmetric waveforms, the outer dynein arms are potential targets of the central pair-radial spoke control system.

Wargo, Matthew J.; Smith, Elizabeth F.

2003-01-01

14

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

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

15

High-frequency vibration in flagellar axonemes with amplitudes reflecting the size of tubulin  

PubMed Central

Flagellar axonemes of sea urchin sperm display high-frequency (approximately 300 Hz) vibration with nanometer-scale amplitudes in the presence of ATP (Kamimura, S., and R. Kamiya. 1989. Nature (Lond.). 340:476-478). The vibration appears to represent normal mechanochemical interaction between dynein and microtubules because the dependence of the frequency on MgATP concentration is similar to that of the axonemal motility, and because it is inhibited by micromolar concentrations of vanadate. In this study a two-dimensional photo-sensor was used to characterize this phenomenon in detail. Several new features were revealed. First, the vibration was found to be due to a back-and-forth movement of the doublet microtubules along the axonemal length. Two beads attached to different parts of the same axoneme vibrated in unison, i.e., synchronized exactly in phase. This suggested that the outer doublet can be regarded as a stiff rod in vibrating axonemes. Second, evidence was obtained that the amplitude of the vibration reflected the number of active dynein arms. Third, under certain conditions, the vibration amplitude took stepwise values of 8 x N + 4 nm (N = 0, 1, 2, 3, or 4), indicating that the amplitude of microtubule sliding was limited by the size of tubulin dimer (8 nm) or monomer (4 nm). To explain this phenomenon, a model is presented based on an assumption that the force production by dynein is turned off when dynein is subjected to tensile force; i.e., dynein is assumed to be equipped with a feedback mechanism necessary for oscillation.

1992-01-01

16

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

PubMed Central

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

1979-01-01

17

Regulation of flagellar dynein by the axonemal central apparatus.  

PubMed

Numerous studies indicate that the central apparatus, radial spokes, and dynein regulatory complex form a signaling pathway that regulates dynein activity in eukaryotic flagella. This regulation involves the action of several kinases and phosphatases anchored to the axoneme. To further investigate the role of the central apparatus in this signaling pathway, we have taken advantage of a microtubule-sliding assay to assess dynein activity in central apparatus defective mutants of Chlamydomonas. Axonemes isolated from both pf18 and pf15 (lacking the entire central apparatus) and from pf16 (lacking the C1 central microtubule) have reduced microtubule-sliding velocity compared with wild-type axonemes. Based on functional analyses of axonemes isolated from radial spokeless mutants, we hypothesized that inhibitors of casein kinase 1 (CK1) and cAMP dependent protein kinase (PKA) would rescue dynein activity and increase microtubule-sliding velocity in central pairless mutants. Treatment of axonemes isolated from both pf18 and pf16 with DRB, a CK1 inhibitor, but not with PKI, a PKA inhibitor, restored dynein activity to wild-type levels. The DRB-induced increase in dynein-driven microtubule sliding was inhibited if axonemes were first incubated with the phosphatase inhibitor, microcystin. Inhibiting CK1 in pf15 axonemes, which lack the central pair as well as PP2A [Yang et al., 2000: J. Cell Sci. 113:91-102], did not increase microtubule-sliding velocity. These data are consistent with a model in which the central apparatus, and specifically the C1 microtubule, regulate dynein through interactions with the radial spokes that ultimately alter the activity of CK1 and PP2A. These data are also consistent with localization of axonemal CK1 and PP2A near the dynein arms. PMID:11977081

Smith, Elizabeth F

2002-05-01

18

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

NASA Astrophysics Data System (ADS)

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

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

1995-03-01

19

Axonemal radial spokes  

PubMed Central

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.

Pigino, Gaia; Ishikawa, Takashi

2012-01-01

20

Microtubule Catastrophe and Rescue  

PubMed Central

Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed “dynamic instability”. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule `catastrophe'), and then by switching back from shrinkage to growth (called microtubule `rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells.

Gardner, Melissa K.; Zanic, Marija; Howard, Jonathon

2012-01-01

21

Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii  

PubMed Central

Chlamydomonas cells excise their flagella in response to a variety of experimental conditions (e.g., extremes of temperature or pH, alcohol or detergent treatment, and mechanical shear). Here, we show that flagellar excision is an active process whereby microtubules are severed at select sites within the transition zone. The transition zone is located between the flagellar axoneme and the basal body; it is characterized by a pair of central cylinders that have an H shape when viewed in longitudinal section. Both central cylinders are connected to the A tubule of each microtubule doublet of the transition zone by fibers (approximately 5 nm diam). When viewed in cross section, these fibers are seen to form a distinctive stellate pattern characteristic of the transition zone (Manton, I. 1964. J. R. Microsc. Soc. 82:279- 285; Ringo. D. L. 1967. J. Cell Biol. 33:543-571). We demonstrate that at the time of flagellar excision these fibers contract and displace the microtubule doublets of the axoneme inward. We believe that the resulting shear force and torsional load act to sever the axonemal microtubules immediately distal to the central cylinder. Structural alterations of the transition zone during flagellar excision occur both in living cells and detergent-extracted cell models, and are dependent on the presence of calcium (greater than or equal to 10(-6) M). Immunolocalization using monoclonal antibodies against the calcium- binding protein centrin demonstrate the presence of centrin in the fiber-based stellate structure of the transition zone of wild-type cells. Examination of the flagellar autotomy mutant, fa-1, which fails to excise its flagella (Lewin, R., and C. Burrascano. 1983. Experientia. 39:1397-1398), demonstrates that the fa-1 lacks the ability to completely contract the fibers of the stellate structure. We conclude that flagellar excision in Chlamydomonas involves microtubule severing that is mediated by the action of calcium-sensitive contractile fibers of the transition zone. These observations have led us to question whether microtubule severing may be a more general phenomenon than previously suspected and to suggest that microtubule severing may contribute to the dynamic behavior of cytoplasmic microtubules in other cells.

1989-01-01

22

Molecular structure of dynein and motility of a giant sperm axoneme provided with only the outer dynein arm.  

PubMed

The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed. PMID:11807935

Mencarelli, C; Lupetti, P; Rosetto, M; Mercati, D; Heuser, J E; Dallai, R

2001-11-01

23

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

24

Regulation of flagellar dynein by calcium and a role for an axonemal calmodulin and calmodulin-dependent kinase.  

PubMed

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. PMID:12221134

Smith, Elizabeth F

2002-09-01

25

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

26

Cloning, Localization, and Axonemal Function of Tetrahymena Centrin  

PubMed Central

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

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

2003-01-01

27

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

28

Ultrastructure of the sperm axoneme and molecular analysis of axonemal dynein in Ephemeroptera (Insecta).  

PubMed

The Ephemeroptera sperm axoneme is devoid of outer dynein arms (ODA) and exhibits a pronounced modification of the central pair complex (CPC), which is substituted by the central sheath (CS): a tubular element of unknown molecular composition. We performed a detailed ultrastructural analysis of sperm axonemes in the genera Cloeon and Ecdyonurus using quick-freeze, deep-etch electron microscopy, showing that the loss of the conventional CPC is not only concomitant with the loss of ODA, but also with a substantial modification in the longitudinal distribution of both radial spokes (RS) and inner dynein arms (IDA). Such structures are no longer distributed following the alternation of different repeats as in the 9?+?2 axoneme, but instead share a 32 nm longitudinal repeat: a multiple of the 8 nm repeat observed along the CS wall. Differently from the conventional CPC, the CS and the surrounding RS possess a ninefold symmetry, coherently with the three-dimensional pattern of motility observed in Cloeon free spermatozoa. Biochemical analyses revealed that ultrastructural modifications are concomitant with a reduced complexity of the IDA heavy chain complement. We propose that these structural and molecular modifications might be related to the relief from the evolutionary constraints imposed by the CPC on the basal 9?+?9?+?2 axoneme and could also represent the minimal set compatible with flagellar beating and progressive motility mechanically regulated as suggested by the geometric clutch hypothesis. © 2014 Wiley Periodicals, Inc. PMID:24668829

Mencarelli, Caterina; Mercati, David; Dallai, Romano; Lupetti, Pietro

2014-05-01

29

Dynamic Instability of Individual Microtubules Analyzed by Video Light Microscopy: Rate Constants and Transition Frequencies  

Microsoft Academic Search

We have developed video microscopy methods to visualize the assembly and disas- sembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37°C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concen- trations between 7 and 15.5 I.tM. Elongation and rapid shortening

R. A. Walker; E. T. O'Brien; N. K. Pryer; M. E Soboeiro; W. A. Voter; H. P. Erickson

2005-01-01

30

Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii.  

PubMed

Mutations at two loci, which cause an altered mobility of the flagella, affected the central pair microtubule complex of Chlamydomonas reinhardtii flagella. The mutations at both loci primarily affected the C1 microtubule of the complex. Three alleles at the PF16 locus affected the stability of the C1 microtubule in isolated axonemes. This phenotype has allowed us to determine that at least ten polypeptides of the central pair complex are unique to the C1 microtubule. The motility defect was correlated with the failure to assemble three of these ten polypeptides in vivo. The structural gene product of the PF16 locus was a polypeptide with molecular weight 57,000 as shown by analysis of five intragenic revertants and by analysis of axonemes from dikaryon rescue experiments. Three alleles at the PF6 locus affected the assembly of one of the two projections of the C1 microtubule and this projection was formed by at least three polypeptide components, which are a subset of polypeptides missing in isolated pf16 axonemes. No structural gene product has been identified for the PF6 locus. The gene product is probably not one of the identified projection constituents as shown by analysis of dikaryon rescue experiments. Chemical extraction of isolated wild-type axonemes suggests that at least seven polypeptide components are unique to the C2 microtubule. PMID:6707088

Dutcher, S K; Huang, B; Luck, D J

1984-01-01

31

The role of central apparatus components in flagellar motility and microtubule assembly.  

PubMed

In order to generate the complex waveforms typical of beating cilia and flagella, the action of the dynein arms must be regulated. This regulation not only depends on the presence of multiple dynein isoforms, but also clearly involves other structures in the axoneme such as the radial spokes and central apparatus; mutants lacking these structures have paralyzed flagella. In this article, we review recent progress in identifying protein components of the central apparatus and discuss the role of these components in regulation of flagellar motility and central apparatus assembly. The central apparatus is composed of two single microtubules and their associated structures which include the central pair projections, the central pair bridges linking the two tubules, and the central pair caps which are attached to the distal or plus ends of the microtubules. To date, the genes encoding four components of the central apparatus have been cloned, PF15, PF16, PF20 and KLP1. PF16, PF20 and KLP1 have been sequenced and their gene products localized. Two additional components have been identified immunologically, a 110 kD polypeptide recognized by an antibody generated against highly conserved kinesin peptide sequence, and a 97 kD polypeptide recognized by CREST antisera. Based on a variety of data, one model that has emerged to explain the role of the central apparatus in flagellar motility is that the central apparatus ultimately regulates dynein through interactions with the radial spokes. The challenge now is to determine the precise mechanism by which the polypeptides comprising the central apparatus and the radial spokes interact to transduce a regulatory signal to the dynein arms. In terms of assembly, the central apparatus microtubules assemble with their plus ends distal to the cell body but, unlike the nine doublet microtubules, they are not nucleated from the basal bodies. Since some central apparatus defective mutants fail to assemble the entire central apparatus, their gene products may eventually prove to have microtubule nucleating or stabilizing properties. By continuing to identify the genes that encode central apparatus components, we will begin to understand the contribution of these microtubules to flagellar motility and gain insight into their nucleation, assembly, and stability. PMID:9295136

Smith, E F; Lefebvre, P A

1997-01-01

32

Mutations in a novel locus on mouse chromosome 11 resulting in male infertility associated with defects in microtubule assembly and sperm tail function.  

PubMed

Traditional gene knock-out approaches using homologous recombination in embryonic stem cells are routinely used to provide functional information about genes involved in reproduction. In the present study, we examined a novel approach using N-ethyl-N-nitrosourea (ENU) together with a balancer chromosome mating strategy to identify new loci with functional roles in male fertility. Our genetic strategy is a forward-genetic approach; thus, our phenotypic investigation begins with the discovery of an abnormal phenotype without previous knowledge of the mutant locus. We isolated eight recessive mutations on chromosome 11 that resulted in male or female infertility from a screen of 184 founder pedigrees from ENU-treated males. After testing the six male infertile and two female infertile mutations for their ability to complement, we found that three independent recessive male infertile mutations failed to complement each other. The male infertility was associated with reduced epididymal sperm count, a block in late-spermatid differentiation, and increased apoptosis. Furthermore, the three male infertile mutants had severe defects in epididymal sperm morphology associated with incorrect microtubule assembly. Electron microscopy revealed unique defects in sperm head and tail morphology for each of the three alleles. One allele had an abnormal manchette assembly of the sperm head. The other two alleles had different abnormalities in the 9+2 patterning of the microtubules in the sperm tail axoneme, with one containing only five of the microtubule doublets and the other containing an extra doublet. The isolation of this allelic series identifies a new locus on mouse chromosome 11 that is required for spermiogenesis and male fertility. PMID:14711786

Clark, Amander T; Firozi, Karen; Justice, Monica J

2004-05-01

33

Drosophila Centrosomin Protein is Required for Male Meiosis and Assembly of the Flagellar Axoneme  

PubMed Central

Centrosomes and microtubules play crucial roles during cell division and differentiation. Spermatogenesis is a useful system for studying centrosomal function since it involves both mitosis and meiosis, and also transformation of the centriole into the sperm basal body. Centrosomin is a protein localized to the mitotic centrosomes in Drosophila melanogaster. We have found a novel isoform of centrosomin expressed during spermatogenesis. Additionally, an anticentrosomin antibody labels both the mitotic and meiotic centrosomes as well as the basal body. Mutational analysis shows that centrosomin is required for spindle organization during meiosis and for organization of the sperm axoneme. These results suggest that centrosomin is a necessary component of the meiotic centrosomes and the spermatid basal body.

Li, Kaijun; Xu, Eugene Yujun; Cecil, Jeffrey K.; Rudolf Turner, F.; Megraw, Timothy L.; Kaufman, Thomas C.

1998-01-01

34

Dissecting the axoneme interactome: the mammalian orthologue of Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue of Chlamydomonas PF16.  

PubMed

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat. PMID:15827353

Zhang, Zhibing; Jones, Brian H; Tang, Waixing; Moss, Stuart B; Wei, Zhangyong; Ho, Clement; Pollack, Michael; Horowitz, Eran; Bennett, Jean; Baker, Michael E; Strauss, Jerome F

2005-07-01

35

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

PubMed

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

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

2014-05-20

36

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

PubMed

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

Bernstein, M; Beech, P L; Katz, S G; Rosenbaum, J L

1994-06-01

37

Taxol-induced bundling of brain-derived microtubules  

PubMed Central

Taxol has two obvious effects in cells. It stabilizes microtubules and it induces microtubule bundling. We have duplicated the microtubule- bundling effect of taxol in vitro and report preliminary characterization of this bundling using electron microscopy, sedimentation, and electrophoretic analyses. Taxol-bundled microtubules from rat brain crude extracts were seen as massive bundles by electron microscopy. Bundled microtubules sedimented through sucrose five times faster than control microtubules. Electrophoretic analysis of control and taxol-bundled microtubules pelleted through sucrose revealed no striking differences between the two samples except for a protein doublet of approximately 100,000 daltons. Taxol-induced microtubule bundling was not produced by using pure tubulin or recycled microtubule protein; this suggested that taxol-induced microtubule bundling was mediated by a factor present in rat brain crude extracts. Taxol cross- linked rat brain crude extract microtubules were entirely labile to ATP in the millimolar range. This ATP-dependent relaxation was also demonstrated in a more purified system, using taxol-bundled microtubules pelleted through sucrose and gently resuspended. Although the bundling factor did not recycle with microtubule protein, it was apparently retained on isolated taxol-stabilized microtubules. The bundling factor was salt extracted from taxol-stabilized microtubules and its retained activity was demonstrated in an add-back experiment with assembled phosphocellulose-purified tubulin.

1984-01-01

38

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

39

Characterization of a Chlamydomonas insertional mutant that disrupts flagellar central pair microtubule-associated structures.  

PubMed

Two alleles at a new locus, central pair-associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke-defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility. PMID:9922455

Mitchell, D R; Sale, W S

1999-01-25

40

Characterization of a Chlamydomonas Insertional Mutant that Disrupts Flagellar Central Pair Microtubule-associated Structures  

PubMed Central

Two alleles at a new locus, central pair–associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke–defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

Mitchell, David R.; Sale, Winfield S.

1999-01-01

41

Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells  

PubMed Central

MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E.H.K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M.H. Bre, G. Griffiths, and E. Karsenti. 1990. 110:1123-1136). In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced double-immunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4 microns/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a t1/2 of less than 30 min, whereas in confluent monolayers, a large population of microtubules had a t1/2 of greater than 2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.

1990-01-01

42

Motility in Echinosphaerium nucleofilum. I. An analysis of particle motions in the axopodia and a direct test of the involvement of the axoneme  

PubMed Central

The motion of particles in the axopodia of Echinosphaerium nucleofilum is saltatory. In the present study, photokymograph records of 123 motions from six axopodia have been analyzed. Particles followed rectilinear paths of from 1 to 15 mum while in continuous motion at an average velocity of 0.66 plus or minus 0.32 mum/s. The velocity of the particles was variable in 36% of the cases measured. Some motions were punctuated by pauses either before continuing in the same direction or reversing. Frequently, several particles were moving at the same velocity, but neighboring particles showed no motion or moved in the opposite direction. Two particles occasionally contacted one another and travelled as a unit for varying lengths of time but subsequently moved independently. These motions reflect the underlying mechanism of motive force production. Furthermore, a glass microneedle can be substituted for the microtubular axoneme in the axopodia. In these artificial axopodia, bidirectional particle motions occurred which were similar to those in normal axopodia. Colchicine, at the threshold dose for axonemal dissolution, had no affect on these particel motions. It is concluded that the microtubular axoneme is not responsible for particle motions and also that individual microtubules are unlikely candidates for motive force production in this system.

1975-01-01

43

Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms  

PubMed Central

The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of alpha- tubulin, termed alpha 1 and alpha 3. alpha 1-Tubulin most likely represents the primary translation product, while alpha 3-tubulin is a posttranslationally acetylated derivative of alpha 1-tubulin. In the pool of soluble cytoplasmic tubulin, alpha 1 is the predominant species, while the very stable flagellar microtubules contain almost exclusively the alpha 3-tubulin isoform. The subpellicular microtubules contain both isoforms. Neither of the two alpha-tubulin isoforms is organelle specific, but the alpha 3 isoform is predominantly located in stable microtubules.

1987-01-01

44

Conserved and specific functions of axoneme components in trypanosome motility.  

PubMed

The Trypanosoma brucei flagellum is unusual as it is attached along the cell body and contains, in addition to an apparently conventional axoneme, a structure called the paraflagellar rod, which is essential for cell motility. Here, we investigated flagellum behaviour in normal and mutant trypanosome cell lines where expression of genes encoding various axoneme proteins (PF16, PF20, DNAI1, LC2) had been silenced by RNAi. First, we show that the propulsive wave (normally used for forward motility) is abolished in the absence of outer dynein arms, whereas the reverse wave (normally used for changing direction) still occurs. Second, in contrast to Chlamydomonas--but like metazoa, the central pair adopts a fixed orientation during flagellum beating. This orientation becomes highly variable in central-pair- and outer-dynein-arm-mutants. Third, the paraflagellar rod contributes to motility by facilitating three-dimensional wave propagation and controlling cell shape. Fourth, motility is required to complete the last stage of cell division in both insect and bloodstream stages of the parasite. Finally, our study also reveals the conservation of molecular components of the trypanosome flagellum. Coupled to the ease of reverse genetics, it raises the interest of trypanosomes as model organisms to study cilia and flagella. PMID:16882690

Branche, Carole; Kohl, Linda; Toutirais, Géraldine; Buisson, Johanna; Cosson, Jacky; Bastin, Philippe

2006-08-15

45

Tubulin and microtubule structure  

Microsoft Academic Search

Our knowledge of microtubule structure and its relationship to microtubule function continues to grow. Cryo-elecron microscopy has given us new images of the microtubule polymerization and depolymerization processes and of the interaction of these polymers with motor proteins. We now know more about the effect of nucleotide state on the structure and dynamic instability of microtubules. The atomic model of

Kenneth H Downing; Eva Nogales

1998-01-01

46

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

47

Analysis of the central pair microtubule complex in Chlamydomonas reinhardtii.  

PubMed

The central pair microtubule complex in Chlamydomonas flagella has been well characterized as a regulator of flagellar dynein activity, but many aspects of this regulation depend on specific interactions between the asymmetric central pair structure and radial spokes, which appear symmetrically arranged along all nine outer doublet microtubules. Relationships between central pair-radial spoke interactions and dynein regulation have been difficult to understand because the Chlamydomonas central pair is twisted in vivo and rotates during bend propagation. Here we describe genetic and biochemical methods of dissecting the Chlamydomonas central pair and electron microscopic methods useful to determine structure-function relationships in this complex. PMID:20409807

Mitchell, David R; Smith, Brandon

2009-01-01

48

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

PubMed

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

Oda, Toshiyuki; Hirokawa, Nobutaka; Kikkawa, Masahide

2007-04-23

49

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

PubMed Central

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

Oda, Toshiyuki; Hirokawa, Nobutaka; Kikkawa, Masahide

2007-01-01

50

Why is the Higgs doublet light.  

National Technical Information Service (NTIS)

We consider a possible mechanism for the explanation of the doublet-triplet mass hierarchy in the grand unified theories. The Higgs doublet is automatically light since it is related by a certain ''custodial'' SU(2)(sub C) symmetry to another doublet whic...

G. Dvali

1993-01-01

51

Microtubule organization in vitro.  

PubMed

Microtubules organize into a set of distinct patterns with the help of associated molecules that control nucleation, polymerization, crosslinking, and transport. These patterns, alone or in combination with each other, define the functional architecture of the microtubule cytoskeleton in living cells. In vitro experiments of increasing complexity help understand, in combination with theoretical models, the basic mechanisms by which elementary microtubule patterns arise, how they are maintained, and how they position themselves with respect to the confining geometry of living cells. PMID:23287583

Dogterom, Marileen; Surrey, Thomas

2013-02-01

52

Dynamic instability of microtubule growth  

Microsoft Academic Search

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

Tim Mitchison; Marc Kirschner

1984-01-01

53

Computer simulation of flagellar movement X: doublet pair splitting and bend propagation modeled using stochastic dynein kinetics.  

PubMed

Experimental observations on cyclic splitting and bending by a flagellar doublet pair are modeled using forces obtained from a model for dynein mechanochemistry, based on ideas introduced by Andrew Huxley and Terrill Hill and extended previously for modeling flagellar movements. The new feature is elastic attachment of dynein to the A doublet, which allows movement perpendicular to the A doublet and provides adhesive force that can strain attached dyneins. This additional strain influences the kinetics of dynein attachment and detachment. Computations using this dynein model demonstrate that very simple and realistic ideas about dynein mechanochemistry are sufficient for explaining the separation and reattachment seen experimentally with flagellar doublet pairs. Additional simulations were performed after adding a "super-adhesion" elasticity. This elastic component is intended to mimic interdoublet connections, normally present in an intact axoneme, that would prevent visible splitting but allow sufficient separation to cause dynein detachment and cessation of shear force generation. This is the situation envisioned by Lindemann's "geometric clutch" hypothesis for control of dynein function in flagella and cilia. The simulations show abrupt disengagement of the "clutch" at one end of a bend, and abrupt reengagement of the "clutch" at the other end of a bend, ensuring that active sliding is only operating where it will cause bend propagation from base to tip. PMID:24574072

Brokaw, Charles J

2014-04-01

54

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

PubMed Central

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

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

2014-01-01

55

Posttranslational modification and microtubule stability  

Microsoft Academic Search

We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after in- jection of biotin-tubulin

Eric Schulze; David J. Asai; Jeannette Chloe Bulinski; Marc Kirschner

1987-01-01

56

Doublet method for very fast autocoding  

PubMed Central

Background Autocoding (or automatic concept indexing) occurs when a software program extracts terms contained within text and maps them to a standard list of concepts contained in a nomenclature. The purpose of autocoding is to provide a way of organizing large documents by the concepts represented in the text. Because textual data accumulates rapidly in biomedical institutions, the computational methods used to autocode text must be very fast. The purpose of this paper is to describe the doublet method, a new algorithm for very fast autocoding. Methods An autocoder was written that transforms plain-text into intercalated word doublets (e.g. "The ciliary body produces aqueous humor" becomes "The ciliary, ciliary body, body produces, produces aqueous, aqueous humor"). Each doublet is checked against an index of doublets extracted from a standard nomenclature. Matching doublets are assigned a numeric code specific for each doublet found in the nomenclature. Text doublets that do not match the index of doublets extracted from the nomenclature are not part of valid nomenclature terms. Runs of matching doublets from text are concatenated and matched against nomenclature terms (also represented as runs of doublets). Results The doublet autocoder was compared for speed and performance against a previously published phrase autocoder. Both autocoders are Perl scripts, and both autocoders used an identical text (a 170+ Megabyte collection of abstracts collected through a PubMed search) and the same nomenclature (neocl.xml, containing over 102,271 unique names of neoplasms). In side-by-side comparison on the same computer, the doublet method autocoder was 8.4 times faster than the phrase autocoder (211 seconds versus 1,776 seconds). The doublet method codes 0.8 Megabytes of text per second on a desktop computer with a 1.6 GHz processor. In addition, the doublet autocoder successfully matched terms that were missed by the phrase autocoder, while the phrase autocoder found no terms that were missed by the doublet autocoder. Conclusions The doublet method of autocoding is a novel algorithm for rapid text autocoding. The method will work with any nomenclature and will parse any ascii plain-text. An implementation of the algorithm in Perl is provided with this article. The algorithm, the Perl implementation, the neoplasm nomenclature, and Perl itself, are all open source materials.

Berman, Jules J

2004-01-01

57

Microtubule Stabilizing Agents  

Microsoft Academic Search

The microtubule cytoskeleton continues to be both an effective and a validated target in the therapy of cancer. Beginning\\u000a with the vinca alkaloids almost 50 years ago and encouraged by the broad activity of first taxol® (paclitaxel) and then taxotere®\\u000a (docetaxel) numerous investigators have identified structurally diverse compounds that interact with the tubulin\\/microtubule\\u000a system displaying antimitotic and anticancer properties. Paclitaxel

Susan Band Horwitz; Tito Fojo

58

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

PubMed Central

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

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

2013-01-01

59

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

60

Singlet-Doublet Dark Matter  

SciTech Connect

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

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

2012-02-15

61

Microtubule Dynamics and the Positioning of Microtubule Organizing Centers  

Microsoft Academic Search

A theoretical analysis is presented that shows how the dynamic instability of microtubules in combination with microtubule polymerization forces provides a microtubule organizing center with a mechanism to position itself at the center of a confining geometry. The rate of approach to the center and the positional preciseness depend on the parameters that characterize dynamic instability, the size of the

Marileen Dogterom; Bernard Yurke

1998-01-01

62

Active Dynamics of Microtubule Bundles  

NASA Astrophysics Data System (ADS)

Microtubule bundles play a central role in a variety of dynamical processes in cells such as cell division, neural growth and blood platelet formation. These processes are driven by the activity of molecular motors that exert sliding forces on the microtubules. In such bundles, motor proteins may crosslink two or more filaments, forming discrete clusters of microtubules whose dynamics is governed by a balance of the motor-generated forces. The connectivity of these microtubule-motor complexes is an essential property of the bundle and dictates its dynamics. We present a systematic computational study of these microtubule bundles based on force-balance computer simulations as well as a simplified analytical theory. This allows us to calculate the characteristic times of microtubule sorting and spreading, as well as the effective diffusion constants and drift coefficients as a function of the microtubule density and the polarity fraction of the microtubules. Application of our theory in the study of blood platelet formation is presented.

Zemel, Assaf; Mogilner, Alex

2008-03-01

63

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

PubMed Central

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

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

1997-01-01

64

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

PubMed

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

Edamatsu, Masaki

2014-05-16

65

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

66

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. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays; however, increasing severing activity alone is not sufficient to drive microtubule alignment. 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

2013-10-01

67

Microtubules and Pathogen Defence  

Microsoft Academic Search

The cytoskeletal network of plant cells represents a dynamic structure that responds to external\\u000a stimuli by changes of organization. An attack of pathogenic microbes represents an external stress that\\u000a seriously threatens plant survival. Growing evidence from recent research indicates that cytoskeletal elements,\\u000a such as microtubules and microfilaments, are central players in plant defence responses. Tubulin and actin\\u000a inhibitors suppress the polarization

Issei Kobayashi; Yuhko Kobayashi

68

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

PubMed Central

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

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

2013-01-01

69

Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella  

PubMed Central

The effect of vanadate on the ATP-induced disruption of trypsin-treated axonemes and the ATP-induced straightening of rigor wave preparations of sea urchin sperm was investigated. Addition of ATP to a suspension of trypsin-treated axonemes results in a rapid decrease in turbidity (optical density measured at 350 nm) concomitant with the disruption of the axonemes by sliding between microtubules to form tangles of connected doublet microtubules (Summers and Gibbons, 1971; Sale and Satir, 1977). For axonemes digested to approximately 93 percent of their initial turbidity, 5 {muM} vanadate completely inhibits the ATP-induced decrease in turbidity and the axonemes maintain their structural integrity. However, with axonemes digested to approximately 80 percent of their initial turbidity, vanadate fails to inhibit the ATP-induced decrease in turbidity and the ATP-induced structural disruption of axonemes, even when the vanadate concentration is raised as high as 100 ?m. For such axonemes digested to 80 percent of their initial turbidity, the form of ATP-induced structural changes, in the presence of 25 ?M vanadate, was observed by dark-field light microscopy and revealed that the axonemes become disrupted into curved, isolated doublet microtubules, small groups of doublet microtubules, and “banana peel” structures in which tubules have peeled back from the axoneme. Addition of 5 ?M ATP to rigor wave sperm, which were prepared by abrupt removal of ATP from reactivated sperm, causes straightening of the rigor waves within 1 min, and addition of more than 10 ?M ATP causes resumption of flagellar beating. Addition of 40 ?M vanadate to the rigor wave sperm does not inhibit straightening of the rigor waves of 2 ?M-1 mM ATP, although oscillatory beating is completely inhibited. These results suggest that vanadate inhibits the mechanochemical cycle of dyein at a step subsequent to the MgATP(2-)-induced release of the bridged dynein arms.

Sale, WS; Gibbon, IR

1979-01-01

70

Doublet III neutral beam test tank design  

SciTech Connect

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

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

1980-03-01

71

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

72

Modeling microtubule oscillations  

SciTech Connect

Synchronization of molecular reactions in a macroscopic volume may cause the volume's physical properties to change dynamically and thus reveal much about the reactions. As an example, experimental time series for so-called microtubule oscillations are analyzed in terms of a minimal model for this complex polymerization-depolymerization cycle. The model reproduces well the qualitatively different time series that result from different experimental conditions, and illuminates the role and importance of individual processes in the cycle. Simple experiments are suggested that can further test and define the model and the polymer's reaction cycle.

Jobs, Elmar [Hoechstleistungsrechenzentrum, Forschungszentrum Juelich GmbH, D-52425 Juelich (Germany); Wolf, Dietrich E. [Theoretical Physics FB10, Gerhard-Mercator-University, D-47048 Duisburg (Germany); Flyvbjerg, Henrik [Condensed Matter Physics and Chemistry Department, Risoe Rise National Laboratory, DK-4000 Roskilde (Denmark); The Niels Bohr Institute, Blegdamsvej 17, DK-2100 Copenhagen Oe (Denmark)

1999-10-05

73

Search for Close-Mass Lepton Doublet.  

National Technical Information Service (NTIS)

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(+)e(-) annihilation data taken with the Mark II detector at a center-of-mass ...

J. K. Riles

1989-01-01

74

Proton Mass Doublet from General Relativity.  

National Technical Information Service (NTIS)

This paper discusses features of the proton mass according to the author's non-linear, spinor field theory in general relativity. Within its context, where mass doublets are generally predicted for all spinor matter fields, it is shown, in a semi-quantita...

M. Sachs

1980-01-01

75

Technicolor with a massless scalar doublet  

Microsoft Academic Search

We consider a minimal technicolor model in which the ordinary and technicolor sectors are coupled by a massless scalar doublet. When technicolor interactions become strong, the resulting technicolor condensate not only breaks the electroweak symmetry, but also causes the scalar to develop a vacuum expectation value. With the appropriate choice of the scalar's Yukawa couplings, fermion masses are generated, giving

Christopher D. Carone; Howard Georgi

1994-01-01

76

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

NASA Astrophysics Data System (ADS)

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

Cibert, Christian

2005-03-01

77

Sustained microtubule treadmilling in Arabidopsis cortical arrays.  

PubMed

Plant cells create highly structured microtubule arrays at the cell cortex without a central organizing center to anchor the microtubule ends. In vivo imaging of individual microtubules in Arabidopsis plants revealed that new microtubules are initiated at the cell cortex and exhibit dynamics at both ends. Polymerization-biased dynamic instability at one end and slow depolymerization at the other end result in sustained microtubule migration across the cell cortex by a hybrid treadmilling mechanism. This motility causes widespread microtubule repositioning and contributes to changes in array organization through microtubule reorientation and bundling. PMID:12714675

Shaw, Sidney L; Kamyar, Roheena; Ehrhardt, David W

2003-06-13

78

Teamwork in microtubule motors.  

PubMed

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

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

2013-11-01

79

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

80

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

2012-08-01

81

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

82

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

83

A proton mass doublet from general relativity  

Microsoft Academic Search

Summary  This paper discusses the features of the proton mass according to the author's nonlinear, spinor field theory in general relativity.\\u000a Within its context, where mass doublets are generally predicted for all spinor matter fields, it is shown, in a semi-quantitative\\u000a fashion, that 1) in addition to the normal (stable) proton there is a heavier proton that has a mass of

M. Sachs

1980-01-01

84

Doublet III: status and future plans  

SciTech Connect

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

Rawls, J.M.

1980-04-01

85

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

PubMed

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

Smith, E F; Lefebvre, P A

1996-02-01

86

Unconventional functions of microtubule motors  

PubMed Central

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.

Muresan, Virgil; Muresan, Zoia

2012-01-01

87

Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy  

PubMed Central

Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, ?-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin ? Glu-tubulin) and then irreversible deglutamination (Glu-tubulin ? ?2-tubulin), such that Glu- and ?2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and ?2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and ?2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and ?2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.

Sato, Hiroshi; Nagai, Toshio; Kuppuswamy, Dhandapani; Narishige, Takahiro; Koide, Masaaki; Menick, Donald R.; IV, George Cooper

1997-01-01

88

Microtubule asters as templates for nanomaterials assembly  

PubMed Central

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

2012-01-01

89

Distinct localization and cell cycle dependence of COOH terminally tyrosinolated alpha-tubulin in the microtubules of Trypanosoma brucei brucei  

PubMed Central

alpha-Tubulin can be posttranslationally modified in that its COOH- terminal amino acid residue, tyrosine, can be selectively removed and replaced again. This reaction cycle involves two enzymes, tubulin carboxypeptidase and tubulin tyrosine ligase. The functional significance of this unusual modification is unclear. The present study demonstrates that posttranslational tyrosinolation of alpha-tubulin does occur in the parasitic hemoflagellate Trypanosoma brucei brucei and that posttranslational tyrosinolation can be detected in both alpha- tubulin isoforms found in this organism. Trypanosomes contain a number of microtubular structures: the flagellar axoneme; the subpellicular layer of singlet microtubules which are closely associated with the cell membrane; the basal bodies; and a cytoplasmic pool of soluble tubulin. Tyrosinolated alpha-tubulin is present in all these populations. However, immunofluorescence studies demonstrate a distinct localization of tyrosinolated alpha-tubulin within individual microtubules and organelles. This localization is subject to a temporal modulation that correlates strongly with progress of a cell through the cell cycle. Our results indicate that the presence of tyrosinolated alpha-tubulin is a marker for newly formed microtubules.

1987-01-01

90

Doublet-triplet fermionic dark matter  

NASA Astrophysics Data System (ADS)

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

Dedes, Athanasios; Karamitros, Dimitrios

2014-06-01

91

Structure of potentials with N Higgs doublets  

SciTech Connect

Extensions of the standard model with N Higgs doublets are simple extensions presenting a rich mathematical structure. An underlying Minkowski structure emerges from the study of both variable space and parameter space. The former can be completely parametrized in terms of two future lightlike Minkowski vectors with spatial parts forming an angle whose cosine is -(N-1){sup -1}. For the parameter space, the Minkowski parametrization enables one to impose sufficient conditions for bounded below potentials, characterize certain classes of local minima, and distinguish charge breaking vacua from neutral vacua. A particular class of neutral minima presents a degenerate mass spectrum for the physical charged Higgs bosons.

Nishi, C. C. [Instituto de Fisica Teorica, UNESP, Sao Paulo State University, Rua Pamplona, 145, 01405-900, Sao Paulo (Brazil) and Instituto de Fisica Gleb Wataghin, UNICAMP, PO Box 6165, 13083-970, Campinas, SP (Brazil)

2007-09-01

92

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

93

Inert Higgs doublet extension of the NMSSM  

NASA Astrophysics Data System (ADS)

We introduce one pair of inert Higgs doublets {Hd,Hu} and singlets {Nc,N}, and consider their couplings with the Higgs doublets of the minimal supersymmetric standard model (MSSM), W ?yNNchuHd+yN'NhdHu. We assign extra U(1)Z' gauge charges only to the extra vectorlike superfields, and so all the MSSM superfields remain neutral under the new U(1)Z'. They can be an extension of the "? term," W ??Shuhd in the next-to MSSM (NMSSM). Because of the U(1)Z', the maximally allowed low energy value of yN can be lifted up to 0.85, avoiding a Landau pole (LP) below the grand unification scale. Such colorless vectorlike superfields remarkably enhance the radiative MSSM Higgs mass particularly for large tan? through the yN term and the corresponding holomorphic soft term. As a result, the lower bound of ? and the upper bound of tan? can be relaxed to disappear from the restricted parameter space of the original NMSSM, 0.6?? ?0.7 and 1m˜t?700 GeV. Thus, the valid parameter space significantly expands up to 0?? ?0.75, 0?yN?0.88, and 2?tan??50, evading the LP problem and also explaining the 126 GeV Higgs mass naturally.

Kyae, Bumseok

2014-04-01

94

Neutrinos from Inert Doublet dark matter  

SciTech Connect

We investigate the signatures of neutrinos produced in the annihilation of WIMP dark matter in the Earth, the Sun and at the Galactic centre within the framework of the Inert Doublet Model and extensions. We consider a dark matter candidate, that we take to be one of the neutral components of an extra Higgs doublet, in three distinct mass ranges, which have all been shown previously to be consistent with both WMAP abundance and direct detection experiments exclusion limits. Specifically, we consider a light WIMP with mass between 4 and 8 GeV (low), a WIMP with mass around 60-70 GeV (middle) and a heavy WIMP with mass above 500 GeV (high). In the first case, we show that capture in the Sun may be constrained using Super-Kamiokande data. In the last two cases, we argue that indirect detection through neutrinos is challenging but not altogether excluded. For middle masses, we try to make the most benefit of the proximity of the so-called 'iron resonance' that might enhance the capture of the dark matter candidate by the Earth. The signal from the Earth is further enhanced if light right-handed Majorana neutrinos are introduced, in which case the scalar dark matter candidate may annihilate into pairs of mono-energetic neutrinos. In the case of high masses, detection of neutrinos from the Galactic centre might be possible, provided the dark matter abundance is substantially boosted.

Andreas, Sarah; Tytgat, Michel H.G.; Swillens, Quentin, E-mail: Sarah.Andreas@rwth-aachen.de, E-mail: mtytgat@ulb.ac.be, E-mail: qswillen@ulb.ac.be [Service de Physique Theorique, Universite Libre de Bruxelles, CP225, Bld du Triomphe, B-1050 Brussels (Belgium)] [Service de Physique Theorique, Universite Libre de Bruxelles, CP225, Bld du Triomphe, B-1050 Brussels (Belgium)

2009-04-15

95

High-Resolution Model of the Microtubule  

Microsoft Academic Search

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

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

1999-01-01

96

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

97

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

98

Microtubule-interacting drugs for cancer treatment  

Microsoft Academic Search

Microtubule-interacting drugs are important agents in cancer chemotherapy. Some of these drugs alter microtubule dynamics and engage the cell cycle surveillance mechanisms to arrest cell division in mitosis. Many cancer cells possess genetic lesions in components of this pathway and thus fail to arrest in mitosis. Therefore, by targeting the spindle microtubules, chemotherapeutic agents can efficiently block cell cycle progression

Paula M Checchi; James H Nettles; Jun Zhou; James P Snyder; Harish C Joshi

2003-01-01

99

Dynamics of interphase microtubules in Schizosaccharomyces pombe  

Microsoft Academic Search

Background: Microtubules in interphase Schizosaccharomyces pombe are essential for maintaining the linear growth habit of these cells. The dynamics of assembly and disassembly of these microtubules are so far uncharacterised.Results: Live cell confocal imaging of ?1 tubulin tagged with enhanced green fluorescent protein revealed longitudinally oriented, dynamically unstable interphase microtubule assemblies (IMAs). The IMAs were uniformly bright along their length

Douglas R. Drummond; Robert A. Cross

2000-01-01

100

A structural view of microtubule dynamics  

Microsoft Academic Search

The essential microtubule property of dynamic instability is based on the binding, hydrolysis and exchange of GTP in each tubulin dimer. The recent high-resolution structures of tubulin and the microtubule have given us the first view at atomic level of properties such as nucleotide exchangeability, the linkage between polymerization and nucleotide hydrolysis, and the origin of microtubule destabilization, as well

E. Nogales

1999-01-01

101

Arabidopsis Cortical Microtubules Are Initiated along, as Well as Branching from, Existing Microtubules[W  

PubMed Central

The principles by which cortical microtubules self-organize into a global template hold important implications for cell wall patterning. Microtubules move along bundles of microtubules, and neighboring bundles tend to form mobile domains that flow in a common direction. The bundles themselves move slowly and for longer than the individual microtubules, with domains describing slow rotary patterns. Despite this tendency for colinearity, microtubules have been seen to branch off extant microtubules at ?45°. To examine this paradoxical behavior, we investigated whether some microtubules may be born on and grow along extant microtubule(s). The plus-end markers Arabidopsis thaliana end binding protein 1a, AtEB1a-GFP, and Arabidopsis SPIRAL1, SPR1-GFP, allowed microtubules of known polarity to be distinguished from underlying microtubules. This showed that the majority of microtubules do branch but in a direction heavily biased toward the plus end of the mother microtubule: few grow backward, consistent with the common polarity of domains. However, we also found that a significant proportion of emergent comets do follow the axes of extant microtubules, both at sites of apparent microtubule nucleation and at cross-over points. These phenomena help explain the persistence of bundles and counterbalance the tendency to branch.

Chan, Jordi; Sambade, Adrian; Calder, Grant; Lloyd, Clive

2009-01-01

102

Irc15 Is a microtubule-associated protein that regulates microtubule dynamics in Saccharomyces cerevisiae.  

PubMed

Microtubules are polymers composed of alpha-beta tubulin heterodimers that assemble into microtubules. Microtubules are dynamic structures that have periods of both growth and shrinkage by addition and removal of subunits from the polymer. Microtubules stochastically switch between periods of growth and shrinkage, termed dynamic instability. Dynamic instability is coupled to the GTPase activity of the beta-tubulin subunit of the tubulin heterodimer. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs) that interact with microtubules to regulate dynamic instability. MAPs in budding yeast have been identified that bind microtubule ends (Bim1), that stabilize microtubule structures (Stu2), that bundle microtubules by forming cross-bridges (Ase1), and that interact with microtubules at the kinetochore (Cin8, Kar3, Kip3). IRC15 was previously identified in four different genetic screens for mutants affecting chromosome transmission or repair [11-14]. Here we present evidence that Irc15 is a microtubule-associated protein, localizing to microtubules in vivo and binding to purified microtubules in vitro. Irc15 regulates microtubule dynamics in vivo and loss of IRC15 function leads to delayed mitotic progression, resulting from failure to establish tension between sister kinetochores. PMID:19285398

Keyes, Brice E; Burke, Daniel J

2009-03-24

103

Tau Protein Diffuses along the Microtubule Lattice*  

PubMed Central

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

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

2012-01-01

104

N ? and ? ? parity doublets in the baryon spectrum  

NASA Astrophysics Data System (ADS)

The N and ? excitation spectrum exhibits parity doublets, i.e. states of equal total angular momentum but with opposite parity being almost degenerate in mass. Among others, it has been suggested by L.Ya. Glozman that the parity-doublet structure may be due to effective chiral restoration.

Credé, V.

105

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

PubMed

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

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

2014-01-01

106

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

PubMed Central

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

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

2014-01-01

107

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

108

Localized modulated waves in microtubules.  

PubMed

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

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

2014-06-01

109

The architecture of outer dynein arms in situ.  

PubMed

Outer dynein arms, the force generators for axonemal motion, form arrays on microtubule doublets in situ, although they are bouquet-like complexes with separated heads of multiple heavy chains when isolated in vitro. To understand how the three heavy chains are folded in the array, we reconstructed the detailed 3D structure of outer dynein arms of Chlamydomonas flagella in situ by electron cryo-tomography and single-particle averaging. The outer dynein arm binds to the A-microtubule through three interfaces on two adjacent protofilaments, two of which probably represent the docking complex. The three AAA rings of heavy chains, seen as stacked plates, are connected in a striking manner on microtubule doublets. The tail of the alpha-heavy chain, identified by analyzing the oda11 mutant, which lacks alpha-heavy chain, extends from the AAA ring tilted toward the tip of the axoneme and towards the inside of the axoneme at 50 degrees , suggesting a three-dimensional power stroke. The neighboring outer dynein arms are connected through two filamentous structures: one at the exterior of the axoneme and the other through the alpha-tail. Although the beta-tail seems to merge with the alpha-tail at the internal side of the axoneme, the gamma-tail is likely to extend at the exterior of the axoneme and join the AAA ring. This suggests that the fold and function of gamma-heavy chain are different from those of alpha and beta-chains. PMID:17391698

Ishikawa, Takashi; Sakakibara, Hitoshi; Oiwa, Kazuhiro

2007-05-18

110

Optically Resolving Individual Microtubules in Live Axons  

PubMed Central

Summary Microtubules are essential cytoskeletal tracks for cargo transportation in axons and also serve as the primary structural scaffold of neurons. Structural assembly, stability, and dynamics of axonal microtubules are of great interest for understanding neuronal functions and pathologies. However, microtubules are so densely packed in axons that their separations are well below the diffraction limit of light, which precludes using optical microscopy for live-cell studies. Here, we present a single-molecule imaging method capable of resolving individual microtubules in live axons. In our method, unlabeled microtubules are revealed by following individual axonal cargos that travel along them. We resolved more than six microtubules in a 1 ?m diameter axon by real-time tracking of endosomes containing quantum dots. Our live-cell study also provided direct evidence that endosomes switch between microtubules while traveling along axons, which has been proposed to be the primary means for axonal cargos to effectively navigate through the crowded axoplasmic environment.

Mudrakola, Harsha V.; Zhang, Kai; Cui, Bianxiao

2010-01-01

111

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

112

Cytoplasmic dynein mediates adenovirus binding to microtubules.  

PubMed

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% +/- 3.5% to 80.7% +/- 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding. PMID:15331745

Kelkar, Samir A; Pfister, K Kevin; Crystal, Ronald G; Leopold, Philip L

2004-09-01

113

Cytoplasmic Dynein Mediates Adenovirus Binding to Microtubules  

PubMed Central

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% ± 3.5% to 80.7% ± 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.

Kelkar, Samir A.; Pfister, K. Kevin; Crystal, Ronald G.; Leopold, Philip L.

2004-01-01

114

Expression Levels of a Kinesin-13 Microtubule Depolymerase Modulates the Effectiveness of Anti-Microtubule Agents  

PubMed Central

Background Chemotheraputic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs. In this report, we test the hypothesis that the cellular levels of microtubule depolymerases, in this case kinesin-13 s, modulate the effectiveness of the microtubule disrupting drug colchicine. Methodology/Principal Findings We used a combination of RNA interference (RNAi), high-throughput microscopy, and time-lapse video microscopy in Drosophila S2 cells to identify a specific MAP, kinesin-like protein 10A (KLP10A), that contributes to the efficacy of the anti-microtubule drug colchicine. KLP10A is an essential microtubule depolymerase throughout the cell cycle. We find that depletion of KLP10A in S2 cells confers resistance to colchicine-induced microtubule depolymerization to a much greater extent than depletion of several other destabilizing MAPs. Using image-based assays, we determined that control cells retained 58% (±2%SEM) of microtubule polymer when after treatment with 2 µM colchicine for 1 hour, while cells depleted of KLP10A by RNAi retained 74% (±1%SEM). Likewise, overexpression of KLP10A-GFP results in increased susceptibility to microtubule depolymerization by colchicine. Conclusions/Significance Our results demonstrate that the efficacy of microtubule destabilization by a pharmacological agent is dependent upon the cellular expression of a microtubule depolymerase. These findings suggest that expression levels of Kif2A, the human kinesin-13 family member, may be an attractive biomarker to assess the effectiveness of anti-microtubule chemotherapies. Knowledge of how MAP expression levels affect the action of anti-microtubule drugs may prove useful for evaluating possible modes of cancer treatment.

Schimizzi, Gregory V.; Currie, Joshua D.; Rogers, Stephen L.

2010-01-01

115

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

116

Microtubule heterogeneity of Ornithogalum umbellatum ovary epidermal cells: non-stable cortical microtubules and stable lipotubuloid microtubules.  

PubMed

Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of ?-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules. PMID:21744330

Kwiatkowska, Maria; St?pi?ski, Dariusz; Polit, Justyna T; Pop?o?ska, Katarzyna; Wojtczak, Agnieszka

2011-01-01

117

Microtubules in the Nervous System  

Microsoft Academic Search

\\u000a Neurons undergo various morphologic changes during development, including neuritogenesis, neurite outgrowth, neurite branching,\\u000a and neurite retraction. Many studies have examined how microtubules (MTs) are reorganized or transported within the neurites\\u000a of developing neurons and have revealed that MT dynamics are regulated by MT-interacting proteins and motor proteins, in concert\\u000a with actin microfilaments. Here, I will describe recent progress in research

Nobuyuki Fukushima

118

CO2 interferometer operation in Doublet III  

NASA Astrophysics Data System (ADS)

A CO2 laser interferometer is used for measuring the average free electron density along a vertical path in Doublet III, a large fusion research tokamak. The CO2 laser is used together with a collinear HeNe laser to form a two-wavelength interferometer capable of measuring plasma fringe shifts which are much less than the fringe shifts due to mechanical vibrations. This diagnostic has been shown to be able to resolve plasma electron fringe shifts smaller than 1/17 of a fringe during the pulsed operation which causes mechanical motion resulting in as many as 10 fringe shifts. The interferometer is operated in the Michelson configuration with a total double path length through the plasma of 6 m. The density resolution then is better than 2 x 10 to the 12th per cu cm. A density resolution of about 0.5 x 10 to the 12th per cu cm has been achieved during relatively quiet operation.

Baker, D. R.

1980-10-01

119

Technicolor with a massless scalar doublet  

SciTech Connect

We consider a minimal technicolor model in which the ordinary and technicolor sectors are coupled by a [ital massless] scalar doublet. When technicolor interactions become strong, the resulting technicolor condensate not only breaks the electroweak symmetry, but also causes the scalar to develop a vacuum expectation value. With the appropriate choice of the scalar's Yukawa couplings, fermion masses are generated, giving us the conventional pattern of flavor symmetry breaking. Although no explicit scalar mass term appears in the full Lagrangian of the model, the pseudoscalar states that remain in the low-energy effective theory gain sufficient mass through technicolor interactions to evade detection. We show that this model does not generate unacceptably large flavor-changing neutral currents, and is consistent with the experimental constraints on oblique electroweak radiative corrections. We determine the experimentally allowed region of the model's parameter space, and discuss the significance of a phenomenologically viable model that has no arbitrary dimensionful parameters. In terms of parameter counting, our model is the simplest possible extension of the standard model.

Carone, C.D.; Georgi, H. (Lyman Laboratory of Physics, Harvard University, Cambridge, Massachusetts 02138 (United States))

1994-02-01

120

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

Microsoft Academic Search

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

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

2010-01-01

121

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

122

Interaction of kinesin motors, microtubules, and MAPs  

Microsoft Academic Search

Kinesins are a family of microtubule-dependent motor proteins that carry cargoes such as vesicles, organelles, or protein complexes along microtubules. Here we summarize structural studies of the “conventional” motor protein kinesin-1 and its interactions with microtubules, as determined by X-ray crystallography and cryo-electron microscopy. In particular, we consider the docking between the kinesin motor domain and tubulin subunits and summarize

A. MARX; J. MÜLLER; E.-M. MANDELKOW; A. HOENGER

2006-01-01

123

Microtubules and the Evolution of Mitosis  

Microsoft Academic Search

The microtubular cytoskeleton of higher plants diverges considerably from its animal counterpart.\\u000a This divergence involves a fundamentally different organization with microtubule arrays, which are\\u000a specific to higher plants, such as cortical microtubules or the phragmoplast. On the other hand, centrioles,\\u000a which are central organizers of microtubules in cells of animals and lower plants, have been progressively\\u000a reduced in the course of

Anne-Catherine Schmit; Peter Nick

124

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

PubMed Central

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.

1985-01-01

125

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules.  

PubMed

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (+/- 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (+/- 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP-expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states. PMID:18230754

Patel-Hett, Sunita; Richardson, Jennifer L; Schulze, Harald; Drabek, Ksenija; Isaac, Natasha A; Hoffmeister, Karin; Shivdasani, Ramesh A; Bulinski, J Chloë; Galjart, Niels; Hartwig, John H; Italiano, Joseph E

2008-05-01

126

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules  

PubMed Central

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (± 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (± 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP–expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states.

Patel-Hett, Sunita; Richardson, Jennifer L.; Schulze, Harald; Drabek, Ksenija; Isaac, Natasha A.; Hoffmeister, Karin; Shivdasani, Ramesh A.; Bulinski, J. Chloe; Galjart, Niels; Hartwig, John H.

2008-01-01

127

The C IV doublet ratio intensity effect in symbiotic stars  

NASA Technical Reports Server (NTRS)

High-resolution UV spectra in the 1200-2000 wavelength range of the symbiotic variable R Aqr and its nebular jet were obtained in July 1987 with the IUE. The line profile structure of the C IV 1548, 1550 doublet in the jet indicates multicomponent velocity structure from an optically thin emitting gas. The C IV doublet profiles in the compact H II region engulfing the Mira and hot companion binary also suggest multicomponent structure with radial velocities up to about -100 km/s. The value of the doublet intensity ratio in the R Aqr H II region has been observed in other similar symbiotic stars, such as RX Pup. It is suggested that the anomalous behavior of the C IV doublet intensities may be useful for studying the spatial structure and temporal nature of winds in symbiotic stars.

Michalitsianos, A. G.; Fahey, M.; Kafatos, M.; Viotti, R.; Cassatella, A.

1988-01-01

128

Low Scale Thermal Leptogenesis in Neutrinophilic Higgs Doublet Models  

NASA Astrophysics Data System (ADS)

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

Haba, N.; Seto, O.

2011-06-01

129

Unique Design of Doublet and Big Dee Vacuum Vessels.  

National Technical Information Service (NTIS)

The Doublet III tokamak now in its fourth year of operation at General Atomic Company, has its plasma contained in a kidney-shaped toroidal vacuum vessel, a configuration that presented unique design challenges. Most tokamak vacuum vessels are constructed...

J. E. Miller

1981-01-01

130

Preliminary results of noncircular plasma experiments in Doublet III  

SciTech Connect

Preliminary results of noncircular plasma experiments in Doublet III are reported. Shaping and discharge characteristics in doublet plasmas with high-Z limiters are described. Electron energy confinement and maximum plasma density are in agreement with standard circular tokamak empirical scaling laws. Chromium and molybdenum appear to be the dominant high-Z contaminants while carbon appears to dominate low-Z contaminants. High-Z impurity radiation does not appear to dominate the central power balance.

Ohkawa, T.

1980-02-01

131

Dynein Light Chain 1 (LC8) Association Enhances Microtubule Stability and Promotes Microtubule Bundling*  

PubMed Central

Dynein light chain 1 (LC8), a highly conserved protein, is known to bind to a variety of different polypeptides. It functions as a dimer, which is inactivated through phosphorylation at the Ser-88 residue. A loss of LC8 function causes apoptosis in Drosophila embryos, and its overexpression induces malignant transformation of breast cancer cells. Here we show that LC8 binds to tubulin, promotes microtubule assembly, and induces the bundling of reconstituted microtubules in vitro. Furthermore, LC8 decorates microtubules both in Drosophila embryos and in HeLa cells, increases the microtubule stability, and promotes microtubule bundling in these cells. Microtubule stability influences a number of different cellular functions including mitosis and cell differentiation. The LC8 overexpression reduces the susceptibility of microtubules to cold and nocodazole-induced depolymerization in tissue-cultured cells and increases microtubule acetylation, suggesting that LC8 stabilizes microtubules. We also show that LC8 knockdown or transfection with inhibitory peptides destabilizes microtubules and inhibits bipolar spindle assembly in HeLa cells. In addition, LC8 knockdown leads to the mitotic block in HeLa cells. Furthermore, molecular docking analysis using the crystal structures of tubulin and LC8 dimer indicated that the latter may bind at ?-? tubulin junction in a protofilament at sites distinct from the kinesin and dynein binding sites. Together, we provide the first evidence of a novel microtubule-associated protein-like function of LC8 that could explain its reported roles in cellular metastasis and differentiation.

Asthana, Jayant; Kuchibhatla, Anuradha; Jana, Swadhin Chandra; Ray, Krishanu; Panda, Dulal

2012-01-01

132

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

PubMed Central

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

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

2014-01-01

133

Mechanics of Microtubules: Effects of Protofilament Orientation  

PubMed Central

Microtubules are hollow cylindrical polymers of the protein tubulin that play a number of important dynamic and structural roles in eukaryotic cells. Both in vivo and in vitro microtubules can exist in several possible configurations, differing in the number of protofilaments, helical rise of tubulin dimers, and protofilament skew angle with respect to the main tube axis. Here, finite element modeling is applied to examine the mechanical response of several known microtubule types when subjected to radial deformation. The data presented here provide an important insight into microtubule stiffness and reveal that protofilament orientation does not affect radial stiffness. Rather, stiffness is primarily dependent on the effective Young's modulus of the polymerized material and the effective radius of the microtubule. These results are also directly correlated to atomic force microscopy nanoindentation measurements to allow a more detailed interpretation of previous experiments. When combined with experimental data that show a significant difference between microtubules stabilized with a slowly hydrolyzable GTP analog and microtubules stabilized with paclitaxel, the finite element data suggest that paclitaxel increases the overall radial flexibility of the microtubule wall.

Donhauser, Zachary J.; Jobs, William B.; Binka, Edem C.

2010-01-01

134

The most important microtubule natural inhibitors.  

PubMed

Natural microtubule inhibitors represent chemically very variegated family of structures with strong effect on cytoskeletal functions and the use of them is one of the most frequent therapeutic strategies for carcinoma treatment. The survey of the most important natural microtubule inhibitors is summarized in this paper. PMID:10566172

Patocka, J; Strunecká, A

1999-01-01

135

Microtubules Underlie Dysfunction in Duchenne Muscular Dystrophy  

PubMed Central

Duchenne muscular dystrophy (DMD) is a fatal X-linked degenerative muscle disease caused by the absence of the microtubule-associated protein dystrophin, which results in a disorganized and denser microtubule cytoskeleton. In addition, mechanotransduction-dependent activation of calcium (Ca2+) and reactive oxygen species (ROS) signaling underpins muscle degeneration in DMD. We show that in muscle from adult mdx mice, a model of DMD, a brief physiologic stretch elicited microtubule-dependent activation of NADPH (reduced-form nicotinamide adenine dinucleotide phosphate) oxidase–dependent production of ROS, termed X-ROS. Further, X-ROS amplified Ca2+ influx through stretch-activated channels in mdx muscle. Consistent with the importance of the microtubules to the dysfunction in mdx muscle, muscle cells with dense microtubule structure, such as those from adult mdx mice or from young wild-type mice treated with Taxol, showed increased X-ROS production and Ca2+ influx, whereas cells with a less dense microtubule network, such as young mdx or adult mdx muscle treated with colchicine or nocodazole, showed little ROS production or Ca2+ influx. In vivo treatments that disrupted the microtubule network or inhibited NADPH oxidase 2 reduced contraction-induced injury in adult mdx mice. Furthermore, transcriptome analysis identified increased expression of X-ROS–related genes in human DMD skeletal muscle. Together, these data show that microtubules are the proximate element responsible for the dysfunction in Ca2+ and ROS signaling in DMD and could be effective therapeutic targets for intervention.

Khairallah, Ramzi J.; Shi, Guoli; Sbrana, Francesca; Prosser, Benjamin L.; Borroto, Carlos; Mazaitis, Mark J.; Hoffman, Eric P.; Mahurkar, Anup; Sachs, Fredrick; Sun, Yezhou; Chen, Yi-Wen; Raiteri, Roberto; Lederer, W. Jonathan; Dorsey, Susan G.; Ward, Christopher W.

2012-01-01

136

Microtubule motors: moving forward on many fronts  

PubMed Central

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

2009-01-01

137

A slow dance for microtubule acetylation.  

PubMed

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

Kull, F Jon; Sloboda, Roger D

2014-06-01

138

Directed microtubule growth, +TIPs and kinesin-2 are required for uniform microtubule polarity in dendrites  

PubMed Central

Summary Background In many differentiated cells microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform polarity minus-end-out microtubules in dendrites, which are often highly branched. As undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, we have used this system to understand how cells can maintain dynamic arrays of polarized microtubules. Results We find that growing microtubules navigate dendrite branch points by turning the same way, towards the cell body, 98% of the time, and that growing microtubules track along stable microtubules towards their plus ends. Using RNAi and genetic approaches, we show that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. Conclusions We conclude that microtubule growth is directed at dendrite branch points, and that kinesin-2, APC and EB1 are likely to play a role in this process. We propose that is recruited to growing microtubules by +TIPS, and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism to maintain polarized arrays of microtubules.

Mattie, Floyd J.; Stackpole, Megan M.; Stone, Michelle C.; Clippard, Jessie R.; Rudnick, David A.; Qiu, Yijun; Tao, Juan; Allender, Dana L.; Parmar, Manpreet; Rolls, Melissa M.

2010-01-01

139

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

2013-09-18

140

Physical aspects of the assembly and function of microtubules  

Microsoft Academic Search

Living cells contain polymers called microtubules. Microtubules are used to control cell shape, to generate force for movement, to transport vesicles, and to separate chromosomes during cell division. Microtubule polymerization is governed by a unique, energy-consuming phenomenon called dynamic instability, which leads to large fluctuations in length. This dissertation reports on physical studies (theory and experiment) of microtubules and their

Timothy Eric Holy

1997-01-01

141

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

142

Membrane-cytoskeleton interactions in the flagellum: a 240,000 Mr surface-exposed glycoprotein is tightly associated with the axoneme in Chlamydomonas moewusii.  

PubMed

The flagellar surface of Chlamydomonas moewusii is a dynamic structure involved in several adhesive and motile events. In this report, we describe for the first time the flagellar membrane components of vegetative C. moewusii. A glycoprotein (or pair of glycoproteins) with an apparent molecular weight of 240 x 10(3) is the dominant flagellar protein (other than the tubulins) in this species of Chlamydomonas. Both a rabbit polyclonal antibody (designated P-19) and the lectin concanavalin A recognize this 240K (K = 10(3) Mr) glycoprotein on nitrocellulose transblots of flagellar proteins. Fluorescence microscopic studies using these same two probes suggest that the 240K glycoprotein is exposed at the flagellar surface. Direct evidence that the 240K glycoprotein is exposed at the flagellar surface is provided by vectorial labelling with a N-hydroxysuccinamide derivitized biotin reagent (NHS-LC-biotin). Nonionic detergent extraction of isolated flagella fails to solubilize most of the 240K glycoprotein, although it completely removes the flagellar membranes as demonstrated by transmission electron microscopy. Furthermore, immunofluorescence microscopy of isolated axonemes demonstrates that both P-19-defined epitopes and surface-biotinylated proteins continue to be associated with the axoneme structure after detergent treatment. These observations demonstrate that the 240K flagellar protein is a glycoprotein that is both exposed at the flagellar surface and tightly coupled to the underlying cytoskeleton (axoneme). Because of its cell surface orientation and axonemal linkage, it is likely that the 240K glycoprotein plays an important role in the adhesive and/or motile phenomena exhibited by the C. moewusii flagellar surface. PMID:3198705

Reinhart, F D; Bloodgood, R A

1988-04-01

143

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

PubMed Central

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

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

2002-01-01

144

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

2013-07-01

145

Passively athermalized broadband optical design using doublet combinations.  

PubMed

In this paper we propose a method for broadband achromatic and passive athermal optical system design using a combination of three doublets. In the implementation of this method, the first-order color dispersion is corrected by distributing proper power to the elements inside the doublets; the thermal and secondary color dispersion are corrected by distributing proper power to the doublet group units. The theoretical deviation is given and we summarize the power distribution equations. A design of an example system using this method, which covers the 350-1200 nm wavelength band, is given and the image quality evaluation is presented. It is shown that the example system maintains high imaging qualities over the required wavelength and temperature range, which proves the effectiveness of the method. PMID:24979421

Li, Ruigang

2014-06-20

146

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

147

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

148

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

149

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

150

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

151

Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella.  

PubMed

The effect of vanadate on the ATP-induced disruption of trypsin-treated axonemes and the ATP-induced straightening of rigor wave preparations of sea urchin sperm was investigated. Addition of ATP to a suspension of trypsin-treated axonemes results in a rapid decrease in turbidity (optical density measured at 350 nm) concomitant with the disruption of the axonemes by sliding between microtubules to form tangles of connected doublet microtubules (Summers and Gibbons, 1971; Sale and Satir, 1977). For axonemes digested to approximately 93 percent of their initial turbidity, 5 {muM} vanadate completely inhibits the ATP-induced decrease in turbidity and the axonemes maintain their structural integrity. However, with axonemes digested to approximately 80 percent of their initial turbidity, vanadate fails to inhibit the ATP-induced decrease in turbidity and the ATP-induced structural disruption of axonemes, even when the vanadate concentration is raised as high as 100 mum. For such axonemes digested to 80 percent of their initial turbidity, the form of ATP-induced structural changes, in the presence of 25 muM vanadate, was observed by dark-field light microscopy and revealed that the axonemes become disrupted into curved, isolated doublet microtubules, small groups of doublet microtubules, and "banana peel" structures in which tubules have peeled back from the axoneme. Addition of 5 muM ATP to rigor wave sperm, which were prepared by abrupt removal of ATP from reactivated sperm, causes straightening of the rigor waves within 1 min, and addition of more than 10 muM ATP causes resumption of flagellar beating. Addition of 40 muM vanadate to the rigor wave sperm does not inhibit straightening of the rigor waves of 2 muM-1 mM ATP, although oscillatory beating is completely inhibited. These results suggest that vanadate inhibits the mechanochemical cycle of dyein at a step subsequent to the MgATP(2-)-induced release of the bridged dynein arms. PMID:158028

Sale, W S; Gibbons, I R

1979-07-01

152

Intrinsic bending of microtubule protofilaments.  

PubMed

The complex polymerization dynamics of the microtubule (MT) plus end are closely linked to the hydrolysis of the GTP nucleotide bound to the ?-tubulin. The destabilization is thought to be associated with the conformational change of the tubulin dimers from the straight conformation in the MT lattice to a curved conformation. It remains under debate whether this transformation is directly related to the nucleotide state, or a consequence of the longitudinal or lateral contacts in the MT lattice. Here, we present large-scale atomistic simulations of short tubulin protofilaments with both nucleotide states, starting from both extreme conformations. Our simulations indicate that both interdimer and intradimer contacts in both GDP and GTP-bound tubulin dimers and protofilaments in solution bend. There are no observable differences between the mesoscopic properties of the contacts in GTP and GDP-bound tubulin or the intradime and interdimer interfaces. PMID:21397191

Grafmüller, Andrea; Voth, Gregory A

2011-03-01

153

Rigidity of microtubules is increased by stabilizing agents  

PubMed Central

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

1995-01-01

154

Haemonchus contortus microtubules are cold resistant.  

PubMed

Haemonchus contortus is an important nematode of livestock that is present in most parts of the world. The life cycle comprises free living stages (egg, L1, L2 and L3 larvae), and parasitic stages (L4, adult and egg) in a ruminant. Microtubules are filamentous structures which are made from polymerization of ?- and ?-tubulin. In vitro polymerization of ?- and ?-tubulin can be achieved by increasing the temperature to 37°C under certain conditions. As part of its normal functioning, in mammals, the microtubules can be depolymerized when the temperature is reduced to 0°C. However, interestingly the microtubules of H. contortus are cold resistant i.e. they do not depolymerize at 0°C. Moreover these microtubules did not depolymerize even in the presence of 5 mM CaCl2 or 50 ?M colchicine. These interesting findings may explain how larvae in the free living stages may survive cold temperatures over winter. PMID:24525483

Ashraf, Shoaib; Prichard, Roger K

2014-01-01

155

Microtubule Dynamics as a Target in Oncology  

PubMed Central

Drugs that affect microtubule dynamics, including the taxanes and vinca alkaloids, have been a mainstay in the treatment of leukemias and solid tumors for decades. New, more effective microtubule-targeting agents continue to enter into clinical trials and some, including the epothilone ixapebilone, have been approved for use. In contrast, several other drugs of this class with promising preclinical data were later shown to be ineffective or intolerable in animal models or clinical trials. In this review we discuss the molecular mechanisms as well as preclinical and clinical results for a variety of microtubule-targeting agents in various stages of development. We also offer a frank discussion of which microtubule-targeting agents are amenable to further development based on their availability, efficacy and toxic profile.

Risinger, April L.; Giles, Francis J.; Mooberry, Susan L.

2009-01-01

156

Bounds on scalar masses in two Higgs doublet models  

Microsoft Academic Search

A thorough analysis of stability and perturbativity bounds is performed in several versions of the two-Higgs doublet model, for both CP-conserving and spontaneously broken CP minima. LEP results further aid in establishing very strict constraints on the mass of the lighter Higgs particle.

P. M. Ferreira; D. R. T. Jones

2009-01-01

157

Mass Terms in Two-Higgs Doublet Models  

Microsoft Academic Search

We take a closer look at the mass terms of all renormalizable and CP conserving two-Higgs doublet models (THDM). We show how some of the dimension two parameters in the potential can be set equal to zero leading to relations among the tree-level parameters of the potential. The different versions of the THDM obtained give rise to different amplitudes for

R. Santos; S. M. Oliveira; A. Barroso

2001-01-01

158

Parity doublets and chiral symmetry restoration in baryon spectrum  

Microsoft Academic Search

It is argued that an appearance of the near parity doublets in the upper part of the light baryon spectrum is an evidence for the chiral symmetry restoration in the regime where a typical momentum of quarks is around the chiral symmetry restoration scale. At high enough baryon excitation energy the nontrivial gap solution, which signals the chiral symmetry breaking

L. Ya. Glozman

2000-01-01

159

Source processes of the 2009 Irian Jaya, Indonesia, earthquake doublet  

NASA Astrophysics Data System (ADS)

On January 3, 2009 UT, an earthquake doublet with moment magnitudes of 7.6 and 7.4 occurred along the New Guinea trench near the northern coast of Irian Jaya, Indonesia. The events were separated by three hours in time and 60 km in space, having similar thrust faulting mechanisms. The Solomon and New Britain Islands regions located east of the source area are well known for the occurrence of earthquake doublets due to the complex tectonic settings. To investigate the source processes of the earthquake doublet, we have performed teleseismic waveform inversions using P-wave displacement records from the IRIS-DMC database. The results of the inversions are consistent with the direction of the subduction along the New Guinea trench for both of the earthquakes. Significant slips exceeding 5.0 m appear near the hypocenters, and the slip distributions look to be complementary to each other. The rupture starting point of the second event is close to the edge of the slip distribution of the first event. Our source models support the triggering mechanism of doublet events suggested by Lay and Kanamori (1980). The results also suggest the state of stress in the Irian Jaya region and the mechanism of earthquakes in this subduction zone.

Poiata, N.; Koketsu, K.; Miyake, H.

2010-05-01

160

Electroweak baryogenesis in the two-doublet model  

Microsoft Academic Search

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

Neil Turok; John Zadrozny

1991-01-01

161

The inert doublet model: an archetype for dark matter  

Microsoft Academic Search

The inert doublet model (IDM), a two Higgs extension of the standard model with an unbroken Z2 symmetry, is a simple and yet rich model of dark matter. We present a systematic analysis of the dark matter abundance and investigate the potentialities for direct and gamma indirect detection. We show that the model should be within the range of future

Laura Lopez Honorez; Emmanuel Nezri; Josep F. Oliver; Michel H. G. Tytgat

2007-01-01

162

Two-Higgs Doublet Model from Quasilocal Quark Interactions  

Microsoft Academic Search

Quark models with four-fermion interaction including derivatives of fields in the strong coupling regime are used to implement composite-Higgs extensions of the standard model. In this approach, the dynamical breaking of chiral symmetry occurs in two (or more) channels (near polycritical values for coupling constants), which gives rise to two (or more) composite Higgs doublets. Two types of models are

A. A. Andrianov; V. A. Andrianov; V. L. Yudichev; R. Rodenberg

2001-01-01

163

Two Higgs Doublet Model From Quasilocal Quark Interactions  

Microsoft Academic Search

Quark models with four-fermion interaction including derivatives of fields in the strong coupling regime are used to implement composite-Higgs extensions of the Standard Model. In this approach the dynamical breaking of chiral symmetry occurs in two (or more) channels (near polycritical values for coupling constants), giving rise to two (or more) composite Higgs doublets. Two types of models are built

A. A. Andrianov; V. A. Andrianov; V. L. Yudichev; R. Rodenberg

1997-01-01

164

Unique design of Doublet and Big Dee vacuum vessels  

Microsoft Academic Search

The Doublet III tokamak now in its fourth year of operation at General Atomic Company, has its plasma contained in a kidney-shaped toroidal vacuum vessel, a configuration that presented unique design challenges. Most tokamak vacuum vessels are constructed of solid walled sections separated by either thin walled bellows (to increase the toroidal resistance) or by poloidal insulation breaks. Such control

J. E. Miller

1982-01-01

165

Masses of a Fourth Generation with Two Higgs Doublets  

SciTech Connect

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

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

2012-05-01

166

Harnessing microtubule dynamic instability for nanostructure assembly.  

SciTech Connect

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 Marie; Osbourn, Gordon Cecil

2004-06-01

167

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

168

Xenopus Meiotic Microtubule-Associated Interactome  

PubMed Central

In metazoan oocytes the assembly of a microtubule-based spindle depends on the activity of a large number of accessory non-tubulin proteins, many of which remain unknown. In this work we isolated the microtubule-bound proteins from Xenopus eggs. Using mass spectrometry we identified 318 proteins, only 43 of which are known to bind microtubules. To integrate our results, we compiled for the first time a network of the meiotic microtubule-related interactome. The map reveals numerous interactions between spindle microtubules and the newly identified non-tubulin spindle components and highlights proteins absent from the mitotic spindle proteome. To validate newly identified spindle components, we expressed as GFP-fusions nine proteins identified by us and for first time demonstrated that Mgc68500, Loc398535, Nif3l1bp1/THOC7, LSM14A/RAP55A, TSGA14/CEP41, Mgc80361 and Mgc81475 are associated with spindles in egg extracts or in somatic cells. Furthermore, we showed that transfection of HeLa cells with siRNAs, corresponding to the human orthologue of Mgc81475 dramatically perturbs spindle formation in HeLa cells. These results show that our approach to the identification of the Xenopus microtubule-associated proteome yielded bona fide factors with a role in spindle assembly.

Winter, Christof; Juhem, Aurelie; Schroeder, Michael; Shevchenko, Andrej; Popov, Andrei V.

2010-01-01

169

Electric field generated by longitudinal axial microtubule vibration modes with high spatial resolution microtubule model  

NASA Astrophysics Data System (ADS)

Microtubules are electrically polar structures fulfilling prerequisites for generation of oscillatory electric field in the kHz to GHz region. Energy supply for excitation of elasto-electrical vibrations in microtubules may be provided from GTP-hydrolysis; motor protein-microtubule interactions; and energy efflux from mitochondria. It recently was determined from anisotropic elastic network modeling of entire microtubules that the frequencies of microtubule longitudinal axial eigenmodes lie in the region of tens of GHz for the physiologically common microtubule lengths. We calculated electric field generated by axial longitudinal vibration modes of microtubule, which model is based on subnanometer precision of charge distribution. Due to elastoelectric nature of the vibrations, the vibration wavelength is million-fold shorter than that of the electromagnetic field in free space and the electric field around the microtubule manifests rich spatial structure with multiple minima. The dielectrophoretic force exerted by electric field on the surrounding molecules will influence the kinetics of reactions via change in the probability of the transport of charge and mass particles. The electric field generated by vibrations of electrically polar cellular structures is expected to play a role in biological self-organization.

Cifra, M.; Havelka, D.; Deriu, M. A.

2011-12-01

170

Shaping and Characteristics of Ohmically Heated Noncircular Plasmas in Doublet III.  

National Technical Information Service (NTIS)

Ohmically heated dee, droplet and doublet plasmas with vertical elongations of up to 3.2 have been produced in Doublet III. Doublet configurations are now obtained by controlled merging of the two current channels of a droplet discharge. All discharges ha...

J. C. Wesley T. Angel C. J. Armentrout

1980-01-01

171

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

PubMed

Microtubules are cylindrical protein polymers that play important roles in a number of cellular functions. The properties of microtubules are dynamically changed by interacting with many microtubule-related proteins and drugs. In this study, we used atomic force microscopy to evaluate the changes in microtubule mechanical properties induced by treatment with nocodazole, which is a microtubule-destabilizing drug. The average spring constant of the microtubules, which was used as a measure of microtubule lateral stiffness, was drastically decreased by treatment with nocodazole within 30 min from 0.052 +/- 0.014 N/m to 0.029 +/- 0.015 N/m. Our findings will aid in the understanding of microtubule dynamics, protein interactions in response to drug treatment, microtubule-related diseases, and drug development. PMID:23755650

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

2013-03-01

172

Molecular linkage underlying microtubule orientation toward cortical sites in yeast.  

PubMed

Selective microtubule orientation toward spatially defined cortical sites is critical to polarized cellular processes as diverse as axon outgrowth and T cell cytotoxicity. In yeast, oriented cytoplasmic microtubules align the mitotic spindle between mother and bud. The cortical marker protein Kar9 localizes to the bud tip and is required for the orientation of microtubules toward this region. Here, we show that Kar9 directs microtubule orientation by acting through Bim1, a conserved microtubule-binding protein. Bim1 homolog EB1 was originally identified through its interaction with adenomatous polyposis coli (APC) tumor suppressor, raising the possibility that an APC-EB1 linkage orients microtubules in higher cells. PMID:10731146

Korinek, W S; Copeland, M J; Chaudhuri, A; Chant, J

2000-03-24

173

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

174

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

175

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

176

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

177

Reconstituting functional microtubule-barrier interactions.  

PubMed

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

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

2014-01-01

178

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

179

Micropattern-guided assembly of overlapping pairs of dynamic microtubules.  

PubMed

Interactions between antiparallel microtubules are essential for the organization of spindles in dividing cells. The ability to form immobilized antiparallel microtubule pairs in vitro, combined with the ability to image them via TIRF microscopy, permits detailed biochemical characterization of microtubule cross-linking proteins and their effects on microtubule dynamics. Here, we describe methods for chemical micropatterning of microtubule seeds on glass surfaces in configurations that specifically promote the formation of antiparallel microtubule overlaps in vitro. We demonstrate that this assay is especially well suited for reconstitution of minimal midzone overlaps stabilized by the antiparallel microtubule cross-linking protein PRC1 and its binding partners. The micropatterning method is suitable for use with a broad range of proteins, and the assay is generally applicable to any microtubule cross-linking protein. PMID:24630116

Fourniol, Franck J; Li, Tai-De; Bieling, Peter; Mullins, R Dyche; Fletcher, Daniel A; Surrey, Thomas

2014-01-01

180

a Search for a Close-Mass Lepton Doublet  

Microsoft Academic Search

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^+e^- 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,

John Keith Riles

1989-01-01

181

Chiral multiplets versus parity doublets in highly excited baryons  

Microsoft Academic Search

It has recently been suggested that the parity doublet structure seen in the\\u000aspectrum of highly excited baryons may be due to effective chiral restoration\\u000afor these states. We argue how the idea of chiral symmetry restoration high in\\u000athe spectrum is consistent with the concept of quark-hadron duality. If chiral\\u000asymmetry is effectively restored for highly-lying states, then the

Thomas D. Cohen; Leonid Glozman

2001-01-01

182

Exclusive decays in general two-Higgs-doublet models  

Microsoft Academic Search

By employing the QCD factorization approach, we calculated the next-to-leading order new physics contributions to the branching ratios, CP asymmetries, isospin and U-spin symmetry breaking of the exclusive decays $B \\\\to V \\\\gamma$ ( $V = K^*, \\\\rho$ ), induced by the charged Higgs penguins in general two-Higgs-doublet models. Within the considered parameter space, we found that (a) the new

Zhenjun Xiao; Ci Zhuang

2004-01-01

183

Baryogenesis in the two-Higgs doublet model  

Microsoft Academic Search

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

Lars Fromme; Stephan J. Huber; Michael Seniuch

2006-01-01

184

FODO/DOUBLET LATTICE FOR THE SNS ACCUMULATOR RING.  

SciTech Connect

Requirements of minimum beam loss for hand-on maintenance and flexibility for future operations are essential for the lattice design of the Spallation Neutron Source (SNS) accumulator ring. In this paper, we present a hybrid lattice that consists of FODO arcs and doublet straights, emphasizing injection and collimation optimization and flexibility, split tunes for coupling control, sextupole families for chromaticity control, and compatibility to future upgrades.

WEI,J.; GARDNER,C.; LEE,Y.Y.; TSOUPAS,N.

2000-06-30

185

? ? + ? - decays in the aligned two-Higgs-doublet model  

NASA Astrophysics Data System (ADS)

The rare decays ? ? + ? - are analyzed within the general framework of the aligned two-Higgs doublet model. We present a complete one-loop calculation of the relevant short-distance Wilson coefficients, giving a detailed technical summary of our results and comparing them with previous calculations performed in particular limits or approximations. We investigate the impact of various model parameters on the branching ratios and study the phenomenological constraints imposed by present data.

Li, Xin-Qiang; Lu, Jie; Pich, Antonio

2014-06-01

186

APC is a component of an organizing template for cortical microtubule networks  

PubMed Central

A microtubule network on the basal cortex of polarized epithelial cells consists of non-centrosomal microtubules of mixed polarity. Here, we investigate the proteins that are involved in organizing this network, and we show that end-binding protein 1 (EB1), adenomatous polyposis coli protein (APC) and p150Glued — although considered to be microtubule plus-end-binding proteins — are localized along the entire length of microtubules within the network, and at T-junctions between microtubules. The network shows microtubule behaviours that arise from physical interactions between microtubules, including microtubule plus-end stabilization on the sides of other microtubules, and sliding of microtubule ends along other microtubules. APC also localizes to the basal cortex. Microtubules grew over and paused at APC puncta; an in vitro reconstituted microtubule network overlaid APC puncta; and microtubule network reconstitution was inhibited by function-blocking APC antibodies. Thus, APC is a component of a cortical template that guides microtubule network formation.

Reilein, Amy; Nelson, W. James

2012-01-01

187

Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins  

PubMed Central

Detergent-extracted BSC-1 monkey cells have been used as a model system to study the Ca2+ sensitivity of in vivo polymerized microtubules under in vitro conditions. The effects of various experimental treatments were observed by immunofluorescence microscopy. Whereas microtubules are completely stable at Ca2+ concentrations below 1 ?M, Ca2+ at greater than 1-4 ?M induces microtubule disassembly that begins in the cell periphery and proceeds towards the cell center. At concentrations of up to 500 ?M, both the pattern and time course of disassembly are not markedly altered, suggesting that, within this concentration range, Ca2+ effects are catalytic rather than stoichiometric. Higher (millimolar) Ca2+ concentration results in rapid destruction of microtubules. Of other divalent cations, only Sr2+ has a slight depolymerizing effect, whereas millimolar Ba2+, Mg2+, or Mn2+ is ineffective. Disassembly induced by micromolar Ca2+ is inhibited by pharmacological agents known to bind to calmodulin and inhibit its function, suggesting that calmodulin mediates Ca2+ effects. Both the addition of exogenous brain microtubule-associated proteins (MAPs) after lysis and the retention of endogenous cellular MAPs normally extracted during the lysis step stabilize microtubules against the depolymerizing effect of micromolar Ca2+. The results indicate that, in this model system, microtubules are sensitive to physiological Ca2+ concentrations and that this sensitivity may be conferred by calmodulin associated with the microtubules. MAPs appear to have a modulating effect on microtubular Ca2+ sensitivity and thus may function as a discriminating factor in cellular functions performed by calmodulin. It is hypothesized that Ca2+-stimulated microtubule disassembly depends on the relative amount of MAPs. Images

Schliwa, Manfred; Euteneuer, Ursula; Bulinski, Jeannette Chloe; Izant, Jonathan G.

1981-01-01

188

Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules.  

PubMed

Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B. PMID:16333575

Kwiatkowska, M; Pop?o?ska, K; Stepi?ski, D

2005-12-01

189

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

190

On the structure of microtubules, tau, and paired helical filaments  

Microsoft Academic Search

Microtubules and their associated proteins form the basis of axonal transport; they are degraded during the neuronal degeneration in Alzheimer's disease. This article surveys recent results on the structure of microtubules, tau protein, and PHFs. Microtubules have been investigated by electron microscopy and image processing after labeling them with the head domain of the motor protein kinesin. This reveals the

E. Mandelkow; Y.-H. Song; O. Schweers; A. Marx

1995-01-01

191

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

192

Gravity-induced reorientation of cortical microtubules observed in vivo  

Microsoft Academic Search

Summary Cortical microtubules play an important role during morphogenesis by determining the direction of cellulose deposition. Although many triggers are known that can induce the reorientation of cortical plant microtubules, the reorientation mechanism has remained obscure. In our approach, we used gravitropic stimulation which is a strong trigger for microtubule reorientation in epidermal cells of maize coleoptiles. To visualize the

Regina Himmelspach; Carol L. Wymer; Clive W. Lloyd; Peter Nick

1999-01-01

193

Nuclear positioning: dynein needed for microtubule shrinkage-coupled movement.  

PubMed

Nuclear movement often requires interactions between the cell cortex and microtubules. A new study has revealed a novel protein interaction linking microtubule plus-ends with the cortex and a role for dynein in microtubule shrinkage-coupled movement. PMID:22720687

Xiang, Xin

2012-06-19

194

Nucleated Assembly of Microtubules in Porcine Brain Extracts  

Microsoft Academic Search

Disk-type structures found in extracts of porcine brain tissue appear to be required for microtubule assembly in vitro. From the morphology of the disks and the dependence of microtubule assembly on the presence of these structures, we propose that the disks are nucleation centers for the polymerization of microtubule protein.

Gary G. Borisy; Joanne B. Olmsted

1972-01-01

195

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

196

Microtubule Self-Organization via Protein-RNA Network Crosstalk.  

PubMed

Microtubule plus-end tracking proteins are crucial for the regulation of microtubule dynamics. Preitner et al. report that one such protein, adenomatous polyposis coli (APC), also binds RNA and identify mRNAs encoding tubulin subunits within the brain APC-RNA interactome, suggesting a new mode of microtubule self-regulation. PMID:25036626

Coles, Charlotte H; Bradke, Frank

2014-07-17

197

Motors and MAPs collaborate to size up microtubules.  

PubMed

Midzone microtubules keep chromosomes apart after segregation and provide a platform for cytokinesis factors. Reporting recently in Cell, Subramanian et al. (2013) describe how the motor protein kinesin-4 and the microtubule-associated protein PRC1 work together to mark microtubule ends for incorporation into the midzone in a length-dependent manner. PMID:23906062

Bechstedt, Susanne; Brouhard, Gary J

2013-07-29

198

A Piconewton Forcemeter Assembled from Microtubules and Kinesins  

Microsoft Academic Search

A forcemeter capable of detecting piconewton forces was assembled from nanoscale building blocks, using a cantilevered microtubule as a beam of known stiffness, which is loaded by a second microtubule transported by kinesin motor proteins adsorbed to the surface and connected to the cantilevered microtubule by the link whose strength is to be determined. Due to the loading rate of

Henry Hess; Jonathon Howard; Viola Vogel

2002-01-01

199

Two Majorana Neutrino Mass Doublets with Thorough Maximal Doublet Mixing from an Analogy with the K^0Meson Oscillations  

Microsoft Academic Search

In the recent note we argued that most of the available positive and negative neutrino oscillation data can be incorporated in a four-neutrino weak interaction model with one physical condition: thorough maximal mixing of the neutrino components in each of the two mass doublets. On the level of CP-invariance an extended Pontecorvo analogy between the neutrino oscillations and the K^0-meson

E. M. Lipmanov

1999-01-01

200

Two Majorana Neutrino Mass Doublets with Thorough Maximal Doublet Mixing from an Analogy with the K^0Meson Oscillations  

Microsoft Academic Search

In the recent note we argued that most of the available positive and negative\\u000aneutrino oscillation data can be incorporated in a four-neutrino weak\\u000ainteraction model with one physical condition: thorough maximal mixing of the\\u000aneutrino components in each of the two mass doublets. On the level of\\u000aCP-invariance an extended Pontecorvo analogy between the neutrino oscillations\\u000aand the K^0-meson

E. M. Lipmanov

1999-01-01

201

Fluorescent microtubules break up under illumination  

PubMed Central

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

1988-01-01

202

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

203

Stathmin regulates microtubule dynamics and microtubule organizing center polarization in activated T cells.  

PubMed

Polarization of T cells involves reorientation of the microtubule organizing center (MTOC). Because activated ERK is localized at the immunological synapse, we investigated its role by showing that ERK activation is important for MTOC polarization. Suspecting that ERK phosphorylates a regulator of microtubules, we next focused on stathmin, a known ERK substrate. Our work indicates that during T cell activation, ERK is recruited to the synapse, allowing it to phosphorylate stathmin molecules near the immunological synapse. Supporting an important role of stathmin phosphorylation in T cell activation, we showed that T cell activation results in increased microtubule growth rate dependent on the presence of stathmin. The significance of this finding was demonstrated by results showing that CTLs from stathmin(-/-) mice displayed defective MTOC polarization and defective target cell cytolysis. These data implicate stathmin as a regulator of the microtubule network during T cell activation. PMID:22529300

Filbert, Erin L; Le Borgne, Marie; Lin, Joseph; Heuser, John E; Shaw, Andrey S

2012-06-01

204

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

205

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

PubMed Central

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- micron 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 examined. MAP 4 antibody also labeled microtubule-like patterns, but the extent of MAP 4 reactivity was cell type-specific within each tissue. MAP 4 antibody labeled microtubules in vascular elements of all tissues and in other cells considered to have supportive functions, including Sertoli cells in the testis and glial elements in the nervous system. Microtubule patterns were also observed in cardiac, smooth, and skeletal (eye) muscle, podocytes in kidney, Kuppfer cells in liver, and spermatid manchettes. The only MAP 4-positive cells in which the pattern was not microtubule-like were the principal cells of the collecting ducts in kidney cortex, in which diffuse fluorescence was seen. MAP 4 antibody did not react with microtubule-rich neuronal elements of the central and peripheral nervous system, skeletal muscle from anterior thigh, liver parenchymal cells, columnar epithelial cells of the small intestine, and absorptive cells of the tubular component of the nephron. These observations indicate that MAP 4 may be associated with only certain kinds of cell functions as demonstrated by the preferential distribution with microtubules of defined cell types.

1984-01-01

206

PLANT SCIENCE: Microtubules Make Tracks for Cellulose  

NSDL National Science Digital Library

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

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

2006-06-09

207

Microtubules in Dendritic Spine Development and Plasticity  

Microsoft Academic Search

Recent studies indicate that microtubules (MTs) may play an important role in spine development and dynam- ics. 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 ap- proaches exert profound influence on the development and plasticity of

Jiaping Gu; James Q. Zheng

2009-01-01

208

Microtubule Structure at 8 Å Resolution  

Microsoft Academic Search

We have obtained a 3D reconstruction of intact microtubules, using cryoelectron microscopy and image processing, at a resolution of about 8 Å, sufficient to resolve much of the secondary structure. The level of detail in the map allows docking of the tubulin structure previously determined by electron crystallography, with very strong constraints, providing several important insights not previously available through

Huilin Li; David J. DeRosier; William V. Nicholson; Eva Nogales; Kenneth H. Downing

2002-01-01

209

Microtubule dynamics control tail retraction in migrating vascular endothelial cells.  

PubMed

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

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

2013-12-01

210

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

211

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

212

The role of microtubule movement in bidirectional organelle transport  

PubMed Central

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

Kulic, Igor M.; Brown, Andre E. X.; Kim, Hwajin; Kural, Comert; Blehm, Benjamin; Selvin, Paul R.; Nelson, Philip C.; Gelfand, Vladimir I.

2008-01-01

213

Both Microtubule-Stabilizing and Microtubule-Destabilizing Drugs Inhibit Hypoxia-Inducible Factor1A Accumulation and Activity by Disrupting Microtubule Function  

Microsoft Academic Search

We have recently identified a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the hypoxia-inducible factor-1 (HIF-1) pathway. Based on this model, we hypothesized that other microtubule-targeting drugs may have a similar effect on HIF- 1A. To test that hypothesis, we studied the effects of different clinically relevant microtubule-disrupting agents, including taxotere, epothilone B,

Daniel Escuin; Erik R. Kline; Paraskevi Giannakakou

2005-01-01

214

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

215

Two scalar doublet models with softly broken symmetries  

NASA Astrophysics Data System (ADS)

A generalized two scalar doublet extension of the standard model is proposed, including a soft scalar term which explicitly breaks CP invariance and the discrete symmetry protecting against scalar mediated, flavour changing neutral currents. The allowed scalar spectrum in the model is discussed together with the top quark mass, based upon the adoption of renormalization group, quasi-fixed point values for the free hard couplings in the lagrangian. The radiative corrections arising from the new scalar sector are calculated and their phenomenological consequences commented upon. In the appendices a complete set of Feynman rules for the theory is given.

Froggatt, C. D.; Moorhouse, R. G.; Knowles, I. G.

1992-11-01

216

Symmetry and mass degeneration in multi-Higgs-doublet models  

Microsoft Academic Search

We investigate possible symmetry properties of the scalar sector of Multi-Higgs-Doublet Models, and, to some extent, the generalization\\u000a of such models to gauge groups other than SU(2)\\u000a L\\u000a ?×?U(1)\\u000a Y\\u000a . In models with C (charge conjugation) invariance, and where certain quartic terms are not present, the scalar potential is invariant under\\u000a a group larger than the gauge group, O(4)

K. Olaussen; P. Osland; M. Aa. Solberg

2011-01-01

217

Symmetry and Mass Degeneration in Multi-Higgs-Doublet Models  

Microsoft Academic Search

We investigate possible symmetry properties of the scalar sector of\\u000aMulti-Higgs-Doublet Models, and, to some extent, the generalization of such\\u000amodels to gauge groups other than $SU(2)_L\\\\times U(1)_Y$. In models where the\\u000a${\\\\cal C}$ (charge conjugation) violating operator $\\\\widehat{C}$ is not\\u000apresent, the scalar potential is invariant under a group larger than the gauge\\u000agroup, $O(4)$ when the Higgs fields

K. Olaussen; P. Osland; M. Aa. Solberg

2010-01-01

218

The timing of division in twin cell doublets.  

PubMed

The fifth stage larval epidermis of Calpodes ethlius (Lepidoptera, Hesperiidae) is a syncytium of doublets where sibling cells are twins connected by residual midbodies between divisions. Twin cells resemble one another more than their other neighbours in such features as the shape and number of nucleolar particles, the number of actin bundles and the position of condensed female chromosomes. We have now found that they also resemble one another in the timing of cell division in preparation for pupation. Twins are more likely to divide together than at random. The paired timing of mitosis is presumed to be a result of the twins sharing a common cytoplasm. PMID:8511769

Jeun, G; Locke, M

1993-01-01

219

Delta wing flutter based on doublet lattice method in NASTRAN  

NASA Technical Reports Server (NTRS)

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

Jew, H.

1975-01-01

220

Binary Asteroids and the Formation of Doublet Craters  

NASA Astrophysics Data System (ADS)

At least 10% (3 out of 28) of the largest known impact craters on Earth and a similar fraction of all impact structures on Venus are doublets (i.e., have a companion crater nearby), formed by the nearly simultaneous impact of objects of comparable size. Mars also has doublet craters, though the fraction found there is smaller (2%). These craters are too large and too far separated to have been formed by the tidal disruption of an asteroid prior to impact, or from asteroid fragments dispersed by aerodynamic forces during entry. We propose that some fast rotating rubble-pile asteroids (e.g., 4769 Castalia), after experiencing a close approach with a planet, undergo tidal breakup and split into multiple co-orbiting fragments. In some cases these fragments evolve into stable binary systems, which re-encounter and impact the planet during a later pass, creating two distinct craters. To test this idea, we modeled close encounters between fast-rotating contact-binary asteroids, our first-order approximation for rubble-pile asteroids, and a chosen planet. Our results show that Earth's tidal forces frequently create binary asteroids, but that the separation distance between the binary's components is almost always too small to produce a doublet crater at impact. However, once the components are orbiting one another, small perturbations from repeated distant Earth enounters, along with mutual tidal forces between the components, frequently increase the separation distance between the components in a random-walk fashion. To model these effects, we combined our numerical model of planetary encounters with a Monte Carlo code that computes the frequency and characteristics of repeated Earth encounters as well as mutual tidal effects occurring between Earth encounters. Our results show that ˜15% of all Earth-crossing asteroids evolve into co-orbiting binary asteroids with well-separated components. Asteroids on solely Mars-crossing orbits produce a smaller fraction of binaries (<5%). Folding these results into another model treating impact encounters between binary asteroids and a chosen planet, we found we could duplicate the observed fraction of doublet craters found on Earth, Venus, and Mars. Our results suggest that any search for asteroid satellites should place emphasis on km-sized Earth-crossing asteroids with short-rotation periods.

Bottke, William F., Jr.; Melosh, H. Jay

1996-12-01

221

Zp scalar dark matter from multi-Higgs-doublet models  

NASA Astrophysics Data System (ADS)

In many models, stability of dark-matter particles is protected by a conserved Z2 quantum number. However, dark matter can be stabilized by other discrete symmetry groups, and examples of such models with custom-tailored field content have been proposed. Here, we show that electroweak symmetry-breaking models with N Higgs doublets can readily accommodate scalar dark matter candidates stabilized by groups Zp with any p?2N-1, leading to a variety of kinds of microscopic dynamics in the dark sector. We give examples in which semiannihilation or multiple semiannihilation processes are allowed or forbidden, which can be especially interesting in the case of asymmetric dark matter.

Ivanov, I. P.; Keus, V.

2012-07-01

222

Observation of a doublet band in the nucleus 128Pr  

NASA Astrophysics Data System (ADS)

The doubly odd nucleus 128Pr was studied via in-beam ?-ray spectroscopy using the 40Ca+92Mo reaction at 190 MeV. Five rotational bands were observed and assigned to configurations involving proton and neutron orbitals close to the Fermi level. The observed bands are discussed in the framework of the three-dimensional (3D) tilted axis cranking (TAC) model. Two of the negative-parity bands have almost degenerate energy levels, which suggests an interpretation in terms of the pseudospin doublet [32~1]. It was impossible, however, to reproduce the observed small level spacing by either 3D TAC or two-quasiparticle-rotor model calculations.

Petrache, C. M.; Lo Bianco, G.; Bazzacco, D.; Lunardi, S.; Menegazzo, R.; Nespolo, M.; de Angelis, G.; Blasi, N.; Dimitrov, V. I.; Frauendorf, S.; Semmes, P.; Zhang, Jing-Ye

2002-05-01

223

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

NASA Technical Reports Server (NTRS)

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

Jukes, T. H.

1977-01-01

224

Spinning Janus doublets driven in uniform ac electric fields.  

PubMed

We provide an experimental proof of concept for a robust, continuously rotating microstructure-consisting of two metallodielectric (gold-polystyrene) Janus particles rigidly attached to each other-which is driven in uniform ac fields by asymmetric induced-charge electro-osmosis. The pairs (doublets) are stabilized on the substrate surface which is parallel to the plane of view and normal to the direction of the applied electric field. We find that the radius of orbit and angular velocity of the pair are predominantly dependent on the relative orientations of the interfaces between the metallic and dielectric hemispheres and that the electrohydrodynamic particle-particle interactions are small. Additionally, we verify that both the angular and linear velocities of the pair are proportional to the square of the applied field which is consistent with the theory for nonlinear electrokinetics. A simple kinematic rigid body model is used to predict the paths and doublet velocities (angular and linear) based on their relative orientations with good agreement. PMID:24580163

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

2014-01-01

225

Energy confinement in Doublet III with high-Z limiters  

SciTech Connect

This report describes the experimental measurements and data analysis techniques used to evaluate the energy confinement in noncircular plasmas produced in Doublet III. Major aspects of the confinement measurements and analysis techniques are summarized. Machine parameters, diagnostic systems and discharge parameters relavent to the confinement measurements are given. Magnetic analysis techniques used to determine the plasma shape are reviewed. Scaling of the on-axis values of electron temperature, confinement time and Z/sub eff/ with plasma density is presented. Comparison with scaling results from other circular tokamaks is discussed. Numerical and analytic techniques developed for calculating the plasma energy confinement time and self-consistent profiles of density, temperature, current, and flux in non-circular geometries are described. These techniques are applied to the data and used to determine the central and global electron energy confinement time for a typical doublet plasma. Additional aspects of the confinement such as the radial dependence of the electron thermal conductivity and the estimated ion temperature are explored with the aid of a non-circular transport simulation code. The results of the confinement measurements are summarized and discussed. A brief summary of the theoretically expected effects of noncircularity on plasma confinement is included for reference as Appendix I.

Marcus, F.B.; Adcock, S.J.; Baker, D.R.; Blau, F.P.; Brooks, N.H.; Chase, R.P.; DeBoo, J.C.; Ejima, S.; Fairbanks, E.S.; Fisher, R.K.

1980-02-01

226

Next-to-minimal two Higgs doublet model  

NASA Astrophysics Data System (ADS)

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

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

2014-04-01

227

Doublet III neutral beam injector test tank cryopanel design  

SciTech Connect

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

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

1980-03-01

228

Spinning Janus doublets driven in uniform ac electric fields  

NASA Astrophysics Data System (ADS)

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

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

2014-01-01

229

Electric dipole moments in two-Higgs-doublet models  

NASA Astrophysics Data System (ADS)

Electric dipole moments are extremely sensitive probes for additional sources of CP violation in new physics models. Specifically, they have been argued in the past to exclude new CP-violating phases in two-Higgs-doublet models. Since recently models including such phases have been discussed widely, we revisit the available constraints in the presence of mechanisms which are typically invoked to evade flavour-changing neutral currents. To that aim, we start by assessing the necessary calculations on the hadronic, nuclear and atomic/molecular level, deriving expressions with conservative error estimates. Their phenomenological analysis in the context of two-Higgs-doublet models yields strong constraints, in some cases weakened by a cancellation mechanism among contributions from neutral scalars. While the corresponding parameter combinations do not yet have to be unnaturally small, the constraints are likely to preclude large effects in other CP-violating observables. Nevertheless, the generically expected contributions to electric dipole moments in this class of models lie within the projected sensitivity of the next-generation experiments.

Jung, Martin; Pich, Antonio

2014-04-01

230

Collisional broadening of alkali doublets by helium perturbers  

NASA Astrophysics Data System (ADS)

We report results for the Lorentzian profiles of the Li I, Na I and K I doublets and the Na I subordinate doublet broadened by helium perturbers for temperatures up to 3000 K. They have been obtained from a fully quantum-mechanical close-coupling description of the colliding atoms, the Baranger theory of line shapes and new ab initio potentials for the alkali-helium interaction. For all lines except the 769.9 nm K I line, the temperature dependence of the widths over the range 70 <= T <= 3000 K is accurately represented by the power law form w = aTbb with 0.38 < b < 0.43. The 769.9 nm K I line has this form for 500 <= T <= 3000 K with b having the higher value of 0.49. Although the shifts have a more complex temperature dependence, they all have the general feature of increasing with temperature above T ~ 500 K apart from the 769.9 K I line whose shift decreases with temperature.

Mullamphy, D. F. T.; Peach, G.; Venturi, V.; Whittingham, I. B.; Gibson, S. J.

2007-03-01

231

Electroweak phase transition in two Higgs doublet models  

SciTech Connect

We reexamine the strength of the first-order phase transition in the electroweak theory supplemented by an extra Higgs doublet. The finite-temperature effective potential V{sub eff} is computed to one-loop order, including the summation of ring diagrams, to study the ratio {phi}{sub c}/T{sub c} of the Higgs field VEV to the critical temperature. We make a number of improvements over previous treatments, including a consistent treatment of Goldstone bosons in V{sub eff}, an accurate analytic approximation to V{sub eff} valid for any mass-to-temperature ratios, and use of the experimentally measured top quark mass. For two-Higgs-doublet models, we identify a significant region of parameter space where {phi}{sub c}/T{sub c} is large enough for electroweak baryogenesis, and we argue that this identification should persist even at higher orders in perturbation theory. In the case of the minimal supersymmetric standard model, our results indicate that the extra Higgs bosons have little effect on the strength of the phase transition. {copyright} {ital 1997} {ital The American Physical Society}

Cline, J.M.; Lemieux, P. [McGill University, Montreal, Quebec H3A 2T8 (Canada)] [McGill University, Montreal, Quebec H3A 2T8 (Canada)

1997-03-01

232

Localized Alteration of Microtubule Polymerization in Response to Guidance Cues  

PubMed Central

Inhibition of microtubule dynamic instability prevents growth cone turning in response to guidance cues, yet specific changes in microtubule polymerization as growth cones encounter boundaries have not been investigated. In this study, we examined the rate and direction of microtubule polymerization in response to soluble nerve growth factor (NGF) and immobilized chondroitin sulfate proteoglycans (CSPGs) by expressing enhanced GFPEB3 in rat pheochromocytoma (PC12) cells. GFP-EB3 comets were monitored in live cells using time-lapse epifluorescent microscopy. Using an automated tracking system, the rate of microtubule polymerization was calculated as the frame-to-frame displacement of EB3 comets. Our results demonstrate that the rate of microtubule polymerization is increased following NGF treatment, while contact with CSPGs decreases microtubule polymerization rates. This reduction in microtubule polymerization rates was specifically localized to neurites in direct contact with CSPGs, and not at non-contacting neurites. Additionally, we found an increase in the percentage of microtubules polymerizing in the retrograde direction in neurites at CSPG boundaries with a concomitant decrease in the rate of retrograde microtubule polymerization. These results implicate localized changes in microtubule dynamics as an important component of the growth cone response to guidance cues.

Kelly, Terri-Ann N.; Katagiri, Yasuhiro; Vartanian, Keri B.; Kumar, Pramukta; Chen, Inn-Inn; Rosoff, William J.; Urbach, Jeffery S.; Geller, Herbert M.

2010-01-01

233

Tumor suppressor NF2/Merlin is a microtubule stabilizer.  

PubMed

Cancer-associated mutations in oncogene products and tumor suppressors contributing to tumor progression manifest themselves, at least in part, by deregulating microtubule-dependent cellular processes that play important roles in many cell biological pathways, including intracellular transport, cell architecture, and primary cilium and mitotic spindle organization. An essential characteristic of microtubules in the performance of these varied cell processes is their ability to continuously remodel, a phenomenon known as dynamic instability. It is therefore conceivable that part of the normal function of certain cancer-causing genes is to regulate microtubule dynamic instability. Here, we report the results of a high-resolution live-cell image-based RNA interference screen targeting a collection of 70 human tumor suppressor genes to uncover cancer genes affecting microtubule dynamic instability. Extraction and computational analysis of microtubule dynamics from EB3-GFP time-lapse image sequences identified the products of the tumor suppressor genes NF1 and NF2 as potent microtubule-stabilizing proteins. Further in-depth characterization of NF2 revealed that it binds to and stabilizes microtubules through attenuation of tubulin turnover by lowering both rates of microtubule polymerization and depolymerization as well as by reducing the frequency of microtubule catastrophes. The latter function appears to be mediated, in part, by inhibition of hydrolysis of tubulin-bound GTP on the growing microtubule plus end. PMID:24282279

Smole, Zlatko; Thoma, Claudio R; Applegate, Kathryn T; Duda, Maria; Gutbrodt, Katrin L; Danuser, Gaudenz; Krek, Wilhelm

2014-01-01

234

Nano monorail for molecular motors: Individually manipulated microtubules for kinesin motion  

Microsoft Academic Search

In a motor protein system (kinesin\\/microtubule), individual microtubules were captured and relocated to build a complex nano-scale transport system. Micro tweezers, attached to a micromanipulator arm, were used to capture polarity marked microtubules. Precise relocation of those microtubules provided an oriented track along which kinesin-coated beads move according to the microtubule polarities.

M. C. Tarhan; L. Jalabert; R. Yokokawa; C. Bottier; D. Collard; H. Fujita

2009-01-01

235

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

PubMed Central

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

1992-01-01

236

Parkin protects dopaminergic neurons against microtubule-depolymerizing toxins by attenuating microtubule-associated protein kinase activation.  

PubMed

Mitogen-activated protein kinases, originally known as microtubule-associated protein (MAP) kinases, are activated in response to a variety of stimuli. Here we report that microtubule-depolymerizing agents such as colchicine or nocodazole induced strong activation of MAP kinases including JNK, ERK, and p38. This effect was markedly attenuated by parkin, whose mutations are linked to Parkinson disease (PD). Our previous study has shown that parkin stabilizes microtubules through strong interactions mediated by three independent domains. We found that each of the three microtubule-binding domains of parkin was sufficient to reduce MAP kinase activation induced by microtubule depolymerization. The ability to attenuate microtubule depolymerization and the ensuing MAP kinase activation was abrogated in B-lymphocytes and fibroblasts derived from PD patients with parkin mutations such as exon 4 deletion. Such mutations produced truncated parkin proteins lacking any microtubule binding domain and prevented parkin from protecting midbrain dopaminergic neurons against microtubule-depolymerizing toxins such as rotenone or colchicine. Consistent with these, blocking MAP kinase activation in midbrain dopaminergic neurons by knocking down MAP kinase kinases (MKK) significantly reduced the selective toxicity of rotenone or colchicine. Conversely, overexpression of MAP kinases caused marked toxicities that were significantly attenuated by parkin. Thus, the results suggest that parkin protects midbrain dopaminergic neurons against microtubule-depolymerizing PD toxins such as rotenone by stabilizing microtubules to attenuate MAP kinase activation. PMID:19074146

Ren, Yong; Jiang, Houbo; Yang, Fang; Nakaso, Kazuhiro; Feng, Jian

2009-02-01

237

MICROBIOLOGY: Bacterial Bushwacking Through a Microtubule Jungle  

NSDL National Science Digital Library

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

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

2006-11-10

238

Microtubule Actin Cross-Linking Factor (Macf)  

PubMed Central

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

Leung, Conrad L.; Sun, Dongming; Zheng, Min; Knowles, David R.; Liem, Ronald K.H.

1999-01-01

239

Drugs That Target Dynamic Microtubules: A New Molecular Perspective  

PubMed Central

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

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

2011-01-01

240

General theory for the mechanics of confined microtubule asters  

NASA Astrophysics Data System (ADS)

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

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

2014-01-01

241

Microtubule Stabilization Leads to Growth Reorientation in Arabidopsis Trichomes  

Microsoft Academic Search

The single-cell trichomes in wild-type Arabidopsis are either unbranched or have two to five branches. Using trans- genic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, which decorates the microtubular cytoskeleton, we observed that during trichome branching, microtubules reorient with re- spect to the longitudinal growth axis. Considering branching to be a localized microtubule-dependent growth reorien- tation event,

Jaideep Mathur; Nam-Hai Chua

2000-01-01

242

Direct observation of single kinesin molecules moving along microtubules  

Microsoft Academic Search

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

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

1996-01-01

243

Microtubule transport, concentration and alignment in enclosed microfluidic channels  

Microsoft Academic Search

The kinesin-microtubule system has emerged as a versatile model system for biologically-derived microscale transport. While\\u000a kinesin motors in cells transport cargo along static microtubule tracks, for in vitro transport applications it is preferable to invert the system and transport cargo-functionalized microtubules along immobilized\\u000a kinesin motors. However, for efficient cargo transport and to enable this novel transport system to be interfaced

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

2007-01-01

244

Microtubule-Actin Cross-talk at Focal Adhesions  

NSDL National Science Digital Library

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

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

2002-07-02

245

Restrictions on Two Higgs Doublet Models and CP Violation at the Unification Scale.  

National Technical Information Service (NTIS)

Bounds on charged Higgs masses and couplings in models with two Higgs doublets are examined that came from CP violation in the neutral K system. Bounds on charged Higgs masses and couplings in two Higgs doublet models are also obtained from their effects ...

G. G. Athanasiu

1987-01-01

246

Relaxation of top mass limits in the two-Higgs-doublet model  

Microsoft Academic Search

The limits on the top quark mass from the recent measurement of the W and Z masses are analysed in the two-Higgs-doublet extension of the standard model. It is found that the addition of a second Higgs doublet relaxes the top mass limit considerably. Work supported by Stifting Volkswagenwerk.

A. Denner; R. J. Guth; J. H. Kühn

1990-01-01

247

A two parameter four neutrino mixing model with an exchange symmetry of the mass doublet neutrinos  

Microsoft Academic Search

In a model with four neutrino mass eigenstates grouping in two narrow doublets the condition of maximal mixing of the doublet neutrino components leads to a simple 4-neutrino mixing matrix which is related to two connected effective two-neutrino mixing patterns. As a result, large values of the neutrino oscillation amplitudes for both the solar and atmospheric neutrino anomalies appear quite

E. M. Lipmanov

1998-01-01

248

Soft supersymmetry breaking masses in a unified model with doublet-triplet splitting  

Microsoft Academic Search

We study soft supersymmetry breaking parameters in a supersymmetric unified model which potentially solves the doublet-triplet splitting problem. In the model the doublet-triplet splitting is solved by the discrete symmetry which is allowed to be introduced due to the direct product structure of the gauge group. The messenger fields for the gauge mediated supersymmetry breaking are naturally embedded in the

Daijiro Suematsu

2003-01-01

249

Observable Parameters of Fermion Mass Matrices in Multi-Higgs-Doublet Models  

Microsoft Academic Search

It is shown that, although the fermionic and scalar sectors of multi-Higgs-doublet models are not independent of each other, the fermion mass matrices, in particular, are invariant under the equivalence class transformations of the scalar doublets. In light of this observation, a method originally formulated for counting the observable parameters of the Yukawa Lagrangian of the standard model is extended

Recai Erdem

1995-01-01

250

Doublet Structure of 41.5 EV Neutron Resonance of exp 185 Re.  

National Technical Information Service (NTIS)

A possible doublet structure of 41.5 eV resonance of exp 185 Re is studied. From the transmission measurements of a thin natural rhenium sample on the neutron spectrometer it has been found that this resonance has a doublet structure which can be describe...

A. B. Popov K. Trzeciak Khvan Cher Gu

1978-01-01

251

Polarized ?b-->Xc?? in the standard model and two-Higgs-doublet model  

NASA Astrophysics Data System (ADS)

The inclusive rate and ? spectrum for a polarized ?b baryon to decay to charm hadronic final states and leptons ?? in the SM and a two-Higgs-doublet model are computed. The O(?s) QCD corrections to the ? spectrum in the two-Higgs-doublet model are also given.

Huang, Chao-Shang; Yan, Hua-Gang

1997-11-01

252

Electroweak unification of Yukawa Couplings in the Extension of the Two Higgs Doublet Model  

Microsoft Academic Search

In the simplest extension of the Standard Model (SM), known as the Two Higgs Doublet Model (2HDM), a second doublet of scalar fields and a new potential are introduced, the last one depends on seven parameters that determine the masses of the five Higgs bosons that are predicted by the model. We use the one loop renormalization group equations (RGE)

D. Morales C; S. R. Juárez W; P. Kielanowski

2011-01-01

253

Pressure Effects of Rare Gases on the Second Doublet of Rubidium Principal Series  

Microsoft Academic Search

The displacement, asymmetry, and broadening of the second doublet of the rubidium principal series perturbed by pure helium, neon and argon up to 13 atmospheres are observed. The displacement as well as the broadening of the doublet components are not the same. The 2P12 component shifts and broadens more conspicuously than the 2P112 component (an exception being the broadening by

Ny Tsi-Zé; Ch'en Shang-Yi

1937-01-01

254

A comparison of micro-mechanical modeling of asphalt materials using finite elements and doublet mechanics  

Microsoft Academic Search

A comparative study is given between two micro-mechanical models that have been developed to simulate the behavior of cemented particulate materials. The first model is a discrete analytical approach called doublet mechanics that represents a solid as an array of particles. This scheme develops analytical expressions for the micro-deformation and stress fields between particle pairs (a doublet). The second approach

Martin H. Sadd; Qingli Dai

2005-01-01

255

The origin of mirror-image symmetry doublet cells in the hypotrich ciliate Paraurostyla weissei  

Microsoft Academic Search

Mirror-image doublets of the hypotrich ciliateParaurostyla weissei were induced by modifying culture conditions. Successive steps of doublet formation involve inhibiting the separation of daughter cells during cell division and the shifting of these to attain a parallel configuration. The posterior part of the adoral band of membranelles in the right component then turns to the left and fuses with the

Maria Jerka-Dziadosz; M. Nencki

1983-01-01

256

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

SciTech Connect

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.

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

1984-10-01

257

Microtubule plus-end tracking proteins in differentiated mammalian cells.  

PubMed

Differentiated mammalian cells are often characterized by highly specialized and polarized structure. Its formation and maintenance depends on cytoskeletal components, among which microtubules play an important role. The shape and dynamic properties of microtubule networks are controlled by multiple microtubule-associated factors. These include molecular motors and non-motor proteins, some of which accumulate specifically at the growing microtubule plus-ends (the so-called microtubule plus-end tracking proteins). Plus-end tracking proteins can contribute to the regulation of microtubule dynamics, mediate the cross-talk between microtubule ends, the actin cytoskeleton and the cell cortex, and participate in transport and positioning of structural and regulatory factors and membrane organelles. Malfunction of these proteins results in various human diseases including some forms of cancer, neurodevelopmental disorders and mental retardation. In this article we discuss recent data on microtubule dynamics and activities of microtubule plus-end binding proteins important for the physiology and pathology of differentiated mammalian cells such as neurons, polarized epithelia, muscle and sperm cells. PMID:18023603

Jaworski, Jacek; Hoogenraad, Casper C; Akhmanova, Anna

2008-01-01

258

Mature Drosophila Meiosis I Spindles Comprise Microtubules of Mixed Polarity  

PubMed Central

Summary New information has been obtained recently regarding microtubule organization in Xenopus extract spindles. These spindles assemble in vitro by chromatin-mediated microtubule nucleation [1] and consist of randomly interspersed long and short microtubules [2] with minus ends distributed throughout the spindle [3]. Fluorescence speckle microscopy has led to the proposal that the Xenopus steady-state spindles contain two overlapping arrays of parallel or antiparallel microtubules with differing poleward flux velocities [4]. Although some of these features have also been reported for C. elegans female meiotic spindles [5], it is not clear whether they are representative of microtubule organization and dynamics in oocyte meiotic spindles. Here we examine anastral meiosis I spindles of live Drosophila oocytes expressing the microtubule plus end-tracking protein, EB1, fused to GFP, and find fluorescent particles throughout the spindle and movement towards both the poles and equator. EB1 particle velocities, corresponding to microtubule growth rates, are similar in both directions, but slower than growth from the poles in mitotic spindles of early embryos. Meiosis I spindles by photobleaching analysis yielded data showing similar microtubule growth rates and dynamics at the poles and equator, consistent with spindle microtubules of mixed polarity, differing from early embryo mitotic spindles.

Liang, Zhang-Yi; Hallen, Mark Andrew; Endow, Sharyn Anne

2009-01-01

259

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

260

In Vitro Microtubule and Motor Protein Motion on Glass  

NASA Astrophysics Data System (ADS)

The intracellular microtubule associated protein kinesin uses adenosine triphosphate (ATP) as an energy source for unidirectional and processive motion on a microtubule filament. In a cell, kinesin motor proteins function as transporters for organelles, macromolecules and various particles. To study the related processes in vitro, we have performed rhodamine-labeled microtubule gliding assays and kinesin-coated quantum dot motility assays on glass surfaces. Motility is observed by fluorescence microscopy. Results from these two assays, as well as the effect of ATP concentration on kinesin velocity will be presented. We will discuss how we use these assays for the manipulation of microtubules on a surface, thus enabling specific particle distribution by kinesin.

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

2011-10-01

261

Microtubule deacetylation sets the stage for successful axon regeneration  

PubMed Central

EMBO J 31: 3063–3078 Successful axon regeneration relies on a sophisticated balance between stable and dynamic microtubules. While it has been established that moderate microtubule stabilization improves regenerative performance, little is known how microtubules are normally regulated during regeneration. A new study by Cho and Cavalli (2012) uncovers an early role for local microtubule deacetylation that seems important for converting a cut axon stump to a regenerating axon. The authors demonstrate that histone deacetylase 5 is responsible for local deacetylation, and that it is locally activated by PKC downstream of axotomy-induced calcium influx.

Chen, Li; Rolls, Melissa M

2012-01-01

262

Dynein and kinesin share an overlapping microtubule-binding site  

PubMed Central

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

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

2004-01-01

263

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

PubMed

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 botanical species and marine organisms has yielded promising new antitubulin agents with novel properties. In the current search for novel microtubule-binding agents, enhanced tumour specificity, reduced neurotoxicity and insensitivity to chemoresistance mechanisms are the three main objectives. PMID:20885410

Dumontet, Charles; Jordan, Mary Ann

2010-10-01

264

The planes of division in twin cell doublets.  

PubMed

The fifth stage larval epidermis of Calpodes ethlius (Lepidoptera, Hesperiidae) is a syncytium of doublets where sibling cells remain connected by residual midbodies between mitoses. These twins resemble one another more than their other neighbours in features such as the shape and number of nucleolar particles, the number of actin bundles, the position of condensed chromosomes in female cells and the timing of mitosis. Although the patterns of arrangement of structures may be similar in twinned nuclei they differ in their orientation. We have now found that twins also differ in the orientation of their division planes with respect to the previous plane of division. Similarity of structural pattern but not orientation is most easily explained if nuclei, together with determinants for the plane of division, are free to rotate in the plane of the epithelium. PMID:8511768

Jeun, G; Locke, M

1993-01-01

265

High power neutral beam heating on Doublet III  

SciTech Connect

Energy confinement studies with up to 7.5 MW of neutral beam heating power have been performed on the Doublet III tokamak. We have found that during beam injection divertor operation results in up to a factor of two improvement in energy confinement as compared to comparable limiter discharges. In both configurations the confinement improves linearly with plasma current. In addition, there are indications that a new plateau in energy confinement time is reached with injection at high power levels. In all configurations electron transport is the major heat loss channel. Transport analysis indicates that the electron thermal conductivity is typically a factor of 2 to 3 lower in diverted discharges as compared to comparable limiter discharges. However, there are indications that the plasma edge may be important since improved confinement is correlated with the separation between the limiter and the separatrix.

Overskei, D.O.; Armentrout, C.J.; Baur, J.F.; Blau, F.P.; Bramson, G.; Burrell, K.H.; Chase, R.P.; DeBoo, J.C.; Ejima, S.; Fairbanks, E.S.

1984-03-01

266

Charge exchange measurements on the Doublet III tokamak  

SciTech Connect

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

Lohr, J.; Armentrout, C.J.

1985-08-01

267

Microtubule-associated protein-4 (MAP4) inhibits microtubule-dependent distribution of mRNA in isolated neonatal cardiocytes  

Microsoft Academic Search

Objectives: Active mRNA distribution in the form of ribonucleoprotein particles moving along microtubules has been shown in several cell types, but not yet in cardiocytes. This study addresses two hypotheses: 1) a similar mRNA distribution mechanism operates in cardiocytes; 2) decoration of microtubules with microtubule-associated proteins compromises this distribution. Methods: To visualize ribonucleoproteins in cultured neonatal rat cardiocytes, they were

Dimitri Scholz; Paul McDermott; Maria Garnovskaya; Thomas N. Gallien; Stefan Huettelmaier; Christina DeRienzo; George Cooper

2006-01-01

268

Microtubules and beta cell function: effect of colchicine on microtubules and insulin secretion in vitro by mouse beta cells  

PubMed Central

A monolayer culture system was developed to study the role of microtubules in insulin secretion. Cultured cells were obtained to study the role of microtubules in insulin secretion. Cultured cells were obtained by enzymatic digestion of pancreases from C57BL-KsJ mice 6-12 wk of age. On day 4 of culture, the medium was changed, control or treatment medium added, and frequent samples were removed for insulin assay. Microtubules and beta cells were identified by indirect immunofluorescence with monospecific antibodies to tubulin and insulin. An extensive microtubule network radiates from the perinuclear region of the beta cell to the plasma membrane. Although alterations in the calcium concentration of the medium did not affect the microtubule pattern, the absence of calcium or glucose in the medium inhibited insulin secretion (P less than 0.001). Optimum insulin release occurred at a calcium concentration of 2.5 mM. Colchicine, in concentrations of 10(-10) M, did not affect the microtubule immunofluorescent pattern, whereas concentrations of 1 and 5 x 10(-7) M decreased the number of microtubules, and microtubules could not be identified in cultures treated with 10(-6) M colchicine for 2 h. After a 2-h preincubation, the prolonged release of insulin at either 2.0 or 4.5 mg/ml of glucose was decreased by 10(-6) M colchicine (P less than 0.02). The immediate release of insulin was similar to that in control plates and occurred in cultures with no identifiable microtubules. Microtubules and insulin secretion were not altered by 10(-6) M lumicolchicine and prolonged insulin secretion recovered 24 h after removal of colchicine. These studies show that the microtubules facilitate sustained secretion of insulin but are not required for the immediate release of the hormone. Alterations in the extracellular calcium concentration which play an essential role in insulin secretion do not alter the microtubule pattern in the beta cell.

1982-01-01

269

The Euglena paraflagellar rod: structure, relationship to other flagellar components and preliminary biochemical characterization.  

PubMed

Demembranated flagella from Euglena gracilis consisted of three distinct components: a 9 + 2 axoneme of microtubules, an extensive surface coating of long and short mastigonemes and a lattice-like axial fibre known as the paraflagellar rod. Negatively stained preparations of isolated paraflagellar rods showed the major structural unit to be a 22 nm filament oriented at approximately 45 degrees to the long axis of the rod in a 7-start left-handed helical arrangement. Attachment of the paraflagellar rod to the flagellar axoneme was via a series of goblet-shaped projections, which anchored the rod to a single outer doublet microtubule. Comparisons of axonemes from E. gracilis bearing the paraflagellar rod with those of Chlamydomonas reinhardtii, in which it is absent, by polyacrylamide gel electrophoresis revealed the presence of two prominent additional peptides with molecular weights of 80,000 and 69,000. Exposure of Euglena axonemes to trypsin selectively solubilized the paraflagellar rod and also removed both proteins, which were therefore tentatively identified as the major subunit proteins of the rod and designed PFR 1 and 2, respectively. In addition to these details of the structure and composition of the paraflagellar rod, the mode of attachment of the axoneme of both long and short mastigonemes was also identified. PMID:6809774

Hyams, J S

1982-06-01

270

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

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

271

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

272

Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells  

PubMed Central

Mechanisms underlying the organization of centrosome-derived microtubule arrays are well understood, but less is known about how acentrosomal microtubule networks are formed. The basal cortex of polarized epithelial cells contains a microtubule network of mixed polarity. We examined how this network is organized by imaging microtubule dynamics in acentrosomal basal cytoplasts derived from these cells. We show that the steady-state microtubule network appears to form by a combination of microtubule–microtubule and microtubule–cortex interactions, both of which increase microtubule stability. We used computational modeling to determine whether these microtubule parameters are sufficient to generate a steady-state acentrosomal microtubule network. Microtubules undergoing dynamic instability without any stabilization points continuously remodel their organization without reaching a steady-state network. However, the addition of increased microtubule stabilization at microtubule–microtubule and microtubule–cortex interactions results in the rapid assembly of a steady-state microtubule network in silico that is remarkably similar to networks formed in situ. These results define minimal parameters for the self-organization of an acentrosomal microtubule network.

Reilein, Amy; Yamada, Soichiro; Nelson, W. James

2005-01-01

273

Microtubules of the Mouse Testis Exhibit Differential Sensitivity to the Microtubule Disruptors Carbendazim and Colchicine  

Microsoft Academic Search

The testicular toxicant benomyl and its metabolite, carbendazim cause reproductive damage to the rat, an early sign of which is sloughing of germ cells with associated Sertoli cell fragments. However, the sensitivity of other mammalian species to these benzimidazole compounds is not clear. In this study, the effects of carbendazim and colchicine, a known microtubule disruptor, on the mouse seminiferous

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

2002-01-01

274

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

275

Direct Observation of Steady-state Microtubule Dynamics  

Microsoft Academic Search

Different types of unusual dynamic behav- ior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic mea- surements of bulk polymerization, we have directly visualized the steady-state addition of subunits to indi- vidual microtubules through the use of tubulin derivi- tized with biotin. Biotinylated tubulin was used both as an internal \\

David Kristofferson; Tim Mitchison; Marc Kirschner

2006-01-01

276

Terminal Warm Blood Cardioplegia Improves Cardiac Function Through Microtubule Repolymerization  

Microsoft Academic Search

Background. To elucidate the mechanisms responsible for the beneficial effects of terminal warm blood cardioplegia, we studied dynamic change in microtubules induced by cold cardioplegia followed by rewarming. Further, we investigated the relationship between cardiac function and morphologic changes in microtubules caused by hyperkalemic, hypocalcemic warm cardioplegia during initial reperfusion.Methods. In protocol 1 isolated rat hearts were perfused at 37°C

Hironori Tenpaku; Koji Onoda; Kyoko Imanaka-Yoshida; Toshimichi Yoshida; Takatsugu Shimono; Hideto Shimpo; Isao Yada

1998-01-01

277

Chromatin-mediated microtubule nucleation in Drosophila syncytial embryos  

PubMed Central

Upon entry into mitosis, many microtubules are nucleated that coordinately integrate into a stable, yet dynamic, mitotic spindle apparatus. In a recent publication, we examined microtubule-generating pathways within a single model system, the Drosophila syncytial embryo. We found that, following depolymerisation of metaphase spindle microtubules by cold treatment, spindles regenerate predominantly from microtubules nucleated within the vicinity of chromatin. We also showed this chromatin-mediated microtubule nucleation is mediated by the Drosophila homolog of a vertebrate spindle assembly factor (SAF), HURP and is dependent on the conserved microtubule amplifying protein complex, Augmin. Here, we expand our investigation into Drosophila SAFs, providing evidence that, in vitro, both D-HURP and D-TPX2 are able to bind to and stabilize microtubules. We show that GFP-D-HURP purified from embryos interacts with Importin-? and Augmin and, consistent with this, demonstrate that the underlying basis of chromatin-mediated microtubule nucleation in Drosophila syncytial embryos is dependent on Ran-GTP.

Hayward, Daniel; Wakefield, James G

2014-01-01

278

Microtubule distribution in gravitropic protonemata of the moss Ceratodon  

NASA Technical Reports Server (NTRS)

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

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

1990-01-01

279

Microtubule dynamics regulates Akt signaling via dynactin p150.  

PubMed

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

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

2014-08-01

280

XMAP215 regulates microtubule dynamics through two distinct domains  

PubMed Central

XMAP215 belongs to a family of proteins involved in the regulation of microtubule dynamics. In this study we analyze the function of different parts of XMAP215 in vivo and in Xenopus egg extracts. XMAP215 has been divided into three fragments, FrN, FrM and FrC (for N-terminal, middle and C-terminal, respectively). FrN co-localizes with microtubules in egg extracts but not in cells, FrC co- localizes with microtubules and centrosomes both in egg extracts and in cells, while FrM does not co- localize with either centrosomes or microtubules. In Xenopus egg extracts, FrN stimulates microtubule growth at plus-ends by inhibiting catastrophes, while FrM has no effect, and FrC suppresses microtubule growth by promoting catastrophes. Our results suggest that XMAP215 is targeted to centrosomes and microtubules mainly through its C-terminal domain, while the evolutionarily conserved N-terminal domain contains its microtubule-stabilizing activity.

Popov, Andrei V.; Pozniakovsky, Andrei; Arnal, Isabelle; Antony, Claude; Ashford, Anthony J.; Kinoshita, Kazuhisa; Tournebize, Regis; Hyman, Anthony A.; Karsenti, Eric

2001-01-01

281

Microtubule-Dependent Spatial Organization of Mitochondria in Fission Yeast  

Microsoft Academic Search

The microtubule cytoskeleton has an important role in the control of mitochondrial distribution in higher eukaryotes. In humans, defects in axonal mitochondrial transport are linked to neurodegenerative diseases. This chapter highlights fission yeast Schizosaccharomyces pombe as a powerful genetic model system for the study of microtubule-dependent mitochondrial movement, dynamics and inheritance.

Maitreyi Das; Stéphane Chiron; Fulvia Verde

2010-01-01

282

Self-organization of microtubules and motors.  

SciTech Connect

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

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

2006-01-01

283

The 2008 May 29 earthquake doublet in SW Iceland  

NASA Astrophysics Data System (ADS)

On 2008 May 29 an earthquake doublet shook the southwestern part of Iceland. The first main shock originated beneath Mt Ingólfsfjall, located near the western margin of the South Iceland Seismic Zone (SISZ) approximately 40 km east of the capital Reykjavík. Immediate aftershock activity was recorded by the SIL seismic network, operated by the Icelandic Meteorological Office (IMO), with both N-S and E-W structures illuminated over a broad area. A continuous GPS (CGPS) network, also operated by the IMO, recorded coseismic offsets with up to 200 mm of horizontal motion at the closest stations. We estimate the coseismic surface deformation observed by campaign and continuous GPS and satellite radar data (InSAR). We invert the geodetic data to find the optimal geometry, location and slip on the main faults, accounting for variation in the elastic parameters of the crust with depth. Our models indicate that most of the slip occurred on two N-S structures spaced ~5 km apart. From a joint inversion of GPS and InSAR data for variable slip models we find that most of the slip for the first (Ingólfsfjall) event was concentrated at 2-4 km depth with a maximum of 1.9 m, whereas the slip on the second (Kross) fault was located deeper, at 3-6 km depth with up to 1.4 m of motion. The models give similar geodetic moments for the two main events, equivalent to a moment magnitude of Mw5.8 and Mw5.9 for the first and second event, respectively. Our estimated composite moment therefore equals a Mw6.1 for the doublet, smaller than the Mw6.3 estimated from teleseismic data (e.g. NEIC and Harvard). The geodetic data support rupture on two main faults and analysis of high-rate (1Hz) CGPS data suggests that slip on the second fault initiated within 3 s of the first main shock. Static Coulomb failure stress calculations indicate that the first event caused a stress increase in the area of the main asperity (i.e. at the location of the largest slip patch) on the second fault. However, we cannot rule out dynamic stress triggering due to the short time between the two main events. The 2008 May 29 earthquake doublet appears to be a continuation of the earthquake sequence that started in 2000 June, when two Mw6.5 events struck the eastern and central part of the South Iceland Seismic Zone, in the span of 81 hr. The 2000 June-2008 May sequence has released about half of the moment accumulated by plate motion since the previous earthquake sequence in 1896-1912. Therefore, continued earthquake activity with moderate size events rupturing N-S faults in the SISZ in the coming decades is likely.

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

2010-05-01

284

Dynamics and organization of cortical microtubules as revealed by superresolution structured illumination microscopy.  

PubMed

Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for the optimal documentation of Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells, dynamic cortical microtubules labeled with green fluorescent protein fused to the microtubule-binding domain of the mammalian microtubule-associated protein MAP4 and with green fluorescent protein-fused to the alpha tubulin6 were comparatively recorded in wild-type Arabidopsis plants and in the mitogen-activated protein kinase mutant mpk4 possessing the former microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of the microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at the subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles. Time-lapse SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid, and nondynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. In conclusion, this study demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization. PMID:24686112

Komis, George; Mistrik, Martin; Samajova, Olga; Dosko?ilova, Anna; Ove?ka, Miroslav; Illés, Peter; Bartek, Jiri; Samaj, Jozef

2014-05-01

285

Microtubule-binding natural products for cancer therapy.  

PubMed

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

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

2010-08-01

286

Structural basis for microtubule binding and release by dynein  

PubMed Central

Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force-generating nucleotide hydrolysis. How this is accomplished given the ~25 nm separating dynein’s track- and nucleotide-binding sites is not understood. Here, we present a sub-nanometer-resolution structure of dynein’s microtubule-binding domain bound to microtubules by cryo-electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single molecule motility assays, which tune dynein’s affinity for microtubules. Our results provide a molecular model for how dynein’s binding to microtubules is communicated to the rest of the motor.

Zou, S.; Huang, J.; Reck-Peterson, S. L.; Leschziner, A. E.

2013-01-01

287

Structural basis for microtubule binding and release by dynein.  

PubMed

Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force-generating nucleotide hydrolysis. How this is accomplished given the ~25 nanometers separating dynein's track- and nucleotide-binding sites is not understood. Here, we present a subnanometer-resolution structure of dynein's microtubule-binding domain bound to microtubules by cryo-electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single-molecule motility assays, which tune dynein's affinity for microtubules. Our results provide a molecular model for how dynein's binding to microtubules is communicated to the rest of the motor. PMID:22997337

Redwine, William B; Hernández-López, Rogelio; Zou, Sirui; Huang, Julie; Reck-Peterson, Samara L; Leschziner, Andres E

2012-09-21

288

b-quark electric dipole moment in the general two- and three-Higgs-doublet models  

NASA Astrophysics Data System (ADS)

We study the electric dipole moment of a b-quark in the general two-Higgs-doublet model (model III) and three-Higgs-doublet model with O(2) symmetry in the Higgs sector. We analyse the dependence of this quantity on the new phase coming from the complex Yukawa couplings and masses of charged and neutral Higgs bosons. We see that the electric dipole moment of a b-quark is of the order of 10-20 e cm, which is an extremely large value compared with that calculated in the standard model and also the two-Higgs-doublet model (model II) with real Yukawa couplings.

Iltan, E. O.

2001-08-01

289

Shaping and characteristics of ohmically heated noncircular plasmas in Doublet III  

SciTech Connect

Ohmically heated dee, droplet and doublet plasmas with vertical elongations of up to 3.2 have been produced in Doublet III. Doublet configurations are now obtained by controlled merging of the two current channels of a droplet discharge. All discharges have low levels of impurities and central radiated power and achieve high density relative to B/sub T//R scaling. The energy confinement follows the standard circular cross-section Alcator scaling law. The lack of significant improvement in the confinement due to vertical elongation is consistent with the high degree of current peaking inferred from magnetic measurements.

Wesley, J.C.; Angel, T.; Armentrout, C.J.

1980-06-01

290

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

Microsoft Academic Search

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

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

1994-01-01

291

Identification of a Novel Microtubule-destabilizing Motif in CPAP That Binds to Tubulin Heterodimers and Inhibits Microtubule AssemblyD?  

PubMed Central

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

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

2004-01-01

292

Dynamic behaviors of microtubules in cytosol.  

PubMed

Highly anisotropic microtubules (MTs) immersed in cytosol are a central part of the cytoskeleton in eukaryotic cells. The dynamic behaviors of an MT-cytosol system are of major interest in biomechanics community. Such a solid-fluid system is characterized by a Reynolds number of the order 10(-3) and a slip ionic layer formed at the MT-cytosol interface. In view of these unique features, an orthotropic shell-Stokes flow model with a slip boundary condition has been developed to explore the distinctive dynamic behaviors of MTs in cytosol. Three types of motions have been identified, i.e., (a) undamped and damped torsional vibration, (b) damped longitudinal vibration, and (c) overdamped bending and radial motions. The exponentially decaying bending motion given by the present model is found to be in qualitative agreement with the existing experimental observation [Felgner et al., 1996. Flexural rigidity of microtubules measured with the use of optical tweezers, Journal of Cell Science 109, 509-516 ]. PMID:19394941

Wang, C Y; Li, C F; Adhikari, S

2009-06-19

293

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

PubMed Central

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

Field, Jessica J.; Pera, Benet; Calvo, Enrique; Canales, Angeles; Zurwerra, Didier; Trigili, Chiara; Rodriguez-Salarichs, Javier; Matesanz, Ruth; Kanakkanthara, Arun; Wakefield, St. John; Singh, A. Jonathan; Jimenez-Barbero, Jesus; Northcote, Peter; Miller, John H.; Lopez, Juan Antonio; Hamel, Ernest; Barasoain, Isabel; Altmann, Karl-Heinz; Diaz, Jose Fernando

2012-01-01

294

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

PubMed

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

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

2013-01-01

295

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

Microsoft Academic Search

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

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

2005-01-01

296

Neuronal Abnormalities in Microtubule-Associated Protein 1B Mutant Mice  

Microsoft Academic Search

Microtubules play an important role in establishing cellular architecture. Neuronal microtubules are considered to have a role in dendrite and axon formation. Different portions of the developing and adult brain microtubules are associated with different microtubule-associated proteins (MAPs). The roles of each of the different MAPs are not well understood. One of these proteins, MAP1B, is expressed in different portions

Winfried Edelmann; Mark Zervas; Pamela Costello; Linda Roback; Itzhak Fischer; James A. Hammarback; Nicholas Cowan; Peter Davies; Bruce Wainer; Raju Kucherlapati

1996-01-01

297

Strange quark contribution to the neutron electric dipole moment in multi-Higgs doublet models.  

National Technical Information Service (NTIS)

The strange quark contribution to the neutron electric dipole moment was studied and compared with other contributions in multi-Higgs doublet models. It was found that the strange quark contribution is significant because the strange quark color dipole mo...

X. G. He H. J. McKeller S. Pakvasa

1990-01-01

298

Instrumentation and Control of the Doublet III Neutral Beam Injector System.  

National Technical Information Service (NTIS)

The hardware and software required for the operation of the Doublet III Neutral Beam Injector System (NBIS) are described. Development and implementation of this Instrumentation and Control System was divided between the major participants - General Atomi...

J. C. Kohli C. D. Moore D. D. Drobnis V. P. Elischer R. Kilgore

1980-01-01

299

Poleward microtubule flux mitotic spindles assembled in vitro  

PubMed Central

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

1991-01-01

300

Kinetics of microtubule catastrophe assessed by probabilistic analysis.  

PubMed Central

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

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

1995-01-01

301

Direct observation of steady-state microtubule dynamics  

PubMed Central

Different types of unusual dynamic behavior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic measurements of bulk polymerization, we have directly visualized the steady-state addition of subunits to individual microtubules through the use of tubulin derivitized with biotin. Biotinylated tubulin was used both as an internal "seed" for polymerization and as a marker for assembly onto the ends of microtubules composed of purified tubulin. Biotinylated segments were distinguished from unmodified tubulin by double-label immunofluorescence. Microtubule lengths, number concentrations, and segment lengths have been monitored with time at steady state under two buffer conditions. The results indicate that the microtubule steady state under these conditions is a balance between a majority of slowly growing microtubules and a minority of rapidly depolymerizing ones as described by the "dynamic instability" model (Mitchison T., and M. Kirschner, 1984, Nature (Lond.)., 312:232-242). Microtubules show no evidence of treadmilling; instead most show progressive growth off both ends at steady state. Although solvent conditions markedly influence the growth rates, qualitatively the behavior is unchanged.

1986-01-01

302

Microtubules in the Cerebral Cortex: Role in Memory and Consciousness  

NASA Astrophysics Data System (ADS)

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

Woolf, Nancy J.

303

Model of ionic currents through microtubule nanopores and the lumen.  

PubMed

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

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

2010-05-01

304

Modulation of Microtubule Interprotofilament Interactions by Modified Taxanes  

PubMed Central

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

Matesanz, Ruth; Rodriguez-Salarichs, Javier; Pera, Benet; Canales, Angeles; Andreu, Jose Manuel; Jimenez-Barbero, Jesus; Bras, Wim; Nogales, Aurora; Fang, Wei-Shuo; Diaz, Jose Fernando

2011-01-01

305

Disruption of microtubule network by Alzheimer abnormally hyperphosphorylated tau  

PubMed Central

Hyperphosphorylated tau has long been proposed as the key molecule disrupting normal neuronal microtubule dynamics and leading to neurofibrillary degeneration in Alzheimer disease. Here we provide a direct evidence of hyperphosphorylated tau-induced disruption of microtubule network. Using Nocodozole-treated and detergent-extracted cells, we created a neuronal environment in mouse embryonic fibroblasts, 3T3 cells, by replacing their cytoplasm with adult rat brain cytosol. By recreating neuronal microtubule network in these cells, we were able to follow the effects of hyperphosphorylated tau on microtubule dynamics in real time. Whereas recombinant human brain tau promoted assembly and bundling of microtubules, abnormally hyperphosphorylated tau isolated from Alzheimer disease brain cytosol (AD P-tau) inhibited the assembly and disrupted preformed microtubule network by sequestering normal brain tau and MAP2. This breakdown of the microtubule network was reversed by treatment of the extracted cells with protein phosphatase-2A. This study, for the first time, provides direct mechanistic insights into the molecular basis of both axonal and dendritic neurodegeneration seen in Alzheimer disease.

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

2011-01-01

306

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

PubMed Central

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

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

2012-01-01

307

Soft SUSY breaking masses in a unified model with doublet-triplet splitting  

Microsoft Academic Search

We study soft supersymmetry breaking parameters in a supersymmetric unified\\u000amodel which potentially solves the doublet-triplet splitting problem. In the\\u000amodel the doublet-triplet splitting is solved by the discrete symmetry which is\\u000aallowed to be introduced due to the direct product structure of the gauge\\u000agroup. The messenger fields for the gauge mediated supersymmetry breaking are\\u000anaturally embedded in the

Daijiro Suematsu

2002-01-01

308

Renormalization of the neutrino mass operators in the multi-Higgs-doublet standard model  

Microsoft Academic Search

We derive the renormalization group equations (RGE) for the flavor coupling matrices of the effective dimension-five operators which yield Majorana neutrino masses in the multi-Higgs-doublet standard model; in particular, we consider the case where two different scalar doublets occur in those operators. We also write down the RGE for the scalar-potential quartic couplings and for the Yukawa couplings of that

Walter Grimus; L. Lavoura

2005-01-01

309

Mass relations in the two Higgs doublet model from the absence of quadratic divergences  

Microsoft Academic Search

By setting the quadratic divergences to zero, four mass relations are obtained for the Standard Model generalized to two Higgs doublets. These four mass relations are obtained most simply in terms of the original fields in the Lagrangian, before spontaneousSU(2)×U(1) symmetry breaking is applied. Unlike the case of the Standard Model, the Higgs tadpoles of the two Higgs doublet theory

Conrad Newton; Tai Tsun Wu

1994-01-01

310

The Yukawa couplings in the Two Higgs Doublet Model and the Froggart-Nielsen mechanism  

SciTech Connect

Mixing of the TeV-scals flavon fields with the electroweak Higgs bosons of the Two Higgs Doublet Model, could induce large Flavor Changing Neutral Curretns (FCNC), interactions which can be tested both at low and high energy experiments. We construct a model of this kind, that involves the Froggart-Nielsen mechanism that TeV-scals flavon field, which mixes with a Two Higgs doublet model and explore its phonomenology.

Noriega-Papaqui, R. [Centro de Investigacion en Matematicas, Universidad Autonoma del Estado de Hidalgo, Carr. Pachuca-Tulancingo Km. 4.5, C.P. 42184, Pachuca, Hgo. (Mexico) and Dual C-P Institute of High Energy Physics (Mexico)

2009-04-20

311

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

PubMed Central

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

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

2009-01-01

312

Centrosome and microtubule instability in aging Drosophila cells  

NASA Technical Reports Server (NTRS)

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

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

1999-01-01

313

Microtubule-Driven Multimerization Recruits ase1p onto Overlapping Microtubules  

Microsoft Academic Search

Microtubule (MT) crosslinking proteins of the ase1p\\/PRC1\\/Map65 family play a major role in the construction of MT networks such as the mitotic spindle. Most homologs in this family have been shown to localize with a remarkable specificity to sets of MTs that overlap with an antiparallel relative orientation []. Regulatory proteins bind to ase1p\\/PRC1\\/Map65 and appear to use the localization

Lukas C. Kapitein; Marcel E. Janson; Siet M. J. L. van den Wildenberg; Casper C. Hoogenraad; Christoph F. Schmidt; Erwin J. G. Peterman

2008-01-01

314

Self-organization of microtubule bundles in anucleate fission yeast cells  

Microsoft Academic Search

Self-organization of cellular structures is an emerging principle underlying cellular architecture. Properties of dynamic microtubules and microtubule-binding proteins contribute to the self-assembly of structures such as microtubule asters. In the fission yeast Schizosaccharomyces pombe, longitudinal arrays of cytoplasmic microtubule bundles regulate cell polarity and nuclear positioning. These bundles are thought to be organized from the nucleus at multiple interphase microtubule

Rafael R. Daga; Kyeng-Gea Lee; Scott Bratman; Silvia Salas-Pino; Fred Chang

2006-01-01

315

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

PubMed

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

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

2010-05-15

316

Purified native microtubule associated protein MAP1A: kinetics of microtubule assembly and MAP1A/tubulin stoichiometry.  

PubMed

In a recent study, we have shown that sulfonate buffers affect microtubule assembly and alter microtubule protein composition (Pedrotti et al., 1993). In particular, we noted that PIPES buffer leads to removal of MAP1 from the microtubule surface without affecting the association of MAP2 with microtubules. This observation has been exploited to develop a simple purification procedure for MAP1A using twice-cycled microtubule protein prepared from whole bovine brain. A single chromatographic step on an ion-exchange column results in > 90% pure MAP1A. Using purified MAP1A, we now show that MAP1A (a) binds in a dose-dependent manner to unpolymerized tubulin and assembled microtubules, (b) binds 13-15 mol of tubulin dimers in assembled microtubules, (c) promotes both nucleation and elongation of tubulin, and (d) promotes incorporation of tubulin dimers at low GTP concentrations and of tubulin dimers and oligomers at high GTP concentrations. MAP1A lowers the critical concentration for assembly, and MAP1A-promoted incorporation of dimers has an association rate constant (K+1) of 39.3 x 10(6) M-1s-1 and a dissociation rate constant (K-1) of 15 s-1; both constants are about 2-3-fold higher compared with MAP2. PMID:7918469

Pedrotti, B; Islam, K

1994-10-18

317

Crowding of molecular motors determines microtubule depolymerization.  

PubMed

The assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT-associated proteins. Here, we investigate how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. Using an individual-based model, we reproduce experimental findings. Moreover, crowding is identified as the key regulatory mechanism of depolymerization dynamics. Our analysis reveals two qualitatively distinct regimes. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise that cancel out the effect of the depolymerization kinetics such that the depolymerization speed is solely determined by the motor density. Because this density changes over the MT length, length-dependent regulation is possible. Remarkably, motor cooperativity affects only the end-residence time of depolymerases and not the depolymerization speed. PMID:22067158

Reese, Louis; Melbinger, Anna; Frey, Erwin

2011-11-01

318

Quantification of asymmetric microtubule nucleation at sub-cellular structures  

PubMed Central

Cell polarization is important for multiple physiological processes. In polarized cells, microtubules (MTs) are organized into a spatially polarized array. Generally, in non-differentiated cells, it is assumed that MTs are symmetrically nucleated exclusively from centrosome (microtubule organizing center, MTOC) and then reorganized into the asymmetric array. We have recently identified the Golgi complex as an additional MTOC that asymmetrically nucleates MTs toward one side of the cell. Methods used for alternative MTOC identification include microtubule re-growth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescence labeled MT +TIP binding proteins in living cells. These approaches can be used for quantification of MT nucleation sites at diverse sub-cellular structures.

Zhu, Xiaodong; Kaverina, Irina

2012-01-01

319

Kinetochores and disease: keeping microtubule dynamics in check!  

PubMed Central

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

Bakhoum, Samuel F.; Compton, Duane A.

2012-01-01

320

Mechanical Models of Microtubule Bundle Collapse in Alzheimer's Disease  

NASA Astrophysics Data System (ADS)

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

Sendek, Austin; Singh, Rajiv; Cox, Daniel

2013-03-01

321

KIF1A Alternately Uses Two Loops to Bind Microtubules  

NASA Astrophysics Data System (ADS)

The motor protein kinesin moves along microtubules, driven by adenosine triphosphate (ATP) hydrolysis. However, it remains unclear how kinesin converts the chemical energy into mechanical movement. We report crystal structures of monomeric kinesin KIF1A with three transition-state analogs: adenylyl imidodiphosphate (AMP-PNP), adenosine diphosphate (ADP)-vanadate, and ADP-AlFx (aluminofluoride complexes). These structures, together with known structures of the ADP-bound state and the adenylyl-(?,?-methylene) diphosphate (AMP-PCP)-bound state, show that kinesin uses two microtubule-binding loops in an alternating manner to change its interaction with microtubules during the ATP hydrolysis cycle; loop L11 is extended in the AMP-PNP structure, whereas loop L12 is extended in the ADP structure. ADP-vanadate displays an intermediate structure in which a conformational change in two switch regions causes both loops to be raised from the microtubule, thus actively detaching kinesin.

Nitta, Ryo; Kikkawa, Masahide; Okada, Yasushi; Hirokawa, Nobutaka

2004-07-01

322

A theory of microtubule catastrophes and their regulation.  

PubMed

Dynamic instability, in which abrupt transitions occur between growing and shrinking states, is an intrinsic property of microtubules that is regulated by both mechanics and specialized proteins. We discuss a model of dynamic instability based on the popular idea that growth is maintained by a cap at the tip of the fiber. The loss of this cap is thought to trigger the transition from growth to shrinkage, called a catastrophe. The model includes longitudinal interactions between the terminal tubulins of each protofilament and "gating rescues" between neighboring protofilaments. These interactions allow individual protofilaments to transiently shorten during a phase of overall microtubule growth. The model reproduces the reported dependency of the catastrophe rate on tubulin concentration, the time between tubulin dilution and catastrophe, and the induction of microtubule catastrophes by walking depolymerases. The model also reproduces the comet tail distribution that is characteristic of proteins that bind to the tips of growing microtubules. PMID:19948965

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

2009-12-15

323

Microfilaments and microtubules in elongating parenchyma cells of Nymphoides indica  

Microsoft Academic Search

Observations on microfilaments and microtubules in elongating parenchyma cells of the central vascular bundle of Nymphoides indica (L.) O. Kuntze petiole are reported and discussed in relation to current concepts of the involvement of cellular organelles in cell wall synthesis.

A. Freundlich

1974-01-01

324

Motion observation and SPR measurements of kinesin motility on microtubules  

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

325

Cortical microtubules in sweet clover columella cells developed in microgravity  

NASA Technical Reports Server (NTRS)

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

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

1995-01-01

326

Insights from an erroneous kinetochore-microtubule attachment state  

PubMed Central

Faithful distribution of the genome requires that sister kinetochores, which assemble on each chromatid during cell division, interact with dynamic microtubules from opposite spindle poles in a configuration called chromosome biorientation. Biorientation produces tension that increases the affinity of kinetochores for microtubules via ill-defined mechanisms. Non-bioriented kinetochore-microtubule (kt-MT) interactions are prevalent but short-lived due to an error correction pathway that reduces the affinity of kinetochores for microtubules. Interestingly, incorrect kt-MT interactions can be stabilized by experimentally applying force to misoriented chromosomes. Here, a live-cell force assay is utilized to characterize the molecular composition of a common type of improper kt-MT attachment. Our force-related studies are also discussed in the context of current models for tension-dependent stabilization of kt-MT interactions.

Cane, Stuart; McGilvray, Philip T.; Maresca, Thomas J.

2013-01-01

327

Intact Microtubules Support Adenovirus and Herpes Simplex Virus Infections  

Microsoft Academic Search

Capsids and the enclosed DNA of adenoviruses, including the species C viruses adenovirus type 2 (Ad2) and Ad5, and herpesviruses, such as herpes simplex virus type 1 (HSV-1), are targeted to the nuclei of epithelial, endothelial, fibroblastic, and neuronal cells. Cytoplasmic transport of fluorophore-tagged Ad2 and immuno- logically detected HSV-1 capsids required intact microtubules and the microtubule-dependent minus-end- directed motor

Hélène Mabit; M. Y. Nakano; U. Prank; B. Saam; K. Dohner; Beate Sodeik; Urs F. Greber

2002-01-01

328

It's a kar9ochore to capture microtubules.  

PubMed

Microtubule orientation to cortical spatial cues is essential for the fidelity of asymmetric cellular processes. A cortical microtubule-capture site, composed of Bim1 and Kar9, has now been identified in yeast. Bim1 is the yeast homologue of EB1, a binding partner of the adenomatous polyposis coli (APC), indicating that important features of this complex may be highly conserved. PMID:10854334

Bloom, K

2000-06-01

329

The MAP2\\/Tau family of microtubule-associated proteins  

Microsoft Academic Search

SUMMARY: Microtubule-associated proteins (MAPs) of the MAP2\\/Tau family include the vertebrate proteins MAP2, MAP4, and Tau and homologs in other animals. All three vertebrate members of the family have alternative splice forms; all isoforms share a conserved carboxy-terminal domain containing microtubule-binding repeats, and an amino-terminal projection domain of varying size. MAP2 and Tau are found in neurons, whereas MAP4 is

Leif Dehmelt; Shelley Halpain

2004-01-01

330

The MAP1 family of microtubule-associated proteins  

Microsoft Academic Search

SUMMARY: MAP1-family proteins are classical microtubule-associated proteins (MAPs) that bind along the microtubule lattice. The founding members, MAP1A and MAP1B, are predominantly expressed in neurons, where they are thought to be important in the formation and development of axons and dendrites. Mammalian genomes usually contain three family members, MAP1A, MAP1B and a shorter, more recently identified gene called MAP1S. By

Shelley Halpain; Leif Dehmelt

2006-01-01

331

Structure of cortical microtubule arrays in plant cells  

Microsoft Academic Search

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

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

1978-01-01

332

Microtubules and control of macronuclear 'amitosis' in Paramecium.  

PubMed

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

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

1980-08-01

333

Transcriptional Regulation of an Axonemal Central Apparatus Gene, Sperm-associated Antigen 6, by a SRY-related High Mobility Group Transcription Factor, S-SOX5*  

PubMed Central

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

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

2010-01-01

334

Mammalian end binding proteins control persistent microtubule growth  

PubMed Central

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

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

2009-01-01

335

Elastic Response, Buckling, and Instability of Microtubules under Radial Indentation  

PubMed Central

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

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

2006-01-01

336

Engineering tubulin: microtubule functionalization approaches for nanoscale device applications  

PubMed Central

With the emergences of engineered devices at microscale and nanoscale dimensions, there is a growing need for controlled actuation and transport at these length scales. The kinesin–microtubule system provides a highly evolved biological transport system well suited for these tasks. Accordingly, there is an ongoing effort to create hybrid nanodevices that integrate biological components with engineered materials for applications such as biological separations, nanoscale assembly, and sensing. Adopting microtubules for these applications generally requires covalent attachment of biotin, fluorophores, or other biomolecules to tubulin enable surface or cargo attachment, or visualization. This review summarizes different strategies for functionalizing microtubules for application-focused as well as basic biological research. These functionalization strategies must maintain the integrity of microtubule proteins so that they do not depolymerize and can be transported by kinesin motors, while adding utility such as the ability to reversibly bind cargo. The relevant biochemical and electrical properties of microtubules are discussed, as well as strategies for microtubule stabilization and long-term storage. Next, attachment strategies, such as antibodies and DNA hybridization that have proven useful to date, are discussed in the context of ongoing hybrid nanodevice research. The review concludes with a discussion of less explored opportunities, such as harnessing the utility of tubulin posttranslational modifications and the use of recombinant tubulin that may enable future progress in nanodevice development.

Malcos, Jennelle L.

2011-01-01

337

Computational Modeling of Axonal Microtubule Bundles under Tension  

PubMed Central

Microtubule bundles cross-linked by tau protein serve a variety of neurological functions including maintaining mechanical integrity of the axon, promoting axonal growth, and facilitating cargo transport. It has been observed that axonal damage in traumatic brain injury leads to bundle disorientation, loss of axonal viability, and cognitive impairment. This study investigates the initial mechanical response of axonal microtubule bundles under uniaxial tension using a discrete bead-spring representation. Mechanisms of failure due to traumatic stretch loading and their impact on the mechanical response and stability are also characterized. This study indicates that cross-linked axonal microtubule bundles in tension display stiffening behavior similar to a power-law relationship from nonaffine network deformations. Stretching of cross-links and microtubule bending were the primary deformation modes at low stresses. Microtubule stretch was negligible up to tensile stresses of ?1 MPa. Bundle failure occurred by failure of cross-links leading to pull-out of microtubules and loss of bundle integrity. This may explain the elongation, undulation, and delayed elasticity of axons following traumatic stretch loading. More extensively cross-linked bundles withstood higher tensile stresses before failing. The bundle mechanical behavior uncovered by these computational techniques should guide future experiments on stretch-injured axons.

Peter, Stephen J.; Mofrad, Mohammad R.K.

2012-01-01

338

Properties of microtubule bundles induced by Glyceraldehyde-3-phosphate dehydrogenase  

NASA Astrophysics Data System (ADS)

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 studied by the addition of 1 mM adenosine 5'-(?, -imidotri-phosphate) (AMPPNP) to bundled microtubules, and is extremely fast. Bundling reduces the rate of association of tubulin dimers to microtubules, as well as the dissocation from the microtubles. Both rates are reduced to the same extent. This is in agreement with the fact that the critical concentration of tubulin is practically not influenced by the binding of the enzyme. Adding microtubule associated proteins (at I=0.1 M) does not appreciably influence the affinity for GAPDH, but reduces bundle formation possibly for sterical reasons.

Somers, Marijke; Engelborghs, Yves

1991-05-01

339

Dimer model for Tau proteins bound in microtubule bundles  

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

340

A functional link between localized Oskar, dynamic microtubules, and endocytosis.  

PubMed

Many cell types including developing oocytes, fibroblasts, epithelia and neurons use mRNA localization as a means to establish polarity. The Drosophila oocyte has served as a useful model in dissecting the mechanism of mRNA localization. The polarity of the oocyte is established by the specific localization of three critical mRNAs-oskar, bicoid and gurken. The localization of these mRNAs requires microtubule integrity, and the activity of microtubule motors. However, the precise organization of the oocyte microtubule cytoskeleton remains an open question. In order to examine the polarity of oocyte microtubules, we visualized the localization of canonical microtubule plus end binding proteins, EB1 and CLIP-190. Both proteins were enriched at the posterior of the oocyte, with additional foci detected within the oocyte cytoplasm and along the cortex. Surprisingly, however, we found that this asymmetric distribution of EB1 and CLIP-190 was not essential for oskar mRNA localization. However, Oskar protein was required for recruiting the plus end binding proteins to the oocyte posterior. Lastly, our results suggest that the enrichment of growing microtubules at the posterior pole functions to promote high levels of endocytosis in this region of the cell. Thus, multiple polarity-determining pathways are functionally linked in the Drosophila oocytes. PMID:22561189

Sanghavi, Paulomi; Lu, Sumin; Gonsalvez, Graydon B

2012-07-01

341

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

PubMed

Wave propagation along the microtubules is one of the issues of major concern in various microtubule cellular functions. In this study, the general wave propagation behavior in protein microtubules is investigated based on a first-order shear deformation shell theory for orthotropic materials, with particular emphasis on the role of strongly anisotropic elastic properties of microtubules. According to experimental observation, the first-order shear deformation theory is used for the modeling of microtubule walls. A general displacement representation is introduced and a type of coupled polynomial eigenvalue problem is developed. Numerical examples describe the effects of shear deformation and rotary inertia on wave velocities in orthotropic microtubules. Finally, the influences of the microtubule shear modulus, axial external force, effective thickness and material temperature dependency on wave velocities along the microtubule protofilaments, helical pathway and radial directions are elucidated. Most results presented in the present investigation have been absent from the literature for the wave propagation in microtubules. PMID:21632054

Daneshmand, Farhang; Ghavanloo, Esmaeal; Amabili, Marco

2011-07-01

342

EBs Recognize a Nucleotide-Dependent Structural Cap at Growing Microtubule Ends  

PubMed Central

Summary Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.

Maurer, Sebastian P.; Fourniol, Franck J.; Bohner, Gergo; Moores, Carolyn A.; Surrey, Thomas

2012-01-01

343

Structural basis for microtubule recognition by the human kinetochore Ska complex  

PubMed Central

The ability of kinetochores (KTs) to maintain stable attachments to dynamic microtubule structures (‘straight’ during microtubule polymerization and ‘curved’ during microtubule depolymerization) is an essential requirement for accurate chromosome segregation. Here we show that the kinetochore-associated Ska complex interacts with tubulin monomers via the carboxy-terminal winged-helix domain of Ska1, providing the structural basis for the ability to bind both straight and curved microtubule structures. This contrasts with the Ndc80 complex, which binds straight microtubules by recognizing the dimeric interface of tubulin. The Ska1 microtubule-binding domain interacts with tubulins using multiple contact sites that allow the Ska complex to bind microtubules in multiple modes. Disrupting either the flexibility or the tubulin contact sites of the Ska1 microtubule-binding domain perturbs normal mitotic progression, explaining the critical role of the Ska complex in maintaining a firm grip on dynamic microtubules.

Abad, Maria Alba; Medina, Bethan; Santamaria, Anna; Zou, Juan; Plasberg-Hill, Carla; Madhumalar, Arumugam; Jayachandran, Uma; Redli, Patrick Marc; Rappsilber, Juri; Nigg, Erich A.; Jeyaprakash, A. Arockia

2014-01-01

344

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

PubMed

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

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

2014-02-01

345

The relative effect of citral on mitotic microtubules in wheat roots and BY2 cells.  

PubMed

The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with ?-tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral's effect on microtubules was both dose- and time-dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of ?-tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP-Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral's effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules. PMID:22039835

Chaimovitsh, D; Rogovoy Stelmakh, O; Altshuler, O; Belausov, E; Abu-Abied, M; Rubin, B; Sadot, E; Dudai, N

2012-03-01

346

Microtubule bundle formation driven by ATP: the effect of concentrations of kinesin, streptavidin and microtubules  

NASA Astrophysics Data System (ADS)

Recently, a method was established for the formation of microtubule (MT) assemblies by an active self-organization (AcSO) process, in which MTs were crosslinked during sliding motion on a kinesin-coated surface, and this was coupled with adenosine triphosphate (ATP) hydrolysis. Streptavidin (ST) was the glue used to crosslink biotin-labeled MTs. Although most of the MT assemblies were in the bundle form, they varied in size, shape and motility, depending on the initial conditions used. In this paper, we systematically examined the effects of the concentrations of kinesin, ST and MT on the formation of MT bundles under the initial conditions of the process.

Kawamura, Ryuzo; Kakugo, Akira; Osada, Yoshihito; Gong, Jian Ping

2010-04-01

347

Crowding of molecular motors determines microtubule depolymerization  

NASA Astrophysics Data System (ADS)

Assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT associated proteins. Here, it is investigated how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. We reproduce experimental findings within the framework of a totally asymmetric simple exclusion process with Langmuir kinetics (TASEP/LK). Thereby, crowding is identified as the key regulatory mechanism of depolymerization dynamics. The analysis gives two qualitatively distinct phases. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise which cancel out the effect of the depolymerization kinetics such that the depolymerization speed is determined by the motor density. Because this density varies over the MT length, length-dependent regulation is possible. The critical length at which MTs start to depolymerize in a length-dependent way is discussed. Reference: Louis Reese, Anna Melbinger and Erwin Frey. Biophys. J. 101, 2190 (2011)

Reese, Louis; Melbinger, Anna; Frey, Erwin

2012-02-01

348

Microtubule-templated biomimetic mineralization of lepidocrocite.  

SciTech Connect

Protein microtubules (MTs) 25 nm in diameter and tens of micrometers long have been used as templates for the biomimetic mineralization of FeOOH. Exposure of MTs to anaerobic aqueous solutions of Fe{sup 2+} buffered to neutral pH followed by aerial oxidation leads to the formation of iron oxide coated MTs. The iron oxide layer was found to grow via a two-step process: initially formed 10-30 nm thick coatings were found to be amorphous in structure and comprised of several iron-containing species. Further growth resulted in MTs coated with highly crystalline layers of lepidocrocite with a controllable thickness of up to 125 nm. On the micrometer size scale, these coated MTs were observed to form large, irregular bundles containing hundreds of individually coated MTs. Iron oxide grew selectively on the MT surface, a result of the highly charged MT surface that provided an interface favorable for iron oxide nucleation. This result illustrates that MTs can be used as scaffolds for the in-situ production of high-aspect-ratio inorganic nanowires.

Bunker, Bruce Conrad; Headley, Thomas Jeffrey; Tissot, Ralph George, Jr.; Boal, Andrew Kiskadden

2003-08-01

349

MAP65/Ase1 promote microtubule flexibility.  

PubMed

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

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

2013-06-01

350

Microtubule assembly and disassembly at alkaline pH  

PubMed Central

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

Regula, CS; Pfeiffer; Berlin, RD

1981-01-01

351

Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters.  

PubMed

Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and Ptdlns3K-dependent formation of LC3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A 1, but not nutrient deprivation or rapamycin treatment, further increased LC3-II generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with SQSTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. Efficient autophagy by hypertonic stress required microtubule remodeling and was DYNC/dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (EHNA) or genetic [DCTN2/dynactin 2 (p50) overexpression] interference of DYNC activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response. PMID:23380587

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

2013-04-01

352

Role of microtubules in the contractile dysfunction of hypertrophied myocardium  

NASA Technical Reports Server (NTRS)

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

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

1999-01-01

353

Microtubule depolymerization promotes particle and chromosome movement in vitro  

PubMed Central

We have developed a system for studying the motions of cellular objects attached to depolymerizing microtubules in vitro. Radial arrays of microtubules were grown from lysed and extracted Tetrahymena cells attached to a glass coverslip that formed the top of a light microscope perfusion chamber. A preparation of chromosomes, which also contained vesicles, was then perfused into the chamber and allowed to bind to the microtubule array. The concentration of tubulin was then reduced by perfusing buffer that lacked both tubulin and nucleotide triphosphates, and the resulting microtubule depolymerization was observed by light microscopy. A fraction of the bound objects detached in the flow and washed away, while others stabilized the microtubules to which they were bound. Some of the particles and chromosomes, however, moved in toward the Tetrahymena ghost as their associated microtubules shortened. The mean speeds for particles and chromosomes were 26 +/- 20 and 15 +/- 12 microns/min, respectively. These motions occurred when nucleotide triphosphate levels were very low, as a result of either dilution or by the action of apyrase. Furthermore, the motions were unaffected by 100 microM sodium orthovanadate, suggesting that these forces are not the result of ATP hydrolysis by a minus end-directed mechanoenzyme. We conclude that microtubule depolymerization provided the free energy for the motions observed. All the objects that we studied in detail moved against a stream of buffer flowing at approximately 100 microns/s, so that the force being developed was at least 10(-7) dynes. This force is large enough to contribute to some forms of motility in living cells.

1991-01-01

354

Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters  

PubMed Central

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

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

2013-01-01

355

Quantum Spin Ices and Topological Phases from Dipolar-Octupolar Doublets on the Pyrochlore Lattice  

NASA Astrophysics Data System (ADS)

We consider a class of d- and f-electron systems in which dipolar-octupolar Kramers doublets arise on the sites of the pyrochlore lattice. For such doublets, two components of the pseudospin transform like a magnetic dipole, while the other transforms like a component of the magnetic octupole tensor. Based on a symmetry analysis, we construct and study models of dipolar-octupolar doublets in itinerant and localized limits. In both limits, the resulting models are of surprisingly simple form. In the itinerant limit, we find topological insulating behavior. In the localized limit, the most general nearest-neighbor spin model is the XYZ model. We show that this XYZ model exhibits two distinct quantum spin ice (QSI) phases, that we dub dipolar QSI, and octupolar QSI. We conclude with a discussion of potential relevance to real material systems.

Huang, Yi-Ping; Chen, Gang; Hermele, Michael

2014-04-01

356

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

SciTech Connect

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

Juarez W, S. R.; Morales C, D. [Departamento de Fisica, Escuela Superior de Fisica y Matematicas, Instituto Politecnico Nacional, Unidad Profesional 'Adolfo Lopez Mateos' C.P. 07738, Mexico, D.F. (Mexico)

2008-07-02

357

Quantum spin ices and topological phases from dipolar-octupolar doublets on the pyrochlore lattice.  

PubMed

We consider a class of d- and f-electron systems in which dipolar-octupolar Kramers doublets arise on the sites of the pyrochlore lattice. For such doublets, two components of the pseudospin transform like a magnetic dipole, while the other transforms like a component of the magnetic octupole tensor. Based on a symmetry analysis, we construct and study models of dipolar-octupolar doublets in itinerant and localized limits. In both limits, the resulting models are of surprisingly simple form. In the itinerant limit, we find topological insulating behavior. In the localized limit, the most general nearest-neighbor spin model is the XYZ model. We show that this XYZ model exhibits two distinct quantum spin ice (QSI) phases, that we dub dipolar QSI, and octupolar QSI. We conclude with a discussion of potential relevance to real material systems. PMID:24815666

Huang, Yi-Ping; Chen, Gang; Hermele, Michael

2014-04-25

358

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

PubMed Central

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

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

2013-01-01

359

Single Molecule Imaging Reveals Differences in Microtubule Track Selection Between Kinesin Motors  

PubMed Central

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

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

2009-01-01

360

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

PubMed Central

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

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

2013-01-01

361

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

PubMed Central

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

Barr, Alexis R.; Gergely, Fanni

2008-01-01

362

Scalable UWB photonic generator based on the combination of doublet pulses.  

PubMed

We propose and experimentally demonstrate a scalable and reconfigurable optical scheme to generate high order UWB pulses. Firstly, various ultra wideband doublets are created through a process of phase-to-intensity conversion by means of a phase modulation and a dispersive media. In a second stage, doublets are combined in an optical processing unit that allows the reconfiguration of UWB high order pulses. Experimental results both in time and frequency domains are presented showing good performance related to the fractional bandwidth and spectral efficiency parameters. PMID:24977794

Moreno, Vanessa; Rius, Manuel; Mora, José; Muriel, Miguel A; Capmany, José

2014-06-30

363

Top quark electric and chromoelectric dipole moments in the general two Higgs doublet model  

NASA Astrophysics Data System (ADS)

We study the electric and chromoelectric dipole moments of the top quark in the general two Higgs doublet model (model III). We analyze the dependency of this quantity on the new phases coming from the complex Yukawa couplings and masses of charged and neutral Higgs bosons. We observe that the electric and chromoelecric dipole moments of the top quark are of the order of 10-21 e cm and 10-20 gs cm, which are extremely large values compared to the ones calculated in the standard model and also in the two Higgs doublet model with real Yukawa couplings.

Iltan, E. O.

2002-04-01

364

Dispersion sensitivity of large-scale axial gradient index glass for spherochromat doublets  

NASA Astrophysics Data System (ADS)

Bi-AGRIN cemented doublets, super corrected for zero axial color, spherical aberration and sphero-chromatism can show polychromatic performance in the range of 0.004 waves PV or better at the red and blue wavelengths for speeds up to F/2. These doublets are comprised of two elements of axial gradient index glass. The crown and flint elements are each designed with separate and distinct gradient glass lines, giving each element an axial gradient in refractive index and dispersion. This paper examines one design and its performance sensitivity to dispersion modeling via the Buchdahl and Sellmeier dispersion equations.

Manhart, Paul K.; Hunter, Boyd V.; Blankenbecler, Richard

1999-10-01

365

Neutral Higgs production on LHC in the two-Higgs-doublet model with spontaneous CP violation  

SciTech Connect

Spontaneous CP violation motivates the introduction of two Higgs doublets in the electroweak theory, such a simple extension of the standard model has five physical Higgs bosons and rich CP-violating sources. Exploration on more than one Higgs boson is a direct evidence for new physics beyond the standard model. The neutral Higgs production at LHC is investigated in such a general two-Higgs-doublet model with spontaneous CP violation, it is shown that the production cross section and decays of the neutral Higgs boson can significantly be different from the predictions from the standard model.

Bao Shoushan; Wu Yueliang [Kavli Institute for Theoretical Physics China (KITPC), Key Laboratory of Frontiers in Theoretical Physics, Institute of Theoretical Physics, Chinese Academy of Science, Beijing, 100190 (China)

2010-04-01

366

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

Microsoft Academic Search

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

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

1997-01-01

367

Molecular crowding creates traffic jams of kinesin motors on microtubules  

PubMed Central

Despite the crowdedness of the interior of cells, microtubule-based motor proteins are able to deliver cargoes rapidly and reliably throughout the cytoplasm. We hypothesize that motor proteins may be adapted to operate in crowded environments by having molecular properties that prevent them from forming traffic jams. To test this hypothesis, we reconstituted high-density traffic of purified kinesin-8 motor protein, a highly processive motor with long end-residency time, along microtubules in a total internal-reflection fluorescence microscopy assay. We found that traffic jams, characterized by an abrupt increase in the density of motors with an associated abrupt decrease in motor speed, form even in the absence of other obstructing proteins. To determine the molecular properties that lead to jamming, we altered the concentration of motors, their processivity, and their rate of dissociation from microtubule ends. Traffic jams occurred when the motor density exceeded a critical value (density-induced jams) or when motor dissociation from the microtubule ends was so slow that it resulted in a pileup (bottleneck-induced jams). Through comparison of our experimental results with theoretical models and stochastic simulations, we characterized in detail under which conditions density- and bottleneck-induced traffic jams form or do not form. Our results indicate that transport kinesins, such as kinesin-1, may be evolutionarily adapted to avoid the formation of traffic jams by moving only with moderate processivity and dissociating rapidly from microtubule ends.

Leduc, Cecile; Padberg-Gehle, Kathrin; Varga, Vladimir; Helbing, Dirk; Diez, Stefan; Howard, Jonathon

2012-01-01

368

Spastin's Microtubule-Binding Properties and Comparison to Katanin  

PubMed Central

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

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

2012-01-01

369

MAP3: characterization of a novel microtubule-associated protein  

PubMed Central

Using monoclonal antibodies we have characterized a brain protein that copurifies with microtubules. We identify it as a microtubule- associated protein (MAP) by the following criteria: it copolymerizes with tubulin through repeated cycles of microtubule assembly in vitro; it is not associated with any brain subcellular fraction other than microtubules; in double-label immunofluorescence experiments antibodies against this protein stain the same fibrous elements in cultured cells as are stained by antitubulin; and this fibrous staining pattern is dispersed when cytoplasmic microtubules are disrupted by colchicine. Because it is distinct from previously described MAPs we designate this novel species MAP3. The MAP3 protein consists of a closely spaced pair of polypeptides on SDS gels, Mr 180,000, which are present in both glial (glioma C6) and neuronal (neuroblastoma B104) cell lines. In brain the MAP3 antigen is present in both neurons and glia. In nerve cells its distribution is strikingly restricted: anti-MAP3 staining is detectable only in neurofilament-rich axons. It is not, however, a component of isolated brain intermediate filaments.

1985-01-01

370

The Fission Yeast Ran Gtpase Is Required for Microtubule Integrity  

PubMed Central

The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.

Fleig, Ursula; Salus, Sandra S.; Karig, Inga; Sazer, Shelley

2000-01-01

371

Molecular crowding creates traffic jams of kinesin motors on microtubules.  

PubMed

Despite the crowdedness of the interior of cells, microtubule-based motor proteins are able to deliver cargoes rapidly and reliably throughout the cytoplasm. We hypothesize that motor proteins may be adapted to operate in crowded environments by having molecular properties that prevent them from forming traffic jams. To test this hypothesis, we reconstituted high-density traffic of purified kinesin-8 motor protein, a highly processive motor with long end-residency time, along microtubules in a total internal-reflection fluorescence microscopy assay. We found that traffic jams, characterized by an abrupt increase in the density of motors with an associated abrupt decrease in motor speed, form even in the absence of other obstructing proteins. To determine the molecular properties that lead to jamming, we altered the concentration of motors, their processivity, and their rate of dissociation from microtubule ends. Traffic jams occurred when the motor density exceeded a critical value (density-induced jams) or when motor dissociation from the microtubule ends was so slow that it resulted in a pileup (bottleneck-induced jams). Through comparison of our experimental results with theoretical models and stochastic simulations, we characterized in detail under which conditions density- and bottleneck-induced traffic jams form or do not form. Our results indicate that transport kinesins, such as kinesin-1, may be evolutionarily adapted to avoid the formation of traffic jams by moving only with moderate processivity and dissociating rapidly from microtubule ends. PMID:22431622

Leduc, Cécile; Padberg-Gehle, Kathrin; Varga, Vladimír; Helbing, Dirk; Diez, Stefan; Howard, Jonathon

2012-04-17

372

SUBPLASMALEMMAL MICROFILAMENTS AND MICROTUBULES IN RESTING AND PHAGOCYTIZING CULTIVATED MACROPHAGES  

PubMed Central

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

Reaven, Eve P.; Axline, Stanton G.

1973-01-01

373

Single molecule studies reveal new mechanisms for microtubule severing  

NASA Astrophysics Data System (ADS)

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

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

2011-03-01

374

Microtubules and Their Role in Cellular Stress in Cancer  

PubMed Central

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

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

2014-01-01

375

Microtubule ionic conduction and its implications for higher cognitive functions.  

PubMed

The neuronal cytoskeleton has been hypothesized to play a role in higher cognitive functions including learning, memory and consciousness. Experimental evidence suggests that both microtubules and actin filaments act as biological electrical wires that can transmit and amplify electric signals via the flow of condensed ion clouds. The potential transmission of electrical signals via the cytoskeleton is of extreme importance to the electrical activity of neurons in general. In this regard, the unique structure, geometry and electrostatics of microtubules are discussed with the expected impact on their specific functions within the neuron. Electric circuit models of ionic flow along microtubules are discussed in the context of experimental data, and the specific importance of both the tubulin C-terminal tail regions, and the nano-pore openings lining the microtubule wall is elucidated. Overall, these recent results suggest that ions, condensed around the surface of the major filaments of the cytoskeleton, flow along and through microtubules in the presence of potential differences, thus acting as transmission lines propagating intracellular signals in a given cell. The significance of this conductance to the functioning of the electrically active neuron, and to higher cognitive function is also discussed. PMID:20589950

Craddock, Travis J A; Tuszynski, Jack A; Priel, Avner; Freedman, Holly

2010-06-01

376

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

PubMed

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

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

2014-06-01

377

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

PubMed Central

The microtubule cytoskeleton is a highly dynamic network. In dividing cells, its complex architecture not only influences cell shape and movement but is also crucial for chromosome segregation. Curiously, nothing is known about the behavior of this cellular machinery in quiescent cells. Here we show that, upon quiescence entry, the Saccharomyces cerevisiae microtubule cytoskeleton is drastically remodeled. Indeed, while cytoplasmic microtubules vanish, the spindle pole body (SPB) assembles a long and stable monopolar array of nuclear microtubules that spans the entire nucleus. Consequently, the nucleolus is displaced. Kinetochores remain attached to microtubule tips but lose SPB clustering and distribute along the microtubule array, leading to a large reorganization of the nucleus. When cells exit quiescence, the nuclear microtubule array slowly depolymerizes and, by pulling attached centromeres back to the SPB, allows the recovery of a typical Rabl-like configuration. Finally, mutants that do not assemble a nuclear array of microtubules are impaired for both quiescence survival and exit.

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

2013-01-01

378

Cell prestress. II. Contribution of microtubules  

NASA Technical Reports Server (NTRS)

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

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

2002-01-01

379

Wave equations, dispersion relations, and van Hove singularities for applications of doublet mechanics to ultrasound propagation in bio- and nanomaterials  

Microsoft Academic Search

A fundamental mathematical framework for applications of Doublet Mechanics to ultrasound propagation in a discrete material is introduced. A multiscale wave equation, dispersion relation for longitudinal waves, and shear waves are derived. The van Hove singularities and corresponding highest frequency limits for the Mth-order wave equations of longitudinal and shear waves are determined for a widely used microbundle structure. Doublet

Junru Wu; Christopher Layman; Jun Liu

2004-01-01

380

Epidemiology of Doublet\\/Multiplet Mutations in Lung Cancers: Evidence that a Subset Arises by Chronocoordinate Events  

Microsoft Academic Search

BackgroundEvidence strongly suggests that spontaneous doublet mutations in normal mouse tissues generally arise from chronocoordinate events. These chronocoordinate mutations sometimes reflect “mutation showers”, which are multiple chronocoordinate mutations spanning many kilobases. However, little is known about mutagenesis of doublet and multiplet mutations (domuplets) in human cancer. Lung cancer accounts for about 25% of all cancer deaths. Herein, we analyze the

Zhenbin Chen; Jinong Feng; Carolyn H. Buzin; Steve S. Sommer; Amanda Ewart Toland

2008-01-01

381

A protein kinase bound to the projection portion of MAP 2 (microtubule-associated protein 2)  

Microsoft Academic Search

In previous work we have demonstrated that the microtubule-associated protein 2 (MAP 2) molecule consistsof two structuralparts.One part of the molecule, referredto as the assembly-promoting domain, binds to the microtubule surface and isresponsible forpromoting microtubule assembly ;the other represents a filamentous projection observed on the micro- tubule surface that may be involved in the interaction of microtubules with other cellular

RICHARD B. VALLEE; WILLIAM E. THEURKAUF

1981-01-01

382

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

PubMed Central

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

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

2012-01-01

383

Msps\\/XMAP215 interacts with the centrosomal protein D-TACC to regulate microtubule behaviour  

Microsoft Academic Search

The XMAP215\\/ch-TOG\\/Msps family of microtubule-associated proteins (MAPs) promote microtubule growth in vitro and are concentrated at centrosomes in vivo. We show here that Msps (mini-spindles protein) interacts with the centrosomal protein D-TACC, and that this interaction strongly influences microtubule behaviour in Drosophila embryos. If D-TACC levels are reduced, Msps does not concentrate at the centrosomes efficiently and the centrosomal microtubules

Michael J. Lee; Fanni Gergely; Kim Jeffers; Sew Yeu Peak-Chew; Jordan W. Raff

2001-01-01

384

Disruption of Microtubule Integrity Initiates Mitosis during CNS Repair  

PubMed Central

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

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

2012-01-01

385

Microtubule-associated nuclear envelope proteins in interphase and mitosis.  

PubMed

The LINC (linker of nucleoskeleton and cytoskeleton) complex forms a transcisternal bridge across the NE (nuclear envelope) that connects the cytoskeleton with the nuclear interior. This enables some proteins of the NE to communicate with the centrosome and the microtubule cytoskeleton. The position of the centrosome relative to the NE is of vital importance for many cell functions, such as cell migration and division, and centrosomal dislocation is a frequent phenotype in laminopathic disorders. Also in mitosis, a small group of transmembrane NE proteins associate with microtubules when they concentrate in a specific membrane domain associated with the mitotic spindle. The present review discusses structural and functional aspects of microtubule association with NE proteins and how this association may be maintained over the cell cycle. PMID:22103526

Figueroa, Ricardo A; Gudise, Santhosh; Hallberg, Einar

2011-12-01

386

Differential mitotic responses to microtubule-stabilizing and -destabilizing drugs.  

PubMed

Although microtubule interacting agents inhibit spindle dynamics, thereby leading to a block in mitosis, we report that low concentrations of these drugs result in differential mitotic effects. Microtubule-stabilizing agents including Taxol, epothilone B, and discodermolide produce aneuploid populations of A549 cells in the absence of a mitotic block. Such aneuploid populations are diminished in an epothilone B-resistant cell line. In contrast, microtubule-destabilizing agents like colchicine, nocodazole, and vinblastine are unable to initiate aneuploidy. The aneuploid cells result from aberrant mitosis as multipolar spindles are induced by the stabilizing drugs, but not by destabilizing agents. The results suggest that the mechanism underlying aberrant mitosis may not be the same as that responsible for mitotic block, and that the former determines the sensitivity of cells to Taxol-like drugs. PMID:11929805

Chen, Jie-Guang; Horwitz, Susan Band

2002-04-01

387

Hypothalamic regulation of microtubule-associated protein phosphorylation in lactotrophs.  

PubMed

Endogenous phosphorylation of microtubule-associated proteins was examined in cultures of anterior pituitary cells from estradiol-treated rats. The cells were incubated for 1 h with 32P-orthophosphate and challenged for different times with removal of dopamine (DA), the addition of TRH in the presence of DA, or the transient removal of DA followed by addition of TRH. Microtubules were bundled by taxol followed by electrophoretic separation of the phosphorylated proteins and autoradiography. Within 10 s to 1 min of any of the treatments increased labeling of eight phosphoproteins (64, 80, 95, 110, 125, 155, 205 and 300 kDa) appeared in autoradiograms. The pattern of labeling in response to DA withdrawal was longer-lasting than that induced by TRH, whose effect disappeared by 10 min. The administration of TRH after a transient 10-min withdrawal of DA increased the magnitude and prolonged the duration of the effect of TRH. The 80-kDa microtubule-associated protein comigrated with the well characterized heat-stable, acid-soluble protein substrate for protein kinase C (PKC). The migration of the proteins following two-dimensional polyacrylamide gel electrophoresis and autoradiography was identical. Furthermore, the sequential extraction of microtubule-associated proteins followed by extraction of heat-stable, acid-soluble proteins showed a phosphoprotein of Mr 80 kDa. These observations suggest that the ubiquitous, heat-stable, acid-soluble 80-kDa phosphoprotein that is a specific substrate for PKC is associated with microtubules in lactotrophs. Furthermore, the levels of microtubule-associated phosphoprotein are increased following hormonal activation of PKC, although it is unclear whether this increase represents translocation of the phosphoprotein or phosphorylation of a previously associated protein. PMID:1501762

Martínez de la Escalera, G; Weiner, R I

1992-03-01

388

Microtubule affinity-regulating kinase 4: structure, function, and regulation.  

PubMed

MAP/Microtubule affinity-regulating kinase 4 (MARK4) belongs to the family of serine/threonine kinases that phosphorylate the microtubule-associated proteins (MAP) causing their detachment from the microtubules thereby increasing microtubule dynamics and facilitating cell division, cell cycle control, cell polarity determination, cell shape alterations, etc. The MARK4 gene encodes two alternatively spliced isoforms, L and S that differ in their C-terminal region. These isoforms are differentially regulated in human tissues including central nervous system. MARK4L is a 752-residue-long polypeptide that is divided into three distinct domains: (1) protein kinase domain (59-314), (2) ubiquitin-associated domain (322-369), and (3) kinase-associated domain (703-752) plus 54 residues (649-703) involved in the proper folding and function of the enzyme. In addition, residues 65-73 are considered to be the ATP-binding domain and Lys88 is considered as ATP-binding site. Asp181 has been proposed to be the active site of MARK4 that is activated by phosphorylation of Thr214 side chain. The isoform MARK4S is highly expressed in the normal brain and is presumably involved in neuronal differentiation. On the other hand, the isoform MARK4L is upregulated in hepatocarcinoma cells and gliomas suggesting its involvement in cell cycle. Several biological functions are also associated with MARK4 including microtubule bundle formation, nervous system development, and positive regulation of programmed cell death. Therefore, MARK4 is considered as the most suitable target for structure-based rational drug design. Our sequence, structure- and function-based analysis should be helpful for better understanding of mechanisms of regulation of microtubule dynamics and MARK4 associated diseases. PMID:23471664

Naz, Farha; Anjum, Farah; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2013-11-01

389

Intact Microtubules Support Adenovirus and Herpes Simplex Virus Infections  

PubMed Central

Capsids and the enclosed DNA of adenoviruses, including the species C viruses adenovirus type 2 (Ad2) and Ad5, and herpesviruses, such as herpes simplex virus type 1 (HSV-1), are targeted to the nuclei of epithelial, endothelial, fibroblastic, and neuronal cells. Cytoplasmic transport of fluorophore-tagged Ad2 and immunologically detected HSV-1 capsids required intact microtubules and the microtubule-dependent minus-end-directed motor complex dynein-dynactin. A recent study with epithelial cells suggested that Ad5 was transported to the nucleus and expressed its genes independently of a microtubule network. To clarify the mechanisms by which Ad2 and, as an independent control, HSV-1 were targeted to the nucleus, we treated epithelial cells with nocodazole (NOC) to depolymerize microtubules and measured viral gene expression at different times and multiplicities of infections. Our results indicate that in NOC-treated cells, viral transgene expression was significantly reduced at up to 48 h postinfection (p.i.). A quantitative analysis of subcellular capsid localization indicated that NOC blocked the nuclear targeting of Ad2 and also HSV-1 by more than 90% at up to 7 h p.i. About 10% of the incoming Texas Red-coupled Ad2 (Ad2-TR) was enriched at the nucleus in microtubule-depleted cells at 5 h p.i. This result is consistent with earlier observations that Ad2-TR capsids move randomly in NOC-treated cells at less than 0.1 ?m/s and over distances of less than 5 ?m, characteristic of Brownian motion. We conclude that fluorophore-tagged Ad2 and HSV-1 particles are infectious and that microtubules play a prominent role in efficient nuclear targeting during entry and gene expression of species C Ads and HSV-1.

Mabit, Helene; Nakano, Michel Y.; Prank, Ute; Saam, Bianca; Dohner, Katinka; Sodeik, Beate; Greber, Urs F.

2002-01-01

390

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

SciTech Connect

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

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

1992-01-14

391

A GFPMAP4 Reporter Gene for Visualizing Cortical Microtubule Rearrangements in Living Epidermal Cells  

Microsoft Academic Search

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed

Jan Marc; Cheryl L. Granger; Jennifer Brincat; Deborah D. Fisher; Teh-hui Kao; Andrew G. Mccubbin; Richard J. Cyr

1998-01-01

392

Nuclear envelope radiating microtubules in plant cells during interphase mitosis transition  

Microsoft Academic Search

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

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

1985-01-01

393

Polarity of kinetochore microtubules in Chinese hamster ovary cells after recovery from a colcemid block  

PubMed Central

The polarity of kinetochore microtubules was determined in a system for which kinetochore-initiated microtubule assembly has been demonstrated. Chinese hamster ovary cells were treated with 0.3 micrograms/ml colcemid for 8 h and then released from the block. Prior to recovery, microtubules were completely absent from the cells. The recovery was monitored using light and electron microscopy to establish that the cells progress through anaphase and that the kinetochore fibers are fully functional. Since early stages of recovery are characterized by short microtubule segments that terminate in the kinetochore fibrous corona rather than on the outer disk, microtubule polarity was determined at later stages of recovery when longer kinetochore bundles had formed, allowing us to establish unambiguously the spatial relationship between microtubules, kinetochores, and chromosomes. The cells were lysed in a detergent mixture containing bovine brain tubulin under conditions that allowed the formation of polarity-revealing hooks. 20 kinetochore bundles were assayed for microtubule polarity in either thick or thin serial sections. We found that 95% of the decorated kinetochore microtubules had the same polarity and that, according to the hook curvature, the plus ends of the microtubules were at the kinetochores. Hence, the polarity of kinetochore microtubules in Chinese hamster ovary cells recovering from a colcemid block is the same as in normal untreated cells. This result suggests that microtubule polarity is likely to be important for spindle function since kinetochore microtubules show the same polarity, regardless of the pattern of spindle formation.

1983-01-01

394

Parkin stabilizes microtubules through strong binding mediated by three independent domains.  

PubMed

Mutations of parkin, a protein-ubiquitin isopeptide ligase (E3), appear to be the most frequent cause of familial Parkinson's disease (PD). Our previous studies have demonstrated that parkin binds strongly to alpha/beta tubulin heterodimers and microtubules. Here we show that the strong binding between parkin and tubulin, as well as that between parkin and microtubules, was mediated by three independent domains: linker, RING1, and RING2. These redundant strong interactions made it virtually impossible to separate parkin from microtubules by high concentrations of salt (3.8 m) or urea (0.5 m). Parkin co-purified with tubulin and was found in highly purified tubulin preparation. Expression of either full-length parkin or any of its three microtubule-binding domains significantly attenuated colchicine-induced microtubule depolymerization. The abilities of parkin to bind to and stabilize microtubules were not affected by PD-linked mutations that abrogate its E3 ligase activity. Thus, the tubulin/microtubule-binding activity of parkin and its E3 ligase activity are independent. The strong binding between parkin and tubulin/microtubules through three redundant interaction domains may not only stabilize microtubules but also guarantee the anchorage of this E3 ligase on microtubules. Because many misfolded proteins are transported on microtubules, the localization of parkin on microtubules may provide an important environment for its E3 ligase activity toward misfolded substrates. PMID:15737990

Yang, Fang; Jiang, Qian; Zhao, Jinghui; Ren, Yong; Sutton, Mark D; Feng, Jian

2005-04-29

395

Profiles of the resonance doublets formed in bipolar winds in symbiotic stars  

NASA Astrophysics Data System (ADS)

We compute the profiles of resonance doublet lines (S1/2-P1/2,3/2) formed in bipolar winds with velocity greater than the doublet separation in symbiotic stars. Particular attention has been paid to the doublet line ratio, where an essential role is played by the conversion of the short-wavelength component arising from the S1/2-P3/2 transition into the long-wavelength component for the transition S1/2-P1/2. We adopted a Monte Carlo technique and the Sobolev approximation. Our bipolar winds take the form of a cone and are characterized by the terminal wind velocity, the mass-loss rate and the opening angle of the cone. When an observer is in the polar direction and the Sobolev optical depth ?Sob~= 1, we mainly obtain profiles with inverted flux line ratios, where the short-wavelength component is weaker than the long-wavelength component. When an observer is in the equatorial direction, we find that the profiles are characterized by two broad components, where the long-wavelength component is the broader and stronger of the two. We conclude that the profiles obtained in our model provide a qualitative understanding of the broad profiles and inverted intensity ratios of the doublets in symbiotic stars.

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

2002-08-01

396

Neutrino mass operator renormalization in two Higgs doublet models and the MSSM  

Microsoft Academic Search

In a recent re-analysis of the Standard Model (SM) (?)-function for the effective neutrino mass operator, we found that the previous results were not entirely correct. Therefore, we consider the analogous dimension five operators in a class of two Higgs doublet models (2HDM's) and the minimal supersymmetric Standard Model (MSSM). Deriving the renormalization group equations for these effective operators, we

Stefan Antusch; Manuel Drees; Jörn Kersten; Manfred Lindner; Michael Ratz

2002-01-01

397

Bounds on Higgs-boson masses in a two-doublet extension of the standard model  

Microsoft Academic Search

The standard electroweak model is extended to include two Higgs-boson doublets. The ratios of the quartic scalar couplings to the U(1) gauge coupling are assumed to remain finite in the ultraviolet limit. The resulting numerical upper bounds on the masses of the four physical Higgs bosons are presented. At least one mass must be less than 86 GeV.

K. S. Babu; Ernest Ma

1985-01-01

398

The Determination of Atomic Mass Doublets by Means of a Mass Spectrometer  

Microsoft Academic Search

A double-focusing mass spectrometer employing a 90° electrostatic analyzer followed by a 60° magnetic analyzer has been constructed having a resolution sufficiently great to permit mass difference measurements by the familiar doublet method. High stability is obtained by use of an auxiliary mass spectrometer tube which compensates for fluctuations in both the magnetic field and the source of potential for

Alfred O. Nier; T. R. Roberts

1951-01-01

399

Lee-Quigg-Thacker bounds for Higgs boson masses in a two-doublet model  

Microsoft Academic Search

Upper bounds for neutral as well as charged Higgs boson masses in a two-doublet model are obtained on the basis of tree unitarity conditions á la Lee, Quigg and Thacker. A wide variety of scattering processes are considered so extensively that our bounds are more restrictive than those obtained previously for neutral Higgs bosons and are also of a new

Shinya Kanemura; Takahiro Kubota; Eiichi Takasugi

1993-01-01

400

Running of the Yukawa Couplings in a Two Higgs Doublet Model  

SciTech Connect

We solve the one loop Renormalization Group Equations (RGE) for the Yukawa couplings in the Standard Model with two Higgs doublets. In the RGE we include the contributions of the up and down quarks. In this approximation we explore universality and unification assumptions to study the mass-hierarchy problem through the running of the vacuum expectation values.

Montes de Oca Y, J. H.; Juarez W, S. R. [Depto. de Fis., Esc. Sup. de Fis. y Mat., Instituto Politecnico Nacional, U.P. 'Adolfo Lopez Mateos' Edif. 9, C.P. 07738, Mexico, D.F. (Mexico); Kielanowski, P. [Dpto. de Fisica, CINVESTAV, Av. IPN 2508, C.P. 07360, Mexico, D.F. (Mexico)

2008-07-02

401

B> Xd l +l - in a CP Softly Broken Two Higgs Doublet Model  

Microsoft Academic Search

We study the differential branching ratio, forward--backward asymmetry, CP violating asymmetry, CP violating asymmetry in the forward-backward asymmetry and polarization asymmetries of the final lepton in the B --> Xd l + l - decays in the context of a CP softly broken two Higgs doublet model. We analyze the dependencies of these observables on the model parameters by paying

Hilal Acar; G. Turan

2004-01-01

402

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

SciTech Connect

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

Schlieder, Joshua E.; Simon, Michal [Department of Physics and Astronomy, Stony Brook University, Stony Brook, NY 11794 (United States); Lepine, Sebastien; Rice, Emily [Department of Astrophysics, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024 (United States); Fielding, Drummond [Department of Physics and Astronomy, Johns Hopkins University, 366 Bloomberg Center, 3400 North Charles Street, Baltimore, MD 21218 (United States); Tomasino, Rachael, E-mail: michal.simon@stonybrook.edu, E-mail: schlieder@mpia-hd.mpg.de, E-mail: lepine@amnh.org, E-mail: erice@amnh.org, E-mail: dfieldi1@jhu.edu, E-mail: tomas1r@cmich.edu [Department of Physics, Central Michigan University, Mount Pleasant, MI 48859 (United States)

2012-05-15

403

Higgs doublet as a Goldstone boson in perturbative extensions of the standard model  

Microsoft Academic Search

We investigate the idea of the Higgs doublet as a pseudo-Goldstone boson in perturbative extensions of the Standard Model, motivated by the desire to ameliorate its hierarchy problem without conflict with the electroweak precision data. Two realistic supersymmetric models with global SU(3) symmetry are proposed, one for large and another for small values of tan beta. The two models demonstrate

Brando Bellazzini; Stefan Pokorski; Vyacheslav S. Rychkov; Alvise Varagnolo

2008-01-01

404

Higgs masses and stability in the standard and the two Higgs doublet models  

Microsoft Academic Search

Within the framework of the standard model (SM) of elementary particles and the two Higgs doublet extension to this model (2DHM), we obtained analytical and numerical solutions for the gauge couplings, the vacuum expectation values (VEV) of the Higgs fields, the quark Yukawa couplings and quark masses, the quartic Higgs couplings, and the running Higgs masses, considering the renormalization group

P. Kielanowski

2010-01-01

405

Reduction of couplings in the two Higgs doublet extension of the electroweak standard model  

Microsoft Academic Search

The method of reduction of couplings is applied to the two Higgs doublet extension of the standard electroweak model. From the solutions of the reduction equations predictions for the top quark and Higgs boson masses are obtained. Present address: Physikalisches Institut der Universität Würzburg, Am Hubland, 8700 Würzburg, FRG.

Ansgar Denner

1990-01-01

406

Absence of spontaneous CP violation in multi-Higgs doublet extension of MSSM  

Microsoft Academic Search

We show that in the multi-Higgs extension of minimal supersymmetric standard model, which has N-pairs of Higgs doublets (called NHMSSM), it is impossible to break CP spontaneously, if we do not allow for fine-tuning relations between parameters. The result holds true even in the presence of spontaneous R-parity breaking.

R. N. Mohapatra; C. C. Nishi

2011-01-01

407

Advances in the Design of the SuperB Final Doublet  

SciTech Connect

SuperB is an asymmetric energy e{sup +}e{sup -} collider operating at the {Upsilon}(4S) peak with a design peak luminosity of 10{sup 36} Hz/cm{sup 2} to be built in Italy in the very near future. The design luminosity is almost a factor hundred higher than that of the present generation comparable facilities. To get the design luminosity a novel collision scheme, the so called 'large Piwinski angle with crab waist', has been designed. The scheme requires a short focus final doublet to reduce the vertical beta function down to {beta}*{sub y} = 0.2mm at the interaction point (IP). The final doublet will be composed by a set of permanent and superconducting (SC) quadrupoles. The SC quadrupole doublets QD0/QF1 will be placed as close to the IP as possible. This layout is critical because the space available for the doublets is very small. An advanced design of the quadrupole has been developed, based on the so-called helical coil concept. The paper discusses the design concept, the construction and the results of test of a model of the superconducting quadrupole based on NbTi technology. Future developments are also presented.

Paoloni, E.; Carmignani, N.; Pilo, F.; /Pisa U. /INFN, Pisa; Bettoni, S.; /CERN; Fabbricatore, P.; Farinon, S.; Musenich, R.; /INFN, Genoa; Bosi, F.; /INFN, Pisa; Biagini, M.E.; Raimondi, P.; /Frascati; Sullivan, M.; /SLAC

2012-04-26

408

The engine of microtubule dynamics comes into focus.  

PubMed

In this issue, Alushin et al. report high-resolution structures of three states of the microtubule lattice: GTP-bound, which is stable to depolymerization; unstable GDP-bound; and stable Taxol and GDP-bound. By comparing these structures at near-atomic resolution, they are able to propose a detailed model for how GTP hydrolysis destabilizes the microtubule and thus powers dynamic instability and chromosome movement. Destabilization of cytoskeleton filaments by nucleotide hydrolysis is an important general principle in cell dynamics, and this work represents a major step forward on a problem with a long history. PMID:24855939

Mitchison, T J

2014-05-22

409

Physical aspects of the assembly and function of microtubules  

NASA Astrophysics Data System (ADS)

Living cells contain polymers called microtubules. Microtubules are used to control cell shape, to generate force for movement, to transport vesicles, and to separate chromosomes during cell division. Microtubule polymerization is governed by a unique, energy-consuming phenomenon called dynamic instability, which leads to large fluctuations in length. This dissertation reports on physical studies (theory and experiment) of microtubules and their roles in living cells. To understand dynamic instability we need to know its mechanism and its function. Experiments on dynamic instability have led to a seeming contradiction as to its mechanism. By introducing a phenomenological model that unites the existing data, we show that this contradiction can be resolved. The model describes the stochastic dynamics of a stabilizing cap which promotes growth, but whose loss leads to disassembly. The theory matches experiments over time scales from seconds to minutes. We address the biological role of dynamic instability, also from a theoretical standpoint. We show that these large length fluctuations are useful: they lead to a rapid search of intracellular space. This search may be an essential step in organizing the cell interior, forging connections between widely-separated components. We show that dynamic instability speeds a search by several orders of magnitude. We also find that the parameters which govern dynamic instability appear to be chosen by the cell so as to minimize the search time. This thesis also reports experiments showing that microtubule polymerization may generate forces for movement and organization. Archetypal movements in living cells (e.g., the movement of the sperm nucleus from the periphery to the center of the egg) were reconstructed in an artificial system. The components of our system are purified proteins and two specially-designed materials: latex beads coated so as to nucleate microtubules, and microscopic chambers fabricated to mimic the confined geometry of cells. With this system, we showed that microtubule growth alone moves beads to the center of chambers. However, once microtubules grow long enough to buckle, the center is de-stabilized and the underlying symmetry is broken. Dynamic instability allows the aster to explore a complex bending-energy landscape.

Holy, Timothy Eric

410

Rectified Brownian motion and kinesin motion along microtubules  

NASA Astrophysics Data System (ADS)

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

Fox, Ronald F.; Choi, Mee Hyang

2001-05-01

411

The incidence of initial doublets in the discharges of motoneurones of two different inspiratory muscles in the cat.  

PubMed Central

1. Trains of action potentials in motoneurones frequently commence with an initial doublet; i.e. a uniquely short interspike interval. Previous authors have speculated on the functional importance of initial doublets. Here we test the hypotheses that these doublets are associated with particular classes of motoneurones or particular physiological conditions. 2. Discharges of inspiratory motoneurones were recorded extracellularly in the thoracic ventral horn of anaesthetized, paralysed cats. Seventy units (35 each with axons in the internal and external intercostal nerves) were classified on the basis of their maximum firing rates, start times in the respiratory cycle and axonal destination. 3. Initial doublets were defined by an interspike interval < 14 ms. Of seventeen units firing initial doublets, fifteen had axons in the external intercostal nerve and two had axons in the internal intercostal nerve. Neither maximum firing rate nor start time during the respiratory cycle predicted the occurrence of doublets. 4. The chemical drive to breathe was manipulated by altering the CO2 content of the inspired gas or by briefly stopping the respiratory pump. Varying the chemical drive to breathe had no consistent effect on the occurrence of initial doublets. 5. These results support the view that initial doublets are part of the normal pattern of discharge of motoneurones. However, because the incidence of doublets does not consistently support previous functional hypotheses, we argue that the occurrence of doublets may not necessarily be dictated by the CNS, but in some circumstances it is an epiphenomenon dependent on the state of the motoneurone, in particular on the statistical properties of its synaptic inputs.

Kirkwood, P A; Munson, J B

1996-01-01

412

New look inside the spindle: microtubule-dependent microtubule generation within the spindle.  

PubMed

The structure, dynamics, and mechanics of mitotic and meiotic spindles have been progressively elucidated through the advancements in microscopic technology, identification of the genes involved, and construction of theoretical frameworks. Here, we review recent works that have utilized quantitative image analysis to advance our understanding of the complex spindle structure of animal cells. In particular, we discuss how microtubules (MTs) are nucleated and distributed inside the spindle. Accumulating evidence supports the presence of MT-dependent MT generation within the spindle. This mechanism would produce dense arrays of intraspindle MTs with various lengths, which may contribute to efficient spindle assembly and stabilize the metaphase spindle. RNA interference (RNAi) screens with quantitative image analysis led to the identification of the augmin complex that plays a key role in this MT generation process. PMID:20022736

Goshima, Gohta; Kimura, Akatsuki

2010-02-01

413

Doublet stimulation protocol to minimize musculoskeletal stress during paralyzed quadriceps muscle testing  

PubMed Central

With long-term electrical stimulation training, paralyzed muscle can serve as an effective load delivery agent for the skeletal system. Muscle adaptations to training, however, will almost certainly outstrip bone adaptations, exposing participants in training protocols to an elevated risk for fracture. Assessing the physiological properties of the chronically paralyzed quadriceps may transmit unacceptably high shear forces to the osteoporotic distal femur. We devised a two-pulse doublet strategy to measure quadriceps physiological properties while minimizing the peak muscle force. The purposes of the study were 1) to determine the repeatability of the doublet stimulation protocol, and 2) to compare this protocol among individuals with and without spinal cord injury (SCI). Eight individuals with SCI and four individuals without SCI underwent testing. The doublet force-frequency relationship shifted to the left after SCI, likely reflecting enhancements in the twitch-to-tetanus ratio known to exist in paralyzed muscle. Posttetanic potentiation occurred to a greater degree in subjects with SCI (20%) than in non-SCI subjects (7%). Potentiation of contractile rate occurred in both subject groups (14% and 23% for SCI and non-SCI, respectively). Normalized contractile speed (rate of force rise, rate of force fall) reflected well-known adaptations of paralyzed muscle toward a fast fatigable muscle. The doublet stimulation strategy provided repeatable and sensitive measurements of muscle force and speed properties that revealed meaningful differences between subjects with and without SCI. Doublet stimulation may offer a unique way to test muscle physiological parameters of the quadriceps in subjects with uncertain musculoskeletal integrity.

Dudley-Javoroski, Shauna; Littmann, Andrew E.; Iguchi, Masaki; Shields, Richard K.

2009-01-01

414

Microtubules and nucleoside diphosphate kinase. Nucleoside diphosphate kinase binds to co-purifying contaminants rather than to microtubule proteins.  

PubMed Central

Nucleoside diphosphate (NDP) kinase has been postulated to generate GTP from the GDP bound to tubulin. The purified chick brain enzyme was studied with respect to its kinetic parameters, and the protein-protein interactions between the NDP kinase and tubulin were examined. No specific interaction is observed between the enzyme and assembled microtubules, tubulin dimers, or tubulin-microtubule-associated protein (MAP) oligomers under a variety of nucleotide conditions. The apparent association is demonstrated to result from NDP kinase binding to a co-purifying contaminant. The absence of detectable NDP kinase-tubulin interactions indicates that NDP kinase does not directly charge up tubulin-GDP.

Islam, K; Burns, R G

1985-01-01

415

Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells  

Microsoft Academic Search

Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978 -1987,

Stephane Honore; Kathy Kamath; Diane Braguer; Susan Band Horwitz; Leslie Wilson; Claudette Briand; Mary Ann Jordan

2004-01-01

416

Poleward Microtubule Flux Is a Major Component of Spindle Dynamics and Anaphase A in Mitotic Drosophila Embryos  

Microsoft Academic Search

During cell division, eukaryotic cells assemble dynamic microtubule-based spindles to segregate replicated chromosomes [1, 2]. Rapid spindle microtubule turnover, likely derived from dynamic instability, has been documented in yeasts [3, 4], plants [5] and vertebrates [6]. Less studied is concerted spindle microtubule poleward translocation (flux) coupled to depolymerization at spindle poles [7]. Microtubule flux has been observed only in vertebrates

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

2002-01-01

417

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

PubMed Central

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 of the microtubule. This transverse force (t-force) is evaluated here using available experimental evidence from sea urchin sperm and bull sperm. At or near the switch point for beat reversal, the t-force is in the range of 0.25–1.0 nN/?m, with 0.5 nN/?m the most likely value. This is the case in both beating and arrested bull sperm and in beating sea urchin sperm. The total force that can be generated (or resisted) by all the dyneins on one micron of outer doublet is also ?0.5 nN. The equivalence of the maximum dynein force/?m and t-force/?m at the switch point may have important consequences. Firstly, the t-force acting on the doublets near the switch point of the flagellar beat is sufficiently strong that it could terminate the action of the dyneins directly by strongly favoring the detached state and precipitating a cascade of detachment from the adjacent doublet. Secondly, after dynein release occurs, the radial spokes and central-pair apparatus are the structures that must carry the t-force. The spokes attached to the central-pair projections will bear most of the load. The central-pair projections are well-positioned for this role, and they are suitably configured to regulate the amount of axoneme distortion that occurs during switching. However, to fulfill this role without preventing flagellar bend formation, moveable attachments that behave like processive motor proteins must mediate the attachment between the spoke heads and the central-pair structure.

Lindemann, Charles B.

2003-01-01

418

Detyrosination of Alpha Tubulin Does Not Stabilize Microtubules In Vivo  

Microsoft Academic Search

The relationship between alpha tubulin detyrosination and microtubule (MT) stability was ex- amined directly in cultured fibroblasts by experimen- tally converting the predominantly tyrosinated MT ar- ray to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence ~1 h after microinjecting antibodies to the tyrosinating en- zyme,

Daniel R. Webster; Juergen Wehland; Klaus Weber; Gary G. Borisy

419