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

PubMed Central

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

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



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


Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Comparative genomics of flagellated and non-flagellated eukaryotes provides one way to identify new putative flagellar proteins. We identified the Parkin co-regulated gene, or PACRG, from such a screen. Male mice deficient in PACRG are sterile, but its function has been little explored. The flagellated protozoan parasite Trypanosoma brucei possesses two homologues of PACRG. We performed RNA interference knockdown experiments of the two genes independently and both together. Simultaneous ablation of both proteins produced slow growth and paralysis of the flagellum with consequent effects on organelle segregation. Moreover, using transmission electron microscopy, structural defects were seen in the axoneme, with microtubule doublets missing from the canonical 9+2 formation. The occurrence of missing doublets increased toward the distal end of the flagellum and sequential loss of doublets was observed along individual axonemes. GFP fusion proteins of both PACRG homologues localised along the full length of the axoneme. Our results provide the first evidence for PACRG function within the axoneme, where we suggest that PACRG acts to maintain functional stability of the axonemal outer doublets of both motile and sensory cilia and flagella. PMID:16278296

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



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


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



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

PubMed Central

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

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



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


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



Rib72, a conserved protein associated with the ribbon compartment of flagellar A-microtubules and potentially involved in the linkage between outer doublet microtubules.  


Ciliary and flagellar axonemes are basically composed of nine outer doublet microtubules and several functional components, e.g. dynein arms, radial spokes, and interdoublet links. Each A-tubule of the doublet contains a specialized "ribbon" of three protofilaments composed of tubulin and other proteins postulated to specify the three-dimensional arrangement of the various axonemal components. The interdoublet links hold the doublet microtubules together and limit their sliding during the flagellar beat. In this study on Chlamydomonas reinhardtii, we cloned a cDNA encoding a 71,985-Da polypeptide with three DM10 repeats, two C-terminal EF-hand motifs, and homologs extending to humans. This polypeptide, designated as Rib72, is a novel component of the ribbon compartment of flagellar microtubules. It remained associated with 9-fold arrays of doublet tubules following extraction under high and low ionic conditions, and anti-Rib72 antibodies revealed an approximately 96-nm periodicity along axonemes, consistent with Rib72 associating with interdoublet links. Following proteolysis- and ATP-dependent disintegration of axonemes, the rate of cleavage of Rib72 correlated closely with the rate of sliding disintegration. These observations identify a ribbon-associated protein that may function in the structural assembly of the axoneme and in the mechanism and regulation of ciliary and flagellar motility. PMID:12435737

Ikeda, Kazuho; Brown, Jennifer A; Yagi, Toshiki; Norrander, Jan M; Hirono, Masafumi; Eccleston, Eric; Kamiya, Ritsu; Linck, Richard W



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


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



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

PubMed Central

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

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



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

PubMed Central

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

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



Beyond 9+0: noncanonical axoneme structures characterize sensory cilia from protists to humans.  


The intracellular amastigote stages of parasites such as Leishmania are often referred to as aflagellate. They do, however, possess a short axoneme of cryptic function. Here, our examination of the structure of this axoneme leads to a testable hypothesis of its role in the cell biology of pathogenicity. We show a striking similarity between the microtubule axoneme structure of the Leishmania mexicana parasite infecting a macrophage and vertebrate primary cilia. In both, the 9-fold microtubule doublet symmetry is broken by the incursion of one or more microtubule doublets into the axoneme core, giving rise to an architecture that we term here the 9v (variable) axoneme. Three-dimensional reconstructions revealed that no particular doublet initiated the symmetry break, and moreover it often involved 2 doublets. The tip of the L. mexicana flagellum was frequently intimately associated with the macrophage vacuole membrane. We propose that the main function of the amastigote flagellum is to act as a sensory organelle with important functions in host-parasite interactions and signaling in the intracellular stage of the L. mexicana life cycle. PMID:20371625

Gluenz, Eva; Höög, Johanna L; Smith, Amy E; Dawe, Helen R; Shaw, Michael K; Gull, Keith



Integrated Control of Axonemal Dynein AAA+ Motors  

PubMed Central

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

King, Stephen M.



Functional diversity of axonemal dyneins as assessed by in vitro and in vivo motility assays of chlamydomonas mutants.  


This review outlines the current knowledge of the functional diversity of axonemal dyneins, as revealed by studies with the model organism Chlamydomonas. Axonemal dyneins, which comprise outer and inner dynein arms, power cilia and flagella beating by producing sliding movements between adjacent outer-doublet microtubules. Outer- and inner-arm dyneins have traditionally been considered similar in structure and function. However, recent evidence suggests that they differ rather strikingly in subunit composition, axonemal arrangement, and molecular motor properties. We posit that these arms make up two largely independent motile systems; whereas outer-arm dynein can generate axonemal beating by itself under certain conditions, inner-arm dynein can generate beating only in cooperation with the central pair/radial spokes. This conclusion is supported by genome analyses of various organisms. Outer-arm dynein appears to be particularly important for nodal cilia of mammalian embryos that function for determination of left-right body asymmetry. PMID:25284382

Kamiya, Ritsu; Yagi, Toshiki



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

PubMed Central

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

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



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


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

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



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

E-print Network

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.

V. Mukundan; P. Sartori; V. F. Geyer; F. Julicher; J. Howard



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


Although the widely shared "9 + 2" structure of axonemes is thought to be highly symmetrical, axonemes show asymmetrical bending during planar and conical motion. In this study, using electron cryotomography and single particle averaging, we demonstrate an asymmetrical molecular arrangement of proteins binding to the nine microtubule doublets in Chlamydomonas reinhardtii flagella. The eight inner arm dynein heavy chains regulate and determine flagellar waveform. Among these, one heavy chain (dynein c) is missing on one microtubule doublet (this doublet also lacks the outer dynein arm), and another dynein heavy chain (dynein b or g) is missing on the adjacent doublet. Some dynein heavy chains either show an abnormal conformation or were replaced by other proteins, possibly minor dyneins. In addition to nexin, there are two additional linkages between specific pairs of doublets. Interestingly, all these exceptional arrangements take place on doublets on opposite sides of the axoneme, suggesting that the transverse functional asymmetry of the axoneme causes an in-plane bending motion. PMID:19667131

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



Axonemal protofilament ribbons, DM10 domains, and the link to juvenile myoclonic epilepsy.  


Juvenile myoclonic epilepsy (JME) is a common neurological disorder that results in short uncontrolled muscle contractions and sometimes more severe seizures. Genetic studies have suggested that JME may be caused by mutations in EFHC1. The Efhc1 protein consists of three DM10 domains and a C-terminal region containing a potential Ca2+ -binding motif. In Chlamydomonas, a protein (Rib72) of almost identical domain structure is a component of the protofilament ribbons within the doublet microtubules of the flagellar axoneme. Here I discuss recent work that supports assignment of human Efhc1 as a ciliary component and the resulting implications for the mechanism of disease causation. PMID:16572395

King, Stephen M



Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase  

PubMed Central

Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild- type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke- deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP- mediated mechanism. PMID:7798320



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

PubMed Central

Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis. PMID:25270598

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



Two anti-radial spoke monoclonal antibodies inhibit Chlamydomonas axonemal motility by different mechanisms.  


In the 9 + 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules. They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Flagella of Chlamydomonas mutants deficient in entire radial spokes or spoke heads are paralyzed. In this study the importance of two radial spoke proteins in the flagellar movement is exemplified by the potent inhibitory action of two monoclonal antibodies on the axonemal motility of demembranated-reactivated Chlamydomonas models. We show that one of these proteins is localized on the stalk of the radial spokes, whereas the other is a component of the head of the same structure and most likely correspond to radial spoke protein 2 and 1, respectively. Fine motility analysis by videomicrography further indicates that these two anti-radial spoke protein antibodies at low concentration affect motility of demembranated-reactivated Chlamydomonas by changing the flagellar waveform without modifying axonemal beat frequency. They also modify wave amplitude differently during motility inhibition. This brings more direct evidence for the involvement of both radial spoke stalk and head in the fine tuning of the waveform during flagellar motility. PMID:15664983

White, Daniel; Aghigh, Soroush; Magder, Ilana; Cosson, Jacky; Huitorel, Philippe; Gagnon, Claude



Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase  

Microsoft Academic Search

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

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



Assessment of inner dynein arm structure and possible function in ciliary and flagellar axonemes.  


The construction and assessment of a three-dimensional computer-generated model of inner dynein arms on a 96-nm repeat unit of an axonemal doublet is described. The model is based on published electron micrographs of axonemes from Tetrahymena cilia and eel sperm, which were prepared using several different techniques: negative stain, freeze etch, and thin section. The inner arm structure is represented as three inner dynein arm complexes containing four inner dynein arms (IDAs), three dyads, and one single-headed arm, each capable of bridging the interdoublet gap. The IDA structures in the model have been correlated with the domains containing dynein heavy-chain isoforms mapped by several authors using genetic analyses of Chlamydomonas mutants. The model is consistent with micrographic evidence from axonemes of cilia and flagella from other organisms that led previously to conflicting structural interpretations. In this reconciling interpretation, the different alignments of the IDAs relative to the corresponding outer dynein arms observed in micrographs of differently prepared samples, result from the IDAs being arrested at different stages of their cycles of activity in each preparation. By interpolating between these positions of arrest, cycles of activity are proposed for each of the IDAs during which the arms attach to the neighbouring doublet microtubule and drive it tipwards. PMID:10379841

Taylor, H C; Satir, P; Holwill, M E



Molecular basis of sperm flagellar axonemes: structural and evolutionary aspects.  


The axonemes serve as motile machineries in sperm flagella. Although atypical axonemal structures are observed in some cases, 9 + 2 microtubule structure of the axoneme is predominant in many organisms. Several structures are bound to these microtubules and comprise a highly organized protein network. Extensive proteomic analysis of the axonemes has led to find several repeats, domains, and motifs in axonemal proteins. Molecular comparison of subunit composition of axonemal substructures between the ascidian Ciona intestinalis and the green algae Chlamydomonas reinhardtti leads to an intriguing molecular aspect concerning the evolution of intracellular functional complex: The architecture of the axonemes has been well conserved through evolution, but the molecular structure of each axonemal component is not always conserved. In light of domain structure in the axonemal proteins, substructures like outer arm dynein and radial spoke contain a set of domain structures, although some domain-containing subunits are different between these two organisms. Thus, conservation of protein domains within a substructure seems to take precedence over that of each protein ("module-dominant conservation"), which may ultimately result in morphological and functional conservation of the axonemes through evolution. PMID:17363437

Inaba, Kazuo



Regulation of flagellar dynein by the axonemal central apparatus.  


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



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. PMID:22754630

Pigino, Gaia; Ishikawa, Takashi



Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme.  


Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA-DC has an ellipsoidal shape ?24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity. PMID:24979786

Owa, Mikito; Furuta, Akane; Usukura, Jiro; Arisaka, Fumio; King, Stephen M; Witman, George B; Kamiya, Ritsu; Wakabayashi, Ken-ichi



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

PubMed Central

We have determined the lesions in a number of mutant alleles of ?Tub85D, the gene that encodes the testis-specific ?2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the ?2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all ?-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t(6) contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate ?-tubulins. Correspondingly, B2t(6) disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that ?3, a developmentally regulated Drosophila ?-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type ?2-tubulin. We show here by complementation analysis that ?3 and the B2t(6) product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the ?2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the ?2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the ?2 variant lacking the carboxy terminus and the B2t(6) variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate ?-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type ?-tubulins. We propose that the integrity of this structure in the Drosophila testis ?2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics. PMID:7705629

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



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


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

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



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


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

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



Outer doublet heterogeneity reveals structural polarity related to beat direction in Chlamydomonas flagella  

PubMed Central

Analysis of serial cross-sections of the Chlamydomonas flagellum reveals several structural asymmetries in the axoneme. One doublet lacks the outer dynein arm, has a beak-like projection in its B-tubule, and bears a two-part bridge that extends from the A-tubule of this doublet to the B-tubule of the adjacent doublet. The two doublets directly opposite the doublet lacking the arm have beak-like projections in their B-tubules. These asymmetries always occur in the same doublets from section to section, indicating that certain doublets have consistent morphological specializations. These unique doublets give the axoneme an inherent structural polarity. All three specializations are present in the proximal portion of the axoneme; based on their frequency in random cross-sections of isolated axonemes, the two-part bridge and the beak-like projections are present in the proximal one quarter and one half of the axoneme, respectively, and the outer arm is absent from the one doublet greater than 90% of the axoneme's length. The outer arm-less doublet of each flagellum faces the other flagellum, indicating that each axoneme has the same rotational orientation relative to the direction of its effective stroke. This strongly suggests that the direction of the effective stroke is controlled by a structural component within the axoneme. The striated fibers are associated with specific triplets in a manner suggesting that they play a role in setting up or maintaining the 180 degrees rotational symmetry of the two flagella. PMID:6224802



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. PMID:12529441

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



Computer Simulation of Flagellar Movement X. Doublet pair splitting and bend propagation modeled using stochastic dynein kinetics.  

E-print Network

the function of structural components that hold the axonemal doublets together. In a working ciliumComputer Simulation of Flagellar Movement X. Doublet pair splitting and bend propagation modeled), Computer Simulation of Flagellar Movement X. Doublet pair splitting and bend propagation modeled using

Low, Steven H.


Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii.  


Previously, we reported that flagellar excision in Chlamydomonas reinhardtii is mediated by an active process whereby microtubules are severed at select sites within the flagellar-basal body transition zone (Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). At the time of flagellar excision, stellate fibers of the transition zone contract and displace the microtubule doublets of the axoneme inward. The resulting shear force and torsional load generated during inward displacement leads to microtubule severing immediately distal to the central cylinder of the transition zone. In this study, we have used a detergent-extracted cell model of Chlamydomonas that allows direct experimental access to the molecular machinery responsible for microtubule severing without the impediment of the plasma membrane. We present four independent lines of experimental evidence for the essential involvement of centrin-based stellate fibers of the transition zone in the process of flagellar excision: (a) Detergent-extracted cell models excise their flagella in response to elevated, yet physiological, levels of free calcium. (b) Extraction of cell models with buffers containing the divalent cation chelator EDTA leads to the disassembly of centrin-based fibers and to the disruption of transition zone stellate fiber structure. This treatment results in a complete loss of flagellar excision competence. (c) Three separate anti-centrin monoclonal antibody preparations, which localize to the stellate fibers of the transition zone, specifically inhibit contraction of the stellate fibers and block calcium-induced flagellar excision, while control antibodies have no inhibitory effect. Finally, (d) cells of the centrin mutant vfl-2 (Taillon, B., S. Adler, J. Suhan, and J. Jarvik. 1992. J. Cell Biol. 119:1613-1624) fail to actively excise their flagella following pH shock in living cells or calcium treatment of detergent-extracted cell models. Taken together, these observations demonstrate that centrin-based fiber contraction plays a fundamental role in microtubule severing at the time of flagellar excision in Chlamydomonas. PMID:8120100

Sanders, M A; Salisbury, J L



Tubulin polyglutamylation regulates axonemal motility by modulating activities of inner-arm dyneins.  


Tubulin polyglutamylation is a modification that adds multiple glutamates to the gamma-carboxyl group of a glutamate residue in the C-terminal tails of alpha- and beta-tubulin [1, 2]. This modification has been implicated in the regulation of axonal transport and ciliary motility. However, its molecular function in cilia remains unknown. Here, using a novel Chlamydomonas reinhardtii mutant (tpg1) that lacks a homolog of human TTLL9, a glutamic acid ligase enzyme [3], we found that the lack of a long polyglutamate side chain in alpha-tubulin moderately weakens flagellar motility without noticeably impairing the axonemal structure. Furthermore, the double mutant of tpg1 with oda2, a mutation that leads to loss of outer-arm dynein, completely lacks motility. More surprisingly, when treated with protease and ATP, the axoneme of this paralyzed double mutant displayed faster microtubule sliding than the motile oda2 axoneme. These and other results suggest that polyglutamylation directly regulates microtubule-dynein interaction mainly by modulating the function of inner-arm dyneins. PMID:20188560

Kubo, Tomohiro; Yanagisawa, Haru-aki; Yagi, Toshiki; Hirono, Masafumi; Kamiya, Ritsu



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

E-print Network

is essential and a glucose oxidase/catalase/glucose system works very well for this purpose. Axonemes (for there are methods for making polar microtubule substrates for DIC, we generally use fluorescent polarity marked taxol-stabilized microtubules to determine polarity, as described later. 1. Coat flow cell

Mitchison, Tim


A new tool improves diagnostic test performance for transmission em evaluation of axonemal dynein arms.  


Abstract Diagnosis of primary ciliary dyskinesia (PCD) by identification of dynein arm loss in transmission electron microscopy (TEM) images can be confounded by high background noise due to random electron-dense material within the ciliary matrix, leading to diagnostic uncertainty even for experienced morphologists. The authors developed a novel image analysis tool to average the axonemal peripheral microtubular doublets, thereby increasing microtubular signal and reducing random background noise. In a randomized, double-blinded study that compared two experienced morphologists and three different diagnostic approaches, they found that use of this tool led to improvement in diagnostic TEM test performance. PMID:23957500

Funkhouser, W Keith; Niethammer, Marc; Carson, Johnny L; Burns, Kimberlie A; Knowles, Michael R; Leigh, Margaret W; Zariwala, Maimoona A; Funkhouser, William K



Model for unidirectional movement of axonemal and cytoplasmic dynein molecules.  


A model for the unidirectional movement of dynein is presented based on the structural observations and biochemical experimental results available. In this model, the binding affinity of dynein for microtubule (MT) is independent of its nucleotide state and the change between strong and weak MT-binding is determined naturally by the variation of relative orientation between the stalk and MT, as the stalk rotates following nucleotide-state transition. Thus the enigmatic communication from the adenosine triphosphate (ATP)-binding site in the globular domain to the far MT-binding site in the tip of the stalk, which is a prerequisite in conventional models, is not required. Using the present model, the previous experimental results such as the effect of ATP and adenosine diphosphate (ADP) bindings on dissociation of dynein from MT, the movement of single-headed axonemal dyneins at saturating ATP concentration, the load dependence of step-size for the movement of two-headed cytoplasmic dyneins and the dependence of stall force on ATP concentration can be well explained. PMID:17033718

Xie, Ping; Dou, Shuo-Xing; Wang, Peng-Ye



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


Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer-inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella. PMID:22945936

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



Microtubules with 15 subunits in cockroach epidermal cells  

PubMed Central

Since Ledbetter and Porter (1964) described the 13 subunits which are visible in cross sections of negatively stained plant microtubules, subsequent observations have generally confirmed this number. By using Mizuhira's fixative composed of tannic acid and glutaraldehyde, it is easyto demonstrate the subunits of microtublules without optical reinforcement Cytoplasmic microtubules and sperm axonemes, fixed with Mizuhira's fixtive, similarly show 13 subunits (Mizuhira's and Futaesaku, 1971, 1972; Futaesaky et al., 1972; Tilney et al., 1973). This paper will describe a particular type of microtubule in insect epidermal cells fixed with the above fixative. The number of the subunits is found to be 15 in tranverse sections. PMID:1109233

Nagano, T; Suzuki, F



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

PubMed Central

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



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


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



Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii  

Microsoft Academic Search

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 tran- sition zone. The transition zone is located between the flagellar axoneme and the basal

M. A. Sanders; J. L. Salisbury



Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis.  


Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle. PMID:17766466

Dawson, Scott C; Sagolla, Meredith S; Mancuso, Joel J; Woessner, David J; House, Susan A; Fritz-Laylin, Lillian; Cande, W Zacheus



Evidence for Axonemal Distortion During the Flagellar Beat of Chlamydomonas  

E-print Network

Evidence for Axonemal Distortion During the Flagellar Beat of Chlamydomonas Charles B. Lindemann1* and David R. Mitchell2 1 Department of Biological Sciences, Oakland University, Rochester, Michigan 2 undergoes distortion during the course of the beat cycle. The rapid fixation method employed by Mitchell

Lindemann, Charles


Calmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus.  


Studies of flagellar motility in Chlamydomonas mutants lacking specific central apparatus components have supported the hypothesis that the inherent asymmetry of this structure provides important spatial cues for asymmetric regulation of dynein activity. These studies have also suggested that specific projections associated with the C1 and C2 central tubules make unique contributions to modulating motility; yet, we still do not know the identities of most polypeptides associated with the central tubules. To identify components of the C1a projection, we took an immunoprecipitation approach using antibodies generated against PF6. The pf6 mutant lacks the C1a projection and possesses flagella that only twitch; calcium-induced modulation of dynein activity on specific doublet microtubules is also defective in pf6 axonemes. Our antibodies specifically precipitated five polypeptides in addition to PF6. Using mass spectrometry, we determined the amino acid identities of these five polypeptides. Most notably, the PF6-containing complex includes calmodulin. Using antibodies generated against each precipitated polypeptide, we confirmed that these polypeptides comprise a single complex with PF6, and we identified specific binding partners for each member of the complex. The finding of a calmodulin-containing complex as an asymmetrically assembled component of the central apparatus implicates the central apparatus in calcium modulation of flagellar waveform. PMID:16188941

Wargo, Matthew J; Dymek, Erin E; Smith, Elizabeth F



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

Microsoft Academic Search

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

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



Three distinct inner dynein arms in Chlamydomonas flagella: molecular composition and location in the axoneme  

Microsoft Academic Search

The molecular composition and organiza- tion of the row of axonemal inner dynein arms were investigated by biochemical and electron microscopic analyses of Chlamydomonas wild-type and mutant axo- nemes. Three inner arm structures could be distin- guished on the basis of their molecular composition and position in the axoneme as determined by analysis of pf30 and pf23 mutants. The three

Gianni Piperno; Zenta Ramanis; Elizabeth E Smith; Winfield S. Sale



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

Microsoft Academic Search

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

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



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


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

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



Microtubules in root hairs.  


The microtubules of root hairs of Raphanus sativus, Lepidium sativum, Equisetum hyemale, Limnobium stoloniferum, Ceratopteris thalictroides, Allium sativum and Urtica dioica were investigated using immunofluorescence and electron microscopy. Arrays of cortical microtubules were observed in all hairs. The microtubules in the hairs show net axial orientations, but in Allium and Urtica helical microtubule patterns are also present. Numerical parameters of microtubules in Raphanus, Equisetum and Limnobium were determined from dry-cleave preparations. The results are discussed with respect to cell wall deposition and cell morphogenesis. PMID:4066793

Traas, J A; Braat, P; Emons, A M; Meekes, H; Derksen, J



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


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



Analysis of the central pair microtubule complex in Chlamydomonas reinhardtii.  


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



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

PubMed Central

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

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



Microtubules, Tubulins and Associated Proteins.  

ERIC Educational Resources Information Center

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

Raxworthy, Michael J.



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

Microsoft Academic Search

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

Charles B. Lindemann



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

Microsoft Academic Search

The fine structure, protein composition, and roles in flagellar movement of specific axonemal components were studied in wild-type Chlamydomonas and paralyzed mutants pf-14, pf-15A, and pf-19. Electron microscope examination of the isolated axoneme of pf-14 showed that it lacks the radial spokes but is otherwise structurally normal. Comparison of isolated axonemes of wild type and pf-14 by sodium dodecyl sulfate-acrylamide

George B. Witman; J. Plummer; Greta Sander



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


Microsporidian extrasporular microtubules  

Microsoft Academic Search

Nosema michaelis spores possess a biologically unique external arrangement of microtubules. The microtubules were studied ultrastructurally with the use of thin sections, freeze-etched and negatively stained preparations. Tubules were sheared from the spores by repetitive freeze-thaw cycles and harvested by high speed centrifugation. Electron microscopic observations of negatively stained and freeze-etched samples revealed the globular subunit construction of the tubules.

Dennis M. Dwyer; Earl Weidner



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

PubMed Central

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



Parity Doublets in Quark Physics  

SciTech Connect

There are numerous examples of very nearly degenerate states of opposite parity in molecular physics. The ammonia maser is based on one such doublet.Theory shows that these parity doublets can occur if the nuclear shape in the molecule is reflection asymmetric because the time scales of the shape and the electronic cloud are well separated. Parity doublets occur in nuclear physics as well for odd A{approximately}219{endash}229. We discuss the theoretical foundation of these doublets and on that basis suggest that parity doublets should occur in particle physics too.In particular they should occur among baryons composed of cbu and cbd quarks. {copyright} {ital 1996} {ital The American Physical Society}

Balachandran, A.; Vaidya, S. [Department of Physics, Syracuse University, Syracuse, New York 13244-1130 (United States)] [Department of Physics, Syracuse University, Syracuse, New York 13244-1130 (United States)



Dark Matter with Two Inert Doublets plus One Higgs Doublet  

E-print Network

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

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



Do prokaryotes contain microtubules?  

NASA Technical Reports Server (NTRS)

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

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



Evidence for axonemal distortion during the flagellar beat ofChlamydomonas  

Microsoft Academic Search

In order to understand the working mechanism that governs the flagellar beat it is essential to know if the axoneme undergoes distortion during the course of the beat cycle. The rapid fixation method employed by Mitchell was able to preserve the waveform of Chlamydomonas flagella much as it appears during normal flag- ellar beating (Mitchell, Cell Motil Cytoskeleton 2003;56:120-129). This

Charles B. Lindemann; David R. Mitchell



Reactivation of outer-arm-depleted lung axonemes: evidence for functional differences between inner and outer dynein arms in situ.  


Demembranated axonemes isolated from newt lung ciliated cells show a complex beat frequency response to varying [MgATP] and temperature [Hard and Cypher, 1992, Cell Motil. Cytoskeleton 21:187-198]. The present study was undertaken to ascertain whether the beat frequency of outer-arm-depleted newt lung axonemes is controlled in a manner similar to that of intact axonemes. Populations of demembranated ciliary axonemes were isolated by Triton X-100 extraction of lungs from the newt, Taricha granulosa. Aliquots of the demembranated axonemes were further treated with solutions containing high salt (0.375 M KC1) and 1.25 mM MgATP. This treatment resulted in the selective removal of outer dynein arms and a concomitant decrease in beat frequency to a stable level, 33-35% of control values. The effects of pH, salt concentration, nucleotides, and temperature on the beat frequency of reactivated outer-arm-depleted axonemes were ascertained and compared with those of intact axonemes. Some reactivation properties, such as nucleotide specificity, the effect of pH on beat frequency and the threshold [MgATP] required for reactivation (approximately 5 microM) were similar to those observed for intact axonemes. Other properties, such as the relationship between beat frequency and varying [MgATP] or salt concentration, differed both qualitatively and quantitatively from those of control axonemes, as did their response to temperature over the range, 5 degrees-32 degrees C. The nature of the results obtained with temperature and MgATP suggests that inner and outer dynein arms are not functionally equivalent in situ. PMID:1533820

Hard, R; Blaustein, K; Scarcello, L



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 PMID:9201709

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



SPIRAL2 determines plant microtubule organization by modulating microtubule severing.  


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



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’. PMID:23255504

Antony, Dinu; Becker-Heck, Anita; Zariwala, Maimoona A; Schmidts, Miriam; Onoufriadis, Alexandros; Forouhan, Mitra; Wilson, Robert; Taylor-Cox, Theresa; Dewar, Ann; Jackson, Claire; Goggin, Patricia; Loges, Niki T; Olbrich, Heike; Jaspers, Martine; Jorissen, Mark; Leigh, Margaret W; Wolf, Whitney E; Daniels, M. Leigh Anne; Noone, 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



Microtubules and the tax payer.  


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

Nick, Peter



The microtubule transistor  

E-print Network

I point out the similarity between the microtubule experiment reported by Priel et al [Biophys. J. 90, 4639 (2006)] and the ZnO nanowire experiment of Wang et al [Nanolett. 6, 2768 (2006)]. It is quite possible that MTs are similar to a piezoelectric field effect transistor for which the role of the control gate electrode is played by the piezo-induced electric field across the width of the MT walls and their elastic bending features

H. C. Rosu



RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins  

PubMed Central

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively-spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of photoreceptor connecting cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, Structural Maintenance of Chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain (RLD) of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal photoreceptors and MDCK cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150Glued and p50-dynamitin. Inhibition of dynein function by over-expressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia. PMID:16043481

Khanna, Hemant; Hurd, Toby W.; Lillo, Concepcion; Shu, Xinhua; Parapuram, Sunil K.; He, Shirley; Akimoto, Masayuki; Wright, Alan F.; Margolis, Ben; Williams, David S.; Swaroop, Anand



Physical Modeling of Microtubules Network  

NASA Astrophysics Data System (ADS)

Microtubules (MT) are highly dynamic tubulin polymers that are involved in many cellular processes such as mitosis, intracellular cell organization and vesicular transport. Nevertheless, the modeling of cytoskeleton and MT dynamics based on physical properties is difficult to achieve. Using the Euler-Bernoulli beam theory, we propose to model the rigidity of microtubules on a physical basis using forces, mass and acceleration. In addition, we link microtubules growth and shrinkage to the presence of molecules (e.g. GTP-tubulin) in the cytosol. The overall model enables linking cytosol to microtubules dynamics in a constant state space thus allowing usage of data assimilation techniques.

Allain, Pierre; Kervrann, Charles



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

SciTech Connect

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

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



Molecular mechanisms of kinetochore microtubule attachment  

E-print Network

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

Schmidt, Jens C. (Jens Christopher)



Anomalous flexural behaviors of microtubules.  


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

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



Stochastic simulation of processive and oscillatory sliding using a two-headed model for axonemal dynein.  


Computer simulations have been used in an attempt to understand experimental observations of processive and oscillatory sliding by one or a few axonemal dyneins. A simple two-headed model has been examined using stochastic simulation methods. To explain the experimental observations, the model must be capable of taking backward steps, as well as forward steps, and there must be hysteresis in switching between forward or backward stepping. When the effects of Brownian movement on motor strain are included, it is not possible to obtain oscillations as regular as the experimental records by using motor strain to regulate switching between forward or backward stepping. PMID:11013391

Brokaw, C J



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


To determine mechanisms of assembly of ciliary dyneins, we focused on the Chlamydomonas inner dynein arm, I1 dynein, also known as dynein f. I1 dynein assembles in the cytoplasm as a 20S complex similar to the 20S I1 dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 dynein preassembly in the cytoplasm. Unlike I1 dynein derived from the axoneme, the cytoplasmic 20S I1 complex will not rebind I1-deficient axonemes in vitro. To test the hypothesis that I1 dynein is transported to the distal tip of the cilia for assembly in the axoneme, we performed cytoplasmic complementation in dikaryons formed between wild-type and I1 dynein mutant cells. Rescue of I1 dynein assembly in mutant cilia occurred first at the distal tip and then proceeded toward the proximal axoneme. Notably, in contrast to other combinations, I1 dynein assembly was significantly delayed in dikaryons formed between ida7 and ida3. Furthermore, rescue of I1 dynein assembly required new protein synthesis in the ida7 × ida3 dikaryons. On the basis of the additional observations, we postulate that IDA3 is required for 20S I1 dynein transport. Cytoplasmic complementation in dikaryons using the conditional kinesin-2 mutant, fla10-1 revealed that transport of I1 dynein is dependent on kinesin-2 activity. Thus, I1 dynein complex assembly depends upon IFT for transport to the ciliary distal tip prior to docking in the axoneme. © 2014 Wiley Periodicals, Inc. PMID:25252184

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



Purification, Cloning, and Sequence Analysis of a Mr 30,000 Protein from Sea Urchin Axonemes That Is Important for  

E-print Network

- lation of flagellar motility. The monoclonal antibody D405-14 inhibited the motility of demembranated of Chlamydomonas reinhardtii. Taken together, these re- sults suggest that we have identified a p28 light chain homolog in sea urchin sperm axoneme and that this protein may play a dynamic role in flagellar motility

Villefranche sur mer


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

PubMed Central

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

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



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

Microsoft Academic Search

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

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



Microtubule defects & Neurodegeneration  

PubMed Central

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

Baird, Fiona J.; Bennett, Craig L



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

PubMed Central

Two related enzymes, katanin and spastin, use the energy from ATP hydrolysis to sever microtubules. Two new studies (one in this issue; see McNally et al., p. 881) show that microtubule severing by katanin provides a means for increasing microtubule density in meiotic spindles. Interestingly, loss of spastin leads to a sparser microtubule array in axons and synaptic boutons. Together, these studies hint at a wider role for microtubule-severing enzymes in the formation and organization of noncentrosomal microtubule arrays by generating new seeds for microtubule growth. PMID:17178905

Roll-Mecak, Antonina; Vale, Ronald D.



Reconstitution of flagellar sliding.  


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

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



Tau Protein Diffuses along the Microtubule Lattice*  

PubMed Central

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

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



Achromatic doublets using group indices of refraction  

NASA Astrophysics Data System (ADS)

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

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



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

Microsoft Academic Search

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

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



Localized modulated waves in microtubules.  


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



Optically resolving individual microtubules in live axons.  


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 microm 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. PMID:19913478

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



Dark Two Higgs Doublet Model  

SciTech Connect

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

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



Inert doublet model and LEP II limits  

SciTech Connect

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

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



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

PubMed Central

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

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



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


Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ-ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo-electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility. PMID:22740634

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



The CSC connects three major axonemal complexes involved in dynein regulation  

PubMed Central

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ­ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility. PMID:22740634

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



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


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



Microtubule assembly in meiotic extract requires glycogen  

PubMed Central

The assembly of microtubules during mitosis requires many identified components, such as ?-tubulin ring complex (?-TuRC), components of the Ran pathway (e.g., TPX2, HuRP, and Rae1), and XMAP215/chTOG. However, it is far from clear how these factors function together or whether more factors exist. In this study, we used biochemistry to attempt to identify active microtubule nucleation protein complexes from Xenopus meiotic egg extracts. Unexpectedly, we found both microtubule assembly and bipolar spindle assembly required glycogen, which acted both as a crowding agent and as metabolic source glucose. By also reconstituting microtubule assembly in clarified extracts, we showed microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly. PMID:21737678

Groen, Aaron C.; Coughlin, Margaret; Mitchison, Timothy J.



Microtubule Interaction Site of the Kinesin Motor  

Microsoft Academic Search

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

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



Flexural Rigidity of a Single Microtubule  

Microsoft Academic Search

Microtubules, which are flexible biopolymers, can be used for nanotechnology applications (e.g., nano-actuator) as they have a rigidity similar to that of plexyglass and other plastic materials. The flexural rigidity, or bending stiffness, of microtubules was measured using a laser trapping technique and dark-field microscopy. One end of a microtubule rod was chemically bound to a glass microsphere, while the

Toru Takasone; Saulius Juodkazis; Yuji Kawagishi; Akira Yamaguchi; Shigeki Matsuo; Hitoshi Sakakibara; Haruto Nakayama; Hiroaki Misawa



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

E-print Network

, such as the biflagellate green alga Chlamydomonas reinhardtii, the interchangeability of the flagellar basal body and flagella and is one of the most iconic structures in cell biology. The origin of the eukaryotic flagellum and the mitotic centriole is evident2,3 . The importance of flagellar biology in sexual development is nicely

Schnaufer, Achim


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. 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.



Microtubule architecture: inspiration for novel carbon nanotube-based  

E-print Network

Microtubule architecture: inspiration for novel carbon nanotube-based biomimetic materials for cell motility, cell division and intra- cellular trafficking. Microtubules have outstanding mechanical that elucidate the relationship between molecular architecture and mechanics in microtubules and examine

Texas at Austin. University of


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.



Radiation shielding design considerations for Doublet III  

SciTech Connect

Calculations and measurements were made of the bremsstrahlung (x-ray) doses resulting from runaway electron shots at Doublet III. The analysis considered direct, wall-scattered, and skyshine contributions. Reasonably good agreement was obtained between calculations and measurements. The x-ray dose in the control room was about 1 mR per runaway shot, while that at the north boundary was undetectable, with a calculated value of 0.05 mR per shot. These low doses attest to the adequacy of the 2 ft concrete shadow shield surrounding the Doublet III room. Exploratory shielding analyses were performed for possible neutron generation if Doublet III were operated with neutral beam injection in an aggressive D-D mode.

Engholm, B.A.



Higgs phenomenology in the Stealth Doublet Model  

E-print Network

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

Rikard Enberg; Johan Rathsman; Glenn Wouda



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

PubMed Central

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

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




PubMed Central

In the first of two companion papers which attempt to correlate microtubules and their nucleating sites with developmental and cell division patterns in the unicellular flagellate, Ochromonas, the distribution of cytoplasmic and mitotic microtubules and various kinetosome-related fibers are detailed. Of the five kinetosome-related fibers, which have been found in Ochromonas, two, the kineto-beak fibers and the rhizoplast fibers are utilized as attachment sites for distinct groups of microtubules. The set of microtubules attached to the kineto-beak fibers apparently shape the anterior beak region of the cell whereas the rhizoplast microtubules appear to extend into and shape the tail in vegetative cells. In mitotic cells a rhizoplast is found at each spindle pole apparently serving as foci for the spindle microtubules. These findings are discussed in relation to the less well defined attachment sites for vegetative and mitotic microtubules in other kinds of cells. It is noted that the effects of depolymerizing microtubules in vivo might be easily quantitated in whole populations since no external wall or pellicle contributes to the maintenance or the biogenesis of the characteristic cell form of Ochromonas. PMID:4682900

Bouck, G. Benjamin; Brown, David L.



Interaction of Actin Filaments with Microtubules Is Mediated by  

E-print Network

Interaction of Actin Filaments with Microtubules Is Mediated by Microtubule-Associated Proteins At the present time, the best evidence for interactions between actin filaments and microtubules comes from biophysical experiments with purified actin and microtubule proteins.'" In cells, actin filaments


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)



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


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

Atkinson, Samantha; Kirik, Angela; Kirik, Viktor



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

PubMed Central

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

Atkinson, Samantha; Kirik, Angela; Kirik, Viktor



Doublet III beamline: as-built  

SciTech Connect

In order to fully exploit Doublet III capabilities and to study new plasma physics regimes, a Neutral Beam Injector System has been constructed. Initially, a two beamline system will supply 7 MW of heat to the plasma. The system is currently being expanded to inject approx. 20 MW of power (6 beamlines). Each beamline is equipped with two Lawrence Berkeley Laboratory type rectangular ion sources with 10 cm x 40 cm extraction grids. These sources will accelerate hydrogen ions to 80 keV, with extracted beam currents in excess of 80 A per source expected. The first completed source is currently being tested and conditioned on the High Voltage Test Stand at Lawrence Livermore Laboratory. This paper pictorially reviews the as-built Doublet III neutral beamline with emphasis on component relation and configuration relative to spatial and source imposed design constraints.

Harder, C.R.; Holland, M.M.; Parker, J.W.; Gunn, J.; Resnick, L.



Dark Matter from the Inert Doublet Model  

E-print Network

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

Laura Lopez Honorez



Microtubules Coordinate VEGFR2 Signaling and Sorting  

PubMed Central

VEGF signaling is a key regulator of vessel formation and function. In vascular endothelial cells, this signaling is mediated through its cognate receptor VEGFR2, which is dynamically sorted in response to ligand. Little is known about the underlying mechanism of this intracellular sorting. Here we examined the role of different components of the cytoskeleton in this process. We found that VEGFR2 mainly associates with microtubule fibers and to a lesser extent with intermediate filaments and actin. Microtubule disruption leads to accumulation of VEGFR2 protein in the membrane and cytoplasm leading to defects in VEGF signaling. In contrast, inhibition of actin filaments results in no accumulation of VEGFR2 total protein or apparent changes in microtubule association. Instead, actin inhibition leads to a more global signaling disruption of the ERK1/2 pathway. This is the first report demonstrating that VEGFR2 associates closely with microtubules in modulating the subcellular sorting and signaling of VEGFR2. PMID:24073279

Czeisler, Catherine; Mikawa, Takashi



Microtubule organization: cell shape is destiny.  


A simple self-assembly pathway generates cytoplasmic microtubule bundles that can locate the cell center and guide spindle assembly in fission yeast. The cylindrical cell shape automatically corrects spindle orientation errors, rendering a checkpoint unnecessary. PMID:17407755

Haase, Steven B; Lew, Daniel J



AMPK attenuates microtubule proliferation in cardiac hypertrophy.  


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

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



The role of dynamic instability in microtubule organization  

PubMed Central

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

Horio, Tetsuya; Murata, Takashi



Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo  

PubMed Central

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

Oladipo, Abiola; Cowan, Ann



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


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

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



Observation of microtubules with scanning force microscopy in liquid  

NASA Astrophysics Data System (ADS)

We present the application of scanning force microscopy using the jumping mode to investigate microtubules adsorbed to glass in air and in liquid. To fix the microtubules the glass surfaces were silanized with aminopropyl-triethoxy-silane. The observed structures ranged from disrupted microtubules in air to intact microtubules in liquid. Intact microtubules show heights between 20 and 24 nm confirming the diameter found in electron microscopy studies. The force applied by the tip was critical for the microtubule height, indicating deformation by the tip. Internal structure, corresponding to protofilaments, was found.

de Pablo, P. J.; Schaap, I. A. T.; Schmidt, C. F.



Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.  

PubMed Central

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis. Images PMID:8019007

Sawin, K E; Mitchison, T J



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. PMID:1629239



Preparation of segmented microtubules to study motions driven by the disassembling microtubule ends.  


Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion. PMID:24686554

Volkov, Vladimir A; Zaytsev, Anatoly V; Grishchuk, Ekaterina L



Spermatozoon cytoarchitecture of Amphilina foliacea (Platyhelminthes, Amphilinidea).  


The mature spermatozoon of Amphilina foliacea Rudolphi, 1819 has been examined using transmission electron microscopy. The male gamete is filiform and tapered at both extremities. Its moderately electron-dense cytoplasm possesses two parallel axonemes of unequal lengths with the 9?+?"1" trepaxonematan pattern, a mitochondrion, a nucleus, parallel cortical microtubules, four electron-dense attachment zones, and electron-dense glycogen granules. A crested body is absent. The anterior extremity of the cell exhibits a single axoneme. The anteriormost cortical microtubules have been observed with the appearance of the second axoneme. The number of cortical microtubules reaches a maximum (up to 25) in the nucleated region III of the spermatozoon. A single mitochondrion extends from the middle of region II to the end of region III of the cell. Both axonemes have become disorganized in a similar way: the axonemal doublets disappear first, followed by the central core. The nucleus is surrounded by a few cortical microtubules in the proximal part of region V. In the distal extremity of the mature spermatozoon, there is only the nucleus. Differences of spermatozoon ultrastructure within Amphilinidea and other Neodermata are discussed. PMID:22932939

Bru?anská, Magdaléna; Poddubnaya, Larisa G; Xylander, Willi E R



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



Multiple chiral doublet bands of identical configuration in 103Rh.  


Three sets of chiral doublet band structures have been identified in the ^{103}Rh nucleus. The properties of the observed chiral doublet bands are in good agreement with theoretical results obtained using constrained covariant density functional theory and particle rotor model calculations. Two of them belong to an identical configuration and provide the first experimental evidence for a novel type of multiple chiral doublets, where an "excited" chiral doublet of a configuration is seen together with the "yrast" one. This observation shows that the chiral geometry in nuclei can be robust against the increase of the intrinsic excitation energy. PMID:25083635

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



Reconstituting functional microtubule-barrier interactions.  


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



Contrasting models for kinetochore microtubule attachment in mammalian cells  

PubMed Central

Kinetochore function is mediated through its interaction with microtubule plus ends embedded in the kinetochore outer plate. Here, we compare and evaluate current models for kinetochore microtubule attachment, beginning with a brief review of the molecular, biochemical, cellular, and structural studies upon which these models are based. The majority of these studies strongly support a model in which the kinetochore outer plate is a network of fibers that form multiple weak attachments to each microtubule, chiefly through the Ndc80 complex. Multiple weak attachments enable kinetochores to remain attached to microtubule plus ends that are continually growing and shrinking. It is unlikely that rings or “kinetochore fibrils” have a significant role in kinetochore microtubule attachment, but such entities could have a role in stabilizing attachment, modifying microtubule dynamics, and harnessing the energy released from microtubule disassembly. It is currently unclear whether kinetochores control and coordinate the dynamics of individual kinetochore microtubules. PMID:20336345

Dong, Yimin



Targeting and transport: How microtubules control focal adhesion dynamics  

PubMed Central

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge. PMID:22908306

Stehbens, Samantha



Improved solution for the cemented doublet  

NASA Astrophysics Data System (ADS)

A method is described that permits the calculation of a cemented doublet with a given spherical aberration and coma at the edge of the lens. In particular the aberrations can be set to zero. Given one glass, the equations reported in this paper permit the determination of a second matching glass that minimizes the spherochromatism and coma of the lens. This result is obtained by the introduction, into the third-order thin-lens formulas, the third-order values of the aberration coefficients, as derived from the equation developed by Mossotti which yields zero finite aberrations for the same lens with added thickness. After a brief historical introduction, the third-order equations are developed and tables for the color-correcting glasses and SI and SII (the Seidel third-order coefficients) are given for objects at infinity and at a magnification of -1, both for flint- and crown-leading cases. The paper closes with a table of corrected doublets. Clairaut-Mossotti equation.

Szulc, Abraham



Microtubule-associated Protein-like Binding of the Kinesin-1 Tail to Microtubules*  

PubMed Central

The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail. PMID:20071331

Seeger, Mark A.; Rice, Sarah E.



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

E-print Network

complex pattern of polypeptides is observed in the 77,000 Mr region of the taxol-microtubule preparation (Fig. 7a,b). Three bands with Mr values of 78,000, 80,000 and 82,000, are observed in L. pictus micro- tubule preparations. The affinity-purified anti.... Suprenant and others Fig. 7. Evidence that the taxol MAP and 80,000 Mr spindle MAP from L. pictus are antigenically related to the 77,000 Mr cycle-purified MAP. SDS-PAGE of taxol microtubules from L. pictus (a) and cycle- purified microtubules from S...

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



Dielectric Measurement of Individual Microtubules Using the Electroorientation Method  

PubMed Central

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

Minoura, Itsushi; Muto, Etsuko



Association of Ebola Virus Matrix Protein VP40 with Microtubules  

Microsoft Academic Search

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola

Gordon Ruthel; Gretchen L. Demmin; George Kallstrom; Melodi P. Javid; Shirin S. Badie; Amy B. Will; Timothy Nelle; Rowena Schokman; Tam L. Nguyen; John H. Carra; Sina Bavari; M. Javad Aman



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


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




Microsoft Academic Search

QCD corrections to b ? s decay in the two Higgs doublet model are calculated from the energy scale of top quark to that of bottom. The constraints on the two Higgs doublet model from the new experimental bounds of b ? s by CLEO and the latest top quark mass by CDF and D0 are reanalyzed. It shows that

Cai-Dian Lu



Microsoft Academic Search

The subperineurial injection of \\/3#'-iminodipropionitrile (IDPN) into rat sciatic nerves resulted in focal disorganization of the axonal cytoskeleton characterized by segregation of neurofilaments and microtubules. Shortly after injection, microtubules clustered together to form a central channel, while neurofilaments became chaotically arrayed between the microtubule channel and axolemma. Electron microscopic autoradiography disclosed that rapidly transported organelles were preferen- tially associated with



Spag16, an axonemal central apparatus gene, encodes a male germ cell nuclear speckle protein that regulates SPAG16 mRNA expression.  


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. PMID:21655194

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



An axonemal dynein particularly important for flagellar movement at high viscosity. Implications from a new Chlamydomonas mutant deficient in the dynein heavy chain gene DHC9.  


Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 radial spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor. PMID:16236707

Yagi, Toshiki; Minoura, Itsushi; Fujiwara, Akiko; Saito, Ryo; Yasunaga, Takuo; Hirono, Masafumi; Kamiya, Ritsu



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

PubMed Central

Microtubules are nucleated in vivo by ?-tubulin complexes. The 300 kDa ?-tubulin small complex (?TuSC), consisting of two molecules of ?-tubulin and one copy each of the accessory proteins Spc97p and Spc98p, is the conserved, essential core of the microtubule nucleating machinery1,2. In metazoa multiple ?TuSCs assemble with other proteins into ?-tubulin ring complexes (?TuRCs). The structure of ?TuRC suggested that it functions as a microtubule template2–5. Because each ?TuSC contains two molecules of ?-tubulin, it was assumed that the ?TuRC-specific proteins are required to organize ?TuSCs to match thirteen-fold microtubule symmetry. Here, we show that ?TuSC forms rings even in the absence of other ?TuRC components. The yeast adaptor protein Spc110p stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8Å cryo-EM reconstruction of the filament reveals thirteen ?-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97p and Spc98p suggest functions for conserved sequence motifs, with implications for the ?TuRC-specific proteins. The ?TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator. PMID:20631709

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



Forces due to curving protofilaments in microtubules  

NASA Astrophysics Data System (ADS)

Microtubules consist of 13 protofilaments arranged in the form of a cylinder. The protofilaments are composed of longitudinally attached tubulin dimers that can exist in either a less curved state [GTP-bound tubulin (T)] or a more curved state [GDP-bound tubulin (D)]. Hydrolysis of T into D leaves the straight and laterally attached protofilaments of the microtubule in a mechanically stressed state, thus leading to their unzipping. The elastic energy in the unzipping protofilaments can be harnessed by a force transducer such as the Dam1-kinetochore ring complex in order to exert pulling force on chromosomes during cell division. In the present paper we develop a simple continuum model to obtain this pulling force as a function of the mechanical properties of protofilaments and the size of the Dam1-kinetochore ring. We also extend this model to investigate the role played by the T subunits found at the plus end of the microtubule (the T cap) on the mechanical stability of microtubules.

Vichare, Shirish; Jain, Ishutesh; Inamdar, Mandar M.; Padinhateeri, Ranjith



Isatin Derivatives as Inhibitors of Microtubule Assembly  

E-print Network

-bromobenzyl)isatin was the most active compound against MCF-7 cells, IC50 = 2.1 µM. To date the most cytotoxic compound tested is 5-methyl-N-(1-propyl)isatin, with an IC50 value of 52 nM (microtubule assembly IC50 = 2.6 µM) in the drug resistant cancer cell...

Beckman, Karen



Reconstituting dynamic microtubule polymerization regulation by TOG domain proteins.  


Microtubules (MTs) polymerize from soluble ??-tubulin and undergo rapid dynamic transitions to depolymerization at their ends. Microtubule-associated regulator proteins modulate polymerization dynamics in vivo by altering microtubule plus end conformations or influencing ??-tubulin incorporation rates. Biochemical reconstitution of dynamic MT polymerization can be visualized with total internal reflection fluorescence (TIRF) microscopy using purified MT regulators. This approach has provided extensive details on the regulation of microtubule dynamics. Here, I describe a general approach to reconstitute MT dynamic polymerization with TOG domain microtubule regulators from the XMAP215/Dis1 and CLASP families using TIRF microscopy. TIRF imaging strategies require nucleation of microtubule polymerization from surface-attached, stabilized MTs. The approaches described here can be used to study the mechanism of a wide variety of microtubule regulatory proteins. PMID:24630105

Al-Bassam, Jawdat



Spindle Assembly and Architecture: From Laser Ablation to Microtubule Nucleation  

NASA Astrophysics Data System (ADS)

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

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



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

Microsoft Academic Search

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

Robert L. Morris; Jonathan M. Scholey



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

PubMed Central

Microtubule plus-end-tracking proteins (+TIPs) localize to growing microtubule plus ends to regulate a multitude of essential microtubule functions. End-binding proteins (EBs) form the core of this network by recognizing a distinct structural feature transiently existing in an extended region at growing microtubule ends and by recruiting other +TIPs to this region. The nature of the conformational difference allowing EBs to discriminate between tubulins in this region and other potential tubulin binding sites farther away from the microtubule end is unknown. By combining in vitro reconstitution, multicolor total internal reflection fluorescence microscopy, and electron microscopy, we demonstrate here that a closed microtubule B lattice with incorporated GTP?S, a slowly hydrolyzable GTP analog, can mimic the natural EB protein binding site. Our findings indicate that the guanine nucleotide ?-phosphate binding site is crucial for determining the affinity of EBs for lattice-incorporated tubulin. This defines the molecular mechanism by which EBs recognize growing microtubule ends. PMID:21368119

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



Inner core differential motion confirmed by earthquake waveform doublets.  


We analyzed 18 high-quality waveform doublets with time separations of up to 35 years in the South Sandwich Islands region, for which the seismic signals have traversed the inner core as PKP(DF). The doublets show a consistent temporal change of travel times at up to 58 stations in and near Alaska, and they show a dissimilarity of PKP(DF) coda. Using waveform doublets avoids artifacts of earthquake mislocations and contamination from small-scale heterogeneities. Our results confirm that Earth's inner core is rotating faster than the mantle and crust at about 0.3 degrees to 0.5 degrees per year. PMID:16123296

Zhang, Jian; Song, Xiaodong; Li, Yingchun; Richards, Paul G; Sun, Xinlei; Waldhauser, Felix



[A functional flagella with a 6 + 0 pattern  

PubMed Central

The male gamete of the Gregarine Lecudina tuzetae has been studied with transmission electron microscopy and microcinematography. It is characterized by a flagellar axoneme of 6 + 0 pattern, a reduction of the chondriome, and the abundance of storage polysaccharide or lipid bodies. The movements of the flagella are of the undulating type and they are performed in the three dimensions of space. They are very slow, with a cycle time of about 2s. The structure of the axoneme components are similar to those of flagella with a 9 + 2 pattern. Each doublet has overall dimensions of 350 x 220 A; the space between the adjacent doublets is about 160 A. The A subfiber bears arms like dynein arms. The diameter of the axoneme is about 1,000 A. The basal body consists of a cylinder of dense material 2,500 A long and 1,300- 1,400 A in diameter; a microtubule 200 A in diameter is present in the axis. This study shows that a 6 + 0 pattern can generate a flagellar movement. The mechanism of the flagellar movement of the male gamete of L. tuzetae does not require the presence of central microtubules and it would include molecular interactions of the dynein-tubulin type between the adjacent peripheric doublets. The slowness of the movements is discussed in terms of the axoneme's structure and its energy supply. Finally, the phylogenetic significance of this flagella is examined on the basis of the morphopoietic potentialities of the centriolar structures. PMID:169268



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

PubMed Central

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

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



Pregnenolone binds to microtubule-associated protein 2 and stimulates microtubule assembly  

PubMed Central

Fetal or adult rat-brain cytosol and fetal rat-brain microtubules contain a high-affinity, low-capacity pregnenolone-binding protein. The equilibrium dissociation constant is in the 30–50 nM range. The best competitors (in decreasing order) are pregnenolone sulfate, progesterone, ?5-pregnene-3?,20?-diol, and 3?-hydroxy-5?-pregnan-20-one. It was hypothesized that the pregnenolone-binding protein pertained to microtubule-associated proteins (MAPs). Indeed, partial purification of fetal brain cytosol by fast pressure liquid chromatography with sequential ion-exchange and gel-filtration columns yielded two fractions, one of very high molecular mass, >200 kDa, and the other of 40–60 kDa, enriched in [3H]pregnenolone-binding activity and in proteins immunolabeled with monoclonal anti-tubulin and anti-MAP2 antibodies. Because many proteins are associated with microtubules, binding assays were repeated with purified calf-brain tubulin, MAP2, and Tau protein. Only the MAP2 fraction showed saturable [3H]pregnenolone binding with an affinity very close to that of rat-brain microtubules, but with a much larger concentration of binding sites (16 pmol/mg MAP2), which was increased more than 8-fold after copolymerization of MAP2 with tubulin. Finally, steroid effects on microtubule-assembly kinetics were assayed. Pregnenolone induced a large, dose-related increase of both the rate and extent of MAP2-induced tubulin assembly, whereas progesterone, inactive per se, counteracted the stimulatory effect of pregnenolone. Electron microscopic analysis confirmed that pregnenolone-increased assembly of microtubules produced a completely normal structure. The stimulatory effect on MAP2–tubulin interaction was also observed in fetal rat-brain neuron cultures. Therefore, we propose a mechanism of neurosteroid action, the control of microtubule or, more generally, of neural cytoskeleton dynamics, with potential roles in brain development, plasticity, and aging. PMID:10737804

Murakami, Koichi; Fellous, Arlette; Baulieu, Etienne-Emile; Robel, Paul



An assay to image neuronal microtubule dynamics in mice  

PubMed Central

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

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



Effects of dynein on microtubule mechanics and centrosome positioning  

PubMed Central

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

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



Effects of dynein on microtubule mechanics and centrosome positioning.  


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

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



Microtubule cortical array organization and plant cell morphogenesis.  


Plant cell cortical microtubule arrays attain a high degree of order without the benefit of an organizing center such as a centrosome. New assays for molecular behaviors in living cells and gene discovery are yielding insight into the mechanisms by which acentrosomal microtubule arrays are created and organized, and how microtubule organization functions to modify cell form by regulating cellulose deposition. Surprising and potentially important behaviors of cortical microtubules include nucleation from the walls of established microtubules, and treadmilling-driven motility leading to polymer interaction, reorientation, and microtubule bundling. These behaviors suggest activities that can act to increase or decrease the local level of order in the array. The SPIRAL1 (SPR1) and SPR2 microtubule-localized proteins and the radial swollen 6 (rsw-6) locus are examples of new molecules and genes that affect both microtubule array organization and cell growth pattern. Functional tagging of cellulose synthase has now allowed the dynamic relationship between cortical microtubules and the cell-wall-synthesizing machinery to be visualized, providing direct evidence that cortical microtubules can organize cellulose synthase complexes and guide their movement through the plasma membrane as they create the cell wall. PMID:17010658

Paradez, Alex; Wright, Amanda; Ehrhardt, David W



Microtubule-associated proteins of neurons  

PubMed Central

Microtubule-associated proteins (MAP) have been identified in cultures of rat sympathetic neurons. In all of the experiments performed here, the cultures consisted of greater than 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 detected in cytoskeletons prepared from cultures depleted of microtubules by treatment with podophyllotoxin, and (d) co-cycled with rat brain microtubule proteins. We conclude that these 26 proteins are associated with microtubules in sympathetic neurons. Two of these proteins have molecular weights of approximately 30,000 and isoelectric points of approximately 6.2; the rest of the proteins range in molecular weight from 60,000 to 76,000 and isoelectric point from 6.3 to 6.9. This latter group of MAPs was heat labile. Several other proteins in the neuronal cultures had the solubility properties and drug-lability expected of MAP. All of these proteins had apparent molecular weights greater than 200,000; one of these putative MAP co-migrated with rat brain MAP-1. We did not detect any putative MAP in these cultures that co-migrated with rat brain MAP-2. In isoelectric focusing-SDS PAGE, the 24 MAP with molecular weights of 60,000-76,000 appeared to comprise four distinct molecular weight classes. Each molecular weight class was in turn composed of several proteins that varied in isoelectric point. In peptide mapping experiments, the isoelectric variants of each molecular weight class gave rise to very similar peptide maps. These observations suggest that each molecular weight class consists of several closely related proteins. It was also determined that all except the most basic member of the four MAP classes could be phosphorylated in vivo, raising the possibility that differential phosphorylation contributed to the variation in the isoelectric points of the members of each MAP class. We performed pulse-chase experiments to further evaluate the contribution of posttranslational modification to the generation of the complex population of MAP in the molecular weight range of 60,000 to 76,000. In cultures labeled for 20 min, only the more basic members of each MAP class were detectably labeled, while in cultures labeled for 20 min and then chased for 220 min the more acidic members of the MAP classes became labeled.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:6619184



A Supersymmetric One Higgs Doublet Model  

E-print Network

We present a supersymmetric extension of the Standard Model in which only one electroweak doublet acquires a vacuum expectation value and gives mass to Standard Model fermions. As well as the novel accommodation of a Standard Model Higgs within a supersymmetric framework, this leads to a very predictive model, with some advantages over the MSSM. In particular, problems with proton decay, flavour changing neutral currents and large CP violation are ameliorated, primarily due to the presence of an anomaly-free R-symmetry. Since supersymmetry must be broken at a low scale, gravity-mediated effects which break the R-symmetry are naturally small. The R-symmetry requires the presence of adjoint chiral superfields, to give Dirac masses to the gauginos; these adjoints are the only non-MSSM fields in the visible sector. The LSP is a very light neutralino, which is mostly bino. Such a light neutralino is not in conflict with experiment, and is a striking prediction of the minimal model. Additional scenarios to raise the mass of this neutralino to the weak scale are also outlined. Prospects for discovery at the LHC are briefly discussed, along with viable scenarios for achieving gauge-coupling unification.

Rhys Davies; John March-Russell; Matthew McCullough



Profile of Two-Higgs-Doublet-Model Parameter Space  

E-print Network

We review recent work on constraining the parameter space of the Two-Higgs-Doublet Model by theoretical and experimental results. Some characteristics of the model, in particular the distribution of masses in the surviving parameter space, are discussed.

Abdul Wahab El Kaffas; Odd Magne Ogreid; Per Osland



Dinitroaniline herbicide resistance and the microtubule cytoskeleton.  


Dinitroaniline herbicides have been used for pre-emergence weed control for the past 25 years in cotton, soybean, wheat and oilseed crops. Considering their long persistence and extensive use, resistance to dinitroanilines is fairly rare. However, the most widespread dinitroaniline-resistant weeds, the highly resistant (R) and the intermediate (I) biotypes of the invasive goosegrass Eleusine indica, are now infesting more than 1000 cotton fields in the southern states of the USA. The molecular basis of this resistance has been identified, and found to be a point mutation in a major microtubule cytoskeletal protein, alpha-tubulin. These studies have served both to explain the establishment of resistance and to reveal fundamental properties of tubulin gene expression and microtubule structure. PMID:10322543

Anthony; Hussey



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)



Beam-correcting holographic doublet for focusing multimode laser diodes  

Microsoft Academic Search

We design and fabricate a beam-correcting, holographic focusing doublet for laser diodes in the presence of a recording-to-readout wavelength shift. This two-hologram assembly compensates for the strong chromatic variation of the spot size and its lateral position, which are typical of a single diffractive element. The doublet, recorded at 488 nm, successfully corrects the astigmatism of a multimode laser diode

A. Aharoni; J. W. Goodman; Y. Amitai



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

Microsoft Academic Search

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

Pinfen Yang; Winfield S. Sale



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. PMID:21381049

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



Manipulation of Microtubules and Actin by Dielectrophoresis  

NASA Astrophysics Data System (ADS)

Myosin and kinesin are ATP powered motor proteins which process along cytoskeletal filaments (actin and microtubules respectively). These motor proteins, which have a motor domain and a payload carrying head domain, are used to transport material within a cell. The filaments possess a structural polarity which dictates the direction of motion of the associated motor protein. By laying down tracks of microtubules or actin, motor protein / filament systems could be utilized in nanodevices for molecular transport. In order for these systems to be useful as nanomechanisms, the transport of material by motor proteins must result in a net flux of material in a specified direction. For maximum efficiency this necessitates that the filaments be aligned parallel, and with a coherent structural polarity orientation. Dielectrophoresis, which utilizes the polarizability of a molecule in an AC field gradient, has been shown to be effective in manipulating biopolymers. In this study, AC electrokinetic manipulation is used to align actin filaments and microtubules. The AC field gradient is achieved using a 2-dimensional quadrapole microelectrode made from indium tin oxide (ITO) deposited on a glass substrate. Since ITO is transparent, the filaments can be manipulated and simultaneously imaged using differential interference contrast (DIC) or fluorescence microscopy.

Meehan, Timothy



Association of ebola virus matrix protein VP40 with microtubules.  


Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus. PMID:15795257

Ruthel, Gordon; Demmin, Gretchen L; Kallstrom, George; Javid, Melodi P; Badie, Shirin S; Will, Amy B; Nelle, Timothy; Schokman, Rowena; Nguyen, Tam L; Carra, John H; Bavari, Sina; Aman, M Javad



The Human Kinetochore Ska1 Complex Facilitates Microtubule Depolymerization-Coupled Motility  

E-print Network

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

Cheeseman, Iain McPherson


Nat Rev Drug Discov . Author manuscript Microtubule-binding agents: a dynamic field of cancer therapeutics  

E-print Network

for decades, and until the advent of targeted therapy microtubules were the only alternative to DNA microtubule binding agents. MESH Keywords Animals ; Antineoplastic Agents ; chemistry ; metabolism therapy ; metabolism ; Protein Binding ; physiology Introduction Microtubules play several key roles

Paris-Sud XI, Université de


?-Tubulin controls neuronal microtubule polarity independently of Golgi outposts  

PubMed Central

Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by ?-tubulin might contribute to polarity, we analyzed neuronal microtubules in Drosophila containing gain- or loss-of-function alleles of ?-tubulin. Both increased and decreased activity of ?-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, we examined the distribution of ?-tubulin. Consistent with local nucleation, tagged and endogenous ?-tubulins were found in specific positions in dendrites and axons. Because the Golgi complex can house nucleation sites, we explored whether microtubule nucleation might occur at dendritic Golgi outposts. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when we dragged the Golgi out of dendrites with an activated kinesin, ?-tubulin remained in dendrites. We conclude that regulated microtubule nucleation controls neuronal microtubule polarity but that the Golgi complex is not directly involved in housing nucleation sites. PMID:24807906

Nguyen, Michelle M.; McCracken, Christie J.; Milner, E. S.; Goetschius, Daniel J.; Weiner, Alexis T.; Long, Melissa K.; Michael, Nick L.; Munro, Sean; Rolls, Melissa M.



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. PMID:14734535

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



Microtubule-binding agents: a dynamic field of cancer therapeutics  

PubMed Central

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

Dumontet, Charles; Jordan, Mary Ann



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


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

Eng, Ryan Christopher; Wasteneys, Geoffrey O



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



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

PubMed Central

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

Eng, Ryan Christopher; Wasteneys, Geoffrey O.



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

E-print Network

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

Backer, Chelsea B.


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

PubMed Central

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

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



A new perspective on microtubules and axon growth  

Microsoft Academic Search

role of the cell body in generating the axonal microtubule ar- ray, whereas the other emphasizes the role of local mecha- nisms within the axon itself. Recent work suggests that events significant to the generation of the axonal microtubule array occur within both of these compartments, and that axon growth is dependent upon the coordinated efforts of several types of

Harish C. Joshi; Peter W. Baas



NMDA receptor activation suppresses microtubule growth and spine entry.  


Dynamic microtubules are important to maintain neuronal morphology and function, but whether neuronal activity affects the organization of dynamic microtubules is unknown. Here, we show that a protocol to induce NMDA-dependent long-term depression (LTD) rapidly attenuates microtubule dynamics in primary rat hippocampal neurons, removing the microtubule-binding protein EB3 from the growing microtubule plus-ends in dendrites. This effect requires the entry of calcium and is mediated by activation of NR2B-containing NMDA-type glutamate receptor. The rapid NMDA effect is followed by a second, more prolonged response, during which EB3 accumulates along MAP2-positive microtubule bundles in the dendritic shaft. MAP2 is both required and sufficient for this activity-dependent redistribution of EB3. Importantly, NMDA receptor activation suppresses microtubule entry in dendritic spines, whereas overexpression of EB3-GFP prevents NMDA-induced spine shrinkage. These results suggest that short-lasting and long-lasting changes in dendritic microtubule dynamics are important determinants for NMDA-induced LTD. PMID:21632941

Kapitein, Lukas C; Yau, Kah Wai; Gouveia, Susana Montenegro; van der Zwan, Wouter A; Wulf, Phebe S; Keijzer, Nanda; Demmers, Jeroen; Jaworski, Jacek; Akhmanova, Anna; Hoogenraad, Casper C



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.



A new directionality tool for assessing microtubule pattern alterations  

PubMed Central

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

Liu, Wenhua; Ralston, Evelyn



Jupiter, a New Drosophila Protein Associated With Microtubules  

E-print Network

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

Villefranche sur mer


Microtubule distribution in gravitropic protonemata of the moss Ceratodon.  


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

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



INTRODUCTION Microtubules are required for a variety of cellular processes,  

E-print Network

by alternative mRNA splicing. Based on shared sequence similarities, two groups of structural MAPs can). Various lines of evidence have indicated that these structural MAPs are made of a carboxy-repeat isoform co-localises with microtubules and induces the formation of microtubule bundles, whereas its

Ahringe, Julie


Mathematics and biophysics of cortical microtubules in plants  

E-print Network

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

Allard, Jun


Microtubule Length Regulation by Molecular Motors  

NASA Astrophysics Data System (ADS)

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

Melbinger, Anna; Reese, Louis; Frey, Erwin



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


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; Samajová, Olga; Dosko?ilová, Anna; Ove?ka, Miroslav; Illés, Peter; Bartek, Jiri; Samaj, Jozef



The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts.  

PubMed Central

The Drosophila gene polo encodes a protein kinase required for progression through mitosis. Wild-type polo protein migrates as a tight doublet of 67 kDa which is converted to a single band by phosphatase treatment, which also inactivates the kinase. We have determined putative polo substrates in a cell-free system derived from mutant embryos. Exogenous polo protein kinase phosphorylates proteins of sizes 220 kDa, 85 kDa and 54 kDa, to a greater extent when added to extracts of polo(1)-derived embryos compared with extracts of wild-type embryos, which in both cases have been subject to mild heat treatment to inactivate endogenous kinases. Proteins of the same size are predominantly phosphorylated by the endogenous kinases present in wild-type extracts, and are either not phosphorylated or are poorly phosphorylated in extracts of polo(1)-derived embryos. We show that a specific monoclonal antibody to beta-tubulin precipitates the phosphorylated 54 kDa protein together with an associated 85 kDa protein also phosphorylated by polo protein kinase. Moreover polo binds to an 85 kDa protein which is enriched in microtubule preparations. We discuss the extent to which these in vitro phosphorylation results reflect the effects of mutations in polo on microtubule behaviour during the mitotic cycle. Images PMID:8890161

Tavares, A A; Glover, D M; Sunkel, C E



Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration.  


Little is known about which components of the axonal cytoskeleton might break during rapid mechanical deformation, such as occurs in traumatic brain injury. Here, we micropatterned neuronal cell cultures on silicone membranes to induce dynamic stretch exclusively of axon fascicles. After stretch, undulating distortions formed along the axons that gradually relaxed back to a straight orientation, demonstrating a delayed elastic response. Subsequently, swellings developed, leading to degeneration of almost all axons by 24 h. Stabilizing the microtubules with taxol maintained the undulating geometry after injury but greatly reduced axon degeneration. Conversely, destabilizing microtubules with nocodazole prevented undulations but greatly increased the rate of axon loss. Ultrastructural analyses of axons postinjury revealed immediate breakage and buckling of microtubules in axon undulations and progressive loss of microtubules. Collectively, these data suggest that dynamic stretch of axons induces direct mechanical failure at specific points along microtubules. This microtubule disorganization impedes normal relaxation of the axons, resulting in undulations. However, this physical damage also triggers progressive disassembly of the microtubules around the breakage points. While the disintegration of microtubules allows delayed recovery of the "normal" straight axon morphology, it comes at a great cost by interrupting axonal transport, leading to axonal swelling and degeneration. PMID:20019243

Tang-Schomer, Min D; Patel, Ankur R; Baas, Peter W; Smith, Douglas H



Singlet scalar Dark Matter in Dark Two Higgs Doublet Model  

E-print Network

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

Gaitan, R; de Oca, J H Montes



Singlet scalar Dark Matter in Dark Two Higgs Doublet Model  

E-print Network

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

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



Doubly Charged Lepton from an Exotic Doublet at the LHC  

E-print Network

We studied the signatures at the LHC of the electroweak SU(2) lepton doublet which embeds a doubly charged lepton. The doubly charged lepton pair and single production rates, which are different from the triplet case, are studied and all the detectable decay modes are considered. We also applied the same kinematic cuts as in triplet case to reduce the background at LHC and the needed integrated luminosities to find the doubly charged leptons with different masses are analyzed. We also suggested a method to distinguish the exotic lepton doublet and triplet.

Teng Ma; Bin Zhang; Giacomo Cacciapaglia



Phenomenology of models with more than two Higgs doublets  

NASA Astrophysics Data System (ADS)

We study the most general Multi-Higgs-Doublet Model (MHDM) with Natural Flavor Conservation (NFC). The couplings of a charged scalar Hi± to up quarks, down quarks and charged leptons depend on three new complex parameters, Xi, Yi, and Zi, respectively. We prove relations among these parameters. We carry out a comprehensive analysis of phenomenological constraints on the couplings of the lightest charged scalar: X, Y and Z. We find that the general MHDM may differ significantly from its minimal version, the Two-Higgs-Doublet Model (2HDM).

Grossman, Yuval



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

PubMed Central

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

Field, Jessica J.; Pera, Benet; Calvo, Enrique; Canales, Angeles; Zurwerra, Didier; Trigili, Chiara; 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



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

Microsoft Academic Search

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

R. C. Brown; B. E. Lemmon



Changes in Pattern of Microtubule Organization During Meiosis in Pollen Mother Cell of Autotetraploid Rice  

Microsoft Academic Search

Changes in microtubule distribution and chromosome behavior in autotetraploid rice during meiosis of pollen mother cell (PMC) were observed in detail using indirect-immunofluorescence laser scanning confocal microscopy. The result showed that microtubule distribution pattern in PMC of the autotetraploid rice was similar to that of the diploid rice during meiosis, but the microtubule behaviors were different, e.g., longer circumferential microtubules

Jin-Hua HE; Xing-An CHENG; Zhi-Xiong CHEN; Hai-Bin GUO; Xiang-Dong LIU; Yong-Gen LU



Dietary antioxidant curcumin inhibits microtubule assembly through tubulin binding.  


Curcumin, a component of turmeric, has potent antitumor activity against several tumor types. However, its molecular target and mechanism of antiproliferative activity are not clear. Here, we identified curcumin as a novel antimicrotubule agent. We have examined the effects of curcumin on cellular microtubules and on reconstituted microtubules in vitro. Curcumin inhibited HeLa and MCF-7 cell proliferation in a concentration-dependent manner with IC(50) of 13.8 +/- 0.7 microm and 12 +/- 0.6 microm, respectively. At higher inhibitory concentrations (> 10 microm), curcumin induced significant depolymerization of interphase microtubules and mitotic spindle microtubules of HeLa and MCF-7 cells. However, at low inhibitory concentrations there were minimal effects on cellular microtubules. It disrupted microtubule assembly in vitro, reduced GTPase activity, and induced tubulin aggregation. Curcumin bound to tubulin at a single site with a dissociation constant of 2.4 +/- 0.4 microm and the binding of curcumin to tubulin induced conformational changes in tubulin. Colchicine and podophyllotoxin partly inhibited the binding of curcumin to tubulin, while vinblastine had no effect on the curcumin-tubulin interactions. The data together suggested that curcumin may inhibit cancer cells proliferation by perturbing microtubule assembly dynamics and may be used to develop efficacious curcumin analogues for cancer chemotherapy. PMID:17069615

Gupta, Kamlesh K; Bharne, Shubhada S; Rathinasamy, Krishnan; Naik, Nishigandha R; Panda, Dulal



An improved quantitative analysis method for plant cortical microtubules.  


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

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



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.



Response of Modulated Doublet Modes to Travelling Wave Excitation  

Microsoft Academic Search

The forced vibration of a rotationally periodic structure when subjected to travelling wave excitation is discussed, with emphasis placed on the steady-state response of doublet modes having either repeated or split frequencies. Such vibration modes have spatially modulated shapes defined by (1) the number of nodal diameters present in the limiting case of axisymmetry, and (2) certain additional Fourier harmonics

J. Y. Chang; J. A. Wickert




Microsoft Academic Search

The forced vibration of a rotationally periodic structure when subjected to travelling wave excitation is discussed, with emphasis placed on the steady-state response of doublet modes having either repeated or split frequencies. Such vibration modes have spatially modulated shapes defined by (1) the number of nodal diameters present in the limiting case of axisymmetry, and (2) certain additional Fourier harmonics





E-print Network

LINEAR COLLIDER FINAL DOUBLET CONSIDERATIONS: ATF2 VIBRATION MEASUREMENTS* B. Bolzon# , N. Geffroy to measure medium frequency vibrations in vertical axis. First, ground motion was measured for 72 hours vibrations (with their supports) were measured to evaluate their rigidity. The correlation of QD0 and QF1

Paris-Sud XI, Université de


Origin and phenomenology of weak-doublet spin-1 bosons  

NASA Astrophysics Data System (ADS)

We study phenomenological consequences of the Standard Model extension by the new spin-1 fields with the internal quantum numbers of the electroweak Higgs doublets. We show, that there are at least three different classes of theories, all motivated by the hierarchy problem, which predict appearance of such vector weak-doublets not far from the weak scale. The common feature for all the models is the existence of an SU(3 gauge extension of the weak SU(2 group, which is broken down to the latter at some energy scale around TeV. The Higgs doublet then emerges as either a pseudo-Nambu-Goldstone boson of a global remnant of SU(3, or as a symmetry partner of the true eaten-up Goldstone boson. In the third class, the Higgs is a scalar component of a high-dimensional SU(3 gauge field. The common phenomenological feature of these theories is the existence of the electroweak doublet vectors (Z,W), which in contrast to well-known Z and W bosons posses only anomalous (magnetic moment type) couplings with ordinary light fermions. This fact leads to some unique signatures for their detection at the hadron colliders.

Chizhov, M. V.; Dvali, Gia



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

Microsoft Academic Search

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

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



Mobility, Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae of Ashbya gossypii  

PubMed Central

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 ?m/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 ?m/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae. PMID:19910487

Lang, Claudia; Grava, Sandrine; van den Hoorn, Tineke; Trimble, Rhonda; Philippsen, Peter



Filamentous microtubules in the neuronal spinous process and the role of microtubule regulatory drugs in neuropathic pain.  


Structural and regulatory components in the spinous processes neurons are still not fully understood. While the presence, regulation and function of actin cytoskeleton are now well established, involvement of microtubule cytoskeleton is only now being studied. In spite of biochemical, genetic and anatomical evidence, neurobiologists have experienced difficulties to even establish the physical presence and to demonstrate the functional relevance of "filamentous microtubules" in the spines. However, recent studies have demonstrated that filamentous microtubules regularly invade most spines. Moreover, functional aspects like stabilization and maturation of spines can be directly correlated with the presence of these microtubules; low doses of microtubule stabilizers can induce as many as 50% more spines. Clinically, knowledge of microtubule active drugs is extremely important, especially in the area of oncology, where they are commonly used as anti-neoplastic agents. Unfortunately, a major dose-limiting side effect of this class of drugs is a painful peripheral neuropathy. We review the recent concept of microtubules in the spine, and also critically analyze their role in chemotherapy-induced neuropathic pain. PMID:20643173

Goswami, Chandan; Goswami, Luna



Diffusion of dextran inside microtubule sample  

NASA Astrophysics Data System (ADS)

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

Prodan, Camelia



Rearrangement of Microtubule Polarity Orientation During Conversion of Dendrites to  

E-print Network

of inhibitors of actin poly- merization, suggesting that actin-dependent transport of microtubules of process, dendrites and a single axon. These two types of process differ in morphology, protein content

Baas, Peter W.


Nek4 Status Differentially Alters Sensitivity to Microtubule Poisons  

E-print Network

Microtubule poisons are widely used in cancer treatment, but the factors determining the relative efficacy of different drugs in this class remain obscure. In this study, we identified the NIMA kinase Nek4 in a genetic ...

Doles, Jason D.


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)



Kinesin-microtubule interactions during gliding assays under magnetic force  

NASA Astrophysics Data System (ADS)

Conventional kinesin is a motor protein capable of converting the chemical energy of ATP into mechanical work. In the cell, this is used to actively transport vesicles through the intracellular matrix. The relationship between the velocity of a single kinesin, as it works against an increasing opposing load, has been well studied. The relationship between the velocity of a cargo being moved by multiple kinesin motors against an opposing load has not been established. A major difficulty in determining the force-velocity relationship for multiple motors is determining the number of motors that are moving a cargo against an opposing load. Here I report on a novel method for detaching microtubules bound to a superparamagnetic bead from kinesin anchor points in an upside down gliding assay using a uniform magnetic field perpendicular to the direction of microtubule travel. The anchor points are presumably kinesin motors bound to the surface which microtubules are gliding over. Determining the distance between anchor points, d, allows the calculation of the average number of kinesins, n, that are moving a microtubule. It is possible to calculate the fraction of motors able to move microtubules as well, which is determined to be ˜ 5%. Using a uniform magnetic field parallel to the direction of microtubule travel, it is possible to impart a uniform magnetic field on a microtubule bound to a superparamagnetic bead. We are able to decrease the average velocity of microtubules driven by multiple kinesin motors moving against an opposing force. Using the average number of kinesins on a microtubule, we estimate that there are an average 2-7 kinesins acting against the opposing force. By fitting Gaussians to the smoothed distributions of microtubule velocities acting against an opposing force, multiple velocities are seen, presumably for n, n-1, n-2, etc motors acting together. When these velocities are scaled for the average number of motors on a microtubule, the force-velocity relationship for multiple motors follows the same trend as for one motor, supporting the hypothesis that multiple motors share the load.

Fallesen, Todd L.


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. PMID:21327409

Malcos, Jennelle L.



Evidence for Opposing Effects of Calmodulin on Cortical Microtubules.  

PubMed Central

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

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



Direct interaction of microtubule- and actin-based transport motors  

NASA Technical Reports Server (NTRS)

The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

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



Functional Elements within the Dynein Microtubule-binding Domain  

PubMed Central

Dynein interacts with microtubules through an ATP-sensitive linkage mapped to a structurally complex region of the heavy chain following the fourth P-loop motif. Virtually nothing is known regarding how binding affinity is achieved and modulated during ATP hydrolysis. We have performed a detailed dissection of the microtubule contact site, using fragment expression, alanine substitution, and peptide competition. Our work identifies three clusters of amino acids important for the physical contact with microtubules; two of these fall within a region sharing sequence homology with MAP1B, the third in a region just downstream. Amino acid substitutions within any one of these regions can eliminate or weaken microtubule binding (KK3379,80, E3385, K3387, K3397, KK3410,11, W3414, RKK3418–20, F3426, R3464, S3466, and K3467), suggesting that their activities are highly coordinated. A peptide that actively displaces MAP1B from microtubules perturbs dynein binding, supporting previous evidence for similar sites of interaction. We have also identified four amino acids whose substitutions affect release of the motor from the microtubule (E3413, R3444, E3460, and C3469). These suggest that nucleotide-sensitive affinity may be locally controlled at the site of contact. Our work is the first detailed description of dynein–tubulin interactions and provides a framework for understanding how affinity is achieved and modulated. PMID:10679011

Koonce, Michael P.; Tikhonenko, Irina



Modulation of host microtubule dynamics by pathogenic bacteria  

PubMed Central

The eukaryotic cytoskeleton is a vulnerable target of many microbial pathogens during the course of infection. Rearrangements of host cytoskeleton benefit microbes in various stages of their infection cycle such as invasion, motility, and persistence. Bacterial pathogens deliver a number of effector proteins into host cells for modulating the dynamics of actin and microtubule cytoskeleton. Alteration of the actin cytoskeleton is generally achieved by bacterial effectors that target the small GTPases of the host. Modulation of microtubule dynamics involves direct interaction of effector proteins with the subunits of microtubules or recruiting cellular proteins that affect microtubule dynamics. This review will discuss effector proteins from animal and human bacterial pathogens that either destabilize or stabilize host micro-tubules to advance the infectious process. A compilation of these research findings will provide an overview of known and unknown strategies used by various bacterial effectors to modulate the host microtubule dynamics. The present review will undoubtedly help direct future research to determine the mechanisms of action of many bacterial effector proteins and contribute to understanding the survival strategies of diverse adherent and invasive bacterial pathogens. PMID:23585820

Radhakrishnan, Girish K.; Splitter, Gary A.



Microtubule Assembly of Isotypically Purified Tubulin and Its Mixtures  

PubMed Central

Numerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubule function in vivo. While this is an attractive hypothesis, there are only a handful of studies demonstrating a direct influence of tubulin isotype composition on the dynamic properties of microtubules. Here, we present the results of experimental assays on the assembly of microtubules from bovine brain tubulin using purified isotypes at various controlled relative concentrations. A novel data analysis is developed using recursive maps which are shown to be related to the master equation formalism. We have found striking similarities between the three isotypes of bovine tubulin studied in regard to their dynamic instability properties, except for subtle differences in their catastrophe frequencies. When mixtures of tubulin isotypes are analyzed, their nonlinear concentration dependence is modeled and interpreted in terms of lower affinities of tubulin dimers belonging to the same isotype than those that represent different isotypes indicating hitherto unsuspected influences of tubulin dimers on each other within a microtubule. Finally, we investigate the fluctuations in microtubule assembly and disassembly rates and conclude that the inherent rate variability may signify differences in the guanosine-5?-triphosphate composition of the growing and shortening microtubule tips. It is the main objective of this article to develop a quantitative model of tubulin polymerization for individual isotypes and their mixtures. The possible biological significance of the observed differences is addressed. PMID:18502790

Rezania, Vahid; Azarenko, Olga; Jordan, Mary Ann; Bolterauer, Hannes; Luduena, Richard F.; Huzil, J. Torin; Tuszynski, Jack A.



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

E-print Network

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

Mirny, Leonid A.


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

PubMed Central

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

Hamada, Takahiro



A doublet microlens array for imaging micron-sized objects  

PubMed Central

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

Tripathi, A; Chronis, N



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.



Controlled propulsion in viscous fluids of magnetically actuated colloidal doublets  

NASA Astrophysics Data System (ADS)

We study the propulsion of a micron-size paramagnetic colloidal doublet dispersed in water and driven above a surface by an external precessing magnetic field. The applied field forces the doublet to precess around an axis parallel to the plane of motion and the rotation of the colloidal assembly is rectified into translation due to a periodic asymmetry in dissipation close to the bounding plate. These recent experimental findings [P. Tierno, R. Golestanian, I. Pagonabarraga, and F. Sagués, Phys. Rev. Lett. 101, 218304 (2008)] are complemented here with a theoretical analysis of the system and extended to more complex magnetic modulations such as elliptical driving fields. Experimental results show a good agreement with numerical simulations with the aim to find the best conditions toward the optimization of propulsion speed and swimming efficiency.

Tierno, Pietro; Güell, Oriol; Sagués, Francesc; Golestanian, Ramin; Pagonabarraga, Ignacio



Controlled propulsion in viscous fluids of magnetically actuated colloidal doublets.  


We study the propulsion of a micron-size paramagnetic colloidal doublet dispersed in water and driven above a surface by an external precessing magnetic field. The applied field forces the doublet to precess around an axis parallel to the plane of motion and the rotation of the colloidal assembly is rectified into translation due to a periodic asymmetry in dissipation close to the bounding plate. These recent experimental findings [P. Tierno, R. Golestanian, I. Pagonabarraga, and F. Sagués, Phys. Rev. Lett. 101, 218304 (2008)] are complemented here with a theoretical analysis of the system and extended to more complex magnetic modulations such as elliptical driving fields. Experimental results show a good agreement with numerical simulations with the aim to find the best conditions toward the optimization of propulsion speed and swimming efficiency. PMID:20365368

Tierno, Pietro; Güell, Oriol; Sagués, Francesc; Golestanian, Ramin; Pagonabarraga, Ignacio



Difference between focal mechanisms of Dayao earthquake doublet sequence  

Microsoft Academic Search

Using the maximum amplitude ratios of vertical component of P and S waves recorded by a regional network, 921 focal mechanisms\\u000a of Dayao earthquake doublet sequence are determined by means of synthetic seismograms of a point source of dislocation in\\u000a a plane layered medium. Among them, 389 focal mechanisms are in the aftershock sequence of M6.2 earthquake occurred on 21

Xiaoshan Wang; Xiangdong Feng; Guiling Diao; Libing Wang; Lingling Cai



On the significance of microtubule flexural behavior in cytoskeletal mechanics.  


Quantitative description of cell mechanics has challenged biological scientists for the past two decades. Various structural models have been attempted to analyze the structure of the cytoskeleton. One important aspect that has been largely ignored in all these modeling approaches is related to the flexural and buckling behavior of microtubular filaments. The objective of this paper is to explore the influence of this flexural and buckling behavior in cytoskeletal mechanics.In vitro the microtubules are observed to buckle in the first mode, reminiscent of a free, simply-supported beam. In vivo images of microtubules, however, indicate that the buckling mostly occurs in higher modes. This buckling mode switch takes place mostly because of the lateral support of microtubules via their connections to actin and intermediate filaments. These lateral loads are exerted throughout the microtubule length and yield a considerable bending behavior that, unless properly accounted for, would produce erroneous results in the modeling and analysis of the cytoskeletal mechanics.One of the promising attempts towards mechanical modeling of the cytoskeleton is the tensegrity model, which simplifies the complex network of cytoskeletal filaments into a combination merely of tension-bearing actin filaments and compression-bearing microtubules. Interestingly, this discrete model can qualitatively explain many experimental observations in cell mechanics. However, evidence suggests that the simplicity of this model may undermine the accuracy of its predictions, given the model's underlying assumption that "every single member bears solely either tensile or compressive behavior," i.e. neglecting the flexural behavior of the microtubule filaments. We invoke an anisotropic continuum model for microtubules and compare the bending energy stored in a single microtubule with its axial strain energy at the verge of buckling. Our results suggest that the bending energy can exceed the axial energy of microtubules by 40 folds. A modification to tensegrity model is, therefore, proved necessary in order to take into account the flexural response of microtubules. The concept of "bendo-tensegrity" is proposed as a modification to contemporary cytoskeletal tensegrity models. PMID:21998675

Mehrbod, Mehrdad; Mofrad, Mohammad R K



Two-Higgs-doublet type-II seesaw model  

NASA Astrophysics Data System (ADS)

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

Chen, Chuan-Hung; Nomura, Takaaki



Considerations of symmetry in the cortical integration of tetrahymena doublets.  


Homopolar doublets of syngen 1, T. pyriformis, may be induced by treatment of conjugating pairs with immobilizing antiserum. These doublets have geometric properties and basal body populations generally indicative of separate autonomous integrative systems in the two halves. The duplex system, though metastable, is transformed through a process of "simplification" back to the simplex state. The transformation is not a single event, but a series involving changes at different times for different structures and processes; for the micronuclei and the macronuclei; for the capacity to generate two oral apparatuses through stomatogenesis and the capacity to develop them through oral replacement; for the structures at the anterior and posterior ends of the cell; for the numbers of ciliary rows and for the numbers of basal bodies which make up the rows. Although the two semicells composing a doublet are in important respects independent of each other, they are coordinated in significant ways. The positions of the contractile vacuole pores and their numbers depend not only on the number of ciliary rows in a semicell, but also on the number of ciliary rows in the opposing twin. Most notably, the probability for dual stomatogenesis, and hence the perpetuation of the doubled oral apparatus, depends on the symmetry of the semicells. The cell as a whole is maintained as an integrated unit over a prolonged interval as various aspects of duplex structure and function are progressively consolidated. PMID:806652

Nanney, D L; Chow, M; Wozencraft, B



Two-Higgs-Doublet Type-II Seesaw Model  

E-print Network

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

Chuan-Hung Chen; Takaaki Nomura



A new viable region of the inert doublet model  

SciTech Connect

The inert doublet model, a minimal extension of the Standard Model by a second Higgs doublet, is one of the simplest and most attractive scenarios that can explain the dark matter. In this paper, we demonstrate the existence of a new viable region of the inert doublet model featuring dark matter masses between M{sub W} and about 160 GeV. Along this previously overlooked region of the parameter space, the correct relic density is obtained thanks to cancellations between different diagrams contributing to dark matter annihilation into gauge bosons (W{sup +}W{sup ?} and Z{sup 0}Z{sup 0}). First, we explain how these cancellations come about and show several examples illustrating the effect of the parameters of the model on the cancellations themselves and on the predicted relic density. Then, we perform a full scan of the new viable region and analyze it in detail by projecting it onto several two-dimensional planes. Finally, the prospects for the direct and the indirect detection of inert Higgs dark matter within this new viable region are studied. We find that present direct detection bounds already rule out a fraction of the new parameter space and that future direct detection experiments, such as Xenon100, will easily probe the remaining part in its entirety.

Honorez, Laura Lopez [Service de Physique Théorique, Université Libre de Bruxelles, 1050 Brussels (Belgium); Yaguna, Carlos E., E-mail:, E-mail: [Departamento de Física Teórica C-XI and Instituto de Física Teórica UAM-CSIC, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid (Spain)



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

PubMed Central

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

Baas, Peter W.; Lin, Shen



Challenging the Role of Microtubules in Tobacco Mosaic Virus Movement by Drug Treatments Is Disputable  

PubMed Central

The movement protein (MP) of Tobacco mosaic virus interacts with microtubules during infection. Although this interaction is correlated with the function of MP in the cell-to-cell transport of viral RNA, a direct role of microtubules in the movement process was recently challenged by studies involving the treatment of plants with inhibitors of microtubule polymerization. Here, we report evidence suggesting that such treatments may not efficiently disrupt all microtubules. Thus, results obtained from studies using microtubule inhibitors may have to remain open to interpretation with regard to the involvement of microtubules in viral RNA trafficking. PMID:16775361

Seemanpillai, Mark; Elamawi, Rabab; Ritzenthaler, Christophe; Heinlein, Manfred



Dynamical Length-Regulation of Microtubules  

NASA Astrophysics Data System (ADS)

Microtubules (MTs) are vital constituents of the cytoskeleton. These stiff filaments are not only needed for mechanical support. They also fulfill highly dynamic tasks. For instance MTs build the mitotic spindle, which pulls the doubled set of chromosomes apart during mitosis. Hence, a well-regulated and adjustable MT length is essential for cell division. Extending a recently introduced model [1], we here study length-regulation of MTs. Thereby we account for both spontaneous polymerization and depolymerization triggered by motor proteins. In contrast to the polymerization rate, the effective depolymerization rate depends on the presence of molecular motors at the tip and thereby on crowding effects which in turn depend on the MT length. We show that these antagonistic effects result in a well-defined MT length. Stochastic simulations and analytic calculations reveal the exact regimes where regulation is feasible. Furthermore, the adjusted MT length and the ensuing strength of fluctuations are analyzed. Taken together, we make quantitative predictions which can be tested experimentally. These results should help to obtain deeper insights in the microscopic mechanisms underlying length-regulation. [4pt] [1] L.Reese, A.Melbinger, E.Frey, Biophys. J., 101, 9, 2190 (2011)

Melbinger, Anna; Reese, Louis; Frey, Erwin



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



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

NASA Astrophysics Data System (ADS)

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

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



Interaction between microtubules and the Drosophila formin Cappuccino and its effect on actin assembly.  


Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte. PMID:24362037

Roth-Johnson, Elizabeth A; Vizcarra, Christina L; Bois, Justin S; Quinlan, Margot E



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

PubMed Central

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

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



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. PMID:23966467

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



SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase  

PubMed Central

The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end–tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2–ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle–specific regulation of MT polymerization and the organization of the MT network. PMID:21646404

van der Vaart, Babet; Manatschal, Cristina; Grigoriev, Ilya; Olieric, Vincent; Gouveia, Susana Montenegro; Bjelic, Sasa; Demmers, Jeroen; Vorobjev, Ivan; Hoogenraad, Casper C.



SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase.  


The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network. PMID:21646404

van der Vaart, Babet; Manatschal, Cristina; Grigoriev, Ilya; Olieric, Vincent; Gouveia, Susana Montenegro; Bjelic, Sasa; Demmers, Jeroen; Vorobjev, Ivan; Hoogenraad, Casper C; Steinmetz, Michel O; Akhmanova, Anna



The feasibility of coherent energy transfer in microtubules.  


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

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



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. PMID:3968174



Diffusive Movement of Processive Kinesin-1 on Microtubules  

PubMed Central

The processive motor kinesin-1 moves unidirectionally toward the plus end of microtubules. This process can be visualized by total internal reflection fluorescence (TIRF) microscopy of kinesin bound to a carboxylated Quantum dot (Qdot), which acts both as cargo and label. Surprisingly, when kinesin is bound to an anti-HIS Qdot, it shows diffusive movement on microtubules, which decreased in favor of processive runs with increasing salt concentration. This observation implies that kinesin movement on microtubules is governed by its conformation, as it is well-established that kinesin undergoes a salt-dependent transition from a folded (inactive) to an extended (active) molecule. A truncated kinesin lacking the last 75 amino acids (kinesin-?C) showed both processive and diffusive movement on microtubules. The extent of each behavior depends on the relative amounts of ADP and ATP, with purely diffusive movement occurring in ADP alone. Taken together, these data imply that folded kinesin.ADP can exist in a state that diffuses along the microtubule lattice without expending energy. This mechanism may facilitate the ability of kinesin to pick up cargo, and/or allow the kinesin/cargo complex to stay bound after encountering obstacles. PMID:19682327

Lu, Hailong; Ali, M. Yusuf; Bookwalter, Carol S.; Warshaw, David M.; Trybus, Kathleen M.



Microtubules contribute to maintain nucleus shape in epithelial cell monolayer  

NASA Astrophysics Data System (ADS)

INTRODUCTION: Tissue strains can result in significant nuclear deformations and may regulate gene expression. However, the precise role of the cytoskeleton in regulating nuclear mechanics remains poorly understood. Here, we investigate the nuclear deformability of Madin-Darky canine kidney cells (MDCK) under various stretching conditions to clarify the role of the microtubules and actin network on the mechanical behavior of the nucleus. METHODS: A custom-built cell-stretching device allowing for real time imaging of MDCK nuclei was used. Cells were seeded on a silicone membrane coated with rat-tail collagen I. A nuclear stain, Hoechst-33342, was used to image nuclei during stretching. We exposed cells to a compressive and non-compressive stretching strain field of 25%. Nocodazole and cytochalasin-D were used to depolymerize the microtubules and actin network. RESULTS: Nuclei in control cells stretched more along their minor axis than major axis with a deformation of 5% and 2% respectively. This anisotropy vanished completely in microtubule-deprived cells and these cells showed a very high nuclear deformability along the minor axis when exposed to a compressive stretching strain field. CONCLUSIONS: The microtubules drive the anisotropic deformability of MDCK nuclei in a monolayer and maintain nuclear shape when exposed to compressive strain. Such intrinsic mechanical behavior indicates that microtubules are essential to maintain nuclear shape and may prevent down regulation of gene expression.

Tremblay, Dominique; Andrzejewski, Lukasz; Pelling, Andrew



Mini spindles, the XMAP215 homologue, suppresses pausing of interphase microtubules in Drosophila  

PubMed Central

Drosophila Mini spindles (Msps) protein belongs to a conserved family of microtubule-associated proteins (MAPs). Intriguingly, this family of MAPs, including Xenopus XMAP215, was reported to have both microtubule stabilising and destabilising activities. While they are shown to regulate various aspects of microtubules, the role in regulating interphase microtubules in animal cells has yet to be established. Here, we show that the depletion or mutation of Msps prevents interphase microtubules from extending to the cell periphery and leads to the formation of stable microtubule bundles. The effect is independent of known Msps regulator or effector proteins, kinesin-13/KinI homologues or D-TACC. Real-time analysis revealed that the depletion of Msps results in a dramatic increase of microtubule pausing with little or no growth. Our study provides the first direct evidence to support a hypothesis that this family of MAPs acts as an antipausing factor to exhibit both microtubule stabilising and destabilising activities. PMID:15775959

Brittle, Amy L; Ohkura, Hiroyuki



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


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

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



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


The microtubule cytoskeleton is a highly dynamic network. In dividing cells, its complex architecture not only influences cell shape and movement but is also crucial for chromosome segregation. Curiously, nothing is known about the behavior of this cellular machinery in quiescent cells. Here we show that, upon quiescence entry, the Saccharomyces cerevisiae microtubule cytoskeleton is drastically remodeled. Indeed, while cytoplasmic microtubules vanish, the spindle pole body (SPB) assembles a long and stable monopolar array of nuclear microtubules that spans the entire nucleus. Consequently, the nucleolus is displaced. Kinetochores remain attached to microtubule tips but lose SPB clustering and distribute along the microtubule array, leading to a large reorganization of the nucleus. When cells exit quiescence, the nuclear microtubule array slowly depolymerizes and, by pulling attached centromeres back to the SPB, allows the recovery of a typical Rabl-like configuration. Finally, mutants that do not assemble a nuclear array of microtubules are impaired for both quiescence survival and exit. PMID:24247429

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



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

PubMed Central

The microtubule cytoskeleton is a highly dynamic network. In dividing cells, its complex architecture not only influences cell shape and movement but is also crucial for chromosome segregation. Curiously, nothing is known about the behavior of this cellular machinery in quiescent cells. Here we show that, upon quiescence entry, the Saccharomyces cerevisiae microtubule cytoskeleton is drastically remodeled. Indeed, while cytoplasmic microtubules vanish, the spindle pole body (SPB) assembles a long and stable monopolar array of nuclear microtubules that spans the entire nucleus. Consequently, the nucleolus is displaced. Kinetochores remain attached to microtubule tips but lose SPB clustering and distribute along the microtubule array, leading to a large reorganization of the nucleus. When cells exit quiescence, the nuclear microtubule array slowly depolymerizes and, by pulling attached centromeres back to the SPB, allows the recovery of a typical Rabl-like configuration. Finally, mutants that do not assemble a nuclear array of microtubules are impaired for both quiescence survival and exit. PMID:24247429

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



Flexural Rigidity of Individual Microtubules Measured by a Buckling Force with Optical Traps  

PubMed Central

We used direct buckling force measurements with optical traps to determine the flexural rigidity of individual microtubules bound to polystyrene beads. To optimize the accuracy of the measurement, we used two optical traps and antibody-coated beads to manipulate each microtubule. We then applied a new analytical model assuming nonaxial buckling. Paclitaxel-stabilized microtubules were polymerized from purified tubulin, and the average microtubule rigidity was calculated as 2.0 × 10?24 Nm2 using this novel microtubule buckling system. This value was not dependent on microtubule length. We also measured the rigidity of paclitaxel-free microtubules, and obtained the value of 7.9 × 10?24 Nm2, which is nearly four times that measured for paclitaxel-stabilized microtubules. PMID:16339879

Kikumoto, Mahito; Kurachi, Masashi; Tosa, Valer; Tashiro, Hideo



Biomolecular motor modulates mechanical property of microtubule.  


The microtubule (MT) is the stiffest cytoskeletal filamentous protein that takes part in a wide range of cellular activities where its mechanical property plays a crucially significant role. How a single biological entity plays multiple roles in cell has been a mystery for long time. Over the recent years, it has been known that modulation of the mechanical property of MT by different cellular agents is the key to performing manifold in vivo activities by MT. Studying the mechanical property of MT thus has been a prerequisite in understanding how MT plays such diversified in vivo roles. However, the anisotropic structure of MT has been an impediment in obtaining a precise description of the mechanical property of MT along its longitudinal and lateral directions that requires employment of distinct experimental approach and has not been demonstrated yet. In this work, we have developed an experimental system that enabled us to investigate the effect of tensile stress on MT. By using our newly developed system, (1) we have determined the Young's modulus of MT considering its deformation under applied tensile stress and (2) a new role of MT associated motor protein kinesin in modulating the mechanical property of MT was revealed for the first time. Decrease in Young's modulus of MT with the increase in interaction with kinesin suggests that kinesin has a softening effect on MT and thereby can modulate the rigidity of MT. This work will be an aid in understanding the modulation of mechanical property of MTs by MT associated proteins and might also help obtain a clear insight of the endurance and mechanical instability of MTs under applied stress. PMID:24697688

Kabir, Arif Md Rashedul; Inoue, Daisuke; Hamano, Yoshimi; Mayama, Hiroyuki; Sada, Kazuki; Kakugo, Akira



Speckled microtubules improve tracking in motor-protein gliding assays  

NASA Astrophysics Data System (ADS)

Gliding assays of motor proteins such as kinesin, dynein and myosin are commonly carried out with fluorescently labeled microtubules or filamentous actin. In this paper, we show that speckled microtubules (MTs), prepared by copolymerizing 98% unlabeled tubulin with 2% rhodamine-labeled tubulin, can be localized to ±7.4 nm (24 measurements) in images acquired every 125 ms. If the speckled MTs move at about 800 nm s-1, ten images are sufficient to determine their velocity to a precision of ±6.8 nm s-1 (6 microtubules, 24 measurements). This velocity precision is four-fold better than manual methods for measuring the gliding velocity of uniformly labeled MTs by end-point localization. The improved velocity precision will permit the determination of velocity-force curves when one, two and three kinesin motors pull a single load in vitro.

Chisena, Ernest N.; Wall, R. Andrew; Macosko, Jed C.; Holzwarth, George



Analysis of microtubule motion due to drag from kinesin walkers  

E-print Network

We analyze the nonlinear waves that propagate on a microtubule that is tethered at its minus end due to kinesin walking on it, as is seen during the fluid mixing caused by cytoplasmic streaming in Drosophila oocytes.The model we use assumes that the microtubule can be modeled as an elastic string in a viscous medium. The effect of the kinesin is to apply a force tangential to the microtubule and we also consider the addition of a uniform cytoplasmic velocity field. We show that travelling wave solutions exist and analyze their properties. There exist scale invariant families of solutions and solutions can exist that are flat or helical. The relationship between the period and wavelength is obtained by both analytic and numerical means. Numerical implementation of the equation of motion verifies our analytical predictions.

J. M. Deutsch; M. E. Brunner; William M. Saxton



TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types.  


Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end-tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics. PMID:25187649

Nwagbara, Belinda U; Faris, Anna E; Bearce, Elizabeth A; Erdogan, Burcu; Ebbert, Patrick T; Evans, Matthew F; Rutherford, Erin L; Enzenbacher, Tiffany B; Lowery, Laura Anne



Microtubule Dynamics in Living Root Hairs: Transient Slowing by Lipochitin Oligosaccharide Nodulation Signals  

Microsoft Academic Search

The incorporation of a fusion of green fluorescent protein and tubulin-a 6 from Arabidopsis thaliana in root hairs of Lotus japonicus has allowed us to visualize and quantify the dynamic parameters of the cortical microtubules in living root hairs. Analysis of individual microtubule turnover in real time showed that only plus polymer ends contributed to overall microtubule dynamicity, exhibiting dynamic

Valya N. Vassileva; Hiroshi Kouchi; Robert W. Ridge



Microtubules in Mesophyll Cells of Nonacclimated and Cold-Acclimated Spinach 1  

PubMed Central

Responses of cortical microtubules in spinach (Spinacia oleracea L. cv Bloomsdale) mesophyll cells to freezing, thawing, supercooling, and dehydration were assessed. Microtubules were visualized using a modified procedure for indirect immunofluorescence microscopy. Leaf sections of nonacclimated and cold-acclimated spinach were slowly frozen to various temperatures, fixed while frozen, and microtubules immunolabelled. Both nonacclimated and cold-acclimated cells exhibited nearly complete microtubule depolymerization after ice formation. After 1 hour thawing at 23°C, microtubules in both nonacclimated and cold-acclimated cells repolymerized. With time, however, microtubules in nonacclimated cells again depolymerized. Since microtubules in cells of leaf tissue frozen slowly are subjected to dehydration as well as subzero temperatures, these stresses were applied separately and their effects on microtubules noted. Supercooling induced microtubule depolymerization in both nonacclimated and cold-acclimated cells, but to a smaller extent than did freezing. Exposing leaf sections to solutions of sorbitol (a cell wall-penetrating osmoticum) or polyethylene glycol 10,000 (a nonpenetrating osmoticum) at room temperature caused microtubule depolymerization. The effects of low temperature and dehydration are roughly additive in producing the observed microtubule responses during freezing. Only small differences in microtubule stability were resolved between nonacclimated and cold-acclimated cells. ImagesFigure 2 PMID:16668366

Bartolo, Michael E.; Carter, John V.



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

Microsoft Academic Search

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

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



MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics  

PubMed Central

Within the mitotic spindle, there are multiple populations of microtubules with different turnover dynamics, but how these different dynamics are maintained is not fully understood. MCAK is a member of the kinesin-13 family of microtubule-destabilizing enzymes that is required for proper establishment and maintenance of the spindle. Using quantitative immunofluorescence and fluorescence recovery after photobleaching, we compared the differences in spindle organization caused by global suppression of microtubule dynamics, by treating cells with low levels of paclitaxel, versus specific perturbation of spindle microtubule subsets by MCAK inhibition. Paclitaxel treatment caused a disruption in spindle microtubule organization marked by a significant increase in microtubules near the poles and a reduction in K-fiber fluorescence intensity. This was correlated with a faster t1/2 of both spindle and K-fiber microtubules. In contrast, MCAK inhibition caused a dramatic reorganization of spindle microtubules with a significant increase in astral microtubules and reduction in K-fiber fluorescence intensity, which correlated with a slower t1/2 of K-fibers but no change in the t1/2 of spindle microtubules. Our data support the model that MCAK perturbs spindle organization by acting preferentially on a subset of microtubules, and they support the overall hypothesis that microtubule dynamics is differentially regulated in the spindle. PMID:19158381

Rizk, Rania S.; Bohannon, Kevin P.; Wetzel, Laura A.; Powers, James; Shaw, Sidney L.



The Dynamic Behavior of Individual Microtubules Associated with Chromosomes In Vitro  

PubMed Central

Mitotic movements of chromosomes are usually coupled to the elongation and shortening of the microtubules to which they are bound. The lengths of kinetochore-associated microtubules change by incorporation or loss of tubulin subunits, principally at their chromosome-bound ends. We have reproduced aspects of this phenomenon in vitro, using a real-time assay that displays directly the movements of individual chromosome-associated microtubules as they elongate and shorten. Chromosomes isolated from cultured Chinese hamster ovary cells were adhered to coverslips and then allowed to bind labeled microtubules. In the presence of tubulin and GTP, these microtubules could grow at their chromosome-bound ends, causing the labeled segments to move away from the chromosomes, even in the absence of ATP. Sometimes a microtubule would switch to shortening, causing the direction of movement to change abruptly. The link between a microtubule and a chromosome was mechanically strong; 15 pN of tension was generally insufficient to detach a microtubule, even though it could add subunits at the kinetochore–microtubule junction. The behavior of the microtubules in vitro was regulated by the chromosomes to which they were bound; the frequency of transitions from polymerization to depolymerization was decreased, and the speed of depolymerization-coupled movement toward chromosomes was only one-fifth the rate of shortening for microtubules free in solution. Our results are consistent with a model in which each microtubule interacts with an increasing number of chromosome-associated binding sites as it approaches the kinetochore. PMID:9763448

Hunt, Alan J.; McIntosh, J. Richard



Molecular Requirements for Kinetochore-Associated Microtubule Formation in Mammalian Cells  

PubMed Central

Summary In centrosome-containing cells, microtubules nucleated at centrosomes are thought to play a major role in spindle assembly [1]. In addition, microtubule formation at kinetochores has also been observed [2-5], most recently under physiological conditions in live cells [6]. The relative contributions of microtubule formation at kinetochores and centrosomes to spindle assembly, and their molecular requirements, remain incompletely understood. Using mammalian cells released from nocodazole-induced disassembly, we observed microtubule formation at centrosomes and at Bub1-positive sites on chromosomes. Kinetochore-associated microtubules rapidly coalesced into pole-like structures in a dynein-dependent manner. Microinjection of excess importin-? or depletion of the Ran-dependent spindle assembly factor, TPX2, blocked kinetochore-associated microtubule formation, enhanced centrosome-associated microtubule formation, but did not prevent chromosome capture by centrosomal microtubules. Depletion of the chromosome passenger protein, survivin, reduced microtubule formation at kinetochores in an MCAK-dependent manner. Microtubule formation in cells depleted of Bub1 or Nuf2 was indistinguishable from that in controls. Our data demonstrate that microtubule assembly at centrosomes and kinetochores is kinetically distinct and differentially regulated. The presence of microtubules at kinetochores provides a mechanism to reconcile the time required for spindle assembly in vivo with that observed in computer simulations of search and capture. PMID:16527751

Tulu, U. Serdar; Fagerstrom, Carey; Ferenz, Nick P.; Wadsworth, Patricia



Estramustine phosphate inhibits germinal vesicle breakdown and induces depolymerization of microtubules  

E-print Network

microtubules in ovo and dispersed non-tubulin antigens associated with microtubules of the metaphase spindle cytoplasmic asters. These results suggest that germinal vesicle breakdown and microtubule polymerization may). At the same time, the critical level of tubulin polymerization decreases since taxol can then induce

Paris-Sud XI, Université de


Pathway of Force Production by the Kinesin-Microtubule ATPase  

NASA Astrophysics Data System (ADS)

Kinesin is the smallest of the molecular motors, consisting of a dimer of motor domains that interact with microtubules and ATP to generate motion towards the plus ends of microtubules for fast axonal transport of membranous organelles. It operates via an alternating site ATPase pathway in which the binding of ATP to one motor domain stimulates the release of ADP from the neighboring domain as the motor walks ``hand over hand'' along the microtubule surface. This alternating site pathway is accomplished in part due to strain that distinguishes the leading from the lagging motor domains when both are bound to the microtubule. This strain leads to a weak nucleotide binding state in the leading motor and a strong nucleotide binding state in the lagging motor. The ATPase activity is linked to alternating weak and strong nucleotide binding states that are coupled to association and dissociation at the microtubule surface to produce a force for forward motion. Strain in the leading motor domain appears to be due to the disruption of the ``neck linker'' in the leading motor. Release of the trailing motor domain from the microtubule surface is the rate-limiting step and, by relaxing the tension, allows the leading domain to bind ATP and continue the cycle and forward motion. Although many of the rate constants for steps in this pathway are known, details regarding the structural and thermodynamic basis for the coupling of ATP hydrolysis to force production remain to be established. I will review our current understanding and describe some of our early attempts to resolve intermediates during movement using single molecule fluorescence methods. In collaboration with Tim Scholz and Bernhard Brenner, Hannover Medical School.

Johnson, Kenneth



Nuclear Matter and Neutron stars in a Parity Doublet Model  

E-print Network

We investigate the properties of isospin-symmetric nuclear matter and neutron stars in a chiral model approach adopting the SU(2) parity doublet formulation. This ansatz explicitly incorporates chiral symmetry restoration with the limit of degenerate masses of the nucleons and their parity partners. Instead of searching for an optimized parameter set we explore the general parameter dependence of nuclear matter and star properties in the model. We are able to get a good description of ground state nuclear matter as well as large values of mass for neutron stars in agreement with observation.

V. Dexheimer; S. Schramm; D. Zschiesche



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.



Cell Microtubules as Cavities: Quantum Coherence and Energy Transfer?  

E-print Network

A model is presented for dissipationless energy transfer in cell microtubules due to quantum coherent states. The model is based on conjectured (hydrated) ferroelectric properties of microtubular arrangements. Ferroelectricity is essential in providing the necessary isolation against thermal losses in thin interior regions, full of ordered water, near the tubulin dimer walls of the microtubule. These play the role of cavity regions, which are similar to electromagnetic cavities of quantum optics. As a result, the formation of (macroscopic) quantum coherent states of electric dipoles on the tubulin dimers may occur. Some experiments, inspired by quantum optics, are suggested for the falsification of this scenario.

N. E. Mavromatos



Microtubule Translocation Properties of Intact and Proteolytically Digested Dyneins from TetrahymenaCilia  

E-print Network

of multi- ple dyneins in axonemes, the functional roles of these mole- cules in ciliary and flagellar also explored the function of the multiple ATPase heads of 22S dynein by preparing one- and two/sec). This finding indicates that the intact three-headed structure of 22S dynein is not essential for generating

Vale, Ronald D.


Disruption of Microtubule Organization and Centrosome Function by Expression of Tobacco Mosaic Virus Movement Protein  

PubMed Central

The movement protein (MP) of Tobacco mosaic virus mediates the cell-to-cell transport of viral RNA through plasmodesmata, cytoplasmic cell wall channels for direct cell-to-cell communication between adjacent cells. Previous in vivo studies demonstrated that the RNA transport function of the protein correlates with its association with microtubules, although the exact role of microtubules in the movement process remains unknown. Since the binding of MP to microtubules is conserved in transfected mammalian cells, we took advantage of available mammalian cell biology reagents and tools to further address the interaction in flat-growing and transparent COS-7 cells. We demonstrate that neither actin, nor endoplasmic reticulum (ER), nor dynein motor complexes are involved in the apparent alignment of MP with microtubules. Together with results of in vitro coprecipitation experiments, these findings indicate that MP binds microtubules directly. Unlike microtubules associated with neuronal MAP2c, MP-associated microtubules are resistant to disruption by microtubule-disrupting agents or cold, suggesting that MP is a specialized microtubule binding protein that forms unusually stable complexes with microtubules. MP-associated microtubules accumulate ER membranes, which is consistent with a proposed role for MP in the recruitment of membranes in infected plant cells and may suggest that microtubules are involved in this process. The ability of MP to interfere with centrosomal ?-tubulin is independent of microtubule association with MP, does not involve the removal of other tested centrosomal markers, and correlates with inhibition of centrosomal microtubule nucleation activity. These observations suggest that the function of MP in viral movement may involve interaction with the microtubule-nucleating machinery. PMID:16731920

Ferralli, Jacqueline; Ashby, Jamie; Fasler, Monika; Boyko, Vitaly; Heinlein, Manfred



Plus-end-tracking proteins and their interactions at microtubule ends.  


Microtubules are cytoskeletal elements that are essential for a large number of intracellular processes, including mitosis, cell differentiation and migration, and vesicle transport. In many cells, the microtubule network is organized in a radial manner, with one end of a microtubule (the minus end) embedded near the nucleus and the other end (the plus end) exploring cytoplasmic space, switching between episodes of growth and shrinkage. Mammalian plus-end-tracking proteins (+TIPs) localize to the ends of growing microtubules and regulate both the dynamic behavior of microtubules as well as the interactions of microtubules with other cellular components. Because of these crucial roles, +TIPs and the mechanisms underlying their association with microtubule ends have been intensively investigated. Results indicate that +TIPs reach microtubule ends by motor-mediated transport or diffusion. Individual +TIP molecules exchange rapidly on microtubule end-binding sites that are formed during microtubule polymerization and that have a slower turnover. Most +TIPs associate with the end-binding (EB) proteins, and appear to require these 'core' +TIPs for localization at microtubule ends. Accumulation of +TIPs may also involve structural features of the microtubule end and interactions with other +TIPs. This complexity makes it difficult to assign discrete roles to specific +TIPs. Given that +TIPs concentrate at microtubule ends and that each +TIP binds in a conformationally distinct manner, I propose that the ends of growing microtubules are 'nano-platforms' for productive interactions between selected proteins and that these interactions might persist and be functional elsewhere in the cytoplasm than at the microtubule end at which they originated. PMID:20620909

Galjart, Niels



Microtubules Accelerate the Kinase Activity of Aurora-B by a Reduction in Dimensionality  

PubMed Central

Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a “reduction in dimensionality.” We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates. PMID:24498282

Noujaim, Michael; Bechstedt, Susanne; Wieczorek, Michal; Brouhard, Gary J.



Electron/muon specific two Higgs doublet model  

NASA Astrophysics Data System (ADS)

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

Kajiyama, Yuji; Okada, Hiroshi; Yagyu, Kei



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.



Nonprocessive motor dynamics at the microtubule membrane tube interface.  


Key cellular processes such as cell division, membrane compartmentalization, and intracellular transport rely on motor proteins. Motors have been studied in detail on the single motor level such that information on their step size, stall force, average run length, and processivity are well known. However, in vivo, motors often work together, so that the question of their collective coordination has raised great interest. Here, we specifically attach motors to giant vesicles and examine collective motor dynamics during membrane tube formation. Image correlation spectroscopy reveals directed motion as processive motors walk at typical speeds (< or = 500 nm/s) along an underlying microtubule and accumulate at the tip of the growing membrane tube. In contrast, nonprocessive motors exhibit purely diffusive behavior, decorating the entire length of a microtubule lattice with diffusion constants at least 1000 times smaller than a freely-diffusing lipid-motor complex in a lipid bilayer (1 microm(2)/s); fluorescence recovery after photobleaching experiments confirm the presence of the slower-moving motor population at the microtubule-membrane tube interface. We suggest that nonprocessive motors dynamically bind and unbind to maintain a continuous interaction with the microtubule. This dynamic and continuous interaction is likely necessary for nonprocessive motors to mediate bidirectional membrane tube dynamics reported previously. PMID:20085722

Shaklee, Paige M; Bourel-Bonnet, Line; Dogterom, Marileen; Schmidt, Thomas



Nonprocessive Motor Dynamics at the Microtubule Membrane Tube Interface  

PubMed Central

Abstract Key cellular processes such as cell division, membrane compartmentalization, and intracellular transport rely on motor proteins. Motors have been studied in detail on the single motor level such that information on their step size, stall force, average run length, and processivity are well known. However, in vivo, motors often work together, so that the question of their collective coordination has raised great interest. Here, we specifically attach motors to giant vesicles and examine collective motor dynamics during membrane tube formation. Image correlation spectroscopy reveals directed motion as processive motors walk at typical speeds (?500 nm/s) along an underlying microtubule and accumulate at the tip of the growing membrane tube. In contrast, nonprocessive motors exhibit purely diffusive behavior, decorating the entire length of a microtubule lattice with diffusion constants at least 1000 times smaller than a freely-diffusing lipid-motor complex in a lipid bilayer (1 ?m2/s); fluorescence recovery after photobleaching experiments confirm the presence of the slower-moving motor population at the microtubule-membrane tube interface. We suggest that nonprocessive motors dynamically bind and unbind to maintain a continuous interaction with the microtubule. This dynamic and continuous interaction is likely necessary for nonprocessive motors to mediate bidirectional membrane tube dynamics reported previously. PMID:20085722

Shaklee, Paige M.; Bourel-Bonnet, Line; Dogterom, Marileen; Schmidt, Thomas



Microtubule polymer assembly and transport during axonal elongation  

PubMed Central

As axons elongate, tubulin, which is synthesized in the cell body, must be transported and assembled into new structures in the axon. The mechanism of transport and the location of assembly are presently unknown. We report here on the use of tubulin tagged with a photoactivatable fluorescent group to investigate these issues. Photoactivatable tubulin, microinjected into frog embryos at the two- cell stage, is incorporated into microtubules in neurons obtained from explants of the neural tube. When activated by light, a fluorescent mark is made on the microtubules in the axon, and transport and turnover can be visualized directly. We find that microtubules are generated in or near the cell body and continually transported distally as a coherent phase of polymer during axon elongation. This vectorial polymer movement was observed at all levels on the axon, even in the absence of axonal elongation. Measurements of the rate of polymer translocation at various places in the axon suggest that new polymer is formed by intercalary assembly along the axon and assembly at the growth cone in addition to transport of polymer from the cell body. Finally, polymer movement near the growth cone appeared to respond in a characteristic manner to growth cone behavior, while polymer proximally in the axon moved more consistently. These results suggest that microtubule translocation is the principal means of tubulin transport and that translocation plays an important role in generating new axon structure at the growth cone. PMID:1717484



The physiology and pathology of microtubule-associated protein tau.  


Tau belongs to the family of microtubule-associated proteins predominantly expressed in neurons where they play an important role in promoting microtubule assembly and stabilizing microtubules. In addition, tau proteins interact with other cytoskeletal elements to allow spacing between microtubules. Recent studies have shown that tau is also actively involved in regulating cell viability and activity. Translated from a single gene located on chromosome 17q21, six isoforms of tau are produced by alternative splicing in adult human brain. Due to multiple post-translational modifications, heterogeneous tau species with a wide range of apparent molecular masses have been observed by denaturing polyacrylamide-gel electrophoresis. Since tau gene mutations and abnormal post-translational modifications have been detected in over 20 neurodegenerative disorders, namely the tauopathies, tau has gained widespread attention as a target protein in Alzheimer's disease and other neurodegenerative disorders. In the present chapter, research progress regarding physiology and pathology of tau is reviewed, particularly in terms of the role of post-translational modification. PMID:25131590

Wang, Jian-Zhi; Gao, Xinya; Wang, Zhi-Hao




PubMed Central

In thin sections through microspikes extending from the surface of isolated cells, a core has been seen which may contain microtubular elements. The differences between these and microtubules seen elsewhere in the cytoplasm are attributed to their rapid growth and exposed location which make them especially vulnerable to injury by preparative treatment. In support of this view it is shown that cytoplasmic microtubules may be altered or even destroyed by exposing the cells to changes in osmotic pressure. Associated with these straight microtubules in the cytoplasm were also found solid microfilaments. The form of these components and their location and alignment in portions of cells which are under tension or in motion suggest that they function in the structural support of the cell and its microspikes and in the transmission of tension in the cytoplasm. A second type of microtubule, smaller in diameter and tortuous in form, was also seen in certain cells and is presumed, from its shape, to have little to do with cytoplasmic support. PMID:5914687

Taylor, A. C.



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

Microsoft Academic Search

Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apopto- sis. Cell growth was stimulated in microgravity by in- creasing serum concentration. After 4 and 48 h, cells filtered



Understanding Microtubule Organizing Centers by Comparing Mutant and  

E-print Network

of their structure and function can benefit from three-dimensional (3D) METHODS IN CELL BIOLOGY, VOL. 79 0091-679X/07 of defects in specific structural components and furthers our understanding of the assembly and functionCHAPTER 5 Understanding Microtubule Organizing Centers by Comparing Mutant and Wild-Type Structures

Wang, Yan Mei


High Rectifying Efficiencies of Microtubule Motility on Kinesin-Coated  

E-print Network

of the well-understood PEG-silane chemistry on SiO2 for the blocking of kinesin, whereas the gold tracks allow nanostructures and that microtubules move on the gold with speeds comparable to that on glass. Second, we present onto a substrate, and microtu- bules are propelled in random directions over the surface

Dekker, Cees


The Ndc80 kinetochore complex directly modulates microtubule dynamics  

E-print Network

Hughes Medical Institute, University of Washington, Seattle, WA 98195 Edited by John Carbon, University. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment chromosome segregation, regulatory mechanisms detect and correct errors in attachments between kinetochores

Eddy, Sean


Doublecortin induces mitotic microtubule catastrophe and inhibits glioma cell invasion  

PubMed Central

Doublecortin (DCX) is a microtubule binding protein that induces growth arrest at the G2-M phase of cell-cycle in glioma and suppresses tumor xenograft in immunocompromised hosts. DCX expression was found in neuronal cells, but lacking in glioma cells. We tested the hypothesis that DCX inhibits glioma U87 cell mitosis and invasion. Our data showed that DCX synthesizing U87 cells underwent mitotic microtubule spindle catastrophe in a neurabin II dependent pathway. Synthesis of both DCX and neurabin II were required to induce apoptosis in U87 and HEK 293T cells. In DCX expressing U87 cells, association of phosphorylated DCX (P-DCX) with protein phosphatase-1 (PP1) in the cytosol disrupted the interaction between kinesin-13 and PP1 in the nucleus and yielded spontaneously active kinesin-13. The activated kinesin-13 caused mitotic microtubule catastrophe in spindle checkpoint. P-DCX induced depolymerization of actin filaments in U87 cells, downregulated matrix metalloproteinase -2 (MMP-2) and MMP-9, and inhibited glioma U87 cell invasion in a neurabin II dependent pathway. Thus, localization of the DCX-neurabin II-PP1 complex in the cytosol of U87 tumor cells inhibited PP1 phosphatase activities leading to anti-glioma effects via 1) mitotic microtubule spindle catastrophe that blocks mitosis, and 2) depolymerization of actin that inhibits glioma cell invasion. PMID:19094064

Santra, Manoranjan; Santra, Sutapa; Roberts, Cindi; Zhang, Rui Lan; Chopp, Michael



On the Significance of Microtubule Flexural Behavior in Cytoskeletal Mechanics  

E-print Network

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

Mofrad, Mohammad R. K.


The unusual microtubule polarity in teleost retinal pigment epithelial cells  

Microsoft Academic Search

In cells of the teleost retinal pigment epi- thelium (RPE), melanin granules disperse into the RPE cell's long apical projections in response to light onset, and aggregate toward the base of the RPE cell in response to dark onset. The RPE cells possess numerous microtubules, which in the apical projec- tions are aligned longitudinally. Nocodazole studies have shown that pigment

Louise Leotta Troutt; Beth Burnside



Microtubules, MAPs and Mitosis: a holistic approach to  

E-print Network

Microtubules, MAPs and Mitosis: a holistic approach to understanding cell division James Wake eld Zitzmann #12;Gene Ontology of MAPs uncharacterised 30% cell cycle/mitosis 9% cytoskeleton binding% cell cycle/mitosis 9% cytoskeleton binding 5% motors 3% vesicle-mediated transport 2% polarity 1

Mumby, Peter J.


A targeted multi-enzyme mechanism for selective microtubule  

E-print Network

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

Paris-Sud XI, Université de


Force on spindle microtubule minus ends moves chromosomes.  


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

Elting, Mary Williard; Hueschen, Christina L; Udy, Dylan B; Dumont, Sophie



Direct observation of single kinesin molecules moving along microtubules  

NASA Astrophysics Data System (ADS)

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 internal reflection fluorescence microscopy4 in the absence of attachment of the motor to a cargo (for example, an organelle or bead). The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a ˜ 1% probability of detachment per mechanical cycle. Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl. Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism.

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



Development/Plasticity/Repair Doublecortin Associates with Microtubules Preferentially in  

E-print Network

the DCX/actin filament patches and collateral branching. Based on the striking resemblance of cytoskeletal orga- nization that constitute the structural basis of neuronal form. A growing body of evidence has been shown in vitro to influ- ence microtubule stability, bundling, and protofilament number

Baas, Peter W.


Prion protein inhibits microtubule assembly by inducing tubulin oligomerization  

SciTech Connect

A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for First time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of {approx}50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers.

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



Microtubules: from classical properties to quantum effects in human cognition  

E-print Network

Theories describing information processing and signaling in microtubules 8 3.1 Cellular automata This seminar paper was motivated by the talk of Professor Dr. Sir Roger Penrose about the Orchestrated objective reduction theory and the potential role of quantum mechanics and quantum gravity in conscious

Â?umer, Slobodan


Microtubule Stabilization by Mdp3 Is Partially Attributed to Its Modulation of HDAC6 in Addition to Its Association with Tubulin and Microtubules  

PubMed Central

Microtubule-mediated cellular events such as intracellular transport and the maintenance of cell polarity are highly dependent upon microtubule stability, which is controlled by a repertoire of microtubule-associated proteins (MAPs) in the cell. MAP7 domain-containing protein 3 (Mdp3) has recently been identified as a critical regulator of microtubule stability. However, it remains elusive how Mdp3 carries out this function. In this study, by examination of tubulin partitioning between the polymer and soluble dimer forms, we found that Mdp3 could protect microtubules from cold- or nocodazole-induced depolymerization. Immunoblotting and immunofluorescence microscopy showed that knockdown of Mdp3 expression significantly reduced the level of tubulin acetylation. In vitro tubulin polymerization assays revealed that the amino-terminal region of Mdp3 was necessary for its ability to stabilize microtubules. Immunoprecipitation and pulldown experiments showed that the amino-terminal region mediated the interaction of Mdp3 with histone deacetylase 6 (HDAC6), in addition to its association with tubulin and microtubules. Immunofluorescence microscopy further demonstrated that endogenous Mdp3 and HDAC6 colocalized in the cytoplasm. Moreover, depletion of Mdp3 dramatically increased the activity of HDAC6 toward tubulin deacetylation. These findings suggest that Mdp3 controls microtubule stability through its binding to tubulin and microtubules as well as its regulation of HDAC6 activity. PMID:24614595

Chen, Jie; Zhang, Linlin; Liu, Ningning; Zhou, Jun; Li, Dengwen; Liu, Min



Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure  

PubMed Central

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

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



Diverse effects of beta-tubulin mutations on microtubule formation and function  

PubMed Central

We have used in vitro mutagenesis and gene replacement to construct five new cold-sensitive mutations in TUB2, the sole gene encoding beta- tubulin in the yeast Saccharomyces cerevisiae. These and one previously isolated tub2 mutant display diverse phenotypes that have allowed us to define the functions of yeast microtubules in vivo. At the restrictive temperature, all of the tub2 mutations inhibit chromosome segregation and block the mitotic cell cycle. However, different microtubule arrays are present in these arrested cells depending on the tub2 allele. One mutant (tub2-401) contains no detectable microtubules, two (tub2-403 and tub2-405) contain greatly diminished levels of both nuclear and cytoplasmic microtubules, one (tub2-104) contains predominantly nuclear microtubules, one (tub2-402) contains predominantly cytoplasmic microtubules, and one (tub2-404) contains prominent nuclear and cytoplasmic microtubule arrays. Using these mutants we demonstrate here that cytoplasmic microtubules are necessary for nuclear migration during the mitotic cell cycle and for nuclear migration and fusion during conjugation; only those mutants that possess cytoplasmic microtubules are able to perform these functions. We also show that microtubules are not required for secretory vesicle transport in yeast; bud growth and invertase secretion occur in cells which contain no microtubules. PMID:3290223



Sites of Glucose Transporter-4 Vesicle Fusion with the Plasma Membrane Correlate Spatially with Microtubules  

PubMed Central

In adipocytes, vesicles containing glucose transporter-4 (GLUT4) redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF) microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin), preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion. PMID:22916292

Dawicki-McKenna, Jennine M.; Goldman, Yale E.; Ostap, E. Michael



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

PubMed Central

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

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



PACSIN1, a Tau-interacting protein, regulates axonal elongation and branching by facilitating microtubule instability.  


Tau is a major member of the neuronal microtubule-associated proteins. It promotes tubulin assembly and stabilizes axonal microtubules. Previous studies have demonstrated that Tau forms cross-bridges between microtubules, with some particles located on cross-bridges, suggesting that some proteins interact with Tau and might be involved in regulating Tau-related microtubule dynamics. This study reports that PACSIN1 interacts with Tau in axon. PACSIN1 blockade results in impaired axonal elongation and a higher number of primary axonal branches in mouse dorsal root ganglia neurons, which is induced by increasing the binding ability of Tau to microtubules. In PACSIN1-blocked dorsal root ganglia neurons, a greater amount of Tau is inclined to accumulate in the central domain of growth cones, and it promotes the stability of the microtubule network. Taken together, these results suggest that PACSIN1 is an important Tau binding partner in regulating microtubule dynamics and forming axonal plasticity. PMID:23035120

Liu, Yingying; Lv, Kaosheng; Li, Zenglong; Yu, Albert C H; Chen, Jianguo; Teng, Junlin



Redistribution of microtubules and pericentriolar material during the development of polarity in mouse blastomeres  

PubMed Central

The distribution of microtubules and microtubule organizing centers (MTOCs) during the development of cell polarity in eight-cell mouse blastomeres was studied by immunofluorescence and immunoelectron microscopy using monoclonal anti-tubulin antibodies and an anti- pericentriolar material (PCM) serum. In early eight-cell blastomeres microtubules were found mainly around the nucleus and in the cell cortex, whereas PCM foci were observed dispersed in the cytoplasm. During the eight-cell stage, microtubules disappeared from the area adjacent to the zone of intercellular contact and accumulated in the apical part of the cell while their number decreased in the basal domain. The PCM also relocalized to the apical domain of the cell, but this occurred after the redistribution of the microtubules by a mechanism that involved the microtubule network. The possible roles of both MTOCs and microtubules in establishing cell polarity are discussed. PMID:3571331



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

PubMed Central

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

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



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


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

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



Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos  

NASA Technical Reports Server (NTRS)

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

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



Doublet Production in the Development of Medieval and Modern Spanish: New Approaches to Phonolexical Duplication  

ERIC Educational Resources Information Center

This dissertation offers new approaches to an old and well-known problem in the study of the development of Romance varieties: duplicate lexis or doublets. Traditional analyses of duplication are narrow in scope both in what qualifies as a doublet (the popular/learned opposition has dominated, to the exclusion of other pairs) and in channels of…

Haney, Darren W.



Microtubule-stabilizing agents based on designed laulimalide analogues  

PubMed Central

Laulimalide is a potent, structurally unique microtubule-stabilizing agent originally isolated from the marine sponge Cacospongia mycofijiensis. Laulimalide exhibits an activity profile different from other microtubule-binding agents, notably including effectiveness against paclitaxel-resistant cells, but it is intrinsically unstable. Five analogues of laulimalide were designed to exhibit enhanced chemical stability yet retain its exceptional biological activities. Evaluations of these analogues showed that all are effective inhibitors of cancer-cell proliferation yet differ substantially in potency with an IC50 range of 0.12–16.5 ?M. Although all of the analogues initiated cellular changes similar to laulimalide, including increased density of interphase microtubules, aberrant mitotic spindles, and ultimately apoptosis, differences among the analogues were apparent. The two most potent analogues, C16-C17-des-epoxy laulimalide and C20-methoxy laulimalide, appear to have a mechanism of action identical to laulimalide. The C16-C17-des-epoxy, C20-methoxy laulimalide derivative, which incorporates both chemical changes of the most potent analogues, was significantly less potent and initiated the formation of unique interphase microtubules unlike the parent compound and other analogues. Two C2-C3-alkynoate derivatives had lower potency, and they initiated abnormal microtubule structures but did not cause micronucleation or extensive G2/M accumulation. Significantly, paclitaxel- and epothilone-resistant cell lines were less resistant to the laulimalide analogues. In summary, analogues of laulimalide designed to minimize or eliminate its intrinsic instability have been synthesized, and some have been found to retain the unique biological activities of laulimalide. PMID:15161976

Mooberry, Susan L.; Randall-Hlubek, Deborah A.; Leal, Rachel M.; Hegde, Sayee G.; Hubbard, Robert D.; Zhang, Lei; Wender, Paul A.



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

PubMed Central

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

Lefevre, Julien; Savarin, Philippe; Gans, Pierre; Hamon, Loic; Clement, Marie-Jeanne; David, Marie-Odile; Bosc, Christophe; Andrieux, Annie; Curmi, Patrick A.



Who Needs Microtubules? Myogenic Reorganization of MTOC, Golgi Complex and ER Exit Sites Persists Despite Lack of Normal Microtubule Tracks  

Microsoft Academic Search

A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol,

Kristien J. M. Zaal; Ericka Reid; Kambiz Mousavi; Tan Zhang; Amisha Mehta; Elisabeth Bugnard; Vittorio Sartorelli; Evelyn Ralston



Detection of single microtubules in living cells: particle transport can occur in both directions along the same microtubule  

PubMed Central

Video-enhanced contrast/differential interference-contrast microscopy was used in conjunction with whole mount electron microscopy to study particle transport along linear elements in fibroblasts. Keratocytes from the corneal stroma of Rana pipiens were grown on gold indicator grids and examined with video microscopy. Video records were taken of the linear elements and associated particle transport until lysis and/or fixation of the cells was completed. The preparations were then processed for whole mount electron microscopy. By combining these two methods, we demonstrated that linear elements detected in the living cell could be identified as single microtubules, and that filaments as small as 10 nm could be detected in lysed and fixed cells. The visibility of different cytoplasmic structures changed after lysis with many more cellular components becoming visible. Microtubules became more difficult to detect after lysis while bundles of microfilaments became more prominent. All particle translocations were observed to take place along linear elements composed of one or more microtubules. Furthermore, particles were observed to translocate in one or both directions on the same microtubule. PMID:6333427



Ultrastructural characters of the spermatozoa in Digeneans of the genus Lecithochirium L?he, 1901 (Digenea, Hemiuridae), parasites of fishes: comparative study of L. microstomum and L. musculus  

PubMed Central

This study provides the first ultrastructural data of spermatozoa in the genus Lecithochirium. The spermatozoa of L. microstomum (from Trichiurus lepturus in Senegal) and L. musculus (from Anguilla anguilla in Corsica) exhibit the general pattern described in the great majority of the Digenea, namely two axonemes with the 9 + “1” pattern typical of the Trepaxonemata, one mitochondrion, a nucleus, parallel cortical microtubules and external ornamentation of the plasma membrane. Spermatozoa of L. microstomum and L. musculus have some specific features such as the presence of a reduced number of cortical microtubules arranged on only one side of the spermatozoon, the lack of spine-like bodies and expansion of the plasma membrane. The external ornamentation of the plasma membrane entirely covers the anterior extremity of the spermatozoa. The ultrastructure of the posterior extremity of the spermatozoa corresponds to the pattern previously described in the Hemiuridae, characterized by only singlets of the second axoneme. A particularity of these spermatozoa is the organization of the microtubule doublets of the second axoneme around the nucleus in the posterior part of the spermatozoon. PMID:25275216

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



Search for multiple chiral doublets in rhodium isotopes  

SciTech Connect

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

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



Tracer transport modeling of the doublet well system  

SciTech Connect

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

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



Coherent and incoherent chaotic tunneling near singlet-doublet crossings  

E-print Network

In the spectrum of systems showing chaos-assisted tunneling, three-state crossings are formed when a chaotic singlet intersects a tunnel doublet. We study the dissipative quantum dynamics in the vicinity of such crossings. A harmonically driven double well coupled to a bath serves as a model. Markov and rotating-wave approximations are introduced with respect to the Floquet spectrum of the time-dependent central system. The resulting master equation is integrated numerically. We find various types of transient tunneling, determined by the relation of the level width to the inherent energy scales of the crossing. The decay of coherent tunneling can be significantly retarded or accelerated. Modifications of the quantum asymptotic state by the crossing are also studied. The comparison with a simple three-state model shows that in contrast to the undamped case, the participation of states outside the crossing cannot be neglected in the presence of dissipation.

Sigmund Kohler; Ralf Utermann; Peter Hanggi; Thomas Dittrich



CPV Phenomenology of Flavor Conserving Two Higgs Doublet Models  

E-print Network

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

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



Active doublet method for measuring small changes in physical properties  


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

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



A Novel Dynein Light Intermediate Chain Colocalizes with the Retrograde Motor for Intraflagellar Transport at Sites of Axoneme Assembly in Chlamydomonas and Mammalian Cells  

PubMed Central

The assembly of cilia and flagella depends on bidirectional intraflagellar transport (IFT). Anterograde IFT is driven by kinesin II, whereas retrograde IFT requires cytoplasmic dynein 1b (cDHC1b). Little is known about how cDHC1b interacts with its cargoes or how it is regulated. Recent work identified a novel dynein light intermediate chain (D2LIC) that colocalized with the mammalian cDHC1b homolog DHC2 in the centrosomal region of cultured cells. To see whether the LIC might play a role in IFT, we characterized the gene encoding the Chlamydomonas homolog of D2LIC and found its expression is up-regulated in response to deflagellation. We show that the LIC subunit copurifies with cDHC1b during flagellar isolation, dynein extraction, sucrose density centrifugation, and immunoprecipitation. Immunocytochemistry reveals that the LIC colocalizes with cDHC1b in the basal body region and along the length of flagella in wild-type cells. Localization of the complex is altered in a collection of retrograde IFT and length control mutants, which suggests that the affected gene products directly or indirectly regulate cDHC1b activity. The mammalian DHC2 and D2LIC also colocalize in the apical cytoplasm and axonemes of ciliated epithelia in the lung, brain, and efferent duct. These studies, together with the identification of an LIC mutation, xbx-1(ok279), which disrupts retrograde IFT in Caenorhabditis elegans, indicate that the novel LIC is a component of the cDHC1b/DHC2 retrograde IFT motor in a variety of organisms. PMID:12802074

Perrone, Catherine A.; Tritschler, Douglas; Taulman, Patrick; Bower, Raqual; Yoder, Bradley K.; Porter, Mary E.



Unusual ciliary abnormalities in three 9/11 response workers.  


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

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



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

PubMed Central

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

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



Tubulin ligands suggest a microtubule-NADPH oxidase relationship in postischemic cardiomyocytes.  


Alterations of the microtubule network, which is involved in many vital processes, occur in several pathological conditions, such as cardiac ischemia. However, the connection between the microtubule assembly state and the factors affecting myocardial reperfusion injury, especially oxidative stress, is unknown. We aimed thus to study the effects of different tubulin ligands on the changes in the microtubule network and in several markers of cell injury and oxidative activity in cardiac muscle cells submitted to a reversible substrate-free, hypoxia-reoxygenation model of ischemia-reperfusion. The microtubule network was visualized by immunocytochemistry. Cell injury was evaluated via lactate dehydrogenase release and the mitochondrial function by the MTT test. Superoxide production was detected using dihydroethidium. The activity of NADPH oxidase and mRNA subunit expression were investigated. The microtubule disassembly induced by simulated ischemia was reversed by placing cardiomyocytes under normoxic conditions. This post-"ischemic" restoration of microtubule assembly was modulated by microtubule stabilizers (taxol: paclitaxel) and by microtubule disrupting drugs (nocodazole, colchicine). In addition, nocodazole decreased superoxide anion production as well as NADPH oxidase activity and mRNA expression of the NADPH oxidase subunit p22phox. These results demonstrated that the "ischemia"-induced microtubule network alteration is reversible and suggest a possible relationship between "reperfusion"-induced reassembly of microtubules and free radical generation in post-"ischemic" cardiomyocytes. PMID:16973157

Devillard, Lisa; Vandroux, David; Tissier, Cindy; Brochot, Amandine; Voisin, Sophie; Rochette, Luc; Athias, Pierre



Nezha/CAMSAP3 and CAMSAP2 cooperate in epithelial-specific organization of noncentrosomal microtubules  

PubMed Central

Major microtubules in epithelial cells are not anchored to the centrosome, in contrast to the centrosomal radiation of microtubules in other cell types. It remains to be discovered how these epithelial microtubules are generated and stabilized at noncentrosomal sites. Here, we found that Nezha [also known as calmodulin-regulated spectrin-associated protein 3 (CAMSAP3)] and its related protein, CAMSAP2, cooperate in organization of noncentrosomal microtubules. These two CAMSAP molecules coclustered at the minus ends of noncentrosomal microtubules and thereby stabilized them. Depletion of CAMSAPs caused a marked reduction of microtubules with polymerizing plus ends, concomitantly inducing the growth of microtubules from the centrosome. In CAMSAP-depleted cells, early endosomes and the Golgi apparatus exhibited irregular distributions. These effects of CAMSAP depletion were maximized when both CAMSAPs were removed. These findings suggest that CAMSAP2 and -3 work together to maintain noncentrosomal microtubules, suppressing the microtubule-organizing ability of the centrosome, and that the network of CAMSAP-anchored microtubules is important for proper organelle assembly. PMID:23169647

Tanaka, Nobutoshi; Meng, Wenxiang; Nagae, Shigenori; Takeichi, Masatoshi



Beyond taxol: microtubule-based treatment of disease and injury of the nervous system  

PubMed Central

Contemporary research has revealed a great deal of information on the behaviours of microtubules that underlie critical events in the lives of neurons. Microtubules in the neuron undergo dynamic assembly and disassembly, bundling and splaying, severing, and rapid transport as well as integration with other cytoskeletal elements such as actin filaments. These various behaviours are regulated by signalling pathways that affect microtubule-related proteins such as molecular motor proteins and microtubule severing enzymes, as well as a variety of proteins that promote the assembly, stabilization and bundling of microtubules. In recent years, translational neuroscientists have earmarked microtubules as a promising target for therapy of injury and disease of the nervous system. Proof-of-principle has come mainly from studies using taxol and related drugs to pharmacologically stabilize microtubules in animal models of nerve injury and disease. However, concerns persist that the negative consequences of abnormal microtubule stabilization may outweigh the positive effects. Other potential approaches include microtubule-active drugs with somewhat different properties, but also expanding the therapeutic toolkit to include intervention at the level of microtubule regulatory proteins. PMID:23811322

Ahmad, Fridoon J.



MTCL1 crosslinks and stabilizes non-centrosomal microtubules on the Golgi membrane.  


Recent studies have revealed the presence of a microtubule subpopulation called Golgi-derived microtubules that support Golgi ribbon formation, which is required for maintaining polarized cell migration. CLASPs and AKAP450/CG-NAP are involved in their formation, but the underlying molecular mechanisms remain unclear. Here, we find that the microtubule-crosslinking protein, MTCL1, is recruited to the Golgi membranes through interactions with CLASPs and AKAP450/CG-NAP, and promotes microtubule growth from the Golgi membrane. Correspondingly, MTCL1 knockdown specifically impairs the formation of the stable perinuclear microtubule network to which the Golgi ribbon tethers and extends. Rescue experiments demonstrate that besides its crosslinking activity mediated by the N-terminal microtubule-binding region, the C-terminal microtubule-binding region plays essential roles in these MTCL1 functions through a novel microtubule-stabilizing activity. These results suggest that MTCL1 cooperates with CLASPs and AKAP450/CG-NAP in the formation of the Golgi-derived microtubules, and mediates their development into a stable microtubule network. PMID:25366663

Sato, Yoshinori; Hayashi, Kenji; Amano, Yoshiko; Takahashi, Mikiko; Yonemura, Shigenobu; Hayashi, Ikuko; Hirose, Hiroko; Ohno, Shigeo; Suzuki, Atsushi



Push-me-pull-you: how microtubules organize the cell interior  

PubMed Central

Dynamic organization of the cell interior, which is crucial for cell function, largely depends on the microtubule cytoskeleton. Microtubules move and position organelles by pushing, pulling, or sliding. Pushing forces can be generated by microtubule polymerization, whereas pulling typically involves microtubule depolymerization or molecular motors, or both. Sliding between a microtubule and another microtubule, an organelle, or the cell cortex is also powered by molecular motors. Although numerous examples of microtubule-based pushing and pulling in living cells have been observed, it is not clear why different cell types and processes employ different mechanisms. This review introduces a classification of microtubule-based positioning strategies and discusses the efficacy of pushing and pulling. The positioning mechanisms based on microtubule pushing are efficient for movements over small distances, and for centering of organelles in symmetric geometries. Mechanisms based on pulling, on the other hand, are typically more elaborate, but are necessary when the distances to be covered by the organelles are large, and when the geometry is asymmetric and complex. Thus, taking into account cell geometry and the length scale of the movements helps to identify general principles of the intracellular layout based on microtubule forces. PMID:18404264



The taccalonolides and paclitaxel cause distinct effects on microtubule dynamics and aster formation  

PubMed Central

Background Microtubule stabilizers suppress microtubule dynamics and, at the lowest antiproliferative concentrations, disrupt the function of mitotic spindles, leading to mitotic arrest and apoptosis. At slightly higher concentrations, these agents cause the formation of multiple mitotic asters with distinct morphologies elicited by different microtubule stabilizers. Results We tested the hypothesis that two classes of microtubule stabilizing drugs, the taxanes and the taccalonolides, cause the formation of distinct aster structures due, in part, to differential effects on microtubule dynamics. Paclitaxel and the taccalonolides suppressed the dynamics of microtubules formed from purified tubulin as well as in live cells. Both agents suppressed microtubule dynamic instability, with the taccalonolides having a more pronounced inhibition of microtubule catastrophe, suggesting that they stabilize the plus ends of microtubules more effectively than paclitaxel. Live cell microscopy was also used to evaluate the formation and resolution of asters after drug treatment. While each drug had similar effects on initial formation, substantial differences were observed in aster resolution. Paclitaxel-induced asters often coalesced over time resulting in fewer, larger asters whereas numerous compact asters persisted once they were formed in the presence of the taccalonolides. Conclusions We conclude that the increased resistance of microtubule plus ends to catastrophe may play a role in the observed inability of taccalonolide-induced asters to coalesce during mitosis, giving rise to the distinct morphologies observed after exposure to these agents. PMID:24576146



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


Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a +TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. PMID:22134091

Iimori, Makoto; Ozaki, Kanako; Chikashige, Yuji; Habu, Toshiyuki; Hiraoka, Yasushi; Maki, Takahisa; Hayashi, Ikuko; Obuse, Chikashi; Matsumoto, Tomohiro



Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles  

NASA Astrophysics Data System (ADS)

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

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



Regulation of microtubule motors by tubulin isotypes and posttranslational modifications  

PubMed Central

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

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



A common pharmacophore for cytotoxic natural products that stabilize microtubules.  


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

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



A Bio-Polymer Transistor: Electrical Amplification by Microtubules  

E-print Network

Microtubules (MTs) are important cytoskeletal structures, engaged in a number of specific cellular activities, including vesicular traffic, cell cyto-architecture and motility, cell division, and information processing within neuronal processes. MTs have also been implicated in higher neuronal functions, including memory, and the emergence of "consciousness". How MTs handle and process electrical information, however, is heretofore unknown. Here we show new electrodynamic properties of MTs. Isolated, taxol-stabilized microtubules behave as bio-molecular transistors capable of amplifying electrical information. Electrical amplification by MTs can lead to the enhancement of dynamic information, and processivity in neurons can be conceptualized as an "ionic-based" transistor, which may impact among other known functions, neuronal computational capabilities.

Avner Priel; Arnolt J. Ramos; Jack A. Tuszynski; Horacio F. Cantiello



Cooperative stabilization of microtubule dynamics by EB1 and CLIP-170 involves displacement of stably bound P(i) at microtubule ends.  


End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 ?M) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [?(32)P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound (32)P(i). Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap. PMID:22424550

Lopus, Manu; Manatschal, Cristina; Buey, Ruben M; Bjeli?, Saša; Miller, Herbert P; Steinmetz, Michel O; Wilson, Leslie



Heat-induced reversible hexagonal packing of spindle microtubules  

Microsoft Academic Search

Epithelial cells cultured from the lung of the Northwest rough-skinned newt (Taricha granulosa granulosa) were subjected to brief (10-15 min) elevated temperature shocks of 33~176 during metaphase. Electron microscope studies on these cells reveal that the spindle microtubules (Mts) are differentially stable to heat treatment. The great majority of nonkinetochore Mts are destroyed within the first few minutes of the




Pentagons, heptagons and negative curvature in graphite microtubule growth  

Microsoft Academic Search

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

Sumio Lijima; Toshinari Ichihashi; Yoshinori Ando



Structure, Function, and Regulation of the Microtubule Associated Protein Tau  

Microsoft Academic Search

The neural protein tau was first identified and purified in 1975 as a protein that copurifies with tubulin and assembles tubulin\\u000a subunits into microtubules (1). Although tau has been studied intensively for almost 30 yr, many aspects of tau structure, function, and regulation remain\\u000a unclear. Whereas tau is widely appreciated to be important in normal neurodevelopment (reviewed in refs. 2,3),

Janis Bunker; Stuart C. Feinstein


Microtubule orientation in globular leaflet cells of Chara inflata  

Microsoft Academic Search

Chara inflata has globular leaflet cells and cylindrical internodal cells. The morphology of the leaflet cells is different from that of\\u000a other Characeae. The orientation of cortical microtubules (MTs) in young leaflet and internodal cells of this species was\\u000a analyzed by immunofluorescence microscopy. MTs with random orientation were observed in leaflet cells, while those relatively\\u000a transverse to the cell axis

Kazuyoshi Iwata; Teruo Shimmen



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

PubMed Central

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

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



RegionSpecific Microtubule Transport in Motile Cells  

Microsoft Academic Search

Photoactivation and photobleaching of fluo- rescence were used to determine the mechanism by which microtubules (MTs) are remodeled in PtK2 cells during fibroblast-like motility in response to hepatocyte growth factor (HGF). The data show that MTs are transported during cell motility in an actomyosin- dependent manner, and that the direction of transport depends on the dominant force in the region

Anne-Marie C. Yvon; Patricia Wadsworth



Enthalpy changes in microtubule assembly from pure tubulin.  


The enthalpy changes that occur in the self-assembly of tubulin into microtubules were examined by adiabatic differential heat capacity microcalorimetry and by isothermal batch microcalorimetry. Tubulin solutions at concentrations between 7 and 17 mg/mL were heated from 0 to 40 degrees C at heating rates of 1 or 2 deg/min in pH 6.8 or 7.0 assembly buffers containing 20 mM MES, 100 mM glutamic acid, 5 mM MgCl2, 3.4 M glycerol, and either 0.5 mM GMP-PCP or 1 mM GTP. The assembly reaction in the presence of GTP was characterized by a complex heat-uptake pattern consisting of a broad endotherm with a sharper exotherm superimposed on it, similar to assembly in a GTP phosphate buffer [Hinz, H.-J., Gorbunoff, M.J., Price, B., & Timasheff, S.N. (1979) Biochemistry 18,3084]. Replacement of GTP by the nonhydrolyzable analogue resulted in a pattern typical for an endothermic reaction only. These results have permitted the assignment of the endothermic process to microtubule assembly and of the exothermic process to the resultant GTP hydrolysis. In these studies equilibration was found to be slow, several hours of cooling being required for the system to return to its original state. Turbidity scans also revealed hysteresis between consecutive scans and a displacement of the depolymerization transition midpoint to a lower temperature than that of assembly. The disassembly of microtubules was examined in batch calorimetry experiments in pH 7.0 phosphate, 1 mM GTP, 16 mM MgCl2, and 3.4 M glycerol, in which tubulin assembled into microtubules was diluted to below the critical concentration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3814584

Hinz, H J; Timasheff, S N



Microtubule distribution in gravitropic protonemata of the moss Ceratodon  

Microsoft Academic Search

Summary Tip cells of dark-grown protonemata of the mossCeratodon purpureus are negatively gravitropic (grow upward). They possess a unique longitudinal zonation: (1) a tip group of amylochloroplasts in the apical dome, (2) a plastid-free zone, (3) a zone of significant plastid sedimentation, and (4) a zone of mostly non-sedimenting plastids. Immunofluorescence of vertical cells showed microtubules distributed throughout the cytoplasm

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



A Common Pharmacophore for Cytotoxic Natural Products That Stabilize Microtubules  

Microsoft Academic Search

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

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



The role of microtubules in contractile ring function  

NASA Technical Reports Server (NTRS)

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

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



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

E-print Network

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

Sthitadhi Roy



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.



The UNI1 and UNI2 Genes Function in the Transition of Triplet to Doublet Microtubules between the Centriole and Cilium in Chlamydomonas  

Microsoft Academic Search

One fundamental role of the centriole in eukaryotic cells is to nucleate the growth of cilia. The unicellular alga Chlamydomonas reinhardtii provides a simple genetic system to study the role of the centriole in ciliogenesis. Wild-type cells are biflagellate, but \\

Brian P. Piasecki; Carolyn D. Silflow



Rearrangement of microtubule network under biochemical and mechanical stimulations  

PubMed Central

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

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



Effects of microtubule mechanics on hydrolysis and catastrophes  

E-print Network

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

Nina Müller; Jan Kierfeld



Symplekin Specifies Mitotic Fidelity by Supporting Microtubule Dynamics ? †  

PubMed Central

Using a pangenomic loss-of-function screening strategy, we have previously identified 76 potent modulators of paclitaxel responsiveness in non-small-cell lung cancer. The top hit isolated from this screen, symplekin, is a well-established component of the mRNA polyadenylation machinery. Here, we performed a high-resolution phenotypic analysis to reveal the mechanistic underpinnings by which symplekin depletion collaborates with paclitaxel. We find that symplekin supports faithful mitosis by contributing to the formation of a bipolar spindle apparatus. Depletion of symplekin attenuates microtubule polymerization activity as well as expression of the critical microtubule polymerization protein CKAP5 (TOGp). Depletion of additional members of the polyadenylation complex induces similar phenotypes, suggesting that polyadenylation machinery is intimately coupled to microtubule function and thus mitotic spindle formation. Importantly, tumor cells depleted of symplekin display reduced fecundity, but the mitotic defects that we observe are not evident in immortalized cells. These results demonstrate a critical connection between the polyadenylation machinery and mitosis and suggest that tumor cells have an enhanced dependency on these components for spindle assembly. PMID:20823274

Cappell, Kathryn M.; Larson, Brittany; Sciaky, Noah; Whitehurst, Angelique W.



Microtubule-dependent redistribution of a cytoplasmic cornified envelope precursor.  


Several cytoplasmic cornified envelope precursors have been described. Nevertheless, the mechanism whereby these proteins are positioned at the site of crosslink formation is not known. In this study, we examine the intracellular distribution of the cornified envelope precursor S100A11 (S100C) and the effects of the physiologic differentiating agent calcium on this distribution. S100A11 is localized in the cytoplasm of resting cultured human keratinocytes. Treatment with calcium causes S100A11 to relocate to the cell periphery. Immunoprecipitation studies reveal that S100A11 associates with microtubules, and inhibitor studies indicate that functional micro-tubules are required for S100A11 peripheral redistribution. Parallel studies indicate that S100A11 is not present in the Golgi or endoplasmic reticulum (ER), suggesting that S100A11 is not moved to the cell periphery via the classical Golgi/ER export pathway. Further evidence that the Golgi/ER is not involved is provided by the observation that the Golgi/ER disruptor brefeldin A does not alter movement. These results suggest that redistribution along microtubules is a mechanism whereby S100A11 is positioned at the cell periphery in preparation for transglutaminase-dependent crosslinking. Staining of epidermal tissue sections from uninvolved and psoriatic epidermis reveals strong staining at the cell periphery in the majority of suprabasal cells, confirming a peripheral distribution of S100A11 in vivo. PMID:14962086

Broome, Ann-Marie; Eckert, Richard L



Elevated polar ejection forces stabilize kinetochore-microtubule attachments  

PubMed Central

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

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



Microtubule reorganization in tobacco BY2 cells stably expressing GFP-MBD  

Microsoft Academic Search

.  ?Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays\\u000a transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term\\u000a observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been

Cheryl L. Granger; Richard J. Cyr



Effect of Microtubule Stabilization on the Freezing Tolerance of Mesophyll Cells of Spinach 1  

PubMed Central

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter, Plant Physiol [1991] 97: 175-181). The objective of this study was to determine whether the LT50 (lethal temperature: the freezing temperature at which 50% of the tissue is killed) of spinach leaf tissue can be changed by diminishing the extent of microtubule depolymerization in response to freezing. Also examined was how tolerance to the components of extracellular freezing, low temperature and dehydration, is affected by microtubule stabilization. Leaf sections of nonacclimated and cold-acclimated spinach were treated with 20 micromolar taxol, a microtubule-stabilizing compound, prior to freezing, supercooling, or dehydration. Taxol stabilized microtubules against depolymerization in cells subjected to these stresses. When pretreated with taxol both nonacclimated and cold-acclimated cells exhibited increased injury during freezing and dehydration. In contrast, supercooling did not injure cells with taxol-stabilized microtubules. Electrolyte leakage, visual appearance of the cells, or a microtubule repolymerization assay were used to assess injury. As leaves were cold-acclimated beyond the normal period of 2 weeks taxol had less of an effect on cell survival during freezing. In leaves acclimated for up to 2 weeks, stabilizing microtubules with taxol resulted in death at a higher freezing temperature. At certain stages of cold acclimation, it appears that if microtubule depolymerization does not occur during a freeze-thaw cycle the plant cell will be killed at a higher temperature than if microtubule depolymerization proceeds normally. An alternative explanation of these results is that taxol may generate abnormal microtubules, and connections between microtubules and the plasma membrane, such that normal cellular responses to freeze-induced dehydration and subsequent rehydration are blocked, with resultant enhanced freezing injury. ImagesFigure 1Figure 2Figure 6 PMID:16668367

Bartolo, Michael E.; Carter, John V.



Polo-like Kinase 1 Regulates Nlp, a Centrosome Protein Involved in Microtubule Nucleation  

Microsoft Academic Search

In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization.

Martina Casenghi; Patrick Meraldi; Ulrike Weinhart; Peter I. Duncan; Roman Körner; Erich A. Nigg



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

Microsoft Academic Search

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

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



Microtubule-Associated Type II Protein Kinase A Is Important for Neurite Elongation  

PubMed Central

Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process. PMID:23967353

Huang, Yung-An; Kao, Jun-Wei; Tseng, Dion Tzu-Huan; Chen, Wen-Shin; Chiang, Ming-Han; Hwang, Eric



The conserved ciliary protein Bug22 controls planar beating of Chlamydomonas flagella.  


Eukaryotic flagella and cilia can exhibit planar and non-planar beating, and the mechanism controlling these beating patterns is not well understood. Chlamydomonas reinhardtii flagella beat in approximately the same plane with either an asymmetric ciliary-type or symmetric flagellar-type waveform. Each B-tubule of the number 1, 5 and 6 doublets of the flagellar axoneme possesses a beak-like structure. The number 5 and 6 beak structures are implicated in conversion of ciliary motion into flagellar motion. Here, we show that in a null mutant of Bug22, the asymmetric ciliary waveform is converted into a three-dimensional (non-planar) symmetric flagellar waveform. Bug22 is localized to approximately the proximal half to two-thirds of the flagellum, similar to localization of beak-like structures. However, as shown by immunogold labeling, Bug22 associates with axonemal microtubules without apparent preference for any particular doublets. Interestingly, bug22 mutants lack all beak-like structures. We propose that one function of Bug22 is to regulate the anchoring of the beak-like structures to the doublet microtubules and confine flagellar beating to a plane. PMID:24259666

Meng, Dan; Cao, Muqing; Oda, Toshiyuki; Pan, Junmin



Prophase Microtubule Arrays Undergo Flux-like Behavior in Mammalian Cells  

PubMed Central

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

Ferenz, Nick P.



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


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

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



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

E-print Network

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

Welburn, Julie P. I.


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

PubMed Central

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

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



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


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

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



In Vivo Analysis of the Microtubule Severing Protein Katanin-60 in the Drosophila Nervous System.  

E-print Network

??Microtubules, dynamic structures that make up the cellular cytoskeleton, are essential for cellular transport, motility, division, and structural stability. Accordingly, their dynamics must be tightly… (more)

Pang, Lisa



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

E-print Network

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

Arthanari, Haribabu


Asymmetric doublets in MAS NMR: coherent and incoherent mechanisms.  


It has been long noted that J-resolved doublets observed in solid-state MAS experiments are asymmetric. The asymmetry has been attributed to a coherent interference effect involving dipolar and CSA interactions. Recently, Bernd Reif and co-workers suggested that under fast MAS conditions the coherent portion of the effect is suppressed and it becomes possible to observe an incoherent mechanism reminiscent of TROSY. The researchers were able to observe the characteristic TROSY-type patterns in (15)N-(1)H(N) spectra of heavily deuterated protein samples (Chevlekov, Diehl, and Reif, previous article in this issue). In the present computer simulation study, we seek to obtain a unified picture of this phenomenon, including both coherent and incoherent aspects. The chosen model focuses on the (15)N-(1)H(N) pair from a polycrystalline sample subject to magic angle spinning. To mimic local dynamics, we assume that the corresponding peptide plane jumps between two orientations. The simulations demonstrate that this simple model reproduces both coherent and incoherent behavior, depending on the MAS speed and the time scale of local dynamics. Furthermore, semianalytical expressions can be derived for both coherent and incoherent (Redfield) limits. Of particular interest is the possibility to use solution-style Redfield results to probe internal protein motions, especially slower motions on the nanosecond time scale. Our simulations show that the differential relaxation measurement permits accurate determination of (15)N dipolar-CSA cross correlations already at moderately high MAS speed (ca 15 kHz). PMID:18157846

Skrynnikov, N R



Type II seesaw mechanism for Higgs doublets and the scale of new physics  

E-print Network

We elaborate on an earlier proposal by Ernest Ma of a type II seesaw mechanism for suppressing the vacuum expectation values of some Higgs doublets. We emphasize that, by nesting this form of seesaw mechanism into various other seesaw mechanisms, one may obtain light neutrino masses in such a way that the new-physics scale present in the seesaw mechanism - the masses of scalar gauge-SU(2) triplets, scalar SU(2) doublets, or right-handed neutrinos - does not need to be higher than a few 10 TeV. We also investigate other usages of the type II seesaw mechanism for Higgs doublets. For instance, the suppression of the vacuum expectation values of Higgs doublets may realize Froggatt-Nielsen suppression factors in some entries of the fermion mass matrices.

W. Grimus; L. Lavoura; B. Radovcic



EGFR somatic doublets in lung cancer are frequent and generally arise from a pair of driver mutations uncommonly seen as singlet mutations: one-third of doublets occur at five pairs of amino acids.  


Doublet mutations in cancer are not well studied. We find that allelic somatic doublet mutations are present at high frequency in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain in lung cancers. When doublets from the literature are added, a total of 96 doublets are available for analysis. The frequency of doublets overall is 6%, which is sevenfold greater than that observed in normal tissue in mouse. All characterized doublets are allelic, and silent mutations occur rarely. About half of all doublets contain one or two of 12 distinct missense mutations at five amino acids: E709, G719, S768, T790 and L861. The mutations at these five amino acids are seldom reported as singlets. Moreover, when the common L858 target is included, more than one-third of EGFR doublets are one of five specific missense pairs: G719/E709, G719/S768, G719/L861, L858/E709 and L858/T790. Structure suggests function: The data imply that most EGFR doublets are NOT consistent with a 'driver and passenger' mutation mechanism. EGFR doublets are highly skewed relative to singlets, consistent with functional selection of two individually suboptimal mutations that, in combination, have enhanced oncogenic potential. PMID:18372921

Chen, Z; Feng, J; Saldivar, J-S; Gu, D; Bockholt, A; Sommer, S S



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

E-print Network

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

Louis Reese; Anna Melbinger; Erwin Frey



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

E-print Network

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

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



Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts  

Microsoft Academic Search

Microtubules are dynamic polymers that move stochastically between periods of growth and shrinkage, a property known as dynamic instability. Here, to investigate the mechanisms regulating microtubule dynamics in Xenopus egg extracts, we have cloned the complementary DNA encoding the microtubule-associated protein XMAP215 and investigated the function of the XMAP215 protein. Immunodepletion of XMAP215 indicated that it is a major microtubule-stabilizing

Régis Tournebize; Andrei Popov; Kazuhisa Kinoshita; Anthony J. Ashford; Sonja Rybina; Andrei Pozniakovsky; Thomas U. Mayer; Claire E. Walczak; Eric Karsenti; Anthony A. Hyman



Katanin Localization Requires Triplet Microtubules in Chlamydomonas reinhardtii  

PubMed Central

Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (?-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization. PMID:23320108

Esparza, Jessica M.; O'Toole, Eileen; Li, Linya; Giddings, Thomas H.; Kozak, Benjamin; Albee, Alison J.; Dutcher, Susan K.



Deficiency of SPAG16L causes male infertility associated with impaired sperm motility.  


The axonemes of cilia and flagella contain a "9+2" structure of microtubules and associated proteins. Proteins associated with the central doublet pair have been identified in Chlamydomonas that result in motility defects when mutated. The murine orthologue of the Chlamydomonas PF20 gene, sperm-associated antigen 16 (Spag16), encodes two proteins of M(r) approximately 71 x 10(3) (SPAG16L) and M(r) approximately 35 x 10(3) (SPAG16S). In sperm, SPAG16L is found in the central apparatus of the axoneme. To determine the function of SPAG16L, gene targeting was used to generate mice lacking this protein but still expressing SPAG16S. Mutant animals were viable and showed no evidence of hydrocephalus, lateralization defects, sinusitis, bronchial infection, or cystic kidneys-symptoms typically associated with ciliary defects. However, males were infertile with a lower than normal sperm count. The sperm had marked motility defects, even though ultrastructural abnormalities of the axoneme were not evident. In addition, the testes of some nullizygous animals showed a spermatogenetic defect, which consisted of degenerated germ cells in the seminiferous tubules. We conclude that SPAG16L is essential for sperm flagellar function. The sperm defect is consistent with the motility phenotype of the Pf20 mutants of Chlamydomonas, but morphologically different in that the mutant algal axoneme lacks the central apparatus. PMID:16382026

Zhang, Zhibing; Kostetskii, Igor; Tang, Waixing; Haig-Ladewig, Lisa; Sapiro, Rossana; Wei, Zhangyong; Patel, Aatish M; Bennett, Jean; Gerton, George L; Moss, Stuart B; Radice, Glenn L; Strauss, Jerome F



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

Microsoft Academic Search

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

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



Microtubule-Induced Pins/Gai Cortical Polarity in Drosophila Neuroblasts  

E-print Network

Microtubule-Induced Pins/Gai Cortical Polarity in Drosophila Neuroblasts Sarah E. Siegrist1.09.043 SUMMARY Cortical polarity regulates cell division, mi- gration, and differentiation. Microtubules induce and Dlg/Pins co- immunoprecipitate, suggesting that micro- tubules induce Pins/Gai cortical polarity

Doe, Chris


Cell divisionMicrotubule-associated proteins in plants — why we need a map  

Microsoft Academic Search

Plants have four main microtubule assemblies. Three are involved in arranging when and where the cell wall is laid down and have no direct homologues in animals. Microtubule-associated proteins are important components of these assemblies, and we are now starting to uncover what these proteins are and how they might work.

Clive Lloyd; Patrick Hussey



TAC1 and ZYG-9 Form a Complex that Promotes Microtubule Assembly in C. elegans Embryos  

Microsoft Academic Search

Background: Modulation of microtubule dynamics is crucial for proper cell division. While a large body of work has made important contributions to our understanding of the mechanisms governing microtubule dynamics in vitro, much remains to be learned about how these mechanisms operate in vivo.Results: We identified TAC-1 as the sole TACC (Transforming Acidic Coiled Coil) protein in C. elegans. TAC-1

Jean-Michel Bellanger; Pierre Gönczy



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

PubMed Central

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

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



The role of MAP65-1 in microtubule bundling during Zinnia tracheary element formation.  


The MAP65 family of microtubule-associated proteins performs various functions at different stages of the cell cycle and differentiation. In this study, we have investigated the synchronous transdifferentiation of Zinnia mesophyll cells into tracheary elements in vitro. This allowed us to examine the role of the microtubule-associated protein MAP65 during the characteristic bunching of cortical microtubules that underlie the developing ribs of secondarily thickened cell wall. Immunofluorescence confirmed the microtubule bundles to be decorated with anti-MAP65 antibodies. Three Zinnia MAP65 genes were examined; the expression of ZeMAP65-1 was found to match that of the differentiation marker TED2 and both were found to be upregulated upon addition of inductive hormones. We cloned the full-length sequence of ZeMAP65-1 and found it to be most similar to other MAP65 isoforms known to bundle microtubules in other plant species. However, not all MAP65 proteins crosslink cortical microtubules and so, to confirm its potential bundling capacity, ZeMAP65-1 was transiently overexpressed in Arabidopsis suspension cells. This resulted in the super-bundling of microtubules in patterns resembling those in differentiating xylem cells. These findings establish that the MAP65-1 group of proteins is responsible for the bundling of cortical microtubules during secondary cell wall formation of xylogenesis as well as during the expansion of primary cell walls. PMID:16449317

Mao, Guojie; Buschmann, Henrik; Doonan, John H; Lloyd, Clive W



Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms  

E-print Network

). The yeast-specific Dam1 subcomplex, and a possible functional homolog in higher eukary- otes, the Ska1Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple-bearing attachments to dynamic microtubule tips, and by participating in phosphoregu- latory error correction

Asbury, Chip


Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape  

Microsoft Academic Search

Microtubules are long, proteinaceous fila- ments that perform structural functions in eukaryotic cells by defining cellular shape and serving as tracks for intracellular motor proteins. We report the first ac- curate measurements of the flexural rigidity of micro- tubules. By analyzing the thermally driven fluctuations in their shape, we estimated the mean flexural rigidity of taxol-stabilized microtubules to be 2.2

Frederick Gittes; Brian Mickey; Jilda Nettleton; Jonathon Howard



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

Microsoft Academic Search

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

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



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


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

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



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

PubMed Central

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

Loong, Herbert H; Yeo, Winnie




Microsoft Academic Search

This study investigated patterns of cytoskeletal organization during microsporogenesis in Chondrilla juncea L., an autonomous apomict with a triploid chromosome number (2n = 15). The distribution of microtubules and organelles is not typical. The microtubules do not form a normal phragmoplast and consequently the organelle equatorial plate observed in many taxons is not present. The organelles are dispersed randomly in



Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells  

Microsoft Academic Search

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30 -100 nM)

Anne-Marie C. Yvon; Patricia Wadsworth; Mary Ann Jordan



Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD  

NASA Technical Reports Server (NTRS)

Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

Granger, C. L.; Cyr, R. J.



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

SciTech Connect

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

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



Mutation of Ser172 in Yeast b Tubulin Induces Defects in Microtubule Dynamics and Cell Division  

E-print Network

Mutation of Ser172 in Yeast b Tubulin Induces Defects in Microtubule Dynamics and Cell Division of both cytoplasmic and spindle MTs and for normal cell division. Citation: Caudron F, Denarier E, Thibout in Microtubule Dynamics and Cell Division. PLoS ONE 5(10): e13553. doi:10.1371/journal.pone.0013553 Editor

Paris-Sud XI, Université de


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

E-print Network

Review Axonal Transport of Microtubules: the Long and Short of It Peter W. Baas*, C. Vidya Nadar. Queen Lane, Philadelphia, PA 19129, USA *Corresponding author: Peter W. Baas, Recent of the microtubule mass in the axon is actually stationary. After a great deal of debate and controversy

Baas, Peter W.


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

Microsoft Academic Search

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

J. B. Mullinax; B. A. Palevitz



Interaction of microtubules with active principles of Xanthium strumarium.  


Indigenous variety of Xanthium strumarium (X. strumarium) was screened for its antimitotic activity using the microtubule-tubulin system isolated from mammalian tissue. A preliminary phytochemical screening of the whole extracts of the plant was carried out followed by partial purification of the whole extract of X.strumarium. The separated fractions obtained were identified and used for in vitro polymerization studies. The whole as well as partially separated chemical constituents of X. strumarium showed effective inhibition of tubulin polymerization. The results thus suggest that X. strumarium may possess antimitotic components. PMID:12002689

Menon, G S; Kuchroo, K; Dasgupta, D



Microtubule-targeting anticancer agents from marine natural substance.  


Effective novel therapeutics is urgently needed due to increasing incidence of malignant cancer and drug multi-resistance. Natural products and their derivatives have historically been a source of pharmaceutical leads and therapeutic drugs. Microtubule-targeting compounds are among the most promising candidates in the combat against cancer. In particular, marine natural products (MNPs) have demonstrated exceptional potency and potential as anticancer agents. Drug discovery from MNPs provides a new pathway to develop original anticancer agents. In this review, seven classes of typical MNPs with diverse structures are summarized. Bioactive marine compounds isolated from different organisms including invertebrate animals, algae, fungi and bacteria are also discussed. PMID:24533652

Liu, Zhiguo; Xu, Pengfei; Wu, Tao; Zeng, Wenbin



Microtubule organization by kinesin motors and microtubule crosslinking protein MAP65 This article has been downloaded from IOPscience. Please scroll down to see the full text article.  

E-print Network

has been downloaded from IOPscience. Please scroll down to see the full text article. 2013 J. Phys the filament-gliding assay with kinesin-1 motors driving microtubule motion. To further enhance the complexity

Ross, Jennifer


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.  


We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit 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 until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 mircon/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione-2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends. PMID:9334345

Waterman-Storer, C M; Salmon, E D



Deletion of the STOP gene, a microtubule stabilizing factor, leads only to discrete cerebral metabolic changes in mice  

E-print Network

and axonal trafficking, and the disturbance of these transports by drugs aimed at microtubules as the members, morphogenesis and vesicle trafficking ( ).Dustin, 1984 They are particularly abundant in neurons and tubulin cells contain microtubule-stabilizing factors that can block microtubule dynamics and prevent

Paris-Sud XI, Université de


Cytomagnetometric study of interactions between microfilaments and microtubules by measuring the energy imparted to magnetic particles within the cells  

NASA Astrophysics Data System (ADS)

Cytomagnetometric measurements of the energy imparted to intracellular organelles were made to study the relationship between microtubules and microfilaments. Depolymerization of microtubules by colchicine resulted in an increase in the energy suggesting that microtubules in control condition suppress the activity of microfilaments.

Nemoto, Iku; Kawamura, Kazuhisa



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

Microsoft Academic Search

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

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



Crystal Structure of the Amino-terminal Microtubule-binding Domain of End-binding Protein 1 (EB1)*  

E-print Network

- main of EB1, which is essential for microtubule binding, and show that it forms a calponin homology (CH to microtubules. Microtubules (MTs)1 are an essential component of the cy- toskeleton, underlying the fundamental led to the hypothesis of a "plus- end complex" (8, 9). The main function of a plus-end complex may

Ikura, Mitsuhiko


On the surface lattice of microtubules: helix starts, protofilament number, seam, and handedness  

PubMed Central

The tubulin monomers of brain microtubules reassembled in vitro are arranged on a 3-start helix, irrespective of whether the number of protofilaments is 13 or 14. The dimer packing is that of the B-lattice described for flagellar microtubules. This implies that the tubulin core of microtubules contains at least one helical discontinuity. Neither 5-start nor 8-start helices have a physical significance and thus cannot be implicated in models of microtubule elongation, but the structure is compatible with elongation of protofilaments by dimers or protofilamentous oligomers. The inner and outer surfaces of the microtubule wall can be visualized by propane jet freezing, freeze fracturing, and metal replication, at a resolution of at least 4 nm. The 3-start helix is left-handed, in contrast to a previous study based on negative staining and shadowing. The reasons for this discrepancy are discussed. PMID:3949873



Interrelation between microtubules and microfilaments in the elongation zone of Arabidopsis root under clinorotation  

NASA Astrophysics Data System (ADS)

Arabidopsis thaliana plants stably transformed with a MAP4-GFP construct were grown under slow clinorotation to characterize the role of tubulin microtubules in cell growth and gravisensing in the distal elongation zone (DEZ) of the root. Plants were treated with actin and tubulin disrupting drugs to investigate the changes in microtubule distribution and orientation between clinostat grown and control plants. It has been shown that organization of microtubules in the meristem and DEZ of Arabidopsis seedling root is interrelated with the organization of microfilaments. Cooperation between cytoskeleton elements is dependent upon the type of cell differential growth and aimed to provide the stabilization of cell growth on earth. In contrast, under clinorotation the cross-talk between microtubules and microfilaments was less pronounced. This phenomenon suggests the existence of complex mechanism for regulation of microtubules and microfilaments which is probably, unmasked in simulated microgravity.

Shevchenko, G. V.; Kalinina, Ya. M.; Kordyum, E. L.


Lithium Decreases Cold-Induced Microtubule Depolymerization in Mesophyll Cells of Spinach 1  

PubMed Central

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter [1991] Plant Physiol 97: 175-181). The objective of this study was to gain insight into the question of whether microtubules depolymerize as a direct response to environmental stresses or as an indirect response to cellular changes that accompany the stresses. Leaf sections of spinach were treated with Li+ before and during exposure to low temperature. Treatment with Li+ decreased the amount of microtubule depolymerization in cells subjected to low temperature, relative to a nontreated control, raising the possibility that the microtubules in these cells may not be inherently cold labile. Rather, microtubule depolymerization may be in response to cold-induced changes in concentration of cytoplasmic components. PMID:16669100

Bartolo, Michael E.; Carter, John V.



Fibrils connect microtubule tips with kinetochores suggesting ways to couple tubulin dynamics to chromosome motion  

PubMed Central

Summary Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving “protofilaments,” strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore “plate” and an encircling, ring-shaped protein complex have been proposed to link protofilament bending to poleward chromosome motion. Here we show by electron tomography that slender fibrils connect curved protofilaments directly to the inner kinetochore. Fibril-protofilament associations correlate with a local straightening of the flared protofilaments. Theoretical analysis reveals that protofilament-fibril connections would be efficient couplers for chromosome motion, and experimental work on two very different kinetochore components suggests that filamentous proteins can couple shortening microtubules to cargo movements. These analyses define a ring-independent mechanism for harnessing microtubule dynamics directly to chromosome movement. PMID:18957206

McIntosh, J.R.; Grishchuk, E.L.; Morphew, M.; Efremov, A.K.; Zhudenkov, K.; Volkov, V.A.; Cheeseman, I.M.; Desai, A.; Mastronarde, D.N.; Ataullakhanov, F.I.



EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B  

PubMed Central

The Aurora B kinase coordinates kinetochore–microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. We find that EB1, a microtubule plus end–tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). Our data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase. PMID:24616220

Banerjee, Budhaditya; Kestner, Cortney A.



Microtubules as key coordinators of nuclear envelope and endoplasmic reticulum dynamics during mitosis.  


During mitosis, cells comprehensively restructure their interior to promote the faithful inheritance of DNA and cytoplasmic contents. In metazoans, this restructuring entails disassembly of the nuclear envelope, redistribution of its components into the endoplasmic reticulum (ER) and eventually nuclear envelope reassembly around the segregated chromosomes. The microtubule cytoskeleton has recently emerged as a critical regulator of mitotic nuclear envelope and ER dynamics. Microtubules and associated molecular motors tear open the nuclear envelope in prophase and remove nuclear envelope remnants from chromatin. Additionally, two distinct mechanisms of microtubule-based regulation of ER dynamics operate later in mitosis. First, association of the ER with microtubules is reduced, preventing invasion of ER into the spindle area, and second, organelle membrane is actively cleared from metaphase chromosomes. However, we are only beginning to understand the role of microtubules in shaping and distributing ER and other organelles during mitosis. PMID:24848719

Schlaitz, Anne-Lore



[Reorganization of microtubules as a response to realization of NO (II) signal pathways in plant cell].  


Effects of exogenic NO donor, sodium nitroprusside, on orientation and organization of cortical microtubules in Arabidopsis thaliana root cells that express GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10-500 microM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in differentiation zone. The influence of sodium nitroprusside revealed in alterations of cortical: microtubules orientation and organization in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of elongation zone where native transverse orientation of cortical microtubules turned into random, oblique or longitudinal relatively to primary root axis. We suppose that NO as one of the intracellular secondary messengers triggers cells differentiation by cortical microtubules reorientation possibly via tubulin nitrotyrosination. PMID:19938620

Emets, A I; Krasilenko, Iu A; Sheremet, Ia A; Blium, Ia B



A sesquiterpene lactone, costunolide, interacts with microtubule protein and inhibits the growth of MCF-7 cells.  


Costunolide is an active sesquiterpene lactone of medicinal herbs with anti-inflammatory and potential anti-cancer activity. Nevertheless, the pharmacological pathways of costunolide have not yet been fully elucidated. In this study we showed that costunolide exerts a dose-dependent antiproliferative activity in the human breast cancer MCF-7 cells. In addition, light microscopy observations indicated that costunolide affected nuclear organization and reorganized microtubule architecture. The antiproliferative and antimicrotubular effects of costunolide were not influenced by paclitaxel, well-known microtubule-stabilizing anticancer agent. The microtubule-interacting activity of costunolide was confirmed by in vitro studies on purified microtubular protein. In fact, costunolide demonstrated polymerizing ability, by inducing the formation of well organized microtubule polymers. Our data suggest an interaction of costunolide with microtubules, which may represent a new intracellular target for this drug. PMID:14726154

Bocca, Claudia; Gabriel, Ludovica; Bozzo, Francesca; Miglietta, Antonella



On the Nature and Shape of Tubulin Trails: Implications on Microtubule Self-Organization  

E-print Network

Microtubules, major elements of the cell skeleton are, most of the time, well organized in vivo, but they can also show self-organizing behaviors in time and/or space in purified solutions in vitro. Theoretical studies and models based on the concepts of collective dynamics in complex systems, reaction-diffusion processes and emergent phenomena were proposed to explain some of these behaviors. In the particular case of microtubule spatial self-organization, it has been advanced that microtubules could behave like ants, self-organizing by 'talking to each other' by way of hypothetic (because never observed) concentrated chemical trails of tubulin that are expected to be released by their disassembling ends. Deterministic models based on this idea yielded indeed like-looking spatio-temporal self-organizing behaviors. Nevertheless the question remains of whether microscopic tubulin trails produced by individual or bundles of several microtubules are intense enough to allow microtubule self-organization at a macr...

Glade, Nicolas



Microtubule sliding in swimming sperm flagella: direct and indirect measurements on sea urchin and tunicate spermatozoa [published erratum appears in J Cell Biol 1991 Nov;115(4):1204  

PubMed Central

Direct measurements of microtubule sliding in the flagella of actively swimming, demembranated, spermatozoa have been made using submicron diameter gold beads as markers on the exposed outer doublet microtubules. With spermatozoa of the tunicate, Ciona, these measurements confirm values of sliding calculated indirectly by measuring angles relative to the axis of the sperm head. Both methods of measurement show a nonuniform amplitude of oscillatory sliding along the length of the flagellum, providing direct evidence that "oscillatory synchronous sliding" can be occurring in the flagellum, in addition to the metachronous sliding that is necessary to propagate a bending wave. Propagation of constant amplitude bends is not accomplished by propagation of a wave of oscillatory sliding of constant amplitude, and therefore appears to require a mechanism for monitoring and controlling the bend angle as bends propagate. With sea urchin spermatozoa, the direct measurements of sliding do not agree with the values calculated by measuring angles relative to the head axis. The oscillation in angular orientation of the sea urchin sperm head as it swims appears to be accommodated by flexure at the head- flagellum junction and does not correspond to oscillation in orientation of the basal end of the flagellum. Consequently, indirect calculations of sliding based on angles measured relative to the longitudinal axis of the sperm head can be seriously inaccurate in this species. PMID:1894694



Alteration of microtubule dynamic instability during preprophase band formation revealed by yellow fluorescent protein-CLIP170 microtubule plus-end labeling.  


At the onset of mitosis, plant cells form a microtubular preprophase band that defines the plane of cell division, but the mechanism of its formation remains a mystery. Here, we describe the use of mammalian yellow fluorescent protein-tagged CLIP170 to visualize the dynamic plus ends of plant microtubules in transfected cowpea protoplasts and in stably transformed and dividing tobacco Bright Yellow 2 cells. Using plus-end labeling, we observed dynamic instability in different microtubular conformations in live plant cells. The interphase plant microtubules grow at 5 micro m/min, shrink at 20 micro m/min, and display catastrophe and rescue frequencies of 0.02 and 0.08 events/s, respectively, exhibiting faster turnover than their mammalian counterparts. Strikingly, during preprophase band formation, the growth rate and catastrophe frequency of plant microtubules double, whereas the shrinkage rate and rescue frequency remain unchanged, making microtubules shorter and more dynamic. Using these novel insights and four-dimensional time-lapse imaging data, we propose a model that can explain the mechanism by which changes in microtubule dynamic instability drive the dramatic rearrangements of microtubules during preprophase band and spindle formation in plant cells. PMID:12615935

Dhonukshe, Pankaj; Gadella, Theodorus W J



Automatic quantification of microtubule dynamics enables RNAi-screening of new mitotic spindle regulators.  


The genetic integrity of every organism depends on the faithful partitioning of its genome between two daughter cells in mitosis. In all eukaryotes, chromosome segregation requires the assembly of the mitotic spindle, a bipolar array of dynamic microtubules. Perturbations in microtubule dynamics affect spindle assembly and maintenance and ultimately result in aberrant cell divisions. To identify new regulators of microtubule dynamics within the hundreds of mitotic hits, reported in RNAi screens performed in C. elegans, Drosophila and mammalian tissue culture cells [Sonnichsen et al., 2005; Goshima et al., 2007; Neumann et al., 2010], we established a fast and quantitative assay to measure microtubule dynamics in living cells. Here we present a fully automated workflow from RNAi transfection, via image acquisition and data processing, to the quantitative characterization of microtubule behaviour. Candidate genes are knocked down by solid-phase reverse transfection with siRNA oligos in HeLa cells stably expressing EB3-EGFP, a microtubule plus end marker. Mitotic cells are selected using an automatic classifier [Conrad et al., 2011] and imaged on a spinning disk confocal microscope at high temporal and spatial resolution. The time-lapse movies are analysed using a multiple particle tracking software, developed in-house, that automatically detects microtubule plus ends, tracks microtubule growth events over consecutive frames and calculates growth speeds, lengths and lifetimes of the tracked microtubules. The entire assay provides a powerful tool to analyse the effect of essential mitotic genes on microtubule dynamics in living cells and to dissect their contribution in spindle assembly and maintenance. PMID:21491614

Sironi, Lucia; Solon, Jérôme; Conrad, Christian; Mayer, Thomas U; Brunner, Damian; Ellenberg, Jan



Arsenic trioxide induces depolymerization of microtubules in an acute promyelocytic leukemia cell line  

PubMed Central

Background Arsenic trioxide (As2O3) is a well-known and effective treatment that can result in clinical remission for patients diagnosed with acute promyelocytic leukemia (APL). The biologic efficacy of As2O3 in APL and solid tumor cells has been explained through its actions on anti-proliferation, anti-angiogenesis, and apoptotic signaling pathways. We theorize that As2O3 activates a pathway that disrupts microtubule dynamics forming abnormal, nonfunctioning mitotic spindles, thus preventing cellular division. In this study, we investigated how As2O3 induces apoptosis by causing microtubule dysfunction. Methods Cultured NB4 cells were treated with As2O3, paclitaxel, and vincristine. Flow cytometric analysis was then performed. An MTT assay was used to determine drug-mediated cytotoxicity. For tubulin polymerization assay, each polymerized or soluble tubulin was measured. Microtubule assembly-disassembly was measured using a tubulin polymerization kit. Cellular microtubules were also observed with fluorescence microscopy. Results As2O3 treatment disrupted tubulin assembly resulting in dysfunctional microtubules that cause death in APL cells. As2O3 markedly enhanced the amount of depolymerized microtubules. The number of microtubule posttranslational modifications on an individual tubulin decreased with As2O3 concentration. Immunocytochemistry revealed changes in the cellular microtubule network and formation of polymerized microtubules in As2O3-treated cells. Conclusion The microtubules alterations found with As2O3 treatment suggest that As2O3 increases the depolymerized forms of tubulin in cells and that this is potentially due to arsenite's negative effects on spindle dynamics. PMID:22783356

Baek, Jin Ho; Moon, Chang Hoon; Cha, Seung Joo; Lee, Hee Soon; Noh, Eui-Kyu; Kim, Hawk; Won, Jong-Ho



The chirality of ciliary beats  

NASA Astrophysics Data System (ADS)

Many eukaryotic cells possess cilia which are motile, whip-like appendages that can oscillate and thereby induce motion and fluid flows. These organelles contain a highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of nine doublets of microtubules. Complex bending waves emerge from the interplay of active internal forces generated by dynein motor proteins within the structure. These bending waves are typically chiral and often exhibit a sense of rotation. In order to study how the shape of the beat emerges from the axonemal structure, we present a three-dimensional description of ciliary dynamics based on the self-organization of dynein motors and microtubules. Taking into account both bending and twisting of the cilium, we determine self-organized beating patterns and find that modes with both a clockwise and anticlockwise sense of rotation exist. Because of the axonemal chirality, only one of these modes is selected dynamically for given parameter values and properties of dynein motors. This physical mechanism, which underlies the selection of a beating pattern with specific sense of rotation, triggers the breaking of the left-right symmetry of developing embryos which is induced by asymmetric fluid flows that are generated by rotating cilia.

Hilfinger, A.; Jülicher, F.



Hepatocyte cotransport of taurocholate and bilirubin glucuronides: Role of microtubules  

SciTech Connect

Modulation of bile pigment excretion by bile salts has been attributed to modification of canalicular membrane transport or a physical interaction in bile. Based on the observation that a microtubule-dependent pathway is involved in the hepatocellular transport of bile salts, the authors investigated the possibility that bilirubin glucuronides are associated with bile salts during intracellular transport. Experiments were conducted in intact rats (basal) or after overnight biliary diversion and intravenous reinfusion of taurocholate (depleted/reinfused). All rats were pretreated with intravenous low-dose colchicine or its inactive isomer lumicolchicine. Biliary excretion of radiolabeled bilirubin glucuronides derived from tracer ({sup 14}C)bilirubin-({sup 3}H)bilirubin monoglucuronide (coinjected iv) was unchanged in basal rats but was consistently delayed in depleted/reinfused rats. This was accompanied by a significant shift toward bilirubin diglucuronide formation from both substrates. In basal Gunn rats, with deficient bilirubin glucuronidation, biliary excretion of intravenous ({sup 14}C)bilirubin monoglucuronide-({sup 3}H)bilirubin diglucuronide was unaffected by colchicine but was retarded in depleted/reinfused Gunn rats. Colchicine had no effect on the rate of bilirubin glucuronidation in vitro in rat liver microsomes. They conclude that a portion of the bilirubin glucuronides generated endogenously in hepatocytes or taken up directly from plasma may be cotransported with bile salts to the bile canalicular membrane via a microtubule-dependent mechanism.

Crawford, J.M.; Gollan, J.L. (Harvard Medical School, Boston, MA (USA))



Phosphorylation of the Yeast ?-Tubulin Tub4 Regulates Microtubule Function  

PubMed Central

The yeast ?-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in ?-tubulins from yeast to human. PMID:21573187

Lin, Tien-chen; Gombos, Linda; Neuner, Annett; Sebastian, Dominik; Olsen, Jesper V.; Hrle, Ajla; Benda, Christian; Schiebel, Elmar



The evolution and diversification of plant microtubule-associated proteins.  


Plant evolution is marked by major advances in structural characteristics that facilitated the highly successful colonization of dry land. Underlying these advances is the evolution of genes encoding specialized proteins that form novel microtubular arrays of the cytoskeleton. This review investigates the evolution of plant families of microtubule-associated proteins (MAPs) through the recently sequenced genomes of Arabidopsis thaliana, Oryza sativa, Selaginella moellendorffii, Physcomitrella patens, Volvox carteri and Chlamydomonas reinhardtii. The families of MAPs examined are AIR9, CLASP, CRIPT, MAP18, MOR1, TON, EB1, AtMAP70, SPR2, SPR1, WVD2 and MAP65 families (abbreviations are defined in the footnote to Table 1). Conjectures are made regarding the evolution of MAPs in plants in relation to the evolution of multicellularity, oriented cell division and vasculature. Angiosperms in particular have high numbers of proteins that are involved in promotion of helical growth or its suppression, and novel plant microtubular structures may have acted as a catalyst for the development of novel plant MAPs. Comparisons of plant MAP gene families with those of animals show that animals may have more flexibility in the structure of their microtubule cytoskeletons than plants, but with both plants and animals possessing many MAP splice variants. PMID:23551562

Gardiner, John



Microtubules restrict plastid sedimentation in protonemata of the moss Ceratodon  

NASA Technical Reports Server (NTRS)

Apical cells of protonemata of the moss Ceratodon purpureus are unusual among plant cells with sedimentation in that only some amyloplasts sediment and these do not fall completely to the bottom of vertical cells. To determine whether the cytoskeleton restricts plastid sedimentation, the effects of amiprophos-methyl (APM) and cytochalasin D (CD) on plastid position were quantified. APM treatments of 30-60 min increased the plastid sedimentation that is normally seen along the length of untreated or control cells. Longer APM treatments often resulted in more dramatic plastid sedimentation, and in some cases almost all plastids sedimented to the lowermost point in the cell. In contrast, the microfilament inhibitor CD did not affect longitudinal plastid sedimentation compared to untreated cells, although it did disturb or eliminate plastid zonation in the tip. These data suggest that microtubules restrict the sedimentation of plastids along the length of the cell and that microtubules are load-bearing for all the plastids in the apical cell. This demonstrates the importance of the cytoskeleton in maintaining organelle position and cell organization against the force of gravity.

Schwuchow, J.; Sack, F. D.



Microtubules restrict plastid sedimentation in protonemata of the moss Ceratodon.  


Apical cells of protonemata of the moss Ceratodon purpureus are unusual among plant cells with sedimentation in that only some amyloplasts sediment and these do not fall completely to the bottom of vertical cells. To determine whether the cytoskeleton restricts plastid sedimentation, the effects of amiprophos-methyl (APM) and cytochalasin D (CD) on plastid position were quantified. APM treatments of 30-60 min increased the plastid sedimentation that is normally seen along the length of untreated or control cells. Longer APM treatments often resulted in more dramatic plastid sedimentation, and in some cases almost all plastids sedimented to the lowermost point in the cell. In contrast, the microfilament inhibitor CD did not affect longitudinal plastid sedimentation compared to untreated cells, although it did disturb or eliminate plastid zonation in the tip. These data suggest that microtubules restrict the sedimentation of plastids along the length of the cell and that microtubules are load-bearing for all the plastids in the apical cell. This demonstrates the importance of the cytoskeleton in maintaining organelle position and cell organization against the force of gravity. PMID:7859298

Schwuchow, J; Sack, F D



Inert Doublet Dark Matter with an additional scalar singlet and 125 GeV Higgs Boson  

E-print Network

In this work we consider a model for particle dark matter where an extra inert Higgs doublet and an additional scalar singlet is added to the Standard Model (SM) Lagrangian. The dark matter candidate is obtained from only the inert doublet. The stability of this one component dark matter is ensured by imposing a $Z_2$ symmetry on this additional inert doublet. The additional singlet scalar has a vacuum expectation value (VEV) and mixes with the Standard Model Higgs doublet resulting in two CP even scalars $h_1$ and $h_2$. We treat one of these scalars, $h_1$, to be consistent with the SM Higgs like boson of mass around 125 GeV reported by the LHC experiment. These two CP even scalars affect the annihilation cross-section of this inert doublet dark matter resulting in a larger dark matter mass region that satisfies the observed relic density. We also investigate the $h_1 \\rightarrow \\gamma\\gamma$ and $h_1 \\rightarrow \\gamma Z$ processes and compared these with LHC results. This is also used to constrain the da...

Banik, Amit Dutta



The effect of human microtubule-associated-protein tau on the assembly structure of microtubules and its ionic strength dependence  

NASA Astrophysics Data System (ADS)

Microtubules (MTs), 25 nm protein nanotubes, are among the major filamentous elements of the eukaryotic cytoskeleton involved in intracellular trafficking, cell division and the establishment and maintenance of cell shape. Microtubule-associated-protein tau regulates tubulin assembly, MT dynamics and stability. Aberrant tau action has long been correlated with numerous neurodegenerative diseases, including Alzheimer's, and fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) Using synchrotron small angle x-ray scattering (SAXS) and binding assay, we examine the effects of tau on the assembly structure of taxol-stabilized MTs. We find that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius of MTs with increasing the tau/tubulin molar ratio. Additionally, tau-MT interactions are mediated to a large extent via electrostatic interactions: the binding affinity of tau to MTs is ionic strength dependent. Supported by DOE-BES DE-FG02-06ER46314, NSF DMR-0803103, NIH NS35010, NIH NS13560. (Ref) M.C. Choi, S.C. Feinstein, and C.R. Safinya et al. Biophys. J. 97; 519 (2009).

Choi, M. C.; Raviv, U.; Miller, H. P.; Gaylord, M. R.; Kiris, E.; Ventimiglia, D.; Needleman, D. J.; Chung, P. J.; Deek, J.; Lapointe, N.; Kim, M. W.; Wilson, L.; Feinstein, S. C.; Safinya, C. R.



Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons.  


The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ?40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP-tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin-microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport. PMID:25165145

Janning, Dennis; Igaev, Maxim; Sündermann, Frederik; Brühmann, Jörg; Beutel, Oliver; Heinisch, Jürgen J; Bakota, Lidia; Piehler, Jacob; Junge, Wolfgang; Brandt, Roland



Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis.  

PubMed Central

Assembly of a mitotic spindle requires the accurate regulation of microtubule dynamics which is accomplished, at least in part, by phosphorylation-dephosphorylation reactions. Here we have investigated the role of serine-threonine phosphatases in the control of microtubule dynamics using specific inhibitors in Xenopus egg extracts. Type 2A phosphatases are required to maintain the short steady-state length of microtubules in mitosis by regulating the level of microtubule catastrophes, in part by controlling the the microtubule-destabilizing activity and phosphorylation of Op18/stathmin. Type 1 phosphatases are only required for control of microtubule dynamics during the transitions into and out of mitosis. Thus, although both type 2A and type 1 phosphatases are involved in the regulation of microtubule dynamics, they have distinct, non-overlapping roles. PMID:9312013

Tournebize, R; Andersen, S S; Verde, F; Dorée, M; Karsenti, E; Hyman, A A



Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity  

PubMed Central

Fast transport of axonal vesicles and organelles is a microtubule- associated movement (Griffin, J. W., K. E. Fahnestock, L. Price, and P. N. Hoffman, 1983, J. Neuroscience, 3:557-566; Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese, 1984, Cell, 40:455-462; Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson, 1985, J. Cell Biol., 100:1736-1752). Proteins that mediate the interactions of axoplasmic vesicles and microtubules were studied using stable complexes of microtubules and vesicles (MtVC). These complexes formed spontaneously in vitro when taxol-stabilized microtubules were mixed with sonically disrupted axoplasm from the giant axon of the squid Loligo pealei. The isolated MtVCs contain a distinct subset of axoplasmic proteins, and are composed primarily of microtubules and attached membranous vesicles. The MtVC also contains nonmitochondrial ATPase activity. The binding of one high molecular mass polypeptide to the complex is significantly enhanced by ATP or adenyl imidodiphosphate. All of the axoplasmic proteins and ATPase activity that bind to microtubules are found in macromolecular complexes and appear to be vesicle-associated. These data allow the identification of several vesicle-associated proteins of the squid giant axon and suggest that one or more of these polypeptides mediates vesicle binding to microtubules. PMID:2943747



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

PubMed Central

Microtubules are hollow cylindrical structures that constitute one of the three major classes of cytoskeletal filaments. On the mesoscopic length scale of a cell, their material properties are characterized by a single stiffness parameter, the persistence length ?p. Its value, in general, depends on the microscopic interactions between the constituent tubulin dimers and the architecture of the microtubule. Here, we use single-particle tracking methods combined with a fluctuation analysis to systematically study the dependence of ?p on the total filament length L. Microtubules are grafted to a substrate with one end free to fluctuate in three dimensions. A fluorescent bead is attached proximally to the free tip and is used to record the thermal fluctuations of the microtubule's end. The position distribution functions obtained with this assay allow the precise measurement of ?p for microtubules of different contour length L. Upon varying L between 2.6 and 47.5 ?m, we find a systematic increase of ?p from 110 to 5,035 ?m. At the same time we verify that, for a given filament length, the persistence length is constant over the filament within the experimental accuracy. We interpret this length dependence as a consequence of a nonnegligible shear deflection determined by subnanometer relative displacement of adjacent protofilaments. Our results may shine new light on the function of microtubules as sophisticated nanometer-sized molecular machines and give a unified explanation of seemingly uncorrelated spreading of microtubules' stiffness previously reported in literature. PMID:16801537

Pampaloni, Francesco; Lattanzi, Gianluca; Jonas, Alexandr; Surrey, Thomas; Frey, Erwin; Florin, Ernst-Ludwig



Collapsin Response Mediator Protein 4 Regulates Growth Cone Dynamics through the Actin and Microtubule Cytoskeleton.  


Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton. PMID:25225289

Khazaei, Mohamad R; Girouard, Marie-Pier; Alchini, Ricardo; Ong Tone, Stephan; Shimada, Tadayuki; Bechstedt, Susanne; Cowan, Mitra; Guillet, Dominique; Wiseman, Paul W; Brouhard, Gary; Cloutier, Jean Francois; Fournier, Alyson E



Mitotic Regulator SKAP Forms a Link between Kinetochore Core Complex KMN and Dynamic Spindle Microtubules*  

PubMed Central

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent study shows that mitotic motor CENP-E cooperates with SKAP to orchestrate an accurate chromosome movement in mitosis. However, it remains elusive how kinetochore core microtubule binding activity KMN (KNL1-MIS12-NDC80) regulates microtubule plus-end dynamics. Here, we identify a novel interaction between MIS13 and SKAP that orchestrates accurate interaction between kinetochore and dynamic spindle microtubules. SKAP physically interacts with MIS13 and specifies kinetochore localization of SKAP. Suppression of MIS13 by small interfering RNA abrogates the kinetochore localization of SKAP. Total internal reflection fluorescence microscopic assays demonstrate that SKAP exhibits an EB1-dependent, microtubule plus-end loading and tracking in vitro. Importantly, SKAP is essential for kinetochore oscillations and dynamics of microtubule plus-ends during live cell mitosis. Based on those findings, we reason that SKAP constitutes a dynamic link between spindle microtubule plus-ends and mitotic chromosomes to achieve faithful cell division. PMID:23035123

Wang, Xiwei; Zhuang, Xiaoxuan; Cao, Dan; Chu, Youjun; Yao, Phil; Liu, Wei; Liu, Lifang; Adams, Gregory; Fang, Guowei; Dou, Zhen; Ding, Xia; Huang, Yuejia; Wang, Dongmei; Yao, Xuebiao



Variational Principles for Buckling of Microtubules Modeled as Nonlocal Orthotropic Shells  

PubMed Central

A variational principle for microtubules subject to a buckling load is derived by semi-inverse method. The microtubule is modeled as an orthotropic shell with the constitutive equations based on nonlocal elastic theory and the effect of filament network taken into account as an elastic surrounding. Microtubules can carry large compressive forces by virtue of the mechanical coupling between the microtubules and the surrounding elastic filament network. The equations governing the buckling of the microtubule are given by a system of three partial differential equations. The problem studied in the present work involves the derivation of the variational formulation for microtubule buckling. The Rayleigh quotient for the buckling load as well as the natural and geometric boundary conditions of the problem is obtained from this variational formulation. It is observed that the boundary conditions are coupled as a result of nonlocal formulation. It is noted that the analytic solution of the buckling problem for microtubules is usually a difficult task. The variational formulation of the problem provides the basis for a number of approximate and numerical methods of solutions and furthermore variational principles can provide physical insight into the problem.



Microtubule Dynamics Regulate Cyclic Stretch-Induced Cell Alignment in Human Airway Smooth Muscle Cells  

PubMed Central

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma. PMID:22022610

Naruse, Keiji; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori; Suki, Béla



CNP/cGMP Signaling Regulates Axon Branching and Growth by Modulating Microtubule Polymerization  

PubMed Central

The peptide hormone CNP has recently been found to positively regulate axon branching and growth via activation of cGMP signaling in embryonic dorsal root ganglion (DRG) neurons, but the cellular mechanisms mediating the regulation of these developmental processes have not been established. In this study, we provide evidence linking CNP/cGMP signaling to microtubule dynamics via the microtubule regulator CRMP2. First, phosphorylation of CRMP2 can be suppressed by cGMP activation in embryonic DRG neurons, and non-phosphorylated CRMP2 promotes axon branching and growth. In addition, real time analysis of growing microtubule ends indicates a similar correlation of CRMP2 phosphorylation and its activity in promoting microtubule polymerization rates and durations in COS cells and DRG growth cones. Moreover, direct activation of cGMP signaling leads to increased assembly of dynamic microtubules in DRG growth cones. Finally, low doses of a microtubule depolymerization drug nocodazole block CNP/cGMP-dependent axon branching and growth. Taken together, our results support a critical role of microtubule dynamics in mediating CNP/cGMP regulation of axonal development. PMID:23420620

Xia, Caihong; Nguyen, Minh; Garrison, Amy K.; Zhao, Zhen; Wang, Zheng; Sutherland, Calum; Ma, Le



Isolation of a 90-kD Microtubule-Associated Protein from Tobacco Membranes.  


The organization and function of microtubules in plant cells are important in key developmental events, including the regulation of directional cellulose deposition. Bridges connecting microtubules to each other and to membranes and other organelles have been documented by electron microscopy; however, the biochemical and molecular nature of these linkages is not known. We have partitioned proteins from a suspension culture of tobacco into cytosolic and membrane fractions, solubilized the membrane fraction with a zwitterionic detergent, and then used affinity chromatography and salt elution to isolate tubulin binding proteins. Dark-field microscopy of in vitro-assembled microtubules showed that the eluted proteins from both fractions induce microtubule bundling and, in the presence of purified tubulin, promote microtubule elongation. Gel electrophoresis of the eluted proteins revealed two distinct sets of polypeptides. Those in the membrane eluate included unique bands with apparent molecular masses of 98, 90, and 75 kD in addition to bands present in both eluates. The cytosolic eluate, in contrast, typically included relatively smaller proteins. The eluted proteins also bound to taxol-stabilized microtubules. Initial immunological characterization using monoclonal antibodies raised against the 90-kD polypeptide showed that it is colocalized in situ with cortical microtubules in tobacco protoplast ghosts. PMID:12239375

Marc, J.; Sharkey, D. E.; Durso, N. A.; Zhang, M.; Cyr, R. J.



Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis  

PubMed Central

During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end–tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II–mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs. PMID:21263030

Myers, Kenneth A.; Applegate, Kathryn T.



The putative RNA-processing protein, THO2, is a microtubule-associated protein in tobacco.  


THO2 is a component of the THO-TREX (transcription and export factor) complex that participates in mRNA metabolism and export from the nucleus in yeast and animal cells. Here we report that tobacco putative THO2-related protein (NtTHO2) is a microtubule-associated protein, which directly binds to microtubules in vitro and co-localizes with cortical microtubules in vivo. We purified endogenous NtTHO2 by cycles of microtubule polymerization-depolymerization from crude extracts of tobacco BY-2 miniprotoplasts. Purified NtTHO2 sedimented with microtubules in vitro. Immunofluorescence revealed that NtTHO2 was present in both the nucleus and cytoplasm. In interphase, cytoplasmic NtTHO2 was localized along cortical microtubules. In the mitotic phase, NtTHO2 was localized to the mitotic spindle but not to either the preprophase band or the phragmoplast. In mature cells of seedling roots, and in BY-2 cells in which proliferation was stopped by removing 2,4-D, NtTHO2 staining was confined mainly to the nucleolus. These results suggest that NtTHO2 is a multifunctional protein that participates in mRNA metabolism, and also functions within the cortical microtubules and mitotic spindle. PMID:19218314

Hamada, Takahiro; Igarashi, Hisako; Taguchi, Ryoichi; Fujiwara, Masayuki; Fukao, Yoichiro; Shimmen, Teruo; Yokota, Etsuo; Sonobe, Seiji



The colchicine derivative CT20126 shows a novel microtubule-modulating activity with apoptosis  

PubMed Central

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5???), the normal thread-like microtubules were disrupted into tubulin dimers within 10?min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20?min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350?nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20?n?, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol. PMID:23598593

Kim, Sung-Kuk; Cho, Sang-Min; Kim, Ho; Seok, Heon; Kim, Soon-Ok; Kyu Kwon, Taeg; Chang, Jong-Soo



Motor protein-driven unidirectional transport of micrometer-sized cargoes across isopolar microtubule arrays  

NASA Astrophysics Data System (ADS)

Conventional kinesin is a motor protein which translocates organelles from cell centre to cell periphery along specialized filamentous tracks, called microtubules. The direction of translocation is determined by microtubule polarity. This process of biological force generation can be simulated outside cells with kinesin-coated particles actively moving along immobilized microtubules. The in vitro approaches of kinesin-mediated transport described so far had the disadvantage that concerning their polarity the microtubules were randomly distributed resulting in random transport direction. The present paper demonstrates the unidirectional translocation of kinesin-coated cargoes across arrays of microtubules aligned not only in a geometrically parallel but also in an isopolar fashion. As cargo, glass, gold, and polystyrene beads with diameters between 1 and 10 µm were used. Independent of material and size, these beads were observed to move unidirectionally with average velocities of 0.3-1.0 µm s-1 over distances up to 2.2 mm. Moreover, the isopolar microtubule arrays even enabled the transport of large flat glass particles with an area of up to 24 µm×12 µm and 2-5 µm thickness which obviously contacted more than one microtubule. The controlling transport direction is considered to be an essential step for future developments of motor protein-based microdevices working in nanometre steps.

Böhm, Konrad J.; Stracke, Roland; Mühlig, Peter; Unger, Eberhard



Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity.  


Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these fin