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Sample records for anterograde microtubule transport

  1. Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

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    Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

    2015-01-01

    The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. PMID:26157166

  2. Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

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    Taylor, Matthew P.; Kratchmarov, Radomir; Enquist, Lynn W.

    2013-01-01

    Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread. PMID:23978901

  3. Anterograde glycoprotein-dependent transport of newly generated rabies virus in dorsal root ganglion neurons.

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    Bauer, Anja; Nolden, Tobias; Schröter, Josephine; Römer-Oberdörfer, Angela; Gluska, Shani; Perlson, Eran; Finke, Stefan

    2014-12-01

    Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of

  4. Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells.

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    De Rossi, María Cecilia; Wetzler, Diana E; Benseñor, Lorena; De Rossi, María Emilia; Sued, Mariela; Rodríguez, Daniela; Gelfand, Vladimir; Bruno, Luciana; Levi, Valeria

    2017-12-01

    Intracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells. We used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion of organelles. The transport performance was studied in cells expressing a slow chimeric plus-end directed motor or the kinesin heavy chain. We also analyzed the influence of peroxisomes membrane fluidity in methyl-β-ciclodextrin treated cells. The experimental data was also confronted with numerical simulations of two well-established tug of war scenarios. The velocity distributions of retrograde and anterograde peroxisomes showed a multimodal pattern suggesting that multiple motor teams drive transport in either direction. The chimeric motors interfered with the performance of anterograde transport and also reduced the speed of the slowest retrograde team. In addition, increasing the fluidity of peroxisomes membrane decreased the speed of the slowest anterograde and retrograde teams. Our results support the existence of a crosstalk between opposed-polarity motor teams. Moreover, the slowest teams seem to mechanically communicate with each other through the membrane to trigger transport. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. KIF5C S176 Phosphorylation Regulates Microtubule Binding and Transport Efficiency in Mammalian Neurons.

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    Artur ePadzik

    2016-03-01

    Full Text Available Increased phosphorylation of the KIF5 anterograde motor is associated with impaired axonal transport and neurodegeneration, but paradoxically also with normal transport, though the details are not fully defined. JNK phosphorylates KIF5C on S176 in the motor domain; a site that we show is phosphorylated in brain. Microtubule pelleting assays demonstrate that phosphomimetic KIF5C(1-560S176D associates weakly with microtubules compared to KIF5C(1-560WT. Consistent with this, 50% of KIF5C(1-560S176D shows diffuse movement in neurons. However the remaining 50% remains microtubule bound and displays decreased pausing and increased bidirectional movement. The same directionality switching is observed with KIF5C(1-560WT in the presence of an active JNK chimera, MKK7-JNK. Yet, in cargo trafficking assays where peroxisome cargo is bound, KIF5C(1-560S176D-GFP-FRB transports normally to microtubule plus ends. We also find that JNK increases the ATP hydrolysis of KIF5C in vitro. These data suggest that phosphorylation of KIF5C-S176 primes the motor to either disengage entirely from microtubule tracks as previously observed in response to stress, or to display improved efficiency. The final outcome may depend on cargo load and motor ensembles.

  6. Herpes Simplex Virus gE/gI and US9 Promote both Envelopment and Sorting of Virus Particles in the Cytoplasm of Neurons, Two Processes That Precede Anterograde Transport in Axons.

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    DuRaine, Grayson; Wisner, Todd W; Howard, Paul; Williams, Melissa; Johnson, David C

    2017-06-01

    Herpes simplex virus (HSV) anterograde transport in neuronal axons is vital, allowing spread from latently infected ganglia to epithelial tissues, where viral progeny are produced in numbers allowing spread to other hosts. The HSV membrane proteins gE/gI and US9 initiate the process of anterograde axonal transport, ensuring that virus particles are transported from the cytoplasm into the most proximal segments of axons. These proteins do not appear to be important once HSV is inside axons. We previously described HSV double mutants lacking both gE and US9 that failed to transport virus particles into axons. Here we show that gE - US9 - double mutants accumulate large quantities of unenveloped and partially enveloped capsids in neuronal cytoplasm. These defects in envelopment can explain the defects in axonal transport of enveloped virions. In addition, the unenveloped capsids that accumulated were frequently bound to cytoplasmic membranes, apparently immobilized in intermediate stages of envelopment. A gE-null mutant produced enveloped virions, but these accumulated in large numbers in the neuronal cytoplasm rather than reaching cell surfaces as wild-type HSV virions do. Thus, in addition to the defects in envelopment, there was missorting of capsids and enveloped particles in the neuronal cytoplasm, which can explain the reduced anterograde transport of unenveloped capsids and enveloped virions. These mechanisms differ substantially from existing models suggesting that gE/gI and US9 function by tethering HSV particles to kinesin microtubule motors. The defects in assembly of gE - US9 - mutant virus particles were novel because they were neuron specific, in keeping with observations that US9 is neuron specific. IMPORTANCE Herpes simplex virus (HSV) and other alphaherpesviruses, such as varicella-zoster virus, depend upon the capacity to navigate in neuronal axons. To do this, virus particles tether themselves to dyneins and kinesins that motor along microtubules

  7. Calsyntenin-1 shelters APP from proteolytic processing during anterograde axonal transport

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    Martin Steuble

    2012-06-01

    Endocytosis of amyloid-β precursor protein (APP is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.

  8. Anterograde transport blockade precedes deficits in retrograde transport in the visual projection of the DBA/2J mouse model of glaucoma

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    Christine M Dengler-Crish

    2014-09-01

    Full Text Available Axonal transport deficits have been reported as an early pathology in several neurodegenerative disorders, including glaucoma. However, the progression and mechanisms of these deficits are poorly understood. Previous work suggests that anterograde transport is affected earlier and to a larger degree than retrograde transport, yet this has never been examined directly in vivo. Using combined anterograde and retrograde tract tracing methods, we examined the time-course of anterograde and retrograde transport deficits in the retinofugal projection in pre-glaucomatous (3 month-old and glaucomatous (9-13 month old DBA/2J mice. DBA/2J-Gpnmb+ mice were used as a control strain and were shown to have similar retinal ganglion cell densities as C57BL/6J control mice—a strain commonly investigated in the field of vision research. Using cholera toxin-B injections into the eye and FluoroGold injections into the superior colliculus (SC, we were able to measure anterograde and retrograde transport in the primary visual projection. In DBA/2J, anterograde transport from the retina to superior colliculus (SC was decreased by 69% in the 9-10 month-old age group, while retrograde transport was only reduced by 23% from levels seen in pre-glaucomatous mice. Despite this minor reduction, retrograde transport remained largely intact in these glaucomatous age groups until 13-months of age. These findings indicate that axonal transport deficits occur in semi-functional axons that are still connected to their brain targets. Structural persistence as determined by presence of estrogen-related receptor beta label in the superficial SC was maintained beyond time-points where reductions in retrograde transport occurred, also supporting that transport deficits may be due to physiological or functional abnormalities as opposed to overt structural loss.

  9. Axonal collateral-collateral transport of tract tracers in brain neurons: false anterograde labelling and useful tool.

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    Chen, S; Aston-Jones, G

    1998-02-01

    It is well established that some neuroanatomical tracers may be taken up by local axonal terminals and transported to distant axonal collaterals (e.g., transganglionic transport in dorsal root ganglion cells). However, such collateral-collateral transport of tracers has not been systematically examined in the central nervous system. We addressed this issue with four neuronal tracers--biocytin, biotinylated dextran amine, cholera toxin B subunit, and Phaseolus vulgaris-leucoagglutinin--in the cerebellar cortex. Labelling of distant axonal collaterals in the cerebellar cortex (indication of collateral-collateral transport) was seen after focal iontophoretic microinjections of each of the four tracers. However, collateral-collateral transport properties differed among these tracers. Injection of biocytin or Phaseolus vulgaris-leucoagglutinin in the cerebellar cortex yielded distant collateral labelling only in parallel fibres. In contrast, injection of biotinylated dextran amine or cholera toxin B subunit produced distant collateral labelling of climbing fibres and mossy fibres, as well as parallel fibres. The present study is the first systematic examination of collateral-collateral transport following injection of anterograde tracers in brain. Such collateral-collateral transport may produce false-positive conclusions regarding neural connections when using these tracers for anterograde transport. However, this property may also be used as a tool to determine areas that are innervated by common distant afferents. In addition, these results may indicate a novel mode of chemical communication in the nervous system.

  10. Anterograde Amnesia

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    Serap Erdogan

    2010-08-01

    Full Text Available Memory can be divided into two categories (i.e. short term memory and long term memory according to time span. Information at our long term memory that can be remembered with conscious effort are placed in declarative memory. Information that can not be remembered conciously are placed in nondeclarative memory. The definition of anterograde amnesia is inability to generate new memories after the event causing amnesia. Episodic and semantic memories are usually unaffected among patients’ who had such amnesia. Anterograde amnesia could mostly result from head trauma but in some cases the cause could be serebrovascular events, Wernicke-Korsakoff Syndrome, santral nervous system enfections, anoxia or various substances. Medial temporal lobe and medial diencephalon are two brain regions mainly related with this condition. Medial temporal lobe is consisted of hippocampus, amygdala, parahippocampal cortex, perirhinal cortex and entorhinal cortex. Hypothalamus, thalamus, mamillary bodies and several thalamic nucleases compose medial diencephalon. Fornix and rarely serebellum damage may also play role in the development of anterograde amnesia. After the famous H.M case, who had anterograde amnesia after an epileptic surgery operation, hippocampus has been placed in the focus of memory researches. In the literature there are several reports evaluating brain tissues of amnesic patients at postmortem stage. Postmortem histological evaluations consistently revealed hippocampal neuronal loss among these patients’ brain tissues. Benzodiazepines usually cause short term anterograde amnesia. Benzodiazepine receptors are allosteric modulatory sites on gamma-aminobutyric acid-A (GABA-A receptors. GABA-A receptors composed of five subunits and anterograde amnesia emerges by means of alfa 1 subunit. Anterograde amnesia has been suggested to occur by the blocking of long term potentiation in hippocampus and piriform cortex. For the treatment of the anterograde

  11. Cortical projection patterns of magnocellular basal nucleus subdivisions as revealed by anterogradely transported Phaseolus vulgaris leucoagglutinin

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    Luiten, P.G.M.; Gaykema, R.P.A.; Traber, J.; Spencer Jr., D.G.

    1987-01-01

    The present paper deals with a detailed analysis of cortical projections from the magnocellular basal nucleus (MBN) and horizontal limb of the diagonal band of Broca (HDB) in the rat. The MBN and HDB were injected iontophoretically with the anterograde tracer Phaseolus vulgaris leucoagglutinin

  12. Unidirectional transport of a bead on a single microtubule immobilized in a submicrometre channel

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    Yokokawa, Ryuji; Yoshida, Yumi; Takeuchi, Shoji; Kon, Takahide; Fujita, Hiroyuki

    2006-01-01

    Artificial nano-scale transportation is demonstrated by reconstructing the intracellular transport in a living cell. The transport system is established on two novel techniques: one is the introduction of a single microtubule filament with controlled polarity in a microfabricated submicrometre channel; the other is the immobilization technique of microtubules by a mercury lamp. To transport a kinesin-coated bead in a designated direction, each microtubule filament is polarly oriented by in vitro gliding assay, in which microtubules are carried by the movement of kinesin immobilized on the channel surface. Then, microtubules are immobilized by exposing kinesin to the mercury lamp, which inactivates kinesin but not microtubules. A kinesin-coated bead is newly introduced and carried on the microtubule from one end of the channel to the other as designed. This is an essential component to build up an integrated nano-scale transport system driven by motor proteins.

  13. Dynamic microtubule organization and mitochondrial transport are regulated by distinct Kinesin-1 pathways

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    Anna Melkov

    2015-12-01

    Full Text Available The microtubule (MT plus-end motor kinesin heavy chain (Khc is well known for its role in long distance cargo transport. Recent evidence showed that Khc is also required for the organization of the cellular MT network by mediating MT sliding. We found that mutations in Khc and the gene of its adaptor protein, kinesin light chain (Klc resulted in identical bristle morphology defects, with the upper part of the bristle being thinner and flatter than normal and failing to taper towards the bristle tip. We demonstrate that bristle mitochondria transport requires Khc but not Klc as a competing force to dynein heavy chain (Dhc. Surprisingly, we demonstrate for the first time that Dhc is the primary motor for both anterograde and retrograde fast mitochondria transport. We found that the upper part of Khc and Klc mutant bristles lacked stable MTs. When following dynamic MT polymerization via the use of GFP-tagged end-binding protein 1 (EB1, it was noted that at Khc and Klc mutant bristle tips, dynamic MTs significantly deviated from the bristle parallel growth axis, relative to wild-type bristles. We also observed that GFP-EB1 failed to concentrate as a focus at the tip of Khc and Klc mutant bristles. We propose that the failure of bristle tapering is due to defects in directing dynamic MTs at the growing tip. Thus, we reveal a new function for Khc and Klc in directing dynamic MTs during polarized cell growth. Moreover, we also demonstrate a novel mode of coordination in mitochondrial transport between Khc and Dhc.

  14. Regulation of long-distance transport of mitochondria along microtubules.

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    Melkov, Anna; Abdu, Uri

    2018-01-01

    Mitochondria are cellular organelles of crucial importance, playing roles in cellular life and death. In certain cell types, such as neurons, mitochondria must travel long distances so as to meet metabolic demands of the cell. Mitochondrial movement is essentially microtubule (MT) based and is executed by two main motor proteins, Dynein and Kinesin. The organization of the cellular MT network and the identity of motors dictate mitochondrial transport. Tight coupling between MTs, motors, and the mitochondria is needed for the organelle precise localization. Two adaptor proteins are involved directly in mitochondria-motor coupling, namely Milton known also as TRAK, which is the motor adaptor, and Miro, which is the mitochondrial protein. Here, we discuss the active mitochondria transport process, as well as motor-mitochondria coupling in the context of MT organization in different cell types. We focus on mitochondrial trafficking in different cell types, specifically neurons, migrating cells, and polarized epithelial cells.

  15. Cytoplasmic Dynein Transports Axonal Microtubules in a Polarity-Sorting Manner

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    Anand N. Rao

    2017-06-01

    Full Text Available Axonal microtubules are predominantly organized into a plus-end-out pattern. Here, we tested both experimentally and with computational modeling whether a motor-based polarity-sorting mechanism can explain this microtubule pattern. The posited mechanism centers on cytoplasmic dynein transporting plus-end-out and minus-end-out microtubules into and out of the axon, respectively. When cytoplasmic dynein was acutely inhibited, the bi-directional transport of microtubules in the axon was disrupted in both directions, after which minus-end-out microtubules accumulated in the axon over time. Computational modeling revealed that dynein-mediated transport of microtubules can establish and preserve a predominantly plus-end-out microtubule pattern as per the details of the experimental findings, but only if a kinesin motor and a static cross-linker protein are also at play. Consistent with the predictions of the model, partial depletion of TRIM46, a protein that cross-links axonal microtubules in a manner that influences their polarity orientation, leads to an increase in microtubule transport.

  16. Myosin-Va and Dynamic Actin Oppose Microtubules to Drive Long-Range Organelle Transport

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    Evans, Richard D.; Robinson, Christopher; Briggs, Deborah A.; Tooth, David J.; Ramalho, Jose S.; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V.; Hume, Alistair N.

    2014-01-01

    Summary In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively [1–8]. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the “highways and local roads” model for transport along microtubule and actin tracks [2]. The “cooperative capture” model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering [5, 9]. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning [10, 11]. PMID:25065759

  17. Aspergillus myosin-V supports polarized growth in the absence of microtubule-based transport.

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    Jun Zhang

    Full Text Available In the filamentous fungus Aspergillus nidulans, both microtubules and actin filaments are important for polarized growth at the hyphal tip. Less clear is how different microtubule-based and actin-based motors work together to support this growth. Here we examined the role of myosin-V (MYOV in hyphal growth. MYOV-depleted cells form elongated hyphae, but the rate of hyphal elongation is significantly reduced. In addition, although wild type cells without microtubules still undergo polarized growth, microtubule disassembly abolishes polarized growth in MYOV-depleted cells. Thus, MYOV is essential for polarized growth in the absence of microtubules. Moreover, while a triple kinesin null mutant lacking kinesin-1 (KINA and two kinesin-3s (UNCA and UNCB undergoes hyphal elongation and forms a colony, depleting MYOV in this triple mutant results in lethality due to a severe defect in polarized growth. These results argue that MYOV, through its ability to transport secretory cargo, can support a significant amount of polarized hyphal tip growth in the absence of any microtubule-based transport. Finally, our genetic analyses also indicate that KINA (kinesin-1 rather than UNCA (kinesin-3 is the major kinesin motor that supports polarized growth in the absence of MYOV.

  18. Cortical compression rapidly trimmed transcallosal projections and altered axonal anterograde transport machinery.

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    Chen, Li-Jin; Wang, Yueh-Jan; Tseng, Guo-Fang

    2017-10-24

    Trauma and tumor compressing the brain distort underlying cortical neurons. Compressed cortical neurons remodel their dendrites instantly. The effects on axons however remain unclear. Using a rat epidural bead implantation model, we studied the effects of unilateral somatosensory cortical compression on its transcallosal projection and the reversibility of the changes following decompression. Compression reduced the density, branching profuseness and boutons of the projection axons in the contralateral homotopic cortex 1week and 1month post-compression. Projection fiber density was higher 1-month than 1-week post-compression, suggesting adaptive temporal changes. Compression reduced contralateral cortical synaptophysin, vesicular glutamate transporter 1 (VGLUT1) and postsynaptic density protein-95 (PSD95) expressions in a week and the first two marker proteins further by 1month. βIII-tubulin and kinesin light chain (KLC) expressions in the corpus callosum (CC) where transcallosal axons traveled were also decreased. Kinesin heavy chain (KHC) level in CC was temporarily increased 1week after compression. Decompression increased transcallosal axon density and branching profuseness to higher than sham while bouton density returned to sham levels. This was accompanied by restoration of synaptophysin, VGLUT1 and PSD95 expressions in the contralateral cortex of the 1-week, but not the 1-month, compression rats. Decompression restored βIII-tubulin, but not KLC and KHC expressions in CC. However, KLC and KHC expressions in the cell bodies of the layer II/III pyramidal neurons partially recovered. Our results show cerebral compression compromised cortical axonal outputs and reduced transcallosal projection. Some of these changes did not recover in long-term decompression. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

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

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    Jason E Duncan

    Full Text Available 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.

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

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

    2013-01-01

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

  1. Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport

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    Barbosa, Daniel J.; Duro, Joana; Prevo, Bram; Cheerambathur, Dhanya K.; Carvalho, Ana X.; Gassmann, Reto

    2017-01-01

    Author summary Animal cells rely on molecular motor proteins to distribute intracellular components and organize their cytoplasmic content. The motor cytoplasmic dynein 1 (dynein) uses microtubule filaments as tracks to transport cargo from the cell periphery to the cell center, where the microtubule minus ends are embedded at the centrosome. Conversely, when dynein is anchored at the cell cortex or on organelles in the cytoplasm, the motor can pull on microtubules to position centrosomes wit...

  2. Microtubule-Based Transport and the Distribution, Tethering, and Organization of Organelles.

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    Barlan, Kari; Gelfand, Vladimir I

    2017-05-01

    SUMMARYMicrotubules provide long tracks along which a broad range of organelles and vesicles are transported by kinesin and dynein motors. Motor protein complexes also tether cargoes to cytoskeletal filaments, helping facilitate their interaction and communication. The generation of biochemically distinct microtubule subpopulations allows subsets of motors to recognize a given microtubule identity, allowing further organization within the cytoplasm. Both transport and tethering are spatiotemporally regulated through multiple modes, including acute modification of both motor-cargo and motor-track associations by various physiological signals. Strict regulation of intracellular transport is particularly important in specialized cell types such as neurons. Here, we review general mechanisms by which cargo transport is controlled and also highlight examples of transport regulated by multiple mechanisms. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  3. Myosin-V opposes microtubule-based cargo transport and drives directional motility on cortical actin.

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    Kapitein, Lukas C; van Bergeijk, Petra; Lipka, Joanna; Keijzer, Nanda; Wulf, Phebe S; Katrukha, Eugene A; Akhmanova, Anna; Hoogenraad, Casper C

    2013-05-06

    Intracellular transport is driven by motor proteins that either use microtubules or actin filaments as their tracks, but the interplay between these transport pathways is poorly understood. Whereas many microtubule-based motors are known to drive long-range transport, several actin-based motors have been proposed to function predominantly in cargo tethering. How these opposing activities are integrated on cargoes that contain both types of motors is unknown. Here we use inducible intracellular transport assays to show that acute recruitment of myosin-V to kinesin-propelled cargo reduces their motility near the cell periphery and enhances their localization at the actin-rich cell cortex. Myosin-V arrests rapid microtubule-based transport without the need for regulated auto- or other inhibition of kinesin motors. In addition, myosin-V, despite being an ineffective long-range transporter, can drive slow, medium-range (1-5 μm), point-to-point transport in cortical cell regions. Altogether, these data support a model in which myosin-V establishes local cortical delivery of kinesin-bound cargos through a combination of tethering and active transport. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Quantitative analysis of microtubule transport in growing nerve processes

    DEFF Research Database (Denmark)

    Ma*, Ytao; Shakiryanova*, Dinara; Vardya, Irina

    2004-01-01

    assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent "stop and go" MT transport has been difficult to detect or to exclude by using direct video microscopy methods. In this study, we measured...

  5. Quantitative analysis of microtubule transport in growing nerve processes

    DEFF Research Database (Denmark)

    Ma*, Ytao; Shakiryanova*, Dinara; Vardya, Irina

    2004-01-01

    the translocation of MT plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required...

  6. Computational modeling reveals optimal strategy for kinase transport by microtubules to nerve terminals.

    Science.gov (United States)

    Koon, Yen Ling; Koh, Cheng Gee; Chiam, Keng-Hwee

    2014-01-01

    Intracellular transport of proteins by motors along cytoskeletal filaments is crucial to the proper functioning of many eukaryotic cells. Since most proteins are synthesized at the cell body, mechanisms are required to deliver them to the growing periphery. In this article, we use computational modeling to study the strategies of protein transport in the context of JNK (c-JUN NH2-terminal kinase) transport along microtubules to the terminals of neuronal cells. One such strategy for protein transport is for the proteins of the JNK signaling cascade to bind to scaffolds, and to have the whole protein-scaffold cargo transported by kinesin motors along microtubules. We show how this strategy outperforms protein transport by diffusion alone, using metrics such as signaling rate and signal amplification. We find that there exists a range of scaffold concentrations for which JNK transport is optimal. Increase in scaffold concentration increases signaling rate and signal amplification but an excess of scaffolds results in the dilution of reactants. Similarly, there exists a range of kinesin motor speeds for which JNK transport is optimal. Signaling rate and signal amplification increases with kinesin motor speed until the speed of motor translocation becomes faster than kinase/scaffold-motor binding. Finally, we suggest experiments that can be performed to validate whether, in physiological conditions, neuronal cells do indeed adopt such an optimal strategy. Understanding cytoskeletal-assisted protein transport is crucial since axonal and cell body accumulation of organelles and proteins is a histological feature in many human neurodegenerative diseases. In this paper, we have shown that axonal transport performance changes with altered transport component concentrations and transport speeds wherein these aspects can be modulated to improve axonal efficiency and prevent or slowdown axonal deterioration.

  7. Computational modeling reveals optimal strategy for kinase transport by microtubules to nerve terminals.

    Directory of Open Access Journals (Sweden)

    Yen Ling Koon

    Full Text Available Intracellular transport of proteins by motors along cytoskeletal filaments is crucial to the proper functioning of many eukaryotic cells. Since most proteins are synthesized at the cell body, mechanisms are required to deliver them to the growing periphery. In this article, we use computational modeling to study the strategies of protein transport in the context of JNK (c-JUN NH2-terminal kinase transport along microtubules to the terminals of neuronal cells. One such strategy for protein transport is for the proteins of the JNK signaling cascade to bind to scaffolds, and to have the whole protein-scaffold cargo transported by kinesin motors along microtubules. We show how this strategy outperforms protein transport by diffusion alone, using metrics such as signaling rate and signal amplification. We find that there exists a range of scaffold concentrations for which JNK transport is optimal. Increase in scaffold concentration increases signaling rate and signal amplification but an excess of scaffolds results in the dilution of reactants. Similarly, there exists a range of kinesin motor speeds for which JNK transport is optimal. Signaling rate and signal amplification increases with kinesin motor speed until the speed of motor translocation becomes faster than kinase/scaffold-motor binding. Finally, we suggest experiments that can be performed to validate whether, in physiological conditions, neuronal cells do indeed adopt such an optimal strategy. Understanding cytoskeletal-assisted protein transport is crucial since axonal and cell body accumulation of organelles and proteins is a histological feature in many human neurodegenerative diseases. In this paper, we have shown that axonal transport performance changes with altered transport component concentrations and transport speeds wherein these aspects can be modulated to improve axonal efficiency and prevent or slowdown axonal deterioration.

  8. Disruption of microtubules in rat skeletal muscle does not inhibit insulin- or contraction-stimulated glucose transport

    DEFF Research Database (Denmark)

    Ai, Hua; Ralston, Evelyn; Lauritzen, Hans P M M

    2003-01-01

    found in all muscle fibers. Here, we test whether microtubules are required mediators of the effect of insulin and contractions. In three different incubated rat muscles with distinct fiber type composition, depolymerization of microtubules with colchicine for ...- or contraction-stimulated 2-deoxyglucose transport or force production. On the contrary, colchicine at least partially prevented the approximately 30% decrease in insulin-stimulated transport that specifically developed during 8 h of incubation in soleus muscle but not in flexor digitorum brevis...... or epitrochlearis muscles. In contrast, nocodazole, another microtubule-disrupting drug, rapidly and dose dependently blocked insulin- and contraction-stimulated glucose transport. A similar discrepancy between colchicine and nocodazole was also found in their ability to block glucose transport in muscle giant...

  9. Microtubule-stabilizing peptides and small molecules protecting axonal transport and brain function: focus on davunetide (NAP).

    Science.gov (United States)

    Magen, Iddo; Gozes, Illana

    2013-12-01

    This review focuses on the therapeutic effects and mechanisms of action of NAP (davunetide), an eight amino acid snippet derived from activity-dependent neuroprotective protein (ADNP) which was discovered in our laboratory. We have recently described the effects of NAP in neurodegenerative disorders, and we now review the beneficial effects of NAP and other microtubule-stabilizing agents on impairments in axonal transport. Experiments in animal models of microtubule-deficiency including tauopathy (spanning from drosophila to mammals) showed protection of axonal transport by microtubule-stabilizers and NAP, which was coupled to motor and cognitive protection. Clinical trials with NAP (davunetide) are reviewed paving the path to future developments. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Dynein is the motor for retrograde axonal transport of organelles

    International Nuclear Information System (INIS)

    Schnapp, B.J.; Reese, T.S.

    1989-01-01

    Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport

  11. Multiple kinesin-14 family members drive microtubule minus end-directed transport in plant cells.

    Science.gov (United States)

    Yamada, Moé; Tanaka-Takiguchi, Yohko; Hayashi, Masahito; Nishina, Momoko; Goshima, Gohta

    2017-06-05

    Minus end-directed cargo transport along microtubules (MTs) is exclusively driven by the molecular motor dynein in a wide variety of cell types. Interestingly, during evolution, plants have lost the genes encoding dynein; the MT motors that compensate for dynein function are unknown. Here, we show that two members of the kinesin-14 family drive minus end-directed transport in plants. Gene knockout analyses of the moss Physcomitrella patens revealed that the plant-specific class VI kinesin-14, KCBP, is required for minus end-directed transport of the nucleus and chloroplasts. Purified KCBP directly bound to acidic phospholipids and unidirectionally transported phospholipid liposomes along MTs in vitro. Thus, minus end-directed transport of membranous cargoes might be driven by their direct interaction with this motor protein. Newly nucleated cytoplasmic MTs represent another known cargo exhibiting minus end-directed motility, and we identified the conserved class I kinesin-14 (ATK) as the motor involved. These results suggest that kinesin-14 motors were duplicated and developed as alternative MT-based minus end-directed transporters in land plants. © 2017 Yamada et al.

  12. A synthetic peptide shows retro- and anterograde neuronal transport before disrupting the chemosensation of plant-pathogenic nematodes.

    Directory of Open Access Journals (Sweden)

    Dong Wang

    2011-03-01

    Full Text Available Cyst nematodes are a group of plant pathogens each with a defined host range that cause major losses to crops including potato, soybean and sugar beet. The infective mobile stage hatches from dormant eggs and moves a short distance through the soil to plant roots, which it then invades. A novel strategy for control has recently been proposed in which the plant is able to secrete a peptide which disorientates the infective stage and prevents invasion of the pathogen. This study provides indirect evidence to support the mechanism by which one such peptide disrupts chemosensory function in nematodes. The peptide is a disulphide-constrained 7-mer with the amino acid sequence CTTMHPRLC that binds to nicotinic acetylcholine receptors. A fluorescently tagged version of this peptide with both epifluorescent and confocal microscopy was used to demonstrate that retrograde transport occurs from an aqueous environment along bare-ending primary cilia of chemoreceptive sensilla. The peptide is transported to the cell bodies of these neurons and on to a limited number of other neurons to which they connect. It appears to be localised in both neuronal processes and organelles adjacent to nuclei of some neurons suggesting it could be transported through the Golgi apparatus. The peptide takes 2.5 h to reach the neuronal cell bodies. Comparative studies established that similar but less abundant uptake occurs for Caenorhabditis elegans along its well studied dye-filling chemoreceptive neurons. Incubation in peptide solution or root-exudate from transgenic plants that secrete the peptide disrupted normal orientation of infective cyst nematodes to host root diffusate. The peptide probably undergoes transport along the dye-filling non-cholinergic chemoreceptive neurons to their synapses where it is taken up by the interneurons to which they connect. Coordinated responses to chemoreception are disrupted when the sub-set of cholinergic interneurons secrete the peptide

  13. The microtubule cytoskeleton does not integrate auxin transport and gravitropism in maize roots

    Science.gov (United States)

    Hasenstein, K. H.; Blancaflor, E. B.; Lee, J. S.

    1999-01-01

    The Cholodny-Went hypothesis of gravitropism suggests that the graviresponse is controlled by the distribution of auxin. However, the mechanism of auxin transport during the graviresponse of roots is still unresolved. To determine whether the microtubule (MT) cytoskeleton is participating in auxin transport, the cytoskeleton was examined and the movement of 3H-IAA measured in intact and excised taxol, oryzalin, and naphthylphthalamic acid (NPA)-treated roots of Zea mays cv. Merit. Taxol and oryzalin did not inhibit the graviresponse of roots but the auxin transport inhibitor NPA greatly inhibited both auxin transport and graviresponse. NPA had no effect on MT organization in vertical roots, but caused MT reorientation in horizontally placed roots. Regardless of treatment, the organization of MTs in intact roots differed from that in root segments. The MT inhibitors, taxol and oryzalin had opposite effects on the MTs, namely, depolymerization (oryzalin) and stabilization and thickening (taxol), but both treatments caused swelling of the roots. The data indicate that the MT cytoskeleton does not directly interfere with auxin transport or auxin-mediated growth responses in maize roots.

  14. Kaposi's sarcoma-associated herpesvirus ORF45 interacts with kinesin-2 transporting viral capsid-tegument complexes along microtubules.

    Directory of Open Access Journals (Sweden)

    Narayanan Sathish

    2009-03-01

    Full Text Available Open reading frame (ORF 45 of Kaposi's sarcoma-associated herpesvirus (KSHV is a tegument protein. A genetic analysis with a null mutant suggested a possible role for this protein in the events leading to viral egress. In this study, ORF45 was found to interact with KIF3A, a kinesin-2 motor protein that transports cargoes along microtubules to cell periphery in a yeast two-hybrid screen. The association was confirmed by both co-immunoprecipitation and immunoflorescence approaches in primary effusion lymphoma cells following virus reactivation. ORF45 principally mediated the docking of entire viral capsid-tegument complexes onto the cargo-binding domain of KIF3A. Microtubules served as the major highways for transportation of these complexes as evidenced by drastically reduced viral titers upon treatment of cells with a microtubule depolymerizer, nocodazole. Confocal microscopic images further revealed close association of viral particles with microtubules. Inhibition of KIF3A-ORF45 interaction either by the use of a headless dominant negative (DN mutant of KIF3A or through shRNA-mediated silencing of endogenous KIF3A expression noticeably decreased KSHV egress reflecting as appreciable reductions in the release of extracellular virions. Both these approaches, however, failed to impact HSV-1 egress, demonstrating the specificity of KIF3A in KSHV transportation. This study thus reports on transportation of KSHV viral complexes on microtubules by KIF3A, a kinesin motor thus far not implicated in virus transportation. All these findings shed light on the understudied but significant events in the KSHV life cycle, delineating a crucial role of a KSHV tegument protein in cellular transport of viral particles.

  15. Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

    Science.gov (United States)

    Jorge-Finnigan, Ana; Kleppe, Rune; Jung-Kc, Kunwar; Ying, Ming; Marie, Michael; Rios-Mondragon, Ivan; Salvatore, Michael F; Saraste, Jaakko; Martinez, Aurora

    2017-08-25

    Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP-tagged 74-kDa intermediate chain (IC74. IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs, suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

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

    Science.gov (United States)

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

    2014-09-10

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

  18. Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesin-dependent microtubule motility

    International Nuclear Information System (INIS)

    Gearhart, Debra A.; Sickles, Dale W.; Buccafusco, Jerry J.; Prendergast, Mark A.; Terry, Alvin V.

    2007-01-01

    Diisopropylfluorophosphate, originally developed as a chemical warfare agent, is structurally similar to nerve agents, and chlorpyrifos has extensive worldwide use as an agricultural pesticide. While inhibition of cholinesterases underlies the acute toxicity of these organophosphates, we previously reported impaired axonal transport in the sciatic nerves from rats treated chronically with subthreshold doses of chlorpyrifos. Those data indicate that chlorpyrifos (and/or its active metabolite, chlorpyrifos-oxon) might directly affect the function of kinesin and/or microtubules-the principal proteins that mediate anterograde axonal transport. The current report describes in vitro assays to assess the concentration-dependent effects of chlorpyrifos (0-10 μM), chlorpyrifos-oxon (0-10 μM), and diisopropylfluorophosphate (0-0.59 nM) on kinesin-dependent microtubule motility. Preincubating bovine brain microtubules with the organophosphates did not alter kinesin-mediated microtubule motility. In contrast, preincubation of bovine brain kinesin with diisopropylfluorophosphate, chlorpyrifos, or chlorpyrifos-oxon produced a concentration-dependent increase in the number of locomoting microtubules that detached from the kinesin-coated glass cover slip. Our data suggest that the organophosphates-chlorpyrifos-oxon, chlorpyrifos, and diisopropylfluorophosphate-directly affect kinesin, thereby disrupting kinesin-dependent transport on microtubules. Kinesin-dependent movement of vesicles, organelles, and other cellular components along microtubules is fundamental to the organization of all eukaryotic cells, especially in neurons where organelles and proteins synthesized in the cell body must move down long axons to pre-synaptic sites in nerve terminals. We postulate that disruption of kinesin-dependent intracellular transport could account for some of the long-term effects of organophosphates on the peripheral and central nervous system

  19. Kinesin-4 Functions in Vesicular Transport on Cortical Microtubules and Regulates Cell Wall Mechanics during Cell Elongation in Plants.

    Science.gov (United States)

    Kong, Zhaosheng; Ioki, Motohide; Braybrook, Siobhan; Li, Shundai; Ye, Zheng-Hua; Julie Lee, Yuh-Ru; Hotta, Takashi; Chang, Anny; Tian, Juan; Wang, Guangda; Liu, Bo

    2015-07-01

    In plants, anisotropic cell expansion depends on cortical microtubules that serve as tracks along which macromolecules and vesicles are transported by the motor kinesins of unknown identities. We used cotton (Gossypium hirsutum) fibers that underwent robust elongation to discover kinesins that are involved in cell elongation and found Gh KINESIN-4A expressed abundantly. The motor was detected by immunofluorescence on vesicle-like structures that were associated with cortical microtubules. In Arabidopsis thaliana, the orthologous motor At KINESIN-4A/FRA1, previously implicated in cellulose deposition during secondary growth in fiber cells, was examined by live-cell imaging in cells expressing the fluorescently tagged functional protein. The motor decorated vesicle-like particles that exhibit a linear movement along cortical microtubules with an average velocity of 0.89 μm/min, which was significantly different from those linked to cellulose biosynthesis. We also discovered that At KINESIN-4A/FRA1 and the related At KINESIN-4C play redundant roles in cell wall mechanics, cell elongation, and the axial growth of various vegetative and reproductive organs, as the loss of At KINESIN-4C greatly enhanced the defects caused by a null mutation at the KINESIN-4A/FRA1 locus. The double mutant displayed a lack of cell wall softening at normal stages of rapid cell elongation. Furthermore, enhanced deposition of arabinose-containing carbohydrate was detected in the kinesin-4 mutants. Our findings established a connection between the Kinesin-4-based transport of cargoes containing non-cellulosic components along cortical microtubules and cell wall mechanics and cell elongation in flowering plants. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  20. Evidence for dynein and astral microtubule-mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells.

    Science.gov (United States)

    Zheng, Zhen; Wan, Qingwen; Liu, Jing; Zhu, Huabin; Chu, Xiaogang; Du, Quansheng

    2013-04-01

    Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gαi-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gαi-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule-mediated transport of Gαi/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gαi/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.

  1. Propagation of kink—antikink pair along microtubules as a control mechanism for polymerization and depolymerization processes

    International Nuclear Information System (INIS)

    Kavitha, L.; Muniyappan, A.; Dhamayanthi, S.; Zdravković, S.; Satarić, M. V.; Marlewski, A.; Gopi, D.

    2014-01-01

    Among many types of proteinaceous filaments, microtubules (MTs) constitute the most rigid components of the cellular cytoskeleton. Microtubule dynamics is essential for many vital cellular processes such as intracellular transport, metabolism, and cell division. We investigate the nonlinear dynamics of inhomogeneous microtubulin systems and the MT dynamics is found to be governed by a perturbed sine-Gordon equation. In the presence of various competing nonlinear inhomogeneities, it is shown that this nonlinear model can lead to the existence of kink and antikink solitons moving along MTs. We demonstrate kink—antikink pair collision in the framework of Hirota's bilinearization method. We conjecture that the collisions of the quanta of energy propagating in the form of kinks and antikinks may offer a new view of the mechanism of the retrograde and anterograde transport direction regulation of motor proteins in microtubulin systems. (interdisciplinary physics and related areas of science and technology)

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

    Science.gov (United States)

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

    2016-08-23

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

  3. Ndel1-derived peptides modulate bidirectional transport of injected beads in the squid giant axon

    Directory of Open Access Journals (Sweden)

    Michal Segal

    2012-01-01

    Bidirectional transport is a key issue in cellular biology. It requires coordination between microtubule-associated molecular motors that work in opposing directions. The major retrograde and anterograde motors involved in bidirectional transport are cytoplasmic dynein and conventional kinesin, respectively. It is clear that failures in molecular motor activity bear severe consequences, especially in the nervous system. Neuronal migration may be impaired during brain development, and impaired molecular motor activity in the adult is one of the hallmarks of neurodegenerative diseases leading to neuronal cell death. The mechanisms that regulate or coordinate kinesin and dynein activity to generate bidirectional transport of the same cargo are of utmost importance. We examined how Ndel1, a cytoplasmic dynein binding protein, may regulate non-vesicular bidirectional transport. Soluble Ndel1 protein, Ndel1-derived peptides or control proteins were mixed with fluorescent beads, injected into the squid giant axon, and the bead movements were recorded using time-lapse microscopy. Automated tracking allowed for extraction and unbiased analysis of a large data set. Beads moved in both directions with a clear bias to the anterograde direction. Velocities were distributed over a broad range and were typically slower than those associated with fast vesicle transport. Ironically, the main effect of Ndel1 and its derived peptides was an enhancement of anterograde motion. We propose that they may function primarily by inhibition of dynein-dependent resistance, which suggests that both dynein and kinesin motors may remain engaged with microtubules during bidirectional transport.

  4. Microtubules as key cytoskeletal elements in cellular transport and shape changes: their expected responses to space environments

    Science.gov (United States)

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

    1992-01-01

    Application of reference standard reagents to alternatively depolymerize or stabilize microtubules in a cell that undergoes very regular cytoskeleton-dependent shape changes provides a model system in which some expected components of the environments of spacecraft and space can be tested on Earth for their effects on the cytoskeleton. The fertilized eggs of Ilyanassa obsoleta undergo polar lobe formation by repeated, dramatic, constriction and relaxation of a microfilamentous band localized in the cortical cytoplasm and activated by microtubules.

  5. Interactions between the Translation Machinery and Microtubules.

    Science.gov (United States)

    Chudinova, E M; Nadezhdina, E S

    2018-01-01

    Microtubules are components of eukaryotic cytoskeleton that are involved in the transport of various components from the nucleus to the cell periphery and back. They also act as a platform for assembly of complex molecular ensembles. Ribonucleoprotein (RNP) complexes, such as ribosomes and mRNPs, are transported over significant distances (e.g. to neuronal processes) along microtubules. The association of RNPs with microtubules and their transport along these structures are essential for compartmentalization of protein biosynthesis in cells. Microtubules greatly facilitate assembly of stress RNP granules formed by accumulation of translation machinery components during cell stress response. Microtubules are necessary for the cytoplasm-to-nucleus transport of proteins, including ribosomal proteins. At the same time, ribosomal proteins and RNA-binding proteins can influence cell mobility and cytoplasm organization by regulating microtubule dynamics. The molecular mechanisms underlying the association between the translation machinery components and microtubules have not been studied systematically; the results of such studies are mostly fragmentary. In this review, we attempt to fill this gap by summarizing and discussing the data on protein and RNA components of the translation machinery that directly interact with microtubules or microtubule motor proteins.

  6. The Neuroinvasive Profiles of H129 (Herpes Simplex Virus Type 1) Recombinants with Putative Anterograde-Only Transneuronal Spread Properties

    Science.gov (United States)

    Wojaczynski, Gregory J.; Engel, Esteban A.; Steren, Karina E.; Enquist, Lynn W.; Card, J. Patrick

    2014-01-01

    The use of viruses as transneuronal tracers has become an increasingly powerful technique for defining the synaptic organization of neural networks. Although a number of recombinant alpha herpesviruses are known to spread selectively in the retrograde direction through neural circuits only one strain, the H129 strain of herpes simplex virus type 1, is reported to selectively spread in the anterograde direction. However, it is unclear from the literature whether there is an absolute block or an attenuation of retrograde spread of H129. Here we demonstrate efficient anterograde spread, and temporally delayed retrograde spread, of H129 and three novel recombinants. In vitro studies revealed no differences in anterograde and retrograde spread of parental H129 and its recombinants through superior cervical ganglion neurons. In vivo injections of rat striatum revealed a clear bias of anterograde spread, although evidence of deficient retrograde transport was also present. Evidence of temporally delayed retrograde transneuronal spread of H129 in the retina was observed following injection of the lateral geniculate nucleus. The data also demonstrated that three novel recombinants efficiently express unique fluorescent reporters and have the capacity to infect the same neurons in dual infection paradigms. From these experiments we conclude that H129 and its recombinants efficiently infect neurons through anterograde transneuronal passage, but also are capable of temporally delayed retrograde transneuronal spread. In addition, the capacity to produce dual infection of projection targets following anterograde transneuronal passage provides an important addition to viral transneuronal tracing technology. PMID:24585022

  7. Microtubules in health and degenerative disease of the nervous system.

    Science.gov (United States)

    Matamoros, Andrew J; Baas, Peter W

    2016-09-01

    Microtubules are essential for the development and maintenance of axons and dendrites throughout the life of the neuron, and are vulnerable to degradation and disorganization in a variety of neurodegenerative diseases. Microtubules, polymers of tubulin heterodimers, are intrinsically polar structures with a plus end favored for assembly and disassembly and a minus end less favored for these dynamics. In the axon, microtubules are nearly uniformly oriented with plus ends out, whereas in dendrites, microtubules have mixed orientations. Microtubules in developing neurons typically have a stable domain toward the minus end and a labile domain toward the plus end. This domain structure becomes more complex during neuronal maturation when especially stable patches of polyaminated tubulin become more prominent within the microtubule. Microtubules are the substrates for molecular motor proteins that transport cargoes toward the plus or minus end of the microtubule, with motor-driven forces also responsible for organizing microtubules into their distinctive polarity patterns in axons and dendrites. A vast array of microtubule-regulatory proteins impart direct and indirect changes upon the microtubule arrays of the neuron, and these include microtubule-severing proteins as well as proteins responsible for the stability properties of the microtubules. During neurodegenerative diseases, microtubule mass is commonly diminished, and the potential exists for corruption of the microtubule polarity patterns and microtubule-mediated transport. These ill effects may be a primary causative factor in the disease or may be secondary effects, but regardless, therapeutics capable of correcting these microtubule abnormalities have great potential to improve the status of the degenerating nervous system. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Teamwork in microtubule motors.

    Science.gov (United States)

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

    2013-11-01

    Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Dynamics of microtubules: highlights of recent computational and experimental investigations

    Science.gov (United States)

    Barsegov, Valeri; Ross, Jennifer L.; Dima, Ruxandra I.

    2017-11-01

    Microtubules are found in most eukaryotic cells, with homologs in eubacteria and archea, and they have functional roles in mitosis, cell motility, intracellular transport, and the maintenance of cell shape. Numerous efforts have been expended over the last two decades to characterize the interactions between microtubules and the wide variety of microtubule associated proteins that control their dynamic behavior in cells resulting in microtubules being assembled and disassembled where and when they are required by the cell. We present the main findings regarding microtubule polymerization and depolymerization and review recent work about the molecular motors that modulate microtubule dynamics by inducing either microtubule depolymerization or severing. We also discuss the main experimental and computational approaches used to quantify the thermodynamics and mechanics of microtubule filaments.

  10. Anterograde and Retrograde Amnesia following Bitemporal Infarction

    Directory of Open Access Journals (Sweden)

    A. Schnider

    1994-01-01

    Full Text Available A patient suffered very severe anterograde and retrograde amnesia following infarction of both medial temporal lobes (hippocampus and adjacent cortex and the left inferior temporo-occipital area. The temporal stem and the amygdala were intact; these structures do not appear to be critical for new learning in humans. Extension of the left-sided infarct into the inferior temporo-occipital lobe, an area critically involved in visual processing, appears to be responsible for our patient's loss of remote memories.

  11. Anterograde Episodic Memory in Korsakoff Syndrome

    Science.gov (United States)

    Fama, Rosemary; Pitel, Anne-Lise; Sullivan, Edith V.

    2016-01-01

    A profound anterograde memory deficit for information, regardless of the nature of the material, is the hallmark of Korsakoff syndrome, an amnesic condition resulting from severe thiamine (vitamin B1) deficiency. Since the late nineteenth century when the Russian physician, S. S. Korsakoff, initially described this syndrome associated with “polyneuropathy,” the observed global amnesia has been a primary focus of neuroscience and neuropsychology. In this review we highlight the historical studies that examined anterograde episodic memory processes in KS, present a timeline and evidence supporting the myriad theories proffered to account for this memory dysfunction, and summarize what is known about the neuroanatomical correlates and neural systems presumed affected in KS. Rigorous study of KS amnesia and associated memory disorders of other etiologies provide evidence for distinct mnemonic component processes and neural networks imperative for normal declarative and nondeclarative memory abilities and for mnemonic processes spared in KS, from whence emerged the appreciation that memory is not a unitary function. Debate continues regarding the qualitative and quantitative differences between KS and other amnesias and what brain regions and neural pathways are necessary and sufficient to produce KS amnesia. PMID:22644546

  12. Anterograde episodic memory in Korsakoff syndrome.

    Science.gov (United States)

    Fama, Rosemary; Pitel, Anne-Lise; Sullivan, Edith V

    2012-06-01

    A profound anterograde memory deficit for information, regardless of the nature of the material, is the hallmark of Korsakoff syndrome, an amnesic condition resulting from severe thiamine (vitamin B1) deficiency. Since the late nineteenth century when the Russian physician, S. S. Korsakoff, initially described this syndrome associated with "polyneuropathy," the observed global amnesia has been a primary focus of neuroscience and neuropsychology. In this review we highlight the historical studies that examined anterograde episodic memory processes in KS, present a timeline and evidence supporting the myriad theories proffered to account for this memory dysfunction, and summarize what is known about the neuroanatomical correlates and neural systems presumed affected in KS. Rigorous study of KS amnesia and associated memory disorders of other etiologies provide evidence for distinct mnemonic component processes and neural networks imperative for normal declarative and nondeclarative memory abilities and for mnemonic processes spared in KS, from whence emerged the appreciation that memory is not a unitary function. Debate continues regarding the qualitative and quantitative differences between KS and other amnesias and what brain regions and neural pathways are necessary and sufficient to produce KS amnesia.

  13. Spastic paraplegia mutation N256S in the neuronal microtubule motor KIF5A disrupts axonal transport in a Drosophila HSP model.

    Directory of Open Access Journals (Sweden)

    Petra Füger

    Full Text Available Hereditary spastic paraplegias (HSPs comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a Drosophila model for HSP type 10 (SPG10, caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying a human SPG10-associated mutation (N256S is sufficient to disturb axonal transport and to induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP-like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.

  14. Loop formation of microtubules during gliding at high density

    International Nuclear Information System (INIS)

    Liu, Lynn; Ross, Jennifer L; Tuezel, Erkan

    2011-01-01

    The microtubule cytoskeleton, including the associated proteins, forms a complex network essential to multiple cellular processes. Microtubule-associated motor proteins, such as kinesin-1, travel on microtubules to transport membrane bound vesicles across the crowded cell. Other motors, such as cytoplasmic dynein and kinesin-5, are used to organize the cytoskeleton during mitosis. In order to understand the self-organization processes of motors on microtubules, we performed filament-gliding assays with kinesin-1 motors bound to the cover glass with a high density of microtubules on the surface. To observe microtubule organization, 3% of the microtubules were fluorescently labeled to serve as tracers. We find that microtubules in these assays are not confined to two dimensions and can cross one other. This causes microtubules to align locally with a relatively short correlation length. At high density, this local alignment is enough to create 'intersections' of perpendicularly oriented groups of microtubules. These intersections create vortices that cause microtubules to form loops. We characterize the radius of curvature and time duration of the loops. These different behaviors give insight into how crowded conditions, such as those in the cell, might affect motor behavior and cytoskeleton organization.

  15. Distinct mechanisms of recognizing endosomal sorting complex required for transport III (ESCRT-III) protein IST1 by different microtubule interacting and trafficking (MIT) domains.

    Science.gov (United States)

    Guo, Emily Z; Xu, Zhaohui

    2015-03-27

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains*

    Science.gov (United States)

    Guo, Emily Z.; Xu, Zhaohui

    2015-01-01

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. PMID:25657007

  17. Dissecting the molecular mechanisms of intraflagellar transport in Chlamydomonas

    DEFF Research Database (Denmark)

    Pedersen, L. B.; Geimer, S.; Rosenbaum, J. L.

    2006-01-01

    Background The assembly and maintenance of eukaryotic cilia and flagella are mediated by intraflagellar transport (IFT), a bidirectional microtubule (MT)-based transport system. The IFT system consists of anterograde (kinesin-2) and retrograde (cDynein1b) motor complexes and IFT particles...... comprising two complexes, A and B. In the current model for IFT, kinesin-2 carries cDynein1b, IFT particles, and axonemal precursors from the flagellar base to the tip, and cDynein1b transports kinesin-2, IFT particles, and axonemal turnover products from the tip back to the base. Most of the components...... of the IFT system have been identified and characterized, but the mechanisms by which these different components are coordinated and regulated at the flagellar base and tip are unclear. Results Using a variety of Chlamydomonas mutants, we confirm that cDynein1b requires kinesin-2 for transport toward the tip...

  18. Intracellular transport proteins: classification, structure and function of kinesins

    Directory of Open Access Journals (Sweden)

    Agnieszka Chudy

    2011-09-01

    Full Text Available Correct cell functioning, division and morphogenesis rely on efficient intracellular transport. Apart from dyneins and myosins, kinesins are the main proteins responsible for intracellular movement. Kinesins are a large, diverse group of motor proteins, which based on phylogenetic similarity were classified into fourteen families. Among these families, due to the location of their motor domains, three groups have been characterized: N-, C- and M-kinesin. As molecular motors, kinesins transport various molecules and vesicles mainly towards the microtubule plus end (from the cell body participating in anterograde transport, although there are also kinesins involved in retrograde transport (C-kinesins. Kinesins are also involved in spindle formation, chromosome segregation, and spermatogenesis. Because of their great importance for the correct functioning of cells, mutations in kinesin coding genes may lead to such neurodegenerative diseases as dominant hereditary spastic paraplegia or Charcot-Marie-Tooth disease.

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

    Science.gov (United States)

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

    2016-01-01

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

  20. A small-molecule activator of kinesin-1 drives remodeling of the microtubule network.

    Science.gov (United States)

    Randall, Thomas S; Yip, Yan Y; Wallock-Richards, Daynea J; Pfisterer, Karin; Sanger, Anneri; Ficek, Weronika; Steiner, Roberto A; Beavil, Andrew J; Parsons, Maddy; Dodding, Mark P

    2017-12-26

    The microtubule motor kinesin-1 interacts via its cargo-binding domain with both microtubules and organelles, and hence plays an important role in controlling organelle transport and microtubule dynamics. In the absence of cargo, kinesin-1 is found in an autoinhibited conformation. The molecular basis of how cargo engagement affects the balance between kinesin-1's active and inactive conformations and roles in microtubule dynamics and organelle transport is not well understood. Here we describe the discovery of kinesore, a small molecule that in vitro inhibits kinesin-1 interactions with short linear peptide motifs found in organelle-specific cargo adaptors, yet activates kinesin-1's function of controlling microtubule dynamics in cells, demonstrating that these functions are mechanistically coupled. We establish a proof-of-concept that a microtubule motor-cargo interface and associated autoregulatory mechanism can be manipulated using a small molecule, and define a target for the modulation of microtubule dynamics.

  1. Modeling microtubule oscillations

    DEFF Research Database (Denmark)

    Jobs, E.; Wolf, D.E.; Flyvbjerg, H.

    1997-01-01

    Synchronization of molecular reactions in a macroscopic volume may cause the volume's physical properties to change dynamically and thus reveal much about the reactions. As an example, experimental time series for so-called microtubule oscillations are analyzed in terms of a minimal model for thi...

  2. Electrodynamic effects on microtubules

    Czech Academy of Sciences Publication Activity Database

    Kučera, Ondřej; Havelka, Daniel; Deriu, M.A.; Cifra, Michal

    2015-01-01

    Roč. 44, Jul (2015), s. 169-169 ISSN 0175-7571. [10th European-Biophysical-Societies-Association (EBSA) European Biophysics Congress. 18.07.2015-22.07.2015, Dresden] R&D Projects: GA ČR(CZ) GA15-17102S Institutional support: RVO:67985882 Keywords : Microtubules * Electric al polarity Subject RIV: JA - Electronics ; Optoelectronics, Electric al Engineering

  3. Microtubule's conformational cap

    DEFF Research Database (Denmark)

    Flyvbjerg, H.

    1999-01-01

    The molecular mechanisms that allow elongation of the unstable microtubule lattice remain unclear. It is usually thought that the GDP-liganded tubulin lattice is capped by a small layer of GTP- or GDP-P(i)-liganded molecules, the so called "GTP-cap". Here, we point-out that the elastic properties...

  4. Mitotic motors coregulate microtubule patterns in axons and dendrites.

    Science.gov (United States)

    Lin, Shen; Liu, Mei; Mozgova, Olga I; Yu, Wenqian; Baas, Peter W

    2012-10-03

    Microtubules are nearly uniformly oriented in the axons of vertebrate neurons but are non-uniformly oriented in their dendrites. Studies to date suggest a scenario for establishing these microtubule patterns whereby microtubules are transported into the axon and nascent dendrites with plus-ends-leading, and then additional microtubules of the opposite orientation are transported into the developing dendrites. Here, we used contemporary tools to confirm that depletion of kinesin-6 (also called CHO1/MKLP1 or kif23) from rat sympathetic neurons causes a reduction in the appearance of minus-end-distal microtubules in developing dendrites, which in turn causes them to assume an axon-like morphology. Interestingly, we observed a similar phenomenon when we depleted kinesin-12 (also called kif15 or HKLP2). Both motors are best known for their participation in mitosis in other cell types, and both are enriched in the cell body and dendrites of neurons. Unlike kinesin-12, which is present throughout the neuron, kinesin-6 is barely detectable in the axon. Accordingly, depletion of kinesin-6, unlike depletion of kinesin-12, has no effect on axonal branching or navigation. Interestingly, depletion of either motor results in faster growing axons with greater numbers of mobile microtubules. Based on these observations, we posit a model whereby these two motors generate forces that attenuate the transport of microtubules with plus-ends-leading from the cell body into the axon. Some of these microtubules are not only prevented from moving into the axon but are driven with minus-ends-leading into developing dendrites. In this manner, these so-called "mitotic" motors coregulate the microtubule patterns of axons and dendrites.

  5. Kinesin-2: a family of heterotrimeric and homodimeric motors with diverse intracellular transport functions.

    Science.gov (United States)

    Scholey, Jonathan M

    2013-01-01

    Kinesin-2 was first purified as a heterotrimeric, anterograde, microtubule-based motor consisting of two distinct kinesin-related subunits and a novel associated protein (KAP) that is currently best known for its role in intraflagellar transport and ciliogenesis. Subsequent work, however, has revealed diversity in the oligomeric state of different kinesin-2 motors owing to the combinatorial heterodimerization of its subunits and the coexistence of both heterotrimeric and homodimeric kinesin-2 motors in some cells. Although the functional significance of the homo- versus heteromeric organization of kinesin-2 motor subunits and the role of KAP remain uncertain, functional studies suggest that cooperation between different types of kinesin-2 motors or between kinesin-2 and a member of a different motor family can generate diverse patterns of anterograde intracellular transport. Moreover, despite being restricted to ciliated eukaryotes, kinesin-2 motors are now known to drive diverse transport events outside cilia. Here, I review the organization, assembly, phylogeny, biological functions, and motility mechanism of this diverse family of intracellular transport motors.

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

    Science.gov (United States)

    Gardiner, John; Overall, Robyn; Marc, Jan

    2012-01-01

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

  7. Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells.

    Science.gov (United States)

    Rymut, Sharon M; Kampman, Claire M; Corey, Deborah A; Endres, Tori; Cotton, Calvin U; Kelley, Thomas J

    2016-08-01

    High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF. Copyright © 2016 the American Physiological Society.

  8. Regulation of GPCR Anterograde Trafficking by Molecular Chaperones and Motifs.

    Science.gov (United States)

    Young, Brent; Wertman, Jaime; Dupré, Denis J

    2015-01-01

    G protein-coupled receptors (GPCRs) make up a superfamily of integral membrane proteins that respond to a wide variety of extracellular stimuli, giving them an important role in cell function and survival. They have also proven to be valuable targets in the fight against various diseases. As such, GPCR signal regulation has received considerable attention over the last few decades. With the amplitude of signaling being determined in large part by receptor density at the plasma membrane, several endogenous mechanisms for modulating GPCR expression at the cell surface have come to light. It has been shown that cell surface expression is determined by both exocytic and endocytic processes. However, the body of knowledge surrounding GPCR trafficking from the endoplasmic reticulum to the plasma membrane, commonly known as anterograde trafficking, has considerable room for growth. We focus here on the current paradigms of anterograde GPCR trafficking. We will discuss the regulatory role of both the general and "nonclassical private" chaperone systems in GPCR trafficking as well as conserved motifs that serve as modulators of GPCR export from the endoplasmic reticulum and Golgi apparatus. Together, these topics summarize some of the known mechanisms by which the cell regulates anterograde GPCR trafficking. © 2015 Elsevier Inc. All rights reserved.

  9. A Case for Microtubule Vulnerability in Amyotrophic Lateral Sclerosis: Altered Dynamics During Disease

    Directory of Open Access Journals (Sweden)

    Jayden A Clark

    2016-09-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is an aggressive multifactorial disease converging on a common pathology: the degeneration of motor neurons, their axons and neuromuscular synapses. This vulnerability and dysfunction of motor neurons highlights the dependency of these large cells on their intracellular machinery. Neuronal microtubules are an intracellular structure that facilitates a myriad of vital neuronal functions, including activity dependent axonal transport. In ALS it is becoming increasingly apparent that microtubules are likely to be a critical component of this disease. Not only are disruptions in this intracellular machinery present in the vast majority of seemingly sporadic cases, recent research has revealed that mutation to a microtubule protein, the tubulin isoform TUBA4A, is sufficient to cause a familial, albeit rare, form of disease. In both sporadic and familial disease, studies have provided evidence that microtubule mediated deficits in axonal transport are the tipping point for motor neuron survivability. Axonal transport deficits would lead to abnormal mitochondrial recycling, decreased vesicle and mRNA transport and limited signalling of key survival factors from the neurons peripheral synapses, causing the characteristic peripheral ‘die back’. This disruption to microtubule dependant transport in ALS has been shown to result from alterations in the phenomenon of microtubule dynamic instability: the rapid growth and shrinkage of microtubule polymers. This is accomplished primarily due to aberrant alterations to microtubule associated proteins (MAPS that regulate microtubule stability. Indeed, the current literature would argue that microtubule stability, particularly alterations in their dynamics, may be the initial driving force behind many familial and sporadic insults in ALS. Pharmacological stabilisation of the microtubule network offers an attractive therapeutic strategy in ALS; indeed it has shown promise in

  10. Microtubule-dependent changes in morphology and localization of chloride transport proteins in gill mitochondria-rich cells of the tilapia, Oreochromis mossambicus.

    Science.gov (United States)

    Yang, Wen-Kai; Wu, Yu-Ching; Tang, Cheng-Hao; Lee, Tsung-Han

    2016-08-01

    The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na(+) /Cl(-) cotransporter (NCC) and Na(+) /K(+) /2Cl(-) cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria-rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time-course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule-dependent cellular regulating processes was used. SW-acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine-FW) for 96 h. Compared with the FW-treatment group, in the MR cells of colchicine-FW-treatment group, (1) the average size was significantly larger, (2) only wavy-convex-subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule-dependent. J. Morphol. 277:1113-1122, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Linking cortical microtubule attachment and exocytosis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Ivar Noordstra

    2017-04-01

    Full Text Available Exocytosis is a fundamental cellular process whereby secreted molecules are packaged into vesicles that move along cytoskeletal filaments and fuse with the plasma membrane. To function optimally, cells are strongly dependent on precisely controlled delivery of exocytotic cargo. In mammalian cells, microtubules serve as major tracks for vesicle transport by motor proteins, and thus microtubule organization is important for targeted delivery of secretory carriers. Over the years, multiple microtubule-associated and cortical proteins have been discovered that facilitate the interaction between the microtubule plus ends and the cell cortex. In this review, we focus on mammalian protein complexes that have been shown to participate in both cortical microtubule capture and exocytosis, thereby regulating the spatial organization of secretion. These complexes include microtubule plus-end tracking proteins, scaffolding factors, actin-binding proteins, and components of vesicle docking machinery, which together allow efficient coordination of cargo transport and release.

  12. Centrosome and microtubule instability in aging Drosophila cells

    Science.gov (United States)

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

    1999-01-01

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

  13. Anterograde and Retrograde Effects of Benzodiazepines on Memory

    Directory of Open Access Journals (Sweden)

    Daniel Beracochea

    2006-01-01

    Full Text Available Benzodiazepines are known as “acquisition-impairing” molecules, and their effects on anterograde memory processes are well described. In contrast, the impact of benzodiazepines on retrograde memory and, more particularly, on retrieval processes, is only marginally studied. This mini-review provides an overlook of the main studies evidencing an effect of benzodiazepines on retrograde memory, both in humans and animals, with special emphasis on retrieval processes. The conditions for the emergence of the benzodiazepine-induced retrieval impairments are also discussed.

  14. Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons.

    Science.gov (United States)

    Niwa, Shinsuke

    2017-10-18

    Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

  15. Multiple sclerosis and anterograde axonal degeneration study by magnetic resonance

    International Nuclear Information System (INIS)

    Martinez Pardo, P.; Capdevila Cirera, A.; Sanz Marin, P.M.; Gili Planas, J.

    1993-01-01

    Multiple sclerosis (MS) is a disease of the central nervous system that affects specifically the myelin. Its diagnosis by imaging techniques is, since the development of magnetic resonance (MR), relatively simple, and its occasional association with anterograde axonal degeneration (WD) has been reported. In both disorders, there is a lengthening of the T1 and T2 relaxation times. In the present report, 76 patients with MS with less than 4 plaques in the typical periventricular position were studied retrospectively, resulting in a rate of association with anterograde axonal degeneration of 8%. We consider that in spite of their same behavior in MR,MS and WD, with moreover represent completely different pathologies, are perfectly differential by MR. The S-E images with longer repetition and echo times in the axial and coronal planes have proved to be those most sensitive for this differentiation. Given that MS is specific pathology of then myelin, the axonal damages in delayed until several plaques adjacent to an axon affect it. We consider that this, added to the restriction of our study group (less than 4 plaques), is the cause of the pow percentage of the MS-WD association in our study. (Author)

  16. Neural Correlate of Anterograde Amnesia in Wernicke-Korsakoff Syndrome.

    Science.gov (United States)

    Nahum, Louis; Pignat, Jean-Michel; Bouzerda-Wahlen, Aurélie; Gabriel, Damien; Liverani, Maria Chiara; Lazeyras, François; Ptak, Radek; Richiardi, Jonas; Haller, Sven; Thorens, Gabriel; Zullino, Daniele F; Guggisberg, Adrian G; Schnider, Armin

    2015-09-01

    The neural correlate of anterograde amnesia in Wernicke-Korsakoff syndrome (WKS) is still debated. While the capacity to learn new information has been associated with integrity of the medial temporal lobe (MTL), previous studies indicated that the WKS is associated with diencephalic lesions, mainly in the mammillary bodies and anterior or dorsomedial thalamic nuclei. The present study tested the hypothesis that amnesia in WKS is associated with a disrupted neural circuit between diencephalic and hippocampal structures. High-density evoked potentials were recorded in four severely amnesic patients with chronic WKS, in five patients with chronic alcoholism without WKS, and in ten age matched controls. Participants performed a continuous recognition task of pictures previously shown to induce a left medial temporal lobe dependent positive potential between 250 and 350 ms. In addition, the integrity of the fornix was assessed using diffusion tensor imaging (DTI). WKS, but not alcoholic patients without WKS, showed absence of the early, left MTL dependent positive potential following immediate picture repetitions. DTI indicated disruption of the fornix, which connects diencephalic and hippocampal structures. The findings support an interpretation of anterograde amnesia in WKS as a consequence of a disconnection between diencephalic and MTL structures with deficient contribution of the MTL to rapid consolidation.

  17. Exploring anterograde associative memory in London taxi drivers

    Science.gov (United States)

    Woollett, Katherine; Maguire, Eleanor A.

    2013-01-01

    London taxi drivers are renowned for their navigation ability, spending a number of years acquiring ‘The Knowledge’ of London’s complex layout and having to pass stringent examinations to obtain an operating licence. In several studies, this navigation skill has been associated with increased posterior but also decreased anterior hippocampal grey matter volume. Neuropsychologically, gain and loss has also been documented in taxi drivers; while very skilled at navigation in London, they are significantly poorer than controls at learning and recalling new object-location associations. Here we tested a group of London taxi drivers and matched control participants on this object-location associations task, while also submitting them to a battery of challenging anterograde associative memory tests involving verbal, visual and auditory material both within and across modalities. Our aim was to assess whether their difficulty in previous studies reflected a general problem with associative memory, or was restricted to the spatial domain. We replicated previous findings of poor learning and memory of object-location associations. By contrast, their performance on the other anterograde associative memory tasks was comparable to controls. This resolves an outstanding question in the memory profile of London taxi drivers following hippocampal plasticity, and underlines the close relationship between space and the hippocampus. PMID:22955143

  18. Anterograde amnesia during electroconvulsive therapy: A prospective pilot-study in patients with major depressive disorder

    NARCIS (Netherlands)

    I.A. Boere (Ingrid); A.M. Kamperman (Astrid); Van't Hoog, A.E. (Arianne E.); W.W. van den Broek (Walter); T.K. Birkenhäger (Tom)

    2016-01-01

    textabstractElectroconvulsive therapy (ECT) is considered an effective treatment for major depression with melancholic features. However, neurocognitive side-effects such as anterograde amnesia still regularly occur. The present study aims to evaluate the severity and course of anterograde amnesia

  19. Microtubule organization during human parthenogenesis.

    Science.gov (United States)

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

    2009-04-01

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

  20. Estimating microtubule distributions from 2D immunofluorescence microscopy images reveals differences among human cultured cell lines.

    Directory of Open Access Journals (Sweden)

    Jieyue Li

    Full Text Available Microtubules are filamentous structures that are involved in several important cellular processes, including cell division, cellular structure and mechanics, and intracellular transportation. Little is known about potential differences in microtubule distributions within and across cell lines. Here we describe a method to estimate information pertaining to 3D microtubule distributions from 2D fluorescence images. Our method allows for quantitative comparisons of microtubule distribution parameters (number of microtubules, mean length between different cell lines. Among eleven cell lines compared, some showed differences that could be accounted for by differences in the total amount of tubulin per cell while others showed statistically significant differences in the balance between number and length of microtubules. We also observed that some cell lines that visually appear different in their microtubule distributions are quite similar when the model parameters are considered. The method is expected to be generally useful for comparing microtubule distributions between cell lines and for a given cell line after various perturbations. The results are also expected to enable analysis of the differences in gene expression underlying the observed differences in microtubule distributions among cell types.

  1. Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity

    NARCIS (Netherlands)

    Kevenaar, Josta T|info:eu-repo/dai/nl/338771042; Bianchi, Sarah; van Spronsen, Myrrhe|info:eu-repo/dai/nl/337616655; Olieric, Natacha; Lipka, Joanna|info:eu-repo/dai/nl/369403142; Frias, Cátia P; Mikhaylova, Marina; Harterink, Martin|info:eu-repo/dai/nl/304075655; Keijzer, Nanda; Wulf, Phebe S; Hilbert, Manuel; Kapitein, Lukas C|info:eu-repo/dai/nl/298806630; de Graaff, Esther|info:eu-repo/dai/nl/148374646; Akhmanova, Anna|info:eu-repo/dai/nl/156410591; Steinmetz, Michel O; Hoogenraad, Casper C|info:eu-repo/dai/nl/227263502

    2016-01-01

    Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human

  2. The feasibility of coherent energy transfer in microtubules

    Science.gov (United States)

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

    2014-01-01

    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

  3. The feasibility of coherent energy transfer in microtubules.

    Science.gov (United States)

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

    2014-11-06

    It was once purported that biological systems were far too 'warm and wet' to support quantum phenomena mainly owing to thermal effects disrupting quantum coherence. However, recent experimental results and theoretical analyses have shown that thermal energy may assist, rather than disrupt, quantum coherent transport, especially in the 'dry' hydrophobic interiors of biomolecules. Specifically, evidence has been accumulating for the necessary involvement of quantum coherent energy transfer between uniquely arranged chromophores in light harvesting photosynthetic complexes. The 'tubulin' subunit proteins, which comprise microtubules, also possess a distinct architecture of chromophores, namely aromatic amino acids, including tryptophan. The geometry and dipolar properties of these aromatics are similar to those found in photosynthetic units indicating that tubulin may support coherent energy transfer. Tubulin aggregated into microtubule geometric lattices may support such energy transfer, which could be important for biological signalling and communication essential to living processes. Here, we perform a computational investigation of energy transfer between chromophoric amino acids in tubulin via dipole excitations coupled to the surrounding thermal environment. We present the spatial structure and energetic properties of the tryptophan residues in the microtubule constituent protein tubulin. Plausibility arguments for the conditions favouring a quantum mechanism of signal propagation along a microtubule are provided. Overall, we find that coherent energy transfer in tubulin and microtubules is biologically feasible. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

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

    NARCIS (Netherlands)

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

    2016-01-01

    Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how

  5. Shaping plant microtubule networks via overlap formation

    NARCIS (Netherlands)

    Keijzer, de Jeroen

    2017-01-01

    Microtubules are long filaments made up from protein building blocks and ubiquitously employed by eukaryotic cells for a wide range of often essential cellular processes. To perform these functions, microtubules are virtually always organized into higher order networks. Microtubule networks in

  6. Native kinesin-1 does not bind preferentially to GTP-tubulin-rich microtubules in vitro.

    Science.gov (United States)

    Li, Qiaochu; King, Stephen J; Xu, Jing

    2017-09-01

    Molecular motors such as kinesin-1 work in small teams to actively shuttle cargos in cells, for example in polarized transport in axons. Here, we examined the potential regulatory role of the nucleotide state of tubulin on the run length of cargos carried by multiple kinesin motors, using an optical trapping-based in vitro assay. Based on a previous report that kinesin binds preferentially to GTP-tubulin-rich microtubules, we anticipated that multiple-kinesin cargos would run substantially greater distances along GMPCPP microtubules than along GDP microtubules. Surprisingly, we did not uncover any significant differences in run length between microtubule types. A combination of single-molecule experiments, comparison with previous theory, and classic microtubule affinity pulldown assays revealed that native kinesin-1 does not bind preferentially to GTP-tubulin-rich microtubules. The apparent discrepancy between our observations and the previous report likely reflects differences in post-translational modifications between the native motors used here and the recombinant motors examined previously. Future investigations will help shed light on the interplay between the motor's post-translational modification and the microtubule's nucleotide-binding state for transport regulation in vivo. © 2017 Wiley Periodicals, Inc.

  7. Optomechanical proposal for monitoring microtubule mechanical vibrations

    Science.gov (United States)

    Barzanjeh, Sh.; Salari, V.; Tuszynski, J. A.; Cifra, M.; Simon, C.

    2017-07-01

    Microtubules provide the mechanical force required for chromosome separation during mitosis. However, little is known about the dynamic (high-frequency) mechanical properties of microtubules. Here, we theoretically propose to control the vibrations of a doubly clamped microtubule by tip electrodes and to detect its motion via the optomechanical coupling between the vibrational modes of the microtubule and an optical cavity. In the presence of a red-detuned strong pump laser, this coupling leads to optomechanical-induced transparency of an optical probe field, which can be detected with state-of-the art technology. The center frequency and line width of the transparency peak give the resonance frequency and damping rate of the microtubule, respectively, while the height of the peak reveals information about the microtubule-cavity field coupling. Our method opens the new possibilities to gain information about the physical properties of microtubules, which will enhance our capability to design physical cancer treatment protocols as alternatives to chemotherapeutic drugs.

  8. Griseofulvin-induced aggregation of microtubule protein.

    Science.gov (United States)

    Roobol, A; Gull, K; Pogson, C I

    1977-01-01

    Griseofulvin (7-chloro-2',4,6-trimethoxy-6'-methylspiro[benzofuran-2(3H),1'-[2]cyclohexene]-3,4'-dione) induces aggregation of microtubule protein at 0 degrees C. This aggregate contains approx. 90% of the microtubule-associated proteins originally present in the microtubule protein. The supernatant obtained after removal of the griseofulvin-induced aggregate does not form microtubules on warming at 37 degrees C. Addition of the griseofulvin-aggregated protein to this supernatant and warming to 37 degrees C gives rise to a limited amount of microtubule assembly. The possible involvement of griseofulvin-induced aggregation of microtubule protein at 0 degrees C in the inhibition by griseofulvin of microtubule assembly in vitro is discussed. Images PLATE 1 PLATE 2 PMID:588267

  9. Microtubule capture by mitotic kinesin centromere protein E (CENP-E).

    Science.gov (United States)

    Sardar, Harjinder S; Gilbert, Susan P

    2012-07-20

    Centromere protein E, CENP-E, is a kinetochore-associated kinesin-7 that establishes the microtubule-chromosome linkage and transports monooriented chromosomes to the spindle equator along kinetochore fibers of already bioriented chromosomes. As a processive kinesin, CENP-E uses a hand-over-hand mechanism, yet a number of studies suggest that CENP-E exhibits mechanistic differences from other processive kinesins that may be important for its role in chromosome congression. The results reported here show that association of CENP-E with the microtubule is unusually slow at 0.08 μM(-1) s(-1) followed by slow ADP release at 0.9 s(-1). ATP binding and hydrolysis are fast with motor dissociation from the microtubule at 1.4 s(-1), suggesting that CENP-E head detachment from the microtubule, possibly controlled by phosphate release, determines the rate of stepping during a processive run because the rate of microtubule gliding corresponds to 1.4 steps/s. We hypothesize that the unusually slow CENP-E microtubule association step favors CENP-E binding of stable microtubules over dynamic ones, a mechanism that would bias CENP-E binding to kinetochore fibers.

  10. Friction on MAP determines its traveling direction on microtubules.

    Science.gov (United States)

    Watanabe, Sadanori; Goshima, Gohta

    2014-04-14

    Microtubule networks generate various forces, and the forces are applied to microtubule-associated proteins (MAPs). Forth et al. (2014) show in a recent issue of Cell that asymmetric frictional force between MAPs and microtubules leads to directional movement of MAPs along microtubules, providing insight into the mechanism of microtubule network self-organization. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Mapping sensory circuits by anterograde trans-synaptic transfer of recombinant rabies virus

    Science.gov (United States)

    Zampieri, Niccolò; Jessell, Thomas M.; Murray, Andrew J.

    2014-01-01

    Summary Primary sensory neurons convey information from the external world to relay circuits within the central nervous system (CNS), but the identity and organization of the neurons that process incoming sensory information remains sketchy. Within the CNS viral tracing techniques that rely on retrograde trans-synaptic transfer provide a powerful tool for delineating circuit organization. Viral tracing of the circuits engaged by primary sensory neurons has, however, been hampered by the absence of a genetically tractable anterograde transfer system. In this study we demonstrate that rabies virus can infect sensory neurons in the somatosensory system, is subject to anterograde trans-synaptic transfer from primary sensory to spinal target neurons, and can delineate output connectivity with third-order neurons. Anterograde trans-synaptic transfer is a feature shared by other classes of primary sensory neurons, permitting the identification and potentially the manipulation of neural circuits processing sensory feedback within the mammalian CNS. PMID:24486087

  12. Microtubule dynamics: Caps, catastrophes, and coupled hydrolysis

    DEFF Research Database (Denmark)

    Flyvbjerg, H.; Holy, T.E.; Leibler, S.

    1996-01-01

    individual tubulin dimers, an ignored. In this cap model, GTP hydrolysis is assumed to be stochastic and uncoupled to microtubule growth. Different rates of hydrolysis are assumed for GTP in the cap's interior and for GTP at its boundary with hydrolyzed parts of the microtubule. Expectation values...... and probability distributions relating to available experimental data are derived. Caps are found to be short and the total rate of hydrolysis at a microtubule end is found to be dynamically coupled to growth. The so-called catastrophe rate is a simple function of the microtubule growth rare and fits experimental...... of microtubule growth before dilution. The GTP content of microtubules is found and its rare of hydrolysis is determined under the circumstances created in an experiment designed to measure this GTP content. It is concluded that this experiment's failure to register any GTP content is consistent with the model...

  13. Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy.

    Science.gov (United States)

    Yan, Si; Guo, Changmiao; Hou, Guangjin; Zhang, Huilan; Lu, Xingyu; Williams, John Charles; Polenova, Tatyana

    2015-11-24

    Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

  14. A dissociation between anterograde and retrograde amnesia after treatment with electroconvulsive therapy: a naturalistic investigation.

    Science.gov (United States)

    O'Connor, Margaret; Lebowitz, Brian K; Ly, Jenny; Panizzon, Matthew S; Elkin-Frankston, Seth; Dey, Sangeeta; Bloomingdale, Kerry; Thall, Mark; Pearlman, Chester

    2008-06-01

    The aim of the present study is to investigate the cumulative effects of a clinically determined course of electroconvulsive therapy (ECT) on anterograde and retrograde amnesia. In this study, mood and memory were examined in the context of a protocol driven by therapeutic response, rather than by preordained research criteria. Twenty-two patients with major depressive disorder and 18 nondepressed controls were taught a series of faces and names before the initiation of ECT, and their retention of this information was examined after the end of treatment. Anterograde (ie, new learning) and retrograde memory (ie, recall of information learned before ECT) were assessed. Eleven ECT patients underwent unilateral (UL) stimulation, and 11 had a combination of UL and bilateral stimulation. Major depressive disorder patients and nondepressed controls participants were matched according to baseline memory abilities. Unilateral and unilateral/bilateral (UB) ECT patients were matched according to baseline depression and memory abilities. Treatment with ECT resulted in a dissociation between anterograde and retrograde memory; after treatment, major depressive disorder patients demonstrated significant retrograde amnesia, whereas there was no change in their anterograde memory. Unilateral and UB ECT patients performed equally well on tasks of anterograde memory. Contrary to our expectation, UB ECT was not associated with greater retrograde memory loss than was UL ECT treatment. However, a trend toward a group difference was present on 1 memory measure. Results of the study suggest that a clinical course of ECT is associated with isolated impairment for information learned before treatment (ie, retrograde memory), whereas there was no effect of ECT on posttreatment learning abilities (ie, anterograde memory).

  15. A Mechanism for Cytoplasmic Streaming: Kinesin-Driven Alignment of Microtubules and Fast Fluid Flows.

    Science.gov (United States)

    Monteith, Corey E; Brunner, Matthew E; Djagaeva, Inna; Bielecki, Anthony M; Deutsch, Joshua M; Saxton, William M

    2016-05-10

    The transport of cytoplasmic components can be profoundly affected by hydrodynamics. Cytoplasmic streaming in Drosophila oocytes offers a striking example. Forces on fluid from kinesin-1 are initially directed by a disordered meshwork of microtubules, generating minor slow cytoplasmic flows. Subsequently, to mix incoming nurse cell cytoplasm with ooplasm, a subcortical layer of microtubules forms parallel arrays that support long-range, fast flows. To analyze the streaming mechanism, we combined observations of microtubule and organelle motions with detailed mathematical modeling. In the fast state, microtubules tethered to the cortex form a thin subcortical layer and undergo correlated sinusoidal bending. Organelles moving in flows along the arrays show velocities that are slow near the cortex and fast on the inward side of the subcortical microtubule layer. Starting with fundamental physical principles suggested by qualitative hypotheses, and with published values for microtubule stiffness, kinesin velocity, and cytoplasmic viscosity, we developed a quantitative coupled hydrodynamic model for streaming. The fully detailed mathematical model and its simulations identify key variables that can shift the system between disordered (slow) and ordered (fast) states. Measurements of array curvature, wave period, and the effects of diminished kinesin velocity on flow rates, as well as prior observations on f-actin perturbation, support the model. This establishes a concrete mechanistic framework for the ooplasmic streaming process. The self-organizing fast phase is a result of viscous drag on kinesin-driven cargoes that mediates equal and opposite forces on cytoplasmic fluid and on microtubules whose minus ends are tethered to the cortex. Fluid moves toward plus ends and microtubules are forced backward toward their minus ends, resulting in buckling. Under certain conditions, the buckling microtubules self-organize into parallel bending arrays, guiding varying directions

  16. The microtubule destabilizing protein stathmin controls the transition from dividing neuronal precursors to postmitotic neurons during adult hippocampal neurogenesis

    NARCIS (Netherlands)

    Boekhoorn, Karin; van Dis, Vera; Goedknegt, Erika; Sobel, André; Lucassen, Paul J; Hoogenraad, Casper C

    2014-01-01

    The hippocampus is one of the two areas in the mammalian brain where adult neurogenesis occurs. Adult neurogenesis is well known to be involved in hippocampal physiological functions as well as pathophysiological conditions. Microtubules (MTs), providing intracellular transport, stability, and

  17. Torsional Behavior of Axonal Microtubule Bundles

    Science.gov (United States)

    Lazarus, Carole; Soheilypour, Mohammad; Mofrad, Mohammad R.K.

    2015-01-01

    Axonal microtubule (MT) bundles crosslinked by microtubule-associated protein (MAP) tau are responsible for vital biological functions such as maintaining mechanical integrity and shape of the axon as well as facilitating axonal transport. Breaking and twisting of MTs have been previously observed in damaged undulated axons. Such breaking and twisting of MTs is suggested to cause axonal swellings that lead to axonal degeneration, which is known as “diffuse axonal injury”. In particular, overstretching and torsion of axons can potentially damage the axonal cytoskeleton. Following our previous studies on mechanical response of axonal MT bundles under uniaxial tension and compression, this work seeks to characterize the mechanical behavior of MT bundles under pure torsion as well as a combination of torsional and tensile loads using a coarse-grained computational model. In the case of pure torsion, a competition between MAP tau tensile and MT bending energies is observed. After three turns, a transition occurs in the mechanical behavior of the bundle that is characterized by its diameter shrinkage. Furthermore, crosslink spacing is shown to considerably influence the mechanical response, with larger MAP tau spacing resulting in a higher rate of turns. Therefore, MAP tau crosslinking of MT filaments protects the bundle from excessive deformation. Simultaneous application of torsion and tension on MT bundles is shown to accelerate bundle failure, compared to pure tension experiments. MAP tau proteins fail in clusters of 10–100 elements located at the discontinuities or the ends of MT filaments. This failure occurs in a stepwise fashion, implying gradual accumulation of elastic tensile energy in crosslinks followed by rupture. Failure of large groups of interconnecting MAP tau proteins leads to detachment of MT filaments from the bundle near discontinuities. This study highlights the importance of torsional loading in axonal damage after traumatic brain injury

  18. Anterograde Amnesia during Electroconvulsive Therapy: A Prospective Pilot-Study in Patients with Major Depressive Disorder.

    Directory of Open Access Journals (Sweden)

    Elvira Boere

    Full Text Available Electroconvulsive therapy (ECT is considered an effective treatment for major depression with melancholic features. However, neurocognitive side-effects such as anterograde amnesia still regularly occur. The present study aims to evaluate the severity and course of anterograde amnesia in severely depressed patients undergoing ECT. In a prospective naturalistic study, anterograde memory function was assessed among inpatients who underwent ECT (n = 11. Subjects met DSM-IV criteria for major depressive disorder. Recruitment took place between March 2010-March 2011 and March 2012-March 2013. Controls treated with antidepressants (n = 9 were matched for age, gender and depression severity. Primary outcome measure was immediate recall; secondary outcome measures were delayed recall, recognition, and visual association. Differences were tested using repeated measures ANOVA and paired t-tests. Correlations with hypothesized covariates were calculated. In patients with major depressive disorder, ECT had a significant effect on delayed memory function (p<0.01 with large effect sizes. Findings on immediate recall were less consistent. Four weeks after treatment discontinuation, these memory functions had recovered. Age was identified as a very important covariate. The main limitations of our study are its naturalistic design, possibly compromising internal validity, and its small sample size. However, if these findings can be reproduced in a more comprehensive study group, then the possible induction of anterograde amnesia is not a justifiable reason for clinicians to disregard ECT as a treatment option.

  19. Behavioral and Functional Neuroanatomical Correlates of Anterograde Autobiographical Memory in Isolated Retrograde Amnesic Patient M. L.

    Science.gov (United States)

    Levine, Brian; Svoboda, Eva; Turner, Gary R.; Mandic, Marina; Mackey, Allison

    2009-01-01

    Patient M. L. [Levine, B., Black, S. E., Cabeza, R., Sinden, M., Mcintosh, A. R., Toth, J. P., et al. (1998). "Episodic memory and the self in a case of isolated retrograde amnesia." "Brain", "121", 1951-1973], lost memory for events occurring before his severe traumatic brain injury, yet his anterograde (post-injury) learning and memory appeared…

  20. Altered microtubule dynamics in neurodegenerative disease: Therapeutic potential of microtubule-stabilizing drugs.

    Science.gov (United States)

    Brunden, Kurt R; Lee, Virginia M-Y; Smith, Amos B; Trojanowski, John Q; Ballatore, Carlo

    2017-09-01

    Many neurodegenerative diseases are characterized by deficiencies in neuronal axonal transport, a process in which cellular cargo is shuttled with the aid of molecular motors from the cell body to axonal termini and back along microtubules (MTs). Proper axonal transport is critical to the normal functioning of neurons, and impairments in this process could contribute to the neuronal damage and death that is characteristic of neurodegenerative disease. Although the causes of axonal transport abnormalities may vary among the various neurodegenerative conditions, in many cases it appears that the transport deficiencies result from a diminution of axonal MT stability. Here we review the evidence of MT abnormalities in a number of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and traumatic brain injury, and highlight the potential benefit of MT-stabilizing agents in improving axonal transport and nerve function in these diseases. Moreover, we discuss the challenges associated with the utilization of MT-stabilizing drugs as therapeutic candidates for neurodegenerative conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells

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    Amber L. Jolly

    2016-01-01

    Full Text Available Long-distance intracellular transport of organelles, mRNA, and proteins (“cargo” occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

  2. The Characteristics of Force Production of Kinesin-5 on MCF7 Microtubules

    Science.gov (United States)

    Shojania Feizabadi, Mitra

    Unlike neural mammalian microtubules with class II of beta tubulin as the major beta tubulin in their compositions, MCF7 microtubules composed of 0% class II beta tubulin isotype, 39.1% class I beta tubulin isotype, 2.5% class III beta tubulin isotype and 58.4% class IV beta tubulin isotype. Recent studies have revealed that function of some of motor proteins can be affected by the structural composition of microtubules. In this work, we will show how the function of mitotic kinesin (Kin-5) under external load changed when moving along bovine versus MCF7 microtubules. Along MCF7 microtubules, the detachment force was reduced and the force-velocity curve was different as compared to those related to bovine brain. We will also show that the elimination of the C-terminal tails made the transport almost similar to the two sets of microtubules. This suggests that the C-terminal tails of tubulin plays a regulatory role in Kinesin-5's function.

  3. A detailed, hierarchical study of Giardia lamblia's ventral disc reveals novel microtubule-associated protein complexes.

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    Cindi L Schwartz

    Full Text Available Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia's two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA, we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is

  4. Kinetochore–microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E

    Science.gov (United States)

    Vitre, Benjamin; Gudimchuk, Nikita; Borda, Ranier; Kim, Yumi; Heuser, John E.; Cleveland, Don W.; Grishchuk, Ekaterina L.

    2014-01-01

    Centromere protein E (CENP-E) is a highly elongated kinesin that transports pole-proximal chromosomes during congression in prometaphase. During metaphase, it facilitates kinetochore–microtubule end-on attachment required to achieve and maintain chromosome alignment. In vitro CENP-E can walk processively along microtubule tracks and follow both growing and shrinking microtubule plus ends. Neither the CENP-E–dependent transport along microtubules nor its tip-tracking activity requires the unusually long coiled-coil stalk of CENP-E. The biological role for the CENP-E stalk has now been identified through creation of “Bonsai” CENP-E with significantly shortened stalk but wild-type motor and tail domains. We demonstrate that Bonsai CENP-E fails to bind microtubules in vitro unless a cargo is contemporaneously bound via its C-terminal tail. In contrast, both full-length and truncated CENP-E that has no stalk and tail exhibit robust motility with and without cargo binding, highlighting the importance of CENP-E stalk for its activity. Correspondingly, kinetochore attachment to microtubule ends is shown to be disrupted in cells whose CENP-E has a shortened stalk, thereby producing chromosome misalignment in metaphase and lagging chromosomes during anaphase. Together these findings establish an unexpected role of CENP-E elongated stalk in ensuring stability of kinetochore–microtubule attachments during chromosome congression and segregation. PMID:24920822

  5. Mechanical and geometrical constraints control kinesin-based microtubule guidance

    NARCIS (Netherlands)

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

    2014-01-01

    Proper organization of microtubule networks depends on microtubule-associated proteins and motors that use different spatial cues to guide microtubule growth [1–3]. For example, it has been proposed that the uniform minus-end-out microtubule organization in dendrites of Drosophila neurons is

  6. Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules.

    Science.gov (United States)

    Haren, Laurence; Merdes, Andreas

    2002-05-01

    In mitosis, NuMA localises to spindle poles where it contributes to the formation and maintenance of focussed microtubule arrays. Previous work has shown that NuMA is transported to the poles by dynein and dynactin. So far, it is unclear how NuMA accumulates at the spindle poles following transport and how it remains associated throughout mitosis. We show here that NuMA can bind to microtubules independently of dynein/dynactin. We characterise a 100-residue domain located within the C-terminal tail of NuMA that mediates a direct interaction with tubulin in vitro and that is necessary for NuMA association with tubulin in vivo. Moreover, this domain induces bundling and stabilisation of microtubules when expressed in cultured cells and leads to formation of abnormal mitotic spindles with increased microtubule asters or multiple poles. Our results suggest that NuMA organises the poles by stable crosslinking of the microtubule fibers.

  7. A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons.

    Science.gov (United States)

    Lo, Liching; Anderson, David J

    2011-12-22

    Neurotropic viruses that conditionally infect or replicate in molecularly defined neuronal subpopulations, and then spread transsynaptically, are powerful tools for mapping neural pathways. Genetically targetable retrograde transsynaptic tracer viruses are available to map the inputs to specific neuronal subpopulations, but an analogous tool for mapping synaptic outputs is not yet available. Here we describe a Cre recombinase-dependent, anterograde transneuronal tracer, based on the H129 strain of herpes simplex virus (HSV). Application of this virus to transgenic or knockin mice expressing Cre in peripheral neurons of the olfactory epithelium or the retina reveals widespread, polysynaptic labeling of higher-order neurons in the olfactory and visual systems, respectively. Polysynaptic pathways were also labeled from cerebellar Purkinje cells. In each system, the pattern of labeling was consistent with classical circuit-tracing studies, restricted to neurons, and anterograde specific. These data provide proof-of-principle for a conditional, nondiluting anterograde transsynaptic tracer for mapping synaptic outputs from genetically marked neuronal subpopulations. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors.

    Science.gov (United States)

    Zingg, Brian; Chou, Xiao-Lin; Zhang, Zheng-Gang; Mesik, Lukas; Liang, Feixue; Tao, Huizhong Whit; Zhang, Li I

    2017-01-04

    To decipher neural circuits underlying brain functions, viral tracers are widely applied to map input and output connectivity of neuronal populations. Despite the successful application of retrograde transsynaptic viruses for identifying presynaptic neurons of transduced neurons, analogous anterograde transsynaptic tools for tagging postsynaptically targeted neurons remain under development. Here, we discovered that adeno-associated viruses (AAV1 and AAV9) exhibit anterograde transsynaptic spread properties. AAV1-Cre from transduced presynaptic neurons effectively and specifically drives Cre-dependent transgene expression in selected postsynaptic neuronal targets, thus allowing axonal tracing and functional manipulations of the latter input-defined neuronal population. Its application in superior colliculus (SC) reveals that SC neuron subpopulations receiving corticocollicular projections from auditory and visual cortex specifically drive flight and freezing, two different types of defense behavior, respectively. Together with an intersectional approach, AAV-mediated anterograde transsynaptic tagging can categorize neurons by their inputs and molecular identity, and allow forward screening of distinct functional neural pathways embedded in complex brain circuits. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Multimotor transport in constitutive exocytosis

    NARCIS (Netherlands)

    Afonso Serra Marques, A.M.

    2015-01-01

    Intracellular transport along cytoskeletal filaments is an essential cellular process that controls numerous cellular functions by promoting correct sorting, transport and delivery of different cargos in the cell. The microtubule system and associated molecular motors, dynein and kinesins, are

  10. Luminal localization of α-tubulin K40 acetylation by cryo-EM analysis of fab-labeled microtubules.

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    Virupakshi Soppina

    Full Text Available The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40 has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

  11. Back on track - On the role of the microtubule for kinesin motility and cellular function

    NARCIS (Netherlands)

    Lakamper, S.; Meyhofer, E.

    2006-01-01

    The evolution of cytoskeletal filaments (actin- and intermediate-filaments, and the microtubules) and their associated motor- and non-motor-proteins has enabled the eukaryotic cell to achieve complex organizational and structural tasks. This ability to control cellular transport processes and

  12. The current of a particle along a microtubule in microscopic plasma

    International Nuclear Information System (INIS)

    Li Wei; Chen Junfang; Wang Teng; Lai Xiuqiong

    2008-01-01

    Transport of a particle along the axis of a microtubule in a plasma-enhanced chemical vapor deposition (PECVD) system is investigated. The current, respectively, as a function of the temperature, the magnetic field and the external force is obtained. The value and direction of the current may be controlled by changing the above parameters

  13. Microtubule-dependent targeting of the exocyst complex is necessary for xylem development in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Vukašinović, Nemanja; Oda, Y.; Pejchar, Přemysl; Synek, Lukáš; Pečenková, Tamara; Rawat, Anamika; Sekereš, Juraj; Potocký, Martin; Žárský, Viktor

    2017-01-01

    Roč. 213, č. 3 (2017), s. 1052-1067 ISSN 0028-646X R&D Projects: GA ČR(CZ) GA15-14886S Grant - others:GA MŠk(CZ) LO1417 Institutional support: RVO:61389030 Keywords : secondary cell-wall * tracheary element differentiation * cortical microtubules * plasma-membrane * vesicle trafficking * secretory pathways * auxin transport * exocytosis * deposition * thaliana * conserved oligomeric Golgi (COG) complex * exocyst * microtubules * secondary cell wall * tracheary elements * xylem Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 7.330, year: 2016

  14. Understanding the guiding of kinesin/microtubule-based microtransporters in microfabricated tracks.

    Science.gov (United States)

    Ishigure, Yuki; Nitta, Takahiro

    2014-10-14

    Microtransporters using cargo-laden microtubules propelled by kinesin motors are attractive for numerous applications in nanotechnology. To improve the efficiency of transport, the movement of microtubules must be guided by microfabricated tracks. However, the mechanisms of the guiding methods used are not fully understood. Here, using computer simulation, we systematically studied the guiding of such microtransporters by three different types of guiding methods: a chemical boundary, a physical barrier, and their combination. The simulation reproduced the probabilities of guiding previously observed experimentally for the three methods. Moreover, the simulation provided further insight into the mechanisms of guiding, which overturn previous assumptions and models.

  15. Erucin, the major isothiocyanate in arugula (Eruca sativa, inhibits proliferation of MCF7 tumor cells by suppressing microtubule dynamics.

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    Olga Azarenko

    Full Text Available Consumption of cruciferous vegetables is associated with reduced risk of various types of cancer. Isothiocyanates including sulforaphane and erucin are believed to be responsible for this activity. Erucin [1-isothiocyanato-4-(methylthiobutane], which is metabolically and structurally related to sulforaphane, is present in large quantities in arugula (Eruca sativa, Mill., kohlrabi and Chinese cabbage. However, its cancer preventive mechanisms remain poorly understood. We found that erucin inhibits proliferation of MCF7 breast cancer cells (IC50 = 28 µM in parallel with cell cycle arrest at mitosis (IC50 = 13 µM and apoptosis, by a mechanism consistent with impairment of microtubule dynamics. Concentrations of 5-15 µM erucin suppressed the dynamic instability of microtubules during interphase in the cells. Most dynamic instability parameters were inhibited, including the rates and extents of growing and shortening, the switching frequencies between growing and shortening, and the overall dynamicity. Much higher erucin concentrations were required to reduce the microtubule polymer mass. In addition, erucin suppressed dynamic instability of microtubules reassembled from purified tubulin in similar fashion. The effects of erucin on microtubule dynamics, like those of sulforaphane, are similar qualitatively to those of much more powerful clinically-used microtubule-targeting anticancer drugs, including taxanes and the vinca alkaloids. The results suggest that suppression of microtubule dynamics by erucin and the resulting impairment of critically important microtubule-dependent cell functions such as mitosis, cell migration and microtubule-based transport may be important in its cancer preventive activities.

  16. Buckling behavior of individual and bundled microtubules.

    Science.gov (United States)

    Soheilypour, Mohammad; Peyro, Mohaddeseh; Peter, Stephen J; Mofrad, Mohammad R K

    2015-04-07

    As the major structural constituent of the cytoskeleton, microtubules (MTs) serve a variety of biological functions that range from facilitating organelle transport to maintaining the mechanical integrity of the cell. Neuronal MTs exhibit a distinct configuration, hexagonally packed bundles of MT filaments, interconnected by MT-associated protein (MAP) tau. Building on our previous work on mechanical response of axonal MT bundles under uniaxial tension, this study is focused on exploring the compression scenarios. Intracellular MTs carry a large fraction of the compressive loads sensed by the cell and therefore, like any other column-like structure, are prone to substantial bending and buckling. Various biological activities, e.g., actomyosin contractility and many pathological conditions are driven or followed by bending, looping, and buckling of MT filaments. The coarse-grained model previously developed in our lab has been used to study the mechanical behavior of individual and bundled in vivo MT filaments under uniaxial compression. Both configurations show tip-localized, decaying, and short-wavelength buckling. This behavior highlights the role of the surrounding cytoplasm and MAP tau on MT buckling behavior, which allows MT filaments to bear much larger compressive forces. It is observed that MAP tau interconnections improve this effect by a factor of two. The enhanced ability of MT bundles to damp buckling waves relative to individual MT filaments, may be interpreted as a self-defense mechanism because it helps axonal MTs to endure harsher environments while maintaining their function. The results indicate that MT filaments in a bundle do not buckle simultaneously implying that the applied stress is not equally shared among the MT filaments, that is a consequence of the nonuniform distribution of MAP tau proteins along the bundle length. Furthermore, from a pathological perspective, it is observed that axonal MT bundles are more vulnerable to failure in

  17. Microtubule binding distinguishes dystrophin from utrophin

    Science.gov (United States)

    Belanto, Joseph J.; Mader, Tara L.; Eckhoff, Michael D.; Strandjord, Dana M.; Banks, Glen B.; Gardner, Melissa K.; Lowe, Dawn A.; Ervasti, James M.

    2014-01-01

    Dystrophin and utrophin are highly similar proteins that both link cortical actin filaments with a complex of sarcolemmal glycoproteins, yet localize to different subcellular domains within normal muscle cells. In mdx mice and Duchenne muscular dystrophy patients, dystrophin is lacking and utrophin is consequently up-regulated and redistributed to locations normally occupied by dystrophin. Transgenic overexpression of utrophin has been shown to significantly improve aspects of the disease phenotype in the mdx mouse; therefore, utrophin up-regulation is under intense investigation as a potential therapy for Duchenne muscular dystrophy. Here we biochemically compared the previously documented microtubule binding activity of dystrophin with utrophin and analyzed several transgenic mouse models to identify phenotypes of the mdx mouse that remain despite transgenic utrophin overexpression. Our in vitro analyses revealed that dystrophin binds microtubules with high affinity and pauses microtubule polymerization, whereas utrophin has no activity in either assay. We also found that transgenic utrophin overexpression does not correct subsarcolemmal microtubule lattice disorganization, loss of torque production after in vivo eccentric contractions, or physical inactivity after mild exercise. Finally, our data suggest that exercise-induced inactivity correlates with loss of sarcolemmal neuronal NOS localization in mdx muscle, whereas loss of in vivo torque production after eccentric contraction-induced injury is associated with microtubule lattice disorganization. PMID:24706788

  18. Ferritin associates with marginal band microtubules

    International Nuclear Information System (INIS)

    Infante, Anthony A.; Infante, Dzintra; Chan, M.-C.; How, P.-C.; Kutschera, Waltraud; Linhartova, Irena; Muellner, Ernst W.; Wiche, Gerhard; Propst, Friedrich

    2007-01-01

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

  19. Mechanical properties of organelles driven by microtubule-dependent molecular motors in living cells.

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    Luciana Bruno

    2011-04-01

    Full Text Available The organization of the cytoplasm is regulated by molecular motors which transport organelles and other cargoes along cytoskeleton tracks. Melanophores have pigment organelles or melanosomes that move along microtubules toward their minus and plus end by the action of cytoplasmic dynein and kinesin-2, respectively. In this work, we used single particle tracking to characterize the mechanical properties of motor-driven organelles during transport along microtubules. We tracked organelles with high temporal and spatial resolutions and characterized their dynamics perpendicular to the cytoskeleton track. The quantitative analysis of these data showed that the dynamics is due to a spring-like interaction between melanosomes and microtubules in a viscoelastic microenvironment. A model based on a generalized Langevin equation explained these observations and predicted that the stiffness measured for the motor complex acting as a linker between organelles and microtubules is ∼ one order smaller than that determined for motor proteins in vitro. This result suggests that other biomolecules involved in the interaction between motors and organelles contribute to the mechanical properties of the motor complex. We hypothesise that the high flexibility observed for the motor linker may be required to improve the efficiency of the transport driven by multiple copies of motor molecules.

  20. Transportation of Aspergillus nidulans Class III and V Chitin Synthases to the Hyphal Tips Depends on Conventional Kinesin.

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    Norio Takeshita

    Full Text Available Cell wall formation and maintenance are crucial for hyphal morphogenesis. In many filamentous fungi, chitin is one of the main structural components of the cell wall. Aspergillus nidulans ChsB, a chitin synthase, and CsmA, a chitin synthase with a myosin motor-like domain (MMD at its N-terminus, both localize predominantly at the hyphal tip regions and at forming septa. ChsB and CsmA play crucial roles in polarized hyphal growth in A. nidulans. In this study, we investigated the mechanism by which CsmA and ChsB accumulate at the hyphal tip in living hyphae. Deletion of kinA, a gene encoding conventional kinesin (kinesin-1, impaired the localization of GFP-CsmA and GFP-ChsB at the hyphal tips. The transport frequency of GFP-CsmA and GFP-ChsB in both anterograde and retrograde direction appeared lower in the kinA-deletion strain compared to wild type, although the velocities of the movements were comparable. Co-localization of GFP-ChsB and GFP-CsmA with mRFP1-KinArigor, a KinA mutant that binds to microtubules but does not move along them, was observed in the posterior of the hyphal tip regions. KinA co-immunoprecipitated with ChsB and CsmA. Co-localization and association of CsmA with KinA did not depend on the MMD. These findings indicate that ChsB and CsmA are transported along microtubules to the subapical region by KinA.

  1. One-parameter nonrelativistic supersymmetry for microtubules

    International Nuclear Information System (INIS)

    Rosu, H.C.; Moran-Mirabal, J.M.; Cornejo, O.

    2003-01-01

    The one-parameter nonrelativistic supersymmetry of Mielnik [J. Math. Phys. 25 (1984) 3387] is applied to the simple supersymmetric model of Caticha [Phys. Rev. A 51 (1995) 4264] in the form used by Rosu [Phys. Rev. E 55 (1997) 2038] for microtubules. By this means, we introduce Montroll double-well potentials with singularities that move along the positive or negative traveling direction depending on the sign of the free parameter of Mielnik's method. Possible interpretations of the singularity are either microtubule associated proteins (motors) or structural discontinuities in the arrangement of the tubulin molecules

  2. Microtubules: A network for solitary waves

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    Zdravković Slobodan

    2017-01-01

    Full Text Available In the present paper we deal with nonlinear dynamics of microtubules. The structure and role of microtubules in cells are explained as well as one of models explaining their dynamics. Solutions of the crucial nonlinear differential equation depend on used mathematical methods. Two commonly used procedures, continuum and semi-discrete approximations, are explained. These solutions are solitary waves usually called as kink solitons, breathers and bell-type solitons. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III45010

  3. Mmb1p binds mitochondria to dynamic microtubules

    Science.gov (United States)

    Fu, Chuanhai; Jain, Deeptee; Costa, Judite; Velve-Casquillas, Guilhem; Tran, Phong T.

    2015-01-01

    Summary Background Mitochondria form a dynamics tubular network within the cell. Proper mitochondria movement and distribution are critical for their localized function in cell metabolism, growth, and survival. In mammalian cells, mechanisms of mitochondria positioning appear dependent on the microtubule cytoskeleton, with kinesin or dynein motors carrying mitochondria as cargos and distributing them throughout the microtubule network. Interestingly, the timescale of microtubule dynamics occurs in seconds, and the timescale of mitochondria distribution occurs in minutes. How does the cell couple these two time constants? Results Fission yeast also relies on microtubules for mitochondria distribution. We report here a new microtubule-dependent but motor-independent mechanism for proper mitochondria positioning in fission yeast. We identify the protein mmb1p, which binds to mitochondria and microtubules. Mmb1p attaches the tubular mitochondria to the microtubule lattice at multiple discrete interaction sites. Mmb1 deletion causes mitochondria to aggregate, with the long-term consequence of defective mitochondria distribution and cell death. Mmb1p decreases microtubule dynamicity. Conclusion Mmb1p is a new microtubule-mitochondria binding protein. We propose that mmb1p act to couple long-term mitochondria distribution to short-term microtubule dynamics by attenuating microtubule dynamics, thus enhancing the mitochondria-microtubule interaction time. PMID:21856157

  4. Vault mobility depends in part on microtubules and vaults can be recruited to the nuclear envelope

    International Nuclear Information System (INIS)

    Zon, Arend van; Mossink, Marieke H.; Houtsmuller, Adriaan B.; Schoester, Martijn; Scheffer, George L.; Scheper, Rik J.; Sonneveld, Pieter; Wiemer, Erik A.C.

    2006-01-01

    Vaults are ribonucleoproteins that may function in intracellular transport processes. We investigated the intracellular distribution and dynamics of vaults in non-small cell lung cancer cells in which vaults are labeled with the green fluorescent protein. Immunofluorescence experiments showed that vaults are dispersed throughout the cytoplasm; a small fraction is found in close proximity to microtubules. Immunoprecipitation experiments corroborated these results showing co-precipitation of MVP and β-tubulin. Using quantitative fluorescence-recovery after photobleaching (FRAP), we demonstrated that vault mobility over longer distances in part depends on intact microtubules; vaults moving slower when microtubules are depolymerized by nocodazole. Biochemical fractionation indicated a small fraction of MVP associated with the nucleus, however, no GFP-tagged vaults could be observed inside the nucleus. We observed an accumulation of vaults at the nuclear envelope upon treatment of cells with the protein synthesis inhibitor cycloheximide. Analysis of nucleo-cytoplasmic transport using a fluorescent substrate containing a classical NLS and NES expressed in MVP +/+ and MVP -/- mouse embryonic fibroblasts indicated no differences in nuclear import/export kinetics, suggesting no role for vaults in these processes. We hypothesize that a subset of vaults moves directionally via microtubules, possibly towards the nucleus

  5. Sortilin Associates with Trk Receptors to Enhance Anterograde Transport and Signaling by Neurotrophins

    DEFF Research Database (Denmark)

    Vægter, Christian Bjerggaard

      Neurotrophins (NT) are neuronal growth factors essential for development and maintenance of the nervous system. They are released in two forms with opposing biological functions. The proforms induce apoptosis by engaging a death signaling complex comprising the p75NTR neurotrophin receptor...... and survival has been extensively studied in knockout mouse models. We recently generated a Sortilin knockout mouse model (Sort1-/-) and showed that the receptor is essential for p75NTR to induce death of neurons during certain stages of development and aging and upon brain injury (Jansen et al, Nat. Neurosci...

  6. FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42

    DEFF Research Database (Denmark)

    Kage, Frieda; Steffen, Anika; Ellinger, Adolf

    2017-01-01

    with Cdc42. Moreover, Golgi association of FMNL2 or -3 induced a phalloidin-detectable actin meshwork around the Golgi. Importantly, functional interference with FMNL2/3 formins by RNAi or CRISPR/Cas9-mediated gene deletion invariably induced Golgi fragmentation in different cell lines. Furthermore...

  7. KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility.

    Directory of Open Access Journals (Sweden)

    Lee B Smith

    Full Text Available Spermatogenesis is a complex process reliant upon interactions between germ cells (GC and supporting somatic cells. Testicular Sertoli cells (SC support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1. We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC from 15.5 days post-coitum (dpc and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

  8. Olesoxime prevents microtubule-targeting drug neurotoxicity: selective preservation of EB comets in differentiated neuronal cells.

    Science.gov (United States)

    Rovini, Amandine; Carré, Manon; Bordet, Thierry; Pruss, Rebecca M; Braguer, Diane

    2010-09-15

    Microtubule-targeting agents (MTAs), anticancer drugs widely used in the clinic, often induce peripheral neuropathy, a main dose-limiting side effect. The mechanism for this neurotoxicity remains poorly understood and there are still no approved therapies for neuropathies triggered by MTAs. Olesoxime (cholest-4-en-3-one, oxime; TRO19622) has shown marked neuroprotective properties in animals treated with paclitaxel and vincristine. The purpose of this study was to investigate its mechanism of neuroprotection against MTA neurotoxicity by using rat and human differentiated neuronal cells. We first showed that olesoxime prevented neurite shrinkage induced by MTAs in differentiated PC-12 and SK-N-SH neuroblastoma cell lines by up to 90%. This neuroprotective effect was correlated with enhanced EB1 accumulation at microtubule plus-ends, increased growth cone microtubule growing rate (20%) and decreased microtubule attenuation duration (54%). The effects of olesoxime on EB comets were specific for differentiated neuronal cells and were not seen either in proliferating neuroblastoma cells, glioblastoma cells or primary endothelial cells. Importantly, olesoxime did not alter MTA cytotoxic properties in a wide range of MTA-sensitive tumor cells, a prerequisite for future clinical application. Finally, olesoxime also counteracted MTA inhibition of microtubule-dependent mitochondria trafficking. These results provide additional insight into the neuroprotective properties of olesoxime, highlighting a role for microtubule dynamics in preservation of neurite architecture and axoplasmic transport, which are both disturbed by MTAs. The neuron-specific protective properties of olesoxime support its further development to treat MTA-induced neuropathy. Copyright 2010 Elsevier Inc. All rights reserved.

  9. Electrostatically biased binding of kinesin to microtubules.

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    Barry J Grant

    2011-11-01

    Full Text Available The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules.

  10. Analysis of microtubule assembly kinetics using turbidimetry.

    Science.gov (United States)

    Gaskin, Felicia

    2011-01-01

    Turbidity measurements are rapid and reliable methods to follow the effects of drugs, proteins, nucleotides, metals, and other factors on the assembly kinetics of tubulin into microtubules in vitro and have been in use since 1974. Improvements in spectrophotometers and software have resulted in the use of less protein, and more curves can be analyzed continuously and almost simultaneously.

  11. NAP (davunetide) modifies disease progression in a mouse model of severe neurodegeneration: protection against impairments in axonal transport.

    Science.gov (United States)

    Jouroukhin, Yan; Ostritsky, Regina; Assaf, Yaniv; Pelled, Galit; Giladi, Eliezer; Gozes, Illana

    2013-08-01

    NAP (davunetide) is a novel neuroprotective compound with mechanism of action that appears to involve microtubule (MT) stabilization and repair. To evaluate, for the first time, the impact of NAP on axonal transport in vivo and to translate it to neuroprotection in a severe neurodegeneration, the SOD1-G93A mouse model for amyotrophic lateral sclerosis (ALS) was used. Manganese-enhanced magnetic resonance imaging (MRI), estimating axonal transport rates, revealed a significant reduction of the anterograde axonal transport in the ALS mice compared to healthy control mice. Acute NAP treatment normalized axonal transport rates in these ALS mice. Tau hyperphosphorylation, associated with MT dysfunction and defective axonal transport, was discovered in the brains of the ALS mice and was significantly reduced by chronic NAP treatment. Furthermore, in healthy wild type (WT) mice, NAP reversed axonal transport disruption by colchicine, suggesting drug-dependent protection against axonal transport impairment through stabilization of the neuronal MT network. Histochemical analysis showed that chronic NAP treatment significantly protected spinal cord motor neurons against ALS-like pathology. Sequential MRI measurements, correlating brain structure with ALS disease progression, revealed a significant damage to the ventral tegmental area (VTA), indicative of impairments to the dopaminergic pathways relative to healthy controls. Chronic daily NAP treatment of the SOD1-G93A mice, initiated close to disease onset, delayed degeneration of the trigeminal, facial and hypoglossal motor nuclei as was significantly apparent at days 90-100 and further protected the VTA throughout life. Importantly, protection of the VTA was significantly correlated with longevity and overall, NAP treatment significantly prolonged life span in the ALS mice. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Integrin-linked kinase regulates interphase and mitotic microtubule dynamics.

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    Simin Lim

    Full Text Available Integrin-linked kinase (ILK localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.

  13. Measuring the Dynamic Parameters of MCF7 Cell Microtubules

    Science.gov (United States)

    Winton, Carly; Shojania Feizabadi, Mitra

    2013-03-01

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

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

    Science.gov (United States)

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

    2010-12-01

    Computer models of microtubule dynamics have provided the basis for many of the theories on the cellular mechanics of the microtubules, their polymerization kinetics, and the diffusion of tubulin and tau. In the three-dimensional model presented here, we include the effects of tau concentration and the hydrolysis of GTP-tubulin to GDP-tubulin and observe the emergence of microtubule dynamic instability. This integrated approach simulates the essential physics of microtubule dynamics in a cellular environment. The model captures the structure of the microtubules as they undergo steady state dynamic instabilities in this simplified geometry, and also yields the average number, length, and cap size of the microtubules. The model achieves realistic geometries and simulates cellular structures found in degenerating neurons in disease states such as Alzheimer disease. Further, this model can be used to simulate microtubule changes following the addition of antimitotic drugs which have recently attracted attention as chemotherapeutic agents. Copyright © 2010 Elsevier Ltd. All rights reserved.

  15. A hereditary spastic paraplegia mutation in kinesin-1A/KIF5A disrupts neurofilament transport

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    Brown Anthony

    2010-11-01

    Full Text Available Abstract Background Hereditary spastic paraplegias are a group of neurological disorders characterized by progressive distal degeneration of the longest ascending and descending axons in the spinal cord, leading to lower limb spasticity and weakness. One of the dominantly inherited forms of this disease (spastic gait type 10, or SPG10 is caused by point mutations in kinesin-1A (also known as KIF5A, which is thought to be an anterograde motor for neurofilaments. Results We investigated the effect of an SPG10 mutation in kinesin-1A (N256S-kinesin-1A on neurofilament transport in cultured mouse cortical neurons using live-cell fluorescent imaging. N256S-kinesin-1A decreased both anterograde and retrograde neurofilament transport flux by decreasing the frequency of anterograde and retrograde movements. Anterograde velocity was not affected, whereas retrograde velocity actually increased. Conclusions These data reveal subtle complexities to the functional interdependence of the anterograde and retrograde neurofilament motors and they also raise the possibility that anterograde and retrograde neurofilament transport may be disrupted in patients with SPG10.

  16. Kinetochore-microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E.

    Science.gov (United States)

    Vitre, Benjamin; Gudimchuk, Nikita; Borda, Ranier; Kim, Yumi; Heuser, John E; Cleveland, Don W; Grishchuk, Ekaterina L

    2014-08-01

    Centromere protein E (CENP-E) is a highly elongated kinesin that transports pole-proximal chromosomes during congression in prometaphase. During metaphase, it facilitates kinetochore-microtubule end-on attachment required to achieve and maintain chromosome alignment. In vitro CENP-E can walk processively along microtubule tracks and follow both growing and shrinking microtubule plus ends. Neither the CENP-E-dependent transport along microtubules nor its tip-tracking activity requires the unusually long coiled-coil stalk of CENP-E. The biological role for the CENP-E stalk has now been identified through creation of "Bonsai" CENP-E with significantly shortened stalk but wild-type motor and tail domains. We demonstrate that Bonsai CENP-E fails to bind microtubules in vitro unless a cargo is contemporaneously bound via its C-terminal tail. In contrast, both full-length and truncated CENP-E that has no stalk and tail exhibit robust motility with and without cargo binding, highlighting the importance of CENP-E stalk for its activity. Correspondingly, kinetochore attachment to microtubule ends is shown to be disrupted in cells whose CENP-E has a shortened stalk, thereby producing chromosome misalignment in metaphase and lagging chromosomes during anaphase. Together these findings establish an unexpected role of CENP-E elongated stalk in ensuring stability of kinetochore-microtubule attachments during chromosome congression and segregation. © 2014 Vitre, Gudimchuk, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. Kinesin-2 motors transport IFT-particles, dyneins and tubulin subunits to the tips of Caenorhabditis elegans sensory cilia: relevance to vision research?

    Science.gov (United States)

    Scholey, Jonathan M

    2012-12-15

    The sensory outer segments (OS) of vertebrate retinal photoreceptors, which detect photons of light, resemble the distal segments of Caenorhabditis elegans sensory cilia, which detect chemical ligands that influence the chemotactic movements of the animal. Based on fluorescence microscopy assays performed in sensory cilia of living, transgenic "wild type" and mutant C. elegans, combined with in vitro motility assays using purified motors, we have proposed that two types of kinesin-2 motor, heterotrimeric kinesin-II and homodimeric OSM-3, cooperate to build amphid and phasmid sensory cilia on chemosensory neurons. Specifically, we propose that these motors function together in a redundant manner to build the axoneme core (aka middle segments (MS)), whereas OSM-3 alone serves to build the distal segments (DS). Furthermore, our data suggest that these motors accomplish this by driving two sequential steps of anterograde transport of cargoes consisting of IFT-particles, retrograde dynein motors, and ciliary tubulin subunits, from the transition zone to the tips of the axonemal microtubules (MTs). Homologs of kinesin-II (KIF3) and OSM-3 (KIF17) are also proposed to contribute to the assembly of vertebrate photoreceptors, although how they do so is currently unclear. Here I review our work on kinesin-2 motors, intraflagellar transport (IFT) and cilium biogenesis in C. elegans sensory cilia, and comment on its possible relevance to current research on vertebrate photoreceptor cilia assembly and function. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. CLASPs link focal adhesion-associated microtubule capture to localized exocytosis and adhesion site turnover

    Science.gov (United States)

    Stehbens, Samantha J.; Paszek, Matthew; Pemble, Hayley; Ettinger, Andreas; Gierke, Sarah; Wittmann, Torsten

    2014-01-01

    Turnover of integrin-based focal adhesions (FAs) with the extracellular matrix (ECM) is essential for coordinated cell movement. In collectively migrating human keratinocytes, FAs assemble near the leading edge, grow and mature as a result of contractile forces, and disassemble underneath the advancing cell body. We report that clustering of microtubule-associated CLASP1 and CLASP2 proteins around FAs temporally correlates with FA turnover. CLASPs and LL5β, which recruits CLASPs to FAs, facilitate FA disassembly. CLASPs are further required for FA-associated ECM degradation, and matrix metalloprotease inhibition slows FA disassembly similar to CLASP or LL5β depletion. Finally, CLASP-mediated microtubuletethering at FAs establishes a FA-directed transport pathway for delivery, docking and localized fusion of exocytic vesicles near FAs. We propose that CLASPs couple microtubule organization, vesicle transport and cell interactions with the ECM, establishing a local secretion pathway that facilitates FA turnover by severing cell-matrix connections. PMID:24859005

  19. Microtubules Growth Rate Alteration in Human Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Irina B. Alieva

    2010-01-01

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

  20. Salvianolic acid A exerts antiamnesic effect on diazepam-induced anterograde amnesia in mice.

    Science.gov (United States)

    Wang, T; Shan, S Y; Han, B; Zhang, L M; Fu, F H

    2011-01-01

    Benzodiazepine was known to produce amnesia. Salvianolic acid A extracted from Salvia miltiorrhiza was an effective antioxidant. The objective of the study was to evaluate the effect of salvianolic acid A on diazepam-induced amnesia in mice. C57BL/6 mice were treated with salvianolic acid A at doses of 10, 20 and 40 mg/kg following administration with diazepam at a dose of 3 mg/kg. Morris water maze was performed to evaluate the effect of salvianolic acid A on amnesia. The antioxidative parameters in hippocampus were measured. The results showed that salvianolic acid A decreased the mean escape latency and increased the percentage of time spent in target quadrant. Salvianolic acid A reduced the content of malondialdehyde and increased the activities of superoxide dismutase, catalase and glutathione peroxidase in hippocampus. The findings demonstrated that salvianolic acid A had antiamnesic effects on diazepam-induced anterograde amnesia in mice, by augmenting the antioxidative capacity of hippocampus.

  1. Anterograde effects of a single electroconvulsive shock on inhibitory avoidance and on cued fear conditioning

    Directory of Open Access Journals (Sweden)

    Oliveira M.G.M.

    1998-01-01

    Full Text Available A single electroconvulsive shock (ECS or a sham ECS was administered to male 3-4-month-old Wistar rats 1, 2, and 4 h before training in an inhibitory avoidance test and in cued classical fear conditioning (measured by means of freezing time in a new environment. ECS impaired inhibitory avoidance at all times and, at 1 or 2 h before training, reduced freezing time before and after re-presentation of the ECS. These results are interpreted as a transient conditioned stimulus (CS-induced anxiolytic or analgesic effect lasting about 2 h after a single treatment, in addition to the known amnesic effect of the stimulus. This suggests that the effect of anterograde learning impairment is demonstrated unequivocally only when the analgesic/anxiolytic effect is over (about 4 h after ECS administration and that this impairment of learning is selective, affecting inhibitory avoidance but not classical fear conditioning to a discrete stimulus.

  2. Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery

    NARCIS (Netherlands)

    Prevo, Bram; Scholey, Jonathan M.; Peterman, Erwin J. G.

    Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance, and length control of cilia, which play critical roles in motility, sensory reception, and signal transduction in virtually all eukaryotic cells. During IFT, anterograde

  3. Hepatocyte handling of immunoglobulin A in the rat: the role of microtubules

    International Nuclear Information System (INIS)

    Goldman, I.S.; Jones, A.L.; Hradek, G.T.; Huling, S.

    1983-01-01

    Plasma-derived dimeric immunoglobulin A is transported through liver parenchymal cells into bile, in association with its glycoprotein receptor secretory component, by a vesicular transport system. This study was designed to determine the effects of colchicine, a microtubule-disrupting agent, and thus the role of microtubules on the uptake, intracellular transport, and subsequent biliary secretion of dimeric immunoglobulin A. In vivo studies in rats showed that colchicine treatment reduced the amount of intraportally injected 125 I-dimeric immunoglobulin A that appeared in the bile. It was also found that although the livers in colchicine-treated animals could sequester and internalize immunoglobulin A, it was not readily secreted into bile. In vitro studies using peroxidase-labeled antisecretory component and 125 I-dimeric immunoglobulin A autoradiography were both used to determine the site of this block in immunoglobulin A secretion. These studies demonstrate that colchicine disruption of microtubules (a) has little initial effect on the binding and internalization of dimeric immunoglobulin A; (b) has a major effect on the translocation of immunoglobulin A-containing vesicles within the hepatocyte, and (c) most likely prevents the translocation of newly synthesized secretory component to the plasma membrane

  4. Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

    NARCIS (Netherlands)

    Schuster, M.; Kilaru, S.; Fink, G.; Collemare, J.A.R.; Roger, Y.; Steinberg, G.

    2011-01-01

    The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to

  5. The Role of Molecular Microtubule Motors and the Microtubule Cytoskeleton in Stress Granule Dynamics

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    Kristen M. Bartoli

    2011-01-01

    Full Text Available Stress granules (SGs are cytoplasmic foci that appear in cells exposed to stress-induced translational inhibition. SGs function as a triage center, where mRNAs are sorted for storage, degradation, and translation reinitiation. The underlying mechanisms of SGs dynamics are still being characterized, although many key players have been identified. The main components of SGs are stalled 48S preinitiation complexes. To date, many other proteins have also been found to localize in SGs and are hypothesized to function in SG dynamics. Most recently, the microtubule cytoskeleton and associated motor proteins have been demonstrated to function in SG dynamics. In this paper, we will discuss current literature examining the function of microtubules and the molecular microtubule motors in SG assembly, coalescence, movement, composition, organization, and disassembly.

  6. In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors.

    Science.gov (United States)

    Fort, Alfredo G; Murray, John W; Dandachi, Nadine; Davidson, Michael W; Dermietzel, Rolf; Wolkoff, Allan W; Spray, David C

    2011-07-01

    Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 μm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.

  7. Microtubules are organized independently of the centrosome in Drosophila neurons

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    Nguyen Michelle M

    2011-12-01

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

  8. The nucleation of microtubules in Aspergillus nidulans germlings

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    Andrade-Monteiro Cristina de

    1999-01-01

    Full Text Available Microtubules are filaments composed of dimers of alpha- and beta-tubulins, which have a variety of functions in living cells. In fungi, the spindle pole bodies usually have been considered to be microtubule-organizing centers. We used the antimicrotubule drug Benomyl in block/release experiments to depolymerize and repolymerize microtubules in Aspergillus nidulans germlings to learn more about the microtubule nucleation process in this filamentous fungus. Twenty seconds after release from Benomyl short microtubules were formed from several bright (immunofluorescent dots distributed along the germlings, suggesting that microtubule nucleation is randomly distributed in A. nidulans germlings. Since nuclear movement is dependent on microtubules in A. nidulans we analyzed whether mutants defective in nuclear distribution along the growing hyphae (nud mutants have some obvious microtubule defect. Cytoplasmic, astral and spindle microtubules were present and appeared to be normal in all nud mutants. However, significant changes in the percentage of short versus long mitotic spindles were observed in nud mutants. This suggests that some of the nuclei of nud mutants do not reach the late stage of cell division at normal temperatures.

  9. Producing Conditional Mutants for Studying Plant Microtubule Function

    Energy Technology Data Exchange (ETDEWEB)

    Richard Cyr

    2009-09-29

    The cytoskeleton, and in particular its microtubule component, participates in several processes that directly affect growth and development in higher plants. Normal cytoskeletal function requires the precise and orderly arrangement of microtubules into several cell cycle and developmentally specific arrays. One of these, the cortical array, is notable for its role in directing the deposition of cellulose (the most prominent polymer in the biosphere). An understanding of how these arrays form, and the molecular interactions that contribute to their function, is incomplete. To gain a better understanding of how microtubules work, we have been working to characterize mutants in critical cytoskeletal genes. This characterization is being carried out at the subcellular level using vital microtubule gene constructs. In the last year of funding colleagues have discovered that gamma-tubulin complexes form along the lengths of cortical microtubules where they act to spawn new microtubules at a characteristic 40 deg angle. This finding complements nicely the finding from our lab (which was funded by the DOE) showing that microtubule encounters are angle dependent; high angles encounters results in catastrophic collisions while low angle encounters result in favorable zippering. The finding of a 40 deg spawn of new microtubules from extant microtubule, together with aforementioned rules of encounters, insures favorable co-alignment in the array. I was invited to write a New and Views essay on this topic and a PDF is attached (News and Views policy does not permit funding acknowledgments and so I was not allowed to acknowledge support from the DOE).

  10. MTB-3, a microtubule plus-end tracking protein (+TIP of Neurospora crassa.

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    Rosa R Mouriño-Pérez

    Full Text Available The microtubule (MT "plus end" constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called "MT plus-end tracking proteins" (+TIPs. +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassaMicroTubule Binding protein-3 (MTB-3 is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.

  11. Emerging microtubule targets in glioma therapy

    Czech Academy of Sciences Publication Activity Database

    Katsetos, C.D.; Reginato, M.J.; Baas, P.W.; D'Agostino, L.; Legido, A.; Tuszynski, J. A.; Dráberová, Eduarda; Dráber, Pavel

    2015-01-01

    Roč. 22, č. 1 (2015), s. 49-72 ISSN 1071-9091 R&D Projects: GA MŠk LH12050; GA MZd NT14467 Grant - others:GA AV ČR M200521203PIPP; NIH(US) R01 NS028785; Philadelphia Health Education Corporation (PHEC)–St. Christopher’s Hospital for Children Reunified Endowment (C.D.K.)(US) 323256 Institutional support: RVO:68378050 Keywords : glioma tumorigenesis * glioblastoma * tubulin * microtubules Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.303, year: 2015

  12. Optomechanical proposal for monitoring microtubule mechanical vibrations

    Czech Academy of Sciences Publication Activity Database

    Barzanjeh, Sh.; Salari, V.; Tuszynski, J. A.; Cifra, Michal; Simon, C.

    2017-01-01

    Roč. 96, č. 1 (2017), č. článku 012404. ISSN 2470-0045 R&D Projects: GA ČR(CZ) GA15-17102S Grant - others:AV ČR(CZ) SAV-15-22 Program:Bilaterální spolupráce Institutional support: RVO:67985882 Keywords : Vibrational modes * Microtubule * Resonance frequencies Subject RIV: BH - Optics, Masers, Laser s OBOR OECD: Optics (including laser optics and quantum optics) Impact factor: 2.366, year: 2016

  13. Optomechanical proposal for monitoring microtubule mechanical vibrations

    Czech Academy of Sciences Publication Activity Database

    Barzanjeh, Sh.; Salari, V.; Tuszynski, J. A.; Cifra, Michal; Simon, C.

    2017-01-01

    Roč. 96, č. 1 (2017), č. článku 012404. ISSN 2470-0045 R&D Projects: GA ČR(CZ) GA15-17102S Grant - others:AV ČR(CZ) SAV-15-22 Program:Bilaterální spolupráce Institutional support: RVO:67985882 Keywords : Vibrational modes * Microtubule * Resonance frequencies Subject RIV: BH - Optics, Masers, Lasers OBOR OECD: Optics (including laser optics and quantum optics) Impact factor: 2.366, year: 2016

  14. CLIP-170 facilitates the formation of kinetochore-microtubule attachments

    NARCIS (Netherlands)

    Tanenbaum, ME; Galjart, N; van Vugt, MATM; Medema, RH

    2006-01-01

    CLIP-170 is a microtubule 'plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have

  15. Structural microtubule cap: Stability, catastrophe, rescue, and third state

    DEFF Research Database (Denmark)

    Flyvbjerg, H.; Chretien, D.; Janosi, I.M.

    2002-01-01

    Microtubules polymerize from GTP-liganded tubulin dinners, but are essentially made of GDP-liganded tubulin. We investigate the tug-of-war resulting from the fact that GDP-liganded tubulin favors a curved configuration, but is forced to remain in a straight one when part of a microtubule. We poin...

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

    Science.gov (United States)

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

    2016-11-04

    Buckling of microtubules observed in cells has been reconstructed on a two-dimensional elastic medium consisting of kinesins grafted over compressible substrates, enabling precise control of experimental conditions and quantitative analysis. However, interpretations of the observations have ambiguities due to inevitable experimental difficulties. In this study, with computer simulations, we investigated importance of the mode of interaction of microtubule with elastic medium in the buckling behavior of microtubule. By taking into consideration of forced-induced detachments of kinesins from microtubules, our simulations reproduced the previous experimental results, and showed deviations from predictions of the elastic foundation model. On the other hand, with hypothetical linkers permanently bound to microtubules, our simulation reproduced the predictions of the elastic foundation model. By analyzing the results of the simulations, we investigated as to why the difference arose. These findings indicate the importance of the mode of interaction of microtubule with the medium in the buckling behavior of microtubule. Our findings would bring new insights on buckling of microtubules in living cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. A CRE-DEPENDENT, ANTEROGRADE TRANS-SYNAPTIC VIRAL TRACER FOR MAPPING OUTPUT PATHWAYS OF GENETICALLY MARKED NEURONS

    Science.gov (United States)

    Lo, Liching; Anderson, David J.

    2012-01-01

    SUMMARY Neurotropic viruses that conditionally infect or replicate in molecularly defined neuronal subpopulations, and then spread trans-synaptically, are powerful tools for mapping neural pathways. Genetically targetable retrograde trans-synaptic tracer viruses are available to map the inputs to specific neuronal subpopulations, but an analogous tool for mapping synaptic outputs is not yet available. Here we describe a Cre recombinase-dependent, anterograde trans-neuronal tracer, based on the H129 strain of herpes simplex virus (HSV). Application of this virus to transgenic or knock-in mice expressing Cre in peripheral neurons of the olfactory epithelium or the retina reveals widespread, polysynaptic labeling of higher-order neurons in the olfactory and visual systems, respectively. Polysynaptic pathways were also labeled from cerebellar Purkinje cells. In each system, the pattern of labeling was consistent with classical circuit-tracing studies, restricted to neurons and anterograde-specific. These data provide proof-of-principle for a conditional, non-diluting anterograde trans-synaptic tracer for mapping synaptic outputs from genetically marked neuronal subpopulations. PMID:22196330

  18. Looped host defense peptide CLP-19 binds to microtubules and inhibits surface expression of TLR4 on mouse macrophages.

    Science.gov (United States)

    Li, Di; Liu, Yao; Yang, Ya; Chen, Jian-hong; Yang, Jie; Zou, Lin-yun; Tian, Zhi-qiang; Lv, Jun; Xia, Pei-yuan

    2013-06-15

    The looped host defense peptide CLP-19 is derived from a highly functional core region of the Limulus anti-LPS factor and exerts robust anti-LPS activity by directly interacting with LPS in the extracellular space. We previously showed that prophylactic administration of CLP-19 even 20 h prior to LPS challenge might significantly increase the survival rate in a lethal endotoxin shock mouse model. Such an effect may be associated with immune regulation of CLP-19. To investigate the underlying mechanisms, peptide affinity chromatography, immunofluorescence, and Western blotting procedures were used to identify α- and β-tubulin as direct and specific binding partners of CLP-19 in the mouse macrophage cell line RAW 264.7. Bioinformatic analysis using the AutoDock Vina molecular docking and PyMOL molecular graphics system predicted that CLP-19 would bind to the functional residues of both α- and β-tubulin and would be located within the groove of microtubules. Tubulin polymerization assay revealed that CLP-19 might induce polymerization of microtubules and prevent depolymerization. The immunoregulatory effect of CLP-19 involving microtubules was investigated by flow cytometry, immunofluorescence, and Western blotting, which showed that CLP-19 prophylactic treatment of RAW 264.7 cells significantly inhibited LPS-induced surface expression of TLR4. Taken together, these results suggest that CLP-19 binding to microtubules disrupts the dynamic equilibrium of microtubules, reducing the efficacy of microtubule-dependent vesicular transport that would otherwise translocate TLR4 from the endoplasmic reticulum to the cell surface.

  19. Vaccinia virus intracellular enveloped virions move to the cell periphery on microtubules in the absence of the A36R protein.

    Science.gov (United States)

    Herrero-Martínez, Esteban; Roberts, Kim L; Hollinshead, Michael; Smith, Geoffrey L

    2005-11-01

    Vaccinia virus (VACV) intracellular enveloped virus (IEV) particles are transported to the cell periphery on microtubules where they fuse with the plasma membrane to form cell-associated enveloped virus (CEV). Two IEV-specific proteins, F12L and A36R, are implicated in mediating transport of IEV. Without F12L, virus morphogenesis halts after formation of IEV, and CEV is not formed, whereas without A36R, IEV was reported not to be transported, yet CEV was formed, To address the roles of A36R and F12L in IEV transport, viruses with deletions of either F12L (vdeltaF12L) or A36R (vdeltaA36R) were labelled with enhanced green fluorescent protein (EGFP) fused to the core protein A5L, and used to follow CEV production with time. Without F12L, CEV production was inhibited by >99 %, whereas without A36R, CEV were produced at approximately 60 % of wild-type levels at 24 h post-infection. Depolymerization of microtubules, but not actin, inhibited CEV formation in vdeltaA36R-infected cells. Moreover, vdeltaA36R IEV labelled with EGFP fused to the B5R protein co-localized with microtubules, showing that the A36R protein is not required for the interaction of IEV with microtubules. Time-lapse confocal microscopy confirmed that both wild-type and vdeltaA36R IEV moved in a stop-start manner at speeds consistent with microtubular movement, although the mean length of vdeltaA36R IEV movement was shorter. These data demonstrate that VACV IEV is transported to the cell surface using microtubules in the absence of A36R, and therefore IEV must attach to microtubule motors using at least one protein other than A36R.

  20. A thermodynamic model of microtubule assembly and disassembly.

    Directory of Open Access Journals (Sweden)

    Bernard M A G Piette

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

  1. Does usnic acid affect microtubules in human cancer cells?

    Directory of Open Access Journals (Sweden)

    MA. O'Neill

    Full Text Available Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 µM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules or taxol (which stabilizes microtubules. Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.

  2. An improved quantitative analysis method for plant cortical microtubules.

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

  4. Dietary antioxidant curcumin inhibits microtubule assembly through tubulin binding.

    Science.gov (United States)

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

    2006-12-01

    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.

  5. Structural insights into microtubule doublet interactions inaxonemes

    Energy Technology Data Exchange (ETDEWEB)

    Downing, Kenneth H.; Sui, Haixin

    2007-06-06

    Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of the axoneme. Recent structural studies of the axoneme have used cryo-electron tomography to reveal new details of the interactions among some of the multitude of proteins that form the axoneme and regulate its movement. Connections among the several sets of dyneins, in particular, suggest ways in which their actions may be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding the doublet's mechanical properties that are related to the bending of the axoneme, and has also offered insight into its potential role in the mechanism of dynein activity regulation.

  6. Microtubule-stabilizing agents as potential therapeutics for neurodegenerative disease.

    Science.gov (United States)

    Brunden, Kurt R; Trojanowski, John Q; Smith, Amos B; Lee, Virginia M-Y; Ballatore, Carlo

    2014-09-15

    Microtubules (MTs), cytoskeletal elements found in all mammalian cells, play a significant role in cell structure and in cell division. They are especially critical in the proper functioning of post-mitotic central nervous system neurons, where MTs serve as the structures on which key cellular constituents are trafficked in axonal projections. MTs are stabilized in axons by the MT-associated protein tau, and in several neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, and Parkinson's disease, tau function appears to be compromised due to the protein dissociating from MTs and depositing into insoluble inclusions referred to as neurofibrillary tangles. This loss of tau function is believed to result in alterations of MT structure and function, resulting in aberrant axonal transport that likely contributes to the neurodegenerative process. There is also evidence of axonal transport deficiencies in other neurodegenerative diseases, including amyotrophic lateral sclerosis and Huntington's disease, which may result, at least in part, from MT alterations. Accordingly, a possible therapeutic strategy for such neurodegenerative conditions is to treat with MT-stabilizing agents, such as those that have been used in the treatment of cancer. Here, we review evidence of axonal transport and MT deficiencies in a number of neurodegenerative diseases, and summarize the various classes of known MT-stabilizing agents. Finally, we highlight the growing evidence that small molecule MT-stabilizing agents provide benefit in animal models of neurodegenerative disease and discuss the desired features of such molecules for the treatment of these central nervous system disorders. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Identification of interphase functions for the NIMA kinase involving microtubules and the ESCRT pathway.

    Directory of Open Access Journals (Sweden)

    Meera Govindaraghavan

    2014-03-01

    Full Text Available The Never in Mitosis A (NIMA kinase (the founding member of the Nek family of kinases has been considered a mitotic specific kinase with nuclear restricted roles in the model fungus Aspergillus nidulans. By extending to A. nidulans the results of a synthetic lethal screen performed in Saccharomyces cerevisiae using the NIMA ortholog KIN3, we identified a conserved genetic interaction between nimA and genes encoding proteins of the Endosomal Sorting Complex Required for Transport (ESCRT pathway. Absence of ESCRT pathway functions in combination with partial NIMA function causes enhanced cell growth defects, including an inability to maintain a single polarized dominant cell tip. These genetic insights suggest NIMA potentially has interphase functions in addition to its established mitotic functions at nuclei. We therefore generated endogenously GFP-tagged NIMA (NIMA-GFP which was fully functional to follow its interphase locations using live cell spinning disc 4D confocal microscopy. During interphase some NIMA-GFP locates to the tips of rapidly growing cells and, when expressed ectopically, also locates to the tips of cytoplasmic microtubules, suggestive of non-nuclear interphase functions. In support of this, perturbation of NIMA function either by ectopic overexpression or through partial inactivation results in marked cell tip growth defects with excess NIMA-GFP promoting multiple growing cell tips. Ectopic NIMA-GFP was found to locate to the plus ends of microtubules in an EB1 dependent manner, while impairing NIMA function altered the dynamic localization of EB1 and the cytoplasmic microtubule network. Together, our genetic and cell biological analyses reveal novel non-nuclear interphase functions for NIMA involving microtubules and the ESCRT pathway for normal polarized fungal cell tip growth. These insights extend the roles of NIMA both spatially and temporally and indicate that this conserved protein kinase could help integrate cell

  8. A Case of Bariatric Surgery-related Wernicke-Korsakoff Syndrome with Persisting Anterograde Amnesia.

    Science.gov (United States)

    Gasquoine, Philip Gerard

    2017-08-01

    To describe the theoretical and clinical implications of the neuropsychological evaluation of a case of bariatric surgery-related Wernicke-Korsakoff syndrome. The patient was a 37-year old, female, bilingual, bachelor's degree educated, Mexican American public relations consultant without preexisting psychiatric, neurological, or substance abuse history. Recovery from laparoscopic sleeve gastrectomy surgery for morbid obesity was complicated by intraabdominal abscess, multibacterial infection, and prolonged nausea and vomiting. About 15 weeks post-surgery she was diagnosed with Wernicke's encephalopathy. She had a positive response to thiamine supplement but was left with persisting self-reported memory problems that were confirmed by family members. Multiple neuroimaging studies were all normal. A neuropsychological evaluation at 14 months post-surgery revealed anterograde amnesia for verbal and visual-perceptual material. There was no clear period of temporally graded retrograde amnesia. Scores on tests of visual-perceptual, language, fine motor, and executive functions were unimpaired. She had awareness of her neurocognitive impairment, but did not exhibit emotional distress. Follow-up neuropsychological evaluation at 17 months showed a similar neurocognitive profile with increased emotional distress. Her preserved executive functioning is theoretically important as it supports arguments that such impairment in alcohol use-related Korsakoff syndrome derives from the toxic effects of the prolonged misuse of alcohol and not vitamin deficiency. From a clinical perspective, neuropsychological evaluation of thiamine treated, bariatric surgery-related, Wernicke's encephalopathy cases is indicated if there is suspicion of residual memory impairment. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Halogenated auxins affect microtubules and root elongation in Lactuca sativa

    Science.gov (United States)

    Zhang, N.; Hasenstein, K. H.

    2000-01-01

    We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.

  10. Effects of the KIF2C neck peptide on microtubules: lateral disintegration of microtubules and β-structure formation.

    Science.gov (United States)

    Shimizu, Youské; Shimizu, Takashi; Nara, Masayuki; Kikumoto, Mahito; Kojima, Hiroaki; Morii, Hisayuki

    2013-04-01

    Members of the kinesin-13 sub-family, including KIF2C, depolymerize microtubules. The positive charge-rich 'neck' region extending from the N-terminus of the catalytic head is considered to be important in the depolymerization activity. Chemically synthesized peptides, covering the basic region (A182-E200), induced a sigmoidal increase in the turbidity of a microtubule suspension. The increase was suppressed by salt addition or by reduction of basicity by amino acid substitutions. Electron microscopic observations revealed ring structures surrounding the microtubules at high peptide concentrations. Using the peptide A182-D218, we also detected free thin straight filaments, probably protofilaments disintegrated from microtubules. Therefore, the neck region, even without the catalytic head domain, may induce lateral disintegration of microtubules. With microtubules lacking anion-rich C-termini as a result of subtilisin treatment, addition of the peptide induced only a moderate increase in turbidity, and rings and protofilaments were rarely detected, while aggregations, also thought to be caused by lateral disintegration, were often observed in electron micrographs. Thus, the C-termini are not crucial for the action of the peptides in lateral disintegration but contribute to structural stabilization of the protofilaments. Previous structural studies indicated that the neck region of KIF2C is flexible, but our IR analysis suggests that the cation-rich region (K190-A204) forms β-structure in the presence of microtubules, which may be of significance with regard to the action of the neck region. Therefore, the neck region of KIF2C is sufficient to cause disintegration of microtubules into protofilaments, and this may contribute to the ability of KIF2C to cause depolymerization of microtubules. © 2013 The Authors Journal compilation © 2013 FEBS.

  11. Direct Cytoplasmic Delivery and Nuclear Targeting Delivery of HPMA-MT Conjugates in a Microtubules Dependent Fashion.

    Science.gov (United States)

    Zhong, Jiaju; Zhu, Xi; Luo, Kui; Li, Lian; Tang, Manlin; Liu, Yanxi; Zhou, Zhou; Huang, Yuan

    2016-09-06

    As the hearts of tumor cells, the nucleus is the ultimate target of many chemotherapeutic agents and genes. However, nuclear drug delivery is always hampered by multiple intracellular obstacles, such as low efficiency of lysosome escape and insufficient nuclear trafficking. Herein, an N-(2-hydroxypropyl) methacrylamide (HPMA) polymer-based drug delivery system was designed, which could achieve direct cytoplasmic delivery by a nonendocytic pathway and transport into the nucleus in a microtubules dependent fashion. A special targeting peptide (MT), derived from an endogenic parathyroid hormone-related protein, was conjugated to the polymer backbone, which could accumulate into the nucleus a by microtubule-mediated pathway. The in vitro studies found that low temperature and NaN3 could not influence the cell internalization of the conjugates. Besides, no obvious overlay of the conjugates with lysosome demonstrated that the polymer conjugates could enter the tumor cell cytoplasm by a nonendocytic pathway, thus avoiding the drug degradation in the lysosome. Furthermore, after suppression of the microtubule dynamics with microtubule stabilizing docetaxel (DTX) and destabilizing nocodazole (Noc), the nuclear accumulation of polymeric conjugates was significantly inhibited. Living cells fluorescence recovery after photobleaching study found that the nuclear import rate of conjugates was 2-fold faster compared with the DTX and Noc treated groups. These results demonstrated that the conjugates transported into the nucleus in a microtubules dependent way. Therefore, in addition to direct cytoplasmic delivery, our peptide conjugated polymeric platform could simultaneously mediate nuclear drug accumulation, which may open a new path for further intracellular genes/peptides delivery.

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

    Directory of Open Access Journals (Sweden)

    Michael A. Lampson

    2017-01-01

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

  13. Association of Ebola Virus Matrix Protein VP40 with Microtubules

    National Research Council Canada - National Science Library

    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. J

    2005-01-01

    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...

  14. Anterograde Headless Cannulated Screw Fixation in the Treatment of Medial Malleolar Fractures: Evaluation of a New Technique and Its Outcomes.

    Science.gov (United States)

    Tekin, Ali Çağrı; Çabuk, Haluk; Dedeoğlu, Süleyman Semih; Saygılı, Mehmet Selçuk; Adaş, Müjdat; Büyükkurt, Cem Dinçay; Gürbüz, Hakan; Çakar, Murat; Tekin, Zeynep Nilüfer

    To evaluate the functional and radiological outcomes of anterograde headless cannulated screw fixation for medial malleolar fractures. This study included 12 patients (8 males, 4 females; age 27-55 years) with medial malleolar type B fractures according to the Herscovici fracture classification who had undergone anterograde headless cannulated screw fixation surgery between 2012 and 2014. Seven had an isolated medial malleolar fracture and 5 a bimalleolar fracture. All of the bimalleolar fractures were classified as 44-B2 based on the Arbeitsgemeinschaft für Osteosynthesefragen/Orthopaedic Trauma Association (AO/OTA) classification. Postoperatively, bone union was evaluated on direct radiographs at the final follow-up examination. The American Orthopaedic Foot and Ankle Society (AOFAS) scoring system was used for clinical evaluation. The mean follow-up period was 17.2 ± 5.3 months (range 12-23). Full union was achieved in all fractures. The mean time to union was 3.4 ± 1.5 months (range 2-5). No instability, loss of reduction, non-union or infection was observed in any patient. The mean AOFAS score was 95.0 ± 5.4 (range 87-99). Based on the AOFAS score, 4 patients showed good results and 8 excellent results. The mean time to return to the previous level of activity was 4.0 ± 2.5 months (range 2-5). In this study, anterograde headless cannulated screw fixation yielded good clinical outcome in the surgical treatment of Herscovici type B fractures. © 2016 S. Karger AG, Basel.

  15. Microtubules and control of macronuclear 'amitosis' in Paramecium.

    Science.gov (United States)

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

    1980-08-01

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

  16. Acentrosomal microtubule assembly in mitosis: the where, when, and how

    OpenAIRE

    Meunier, Sylvain; Vernos, Isabelle, 1959-

    2016-01-01

    In mitosis the cell assembles the bipolar spindle, a microtubule (MT)-based apparatus that segregates the duplicated chromosomes into two daughter cells. Most animal cells enter mitosis with duplicated centrosomes that provide an active source of dynamic MTs. However, it is now established that spindle assembly relies on the nucleation of acentrosomal MTs occurring around the chromosomes after nuclear envelope breakdown, and on pre-existing microtubules. Where chromosome-dependent MT nucleati...

  17. Serotonergic projections from the raphe nuclei to the subthalamic nucleus; a retrograde- and anterograde neuronal tracing study

    DEFF Research Database (Denmark)

    Reznitsky, Martin; Plenge, Per; Hay-Schmidt, Anders

    2016-01-01

    the 5-HT1A and 5-HT2A not were present. Retrograde tracer FluoroGold or Choleratoxin subunit B were iontophoretically delivered in the STN and combined with immunohistochemistry for 5-HT in order to map the topographic organization in the dorsal raphe system. The study showed that approximately 320......+/-137 neurons were retrogradely traced from each STN to the DRN, located mainly in the dorsal- and ventrolateral DRN, and of these 108+/-42 or 34% co-localized 5-HT. Additionally anterograde tracer PHA-L was injected in the DRN to confirm projections to STN and accordingly only a sparse number of axon terminals...

  18. Nonlinear dynamics of C–terminal tails in cellular microtubules

    Energy Technology Data Exchange (ETDEWEB)

    Sekulic, Dalibor L., E-mail: dalsek@uns.ac.rs; Sataric, Bogdan M.; Sataric, Miljko V. [University of Novi Sad, Faculty of Technical Sciences, Novi Sad (Serbia); Zdravkovic, Slobodan [University of Belgrade, Institute of Nuclear Sciences Vinca, Belgrade (Serbia); Bugay, Aleksandr N. [Laboratory of Radiation Biology, Joint Institute for Nuclear Research, Dubna (Russian Federation)

    2016-07-15

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

  19. Kinematics of turnaround and retrograde axonal transport

    International Nuclear Information System (INIS)

    Snyder, R.E.

    1986-01-01

    Rapid axonal transport of a pulse of 35 S-methionine-labelled material was studied in vitro in the sensory neurons of amphibian sciatic nerve using a position-sensitive detector. For 10 nerves studied at 23.0 +/- 0.2 degrees C it was found that a pulse moved in the anterograde direction characterized by front edge, peak, and trailing edge transport rates of (mm/d) 180.8 +/- 2.2 (+/- SEM), 176.6 +/- 2.3, and 153.7 +/- 3.0, respectively. Following its arrival at a distal ligature, a smaller pulse was observed to move in the retrograde direction characterized by front edge and peak transport rates of 158.0 +/- 7.3 and 110.3 +/- 3.5, respectively, indicating that retrograde transport proceeds at a rate of 0.88 +/- 0.04 that of anterograde. The retrograde pulse was observed to disperse at a rate greater than the anterograde. Reversal of radiolabel at the distal ligature began 1.49 +/- 0.15 h following arrival of the first radiolabel. Considerable variation was seen between preparations in the way radiolabel accumulated in the end (ligature) regions of the nerve. Although a retrograde pulse was seen in all preparations, in 7 of 10 preparations there was no evidence of this pulse accumulating within less than 2-3 mm of a proximal ligature; however, accumulation was observed within less than 5 mm in all preparations

  20. Crowding of molecular motors determines microtubule depolymerization.

    Science.gov (United States)

    Reese, Louis; Melbinger, Anna; Frey, Erwin

    2011-11-02

    The assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT-associated proteins. Here, we investigate how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. Using an individual-based model, we reproduce experimental findings. Moreover, crowding is identified as the key regulatory mechanism of depolymerization dynamics. Our analysis reveals two qualitatively distinct regimes. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise that cancel out the effect of the depolymerization kinetics such that the depolymerization speed is solely determined by the motor density. Because this density changes over the MT length, length-dependent regulation is possible. Remarkably, motor cooperativity affects only the end-residence time of depolymerases and not the depolymerization speed. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Microtubule Stabilizing Agents as Potential Treatment for Alzheimer’s Disease and Related Neurodegenerative Tauopathies

    Science.gov (United States)

    Ballatore, Carlo; Brunden, Kurt R.; Huryn, Donna M.; Trojanowski, John Q.; Lee, Virginia M.-Y.; Smith, Amos B.

    2012-01-01

    The microtubule (MT)-associated protein tau, which is highly expressed in the axons of neurons, is an endogenous MT-stabilizing agent that plays an important role in the axonal transport. Loss of MT-stabilizing tau function, caused by misfolding, hyperphosphorylation and sequestration of tau into insoluble aggregates, leads to axonal transport deficits with neuropathological consequences. Several in vitro and preclinical in vivo studies have shown that MT-stabilizing drugs can be utilized to compensate for the loss of tau function and to maintain/restore an effective axonal transport. These findings indicate that MT-stabilizing compounds hold considerable promise for the treatment of Alzheimer disease and related tauopathies. The present article provides a synopsis of the key findings demonstrating the therapeutic potential of MT-stabilizing drugs in the context of neurodegenerative tauopathies, as well as an overview of the different classes of MT-stabilizing compounds. PMID:23020671

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

    Science.gov (United States)

    Coffe, G; Raymond, M N

    1990-01-01

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

  3. Dauer pheromone and G-protein signaling modulate the coordination of intraflagellar transport kinesin motor proteins in C. elegans

    NARCIS (Netherlands)

    J.A. Burghoorn (Jan); M.P.J. Dekkers (Martijn); S. Rademakers (Suzanne); A.A.W. de Jong (Ton); R. Willemsen (Rob); P. Swoboda (Peter); J. McCafferty (Gert)

    2010-01-01

    textabstractCilia length and function are dynamically regulated by modulation of intraflagellar transport (IFT). The cilia of C. elegans amphid channel neurons provide an excellent model to study this process, since they use two different kinesins for anterograde transport: kinesin-II and OSM-3

  4. Transportation

    International Nuclear Information System (INIS)

    Anon.

    1998-01-01

    Here is the decree of the thirtieth of July 1998 relative to road transportation, to trade and brokerage of wastes. It requires to firms which carry out a road transportation as well as to traders and to brokers of wastes to declare their operations to the prefect. The declaration has to be renewed every five years. (O.M.)

  5. Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

    Energy Technology Data Exchange (ETDEWEB)

    Ti, Shih-Chieh; Pamula, Melissa C.; Howes, Stuart C.; Duellberg, Christian; Cade, Nicholas I.; Kleiner, Ralph E.; Forth, Scott; Surrey, Thomas; Nogales, Eva; Kapoor, Tarun M.

    2016-04-01

    The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. In this research, we have purified and characterized tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

  6. Longitudinal evidence for anterograde trans-synaptic degeneration after optic neuritis

    Science.gov (United States)

    Goodkin, Olivia; Altmann, Daniel R.; Jenkins, Thomas M.; Miszkiel, Katherine; Mirigliani, Alessia; Fini, Camilla; Gandini Wheeler-Kingshott, Claudia A. M.; Thompson, Alan J.; Ciccarelli, Olga; Toosy, Ahmed T.

    2016-01-01

    Abstract In multiple sclerosis, microstructural damage of normal-appearing brain tissue is an important feature of its pathology. Understanding these mechanisms is vital to help develop neuroprotective strategies. The visual pathway is a key model to study mechanisms of damage and recovery in demyelination. Anterograde trans-synaptic degeneration across the lateral geniculate nuclei has been suggested as a mechanism of tissue damage to explain optic radiation abnormalities seen in association with demyelinating disease and optic neuritis, although evidence for this has relied solely on cross-sectional studies. We therefore aimed to assess: (i) longitudinal changes in the diffusion properties of optic radiations after optic neuritis suggesting trans-synaptic degeneration; (ii) the predictive value of early optic nerve magnetic resonance imaging measures for late optic radiations changes; and (iii) the impact on visual outcome of both optic nerve and brain post-optic neuritis changes. Twenty-eight consecutive patients with acute optic neuritis and eight healthy controls were assessed visually (logMAR, colour vision, and Sloan 1.25%, 5%, 25%) and by magnetic resonance imaging, at baseline, 3, 6, and 12 months. Magnetic resonance imaging sequences performed (and metrics obtained) were: (i) optic nerve fluid-attenuated inversion-recovery (optic nerve cross-sectional area); (ii) optic nerve proton density fast spin-echo (optic nerve proton density-lesion length); (iii) optic nerve post-gadolinium T 1 -weighted (Gd-enhanced lesion length); and (iv) brain diffusion-weighted imaging (to derive optic radiation fractional anisotropy, radial diffusivity, and axial diffusivity). Mixed-effects and multivariate regression models were performed, adjusting for age, gender, and optic radiation lesion load. These identified changes over time and associations between early optic nerve measures and 1-year global optic radiation/clinical measures. The fractional anisotropy in patients

  7. Transportation

    National Research Council Canada - National Science Library

    Allshouse, Michael; Armstrong, Frederick Henry; Burns, Stephen; Courts, Michael; Denn, Douglas; Fortunato, Paul; Gettings, Daniel; Hansen, David; Hoffman, Douglas; Jones, Robert

    2007-01-01

    .... The ability of the global transportation industry to rapidly move passengers and products from one corner of the globe to another continues to amaze even those wise to the dynamics of such operations...

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

    Science.gov (United States)

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

    2011-01-01

    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, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis. PMID:22216166

  9. CENTROSOMES AND MICROTUBULES DURING MEIOSIS IN THE MUSHROOM BOLETUS RUBINELLUS

    Science.gov (United States)

    McLaughlin, David J.

    1971-01-01

    The double centrosome in the basidium of Boletus rubinellus has been observed in three planes with the electron microscope at interphase preceding nuclear fusion, at prophase I, and at interphase I. It is composed of two components connected by a band-shaped middle part. At anaphase I a single, enlarged centrosome is found at the spindle pole, which is attached to the cell membrane. Microtubules mainly oriented parallel to the longitudinal axis of the basidium are present at prefusion, prophase I and interphase I. Cytoplasmic microtubules are absent when the spindle is present. The relationship of the centrosome in B. rubinellus to that in other organisms and the role of the cytoplasmic microtubules are discussed. PMID:4329156

  10. A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition

    Directory of Open Access Journals (Sweden)

    Hannah R. Belsham

    2017-12-01

    Full Text Available The microtubule depolymerising kinesin-13, MCAK, is phosphorylated at residue T537 by Cdk1. This is the only known phosphorylation site within MCAK’s motor domain. To understand the impact of phosphorylation by Cdk1 on microtubule depolymerisation activity, we have investigated the molecular mechanism of the phosphomimic mutant T537E. This mutant significantly impairs microtubule depolymerisation activity and when transfected into cells causes metaphase arrest and misaligned chromosomes. We show that the molecular mechanism underlying the reduced depolymerisation activity of this phosphomimic mutant is an inability to recognise the microtubule end. The microtubule-end residence time is reduced relative to wild-type MCAK, whereas the lattice residence time is unchanged by the phosphomimic mutation. Further, the microtubule-end specific stimulation of ADP dissociation, characteristic of MCAK, is abolished by this mutation. Our data shows that T537E is unable to distinguish between the microtubule end and the microtubule lattice.

  11. Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles.

    Science.gov (United States)

    Decker, Franziska; Oriola, David; Dalton, Benjamin; Brugués, Jan

    2018-01-11

    Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting. © 2018, Decker et al.

  12. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking.

    Directory of Open Access Journals (Sweden)

    Christopher Arnette

    Full Text Available The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.

  13. Septins localize to microtubules during nutritional limitation in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Vázquez de Aldana Carlos R

    2008-10-01

    Full Text Available Abstract Background In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. Results Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11 from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. Conclusion Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources.

  14. Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1.

    Science.gov (United States)

    VanDelinder, Virginia; Adams, Peter G; Bachand, George D

    2016-12-21

    The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.

  15. Anterograde axonal transport and intercellular transfer of WGA-HRP in trigeminal-innervated sensory receptors of rat incisive papilla.

    Science.gov (United States)

    Chan, K Y; Byers, M R

    1985-04-08

    The ultrastructure and identification of WGA-HRP-labeled sensory receptors in the rat incisive papilla (the most anterior part of hard palate) were studied using semiserial thin sections. Various sensory receptors were organized according to three locations: dome region (ventral), chemosensory corpuscle region (medial to orifice of incisive canal), and lateral labium (apposing the incisive canal). In the dome region, the sensory receptors were localized in three sensory zones that were associated with surface ridges (one medial and two lateral). In each of these zones, intraepithelial receptor axons and Merkel receptors occurred in the epithelium, while simple unencapsulated corpuscles, glomerular-Meissner corpuscles, and incisive (encapsulated) corpuscles occurred in the lamina propria. In the chemosensory corpuscle region, chemosensory corpuscles and intraepithelial receptor axons were located in the epithelium, and incisive corpuscles were present in the lamina propria. In the lateral labium, only intraepithelial receptor axons were prominent. In all these sensory receptors, the preterminal axons and axon terminals were labeled with the tracer protein. In addition, some nonneuronal cells closely associated with the axon terminals were selectively labeled, e.g., terminal Schwann cells, lamellar Schwann cells, Merkel cells, corpuscular basal cells and chemosensory cells. Other adjacent cells were not labeled, e.g., unspecialized epithelial cells, capsular cells, corpuscular sustentacular cells, and fibroblasts. In both labeled axons and cells, WGA-HRP was incorporated into vesicles, tubules, and vacuolar organelles. The specific intercellular transfer of tracer protein may indicate trophic interactions between axon terminals and support cells in sensory receptors. The specific organization of multiple sensory receptors in the rat incisive papilla may provide a useful alternative system for studying somatosensory physiology.

  16. Cell Microtubules as Cavities Quantum Coherence and Energy Transfer?

    CERN Document Server

    Mavromatos, Nikolaos E

    2000-01-01

    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.

  17. Regulation of microtubule nucleation mediated by gamma-tubulin complexes

    Czech Academy of Sciences Publication Activity Database

    Sulimenko, Vadym; Hájková, Zuzana; Klebanovych, Anastasiya; Dráber, Pavel

    2017-01-01

    Roč. 254, č. 3 (2017), s. 1187-1199 ISSN 0033-183X R&D Projects: GA MŠk(CZ) LD13015 Institutional support: RVO:68378050 Keywords : mitotic spindle formation * ring complex * fission yeast * organizing centers * protein complex * golgi-complex * cell -cycle * pole body * augmin * centrosome * Centrosomes * Microtubule nucleation * Microtubule-organizing centers * Non-centrosomal nucleation sites * Spindle pole bodies * gamma-Tubulin complexes Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 2.870, year: 2016

  18. S. pombe kinesins-8 promote both nucleation and catastrophe of microtubules.

    Directory of Open Access Journals (Sweden)

    Muriel Erent

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

  19. Electrostatic differences: A possible source for the functional differences between MCF7 and brain microtubules.

    Science.gov (United States)

    Feizabadi, Mitra Shojania; Rosario, Brandon; Hernandez, Marcos A V

    2017-11-04

    Recent studies suggested a link between diversity of beta tubulin isotypes in microtubule structures and the regulatory roles that they play not only on microtubules' intrinsic dynamic, but also on the translocation characteristics of some of the molecular motors along microtubules. Remarkably, unlike porcine brain microtubules, MCF7 microtubules are structured from a different beta tubulin distribution. These types of cancer microtubules show a relatively stable and slow dynamic. In addition, the translocation parameters of some molecular motors are distinctly different along MCF7 as compared to those parameters on brain microtubules. It is known that the diversity of beta tubulin isotypes differ predominantly in the specifications and the electric charge of their carboxy-terminal tails. A key question is to identify whether the negative electrostatic charge of tubulin isotypes and, consequently, microtubules, can potentially be considered as one of the sources of functional differences in MCF7 vs. brain microtubules. We tested this possibility experimentally by monitoring the electro-orientation of these two types of microtubules inside a uniform electric field. Through this evaluation, we quantified and compared the average normalized polarization coefficient of MCF7 vs. Porcine brain microtubules. The higher value obtained for the polarization of MCF7 microtubules, which is associated to the higher negative charge of these types of microtubules, is significant as it can further explain the slow intrinsic dynamic that has been recently reported for single MCF7 microtubules in vitro. Furthermore, it can be potentially considered as a factor that can directly impact the translocation parameters of some molecular motors along MCF7 microtubules, by altering the mutual electrostatic interactions between microtubules and molecular motors. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Sliding of microtubules by a team of dynein motors: Understanding the effect of spatial distribution of motor tails and mutual exclusion of motor heads on microtubules

    Science.gov (United States)

    Singh, Hanumant Pratap; Takshak, Anjneya; Mall, Utkarsh; Kunwar, Ambarish

    2016-06-01

    Molecular motors are natural nanomachines that use the free energy released from ATP hydrolysis to generate mechanical forces. Cytoplasmic dynein motors often work collectively as a team to drive important processes such as axonal growth, proplatelet formation and mitosis, as forces generated by single motors are insufficient. A large team of dynein motors is used to slide cytoskeletal microtubules with respect to one another during the process of proplatelet formation and axonal growth. These motors attach to a cargo microtubule via their tail domains, undergo the process of detachment and reattachment of their head domains on another track microtubule, while sliding the cargo microtubule along the track. Traditional continuum/mean-field approaches used in the past are not ideal for studying the sliding mechanism of microtubules, as they ignore spatial and temporal fluctuations due to different possible distributions of motor tails on cargo filament, as well as binding/unbinding of motors from their track. Therefore, these models cannot be used to address important questions such as how the distribution of motor tails on microtubules, or how the mutual exclusion of motor heads on microtubule tracks affects the sliding velocity of cargo microtubule. To answer these, here we use a computational stochastic model where we model each dynein motor explicitly. In our model, we use both random as well as uniform distributions of dynein motors on cargo microtubule, as well as mutual exclusion of motors on microtubule tracks. We find that sliding velocities are least affected by the distribution of motor tails on microtubules, whereas they are greatly affected by mutual exclusion of motor heads on microtubule tracks. We also find that sliding velocity depends on the length of cargo microtubule if mutual exclusion among motor heads is considered.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1992-01-14

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

  2. Tau can switch microtubule network organizations: from random networks to dynamic and stable bundles.

    Science.gov (United States)

    Prezel, Elea; Elie, Auréliane; Delaroche, Julie; Stoppin-Mellet, Virginie; Bosc, Christophe; Serre, Laurence; Fourest-Lieuvin, Anne; Andrieux, Annie; Vantard, Marylin; Arnal, Isabelle

    2018-01-15

    In neurons, microtubule networks alternate between single filaments and bundled arrays under the influence of effectors controlling their dynamics and organization. Tau is a microtubule bundler that stabilizes microtubules by stimulating growth and inhibiting shrinkage. The mechanisms by which tau organizes microtubule networks remain poorly understood. Here, we studied the self-organization of microtubules growing in the presence of tau isoforms and mutants. The results show that tau's ability to induce stable microtubule bundles requires two hexapeptides located in its microtubule-binding domain and is modulated by its projection domain. Site-specific pseudophosphorylation of tau promotes distinct microtubule organizations: stable single microtubules, stable bundles, or dynamic bundles. Disease-related tau mutations increase the formation of highly dynamic bundles. Finally, cryo-electron microscopy experiments indicate that tau and its variants similarly change the microtubule lattice structure by increasing both the protofilament number and lattice defects. Overall, our results uncover novel phosphodependent mechanisms governing tau's ability to trigger microtubule organization and reveal that disease-related modifications of tau promote specific microtubule organizations that may have a deleterious impact during neurodegeneration. © 2018 Prezel, Elie, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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

    DEFF Research Database (Denmark)

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob

    2013-01-01

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

  4. The Aspergillus nidulans kinesin-3 tail is necessary and sufficient to recognize modified microtubules.

    Directory of Open Access Journals (Sweden)

    Constanze Seidel

    Full Text Available Posttranslational microtubule modifications (PTMs are numerous; however, the biochemical and cell biological roles of those modifications remain mostly an enigma. The Aspergillus nidulans kinesin-3 UncA uses preferably modified microtubules (MTs as tracks for vesicle transportation. Here, we show that a positively charged region in the tail of UncA (amino acids 1316 to 1402 is necessary for the recognition of modified MTs. Chimeric proteins composed of the kinesin-1 motor domain and the UncA tail displayed the same specificity as UncA, suggesting that the UncA tail is sufficient to establish specificity. Interaction between the UncA tail and alpha-tubulin was shown using a yeast two-hybrid assay and in A. nidulans by bimolecular fluorescence complementation. This is the first demonstration of how a kinesin-3 motor protein distinguishes among different MT populations in fungal cells, and how specificity determination depends on the tail rather than the motor domain, as has been demonstrated for kinesin 1 in neuronal cells.

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

    Science.gov (United States)

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

    2014-01-01

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

  6. Microtubules accelerate the kinase activity of Aurora-B by a reduction in dimensionality.

    Science.gov (United States)

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

    2014-01-01

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

  7. Microtubules accelerate the kinase activity of Aurora-B by a reduction in dimensionality.

    Directory of Open Access Journals (Sweden)

    Michael Noujaim

    Full Text Available 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.

  8. Microtubule depolymerization induces traction force increase through two distinct pathways

    Science.gov (United States)

    Rape, Andrew; Guo, Wei-hui; Wang, Yu-li

    2011-01-01

    Traction forces increase after microtubule depolymerization; however, the signaling mechanisms underlying this, in particular the dependence upon myosin II, remain unclear. We investigated the mechanism of traction force increase after nocodazole-induced microtubule depolymerization by applying traction force microscopy to cells cultured on micropatterned polyacrylamide hydrogels to obtain samples of homogeneous shape and size. Control cells and cells treated with a focal adhesion kinase (FAK) inhibitor showed similar increases in traction forces, indicating that the response is independent of FAK. Surprisingly, pharmacological inhibition of myosin II did not prevent the increase of residual traction forces upon nocodazole treatment. This increase was abolished upon pharmacological inhibition of FAK. These results suggest two distinct pathways for the regulation of traction forces. First, microtubule depolymerization activates a myosin-II-dependent mechanism through a FAK-independent pathway. Second, microtubule depolymerization also enhances traction forces through a myosin-II-independent, FAK-regulated pathway. Traction forces are therefore regulated by a complex network of complementary signals and force-generating mechanisms. PMID:22193960

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

    Science.gov (United States)

    Lewis, M. L.; Reynolds, J. L.; Cubano, L. A.; Hatton, J. P.; Lawless, B. D.; Piepmeier, E. H.

    1998-01-01

    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 apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.

  10. Lamin A and microtubules collaborate to maintain nuclear morphology.

    Science.gov (United States)

    Tariq, Zeshan; Zhang, Haoyue; Chia-Liu, Alexander; Shen, Yang; Gete, Yantenew; Xiong, Zheng-Mei; Tocheny, Claire; Campanello, Leonard; Wu, Di; Losert, Wolfgang; Cao, Kan

    2017-07-04

    Lamin A (LA) is a critical structural component of the nuclear lamina. Mutations within the LA gene (LMNA) lead to several human disorders, most striking of which is Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disorder. HGPS cells are best characterized by an abnormal nuclear morphology known as nuclear blebbing, which arises due to the accumulation of progerin, a dominant mutant form of LA. The microtubule (MT) network is known to mediate changes in nuclear morphology in the context of specific events such as mitosis, cell polarization, nucleus positioning and cellular migration. What is less understood is the role of the microtubule network in determining nuclear morphology during interphase. In this study, we elucidate the role of the cytoskeleton in regulation and misregulation of nuclear morphology through perturbations of both the lamina and the microtubule network. We found that LA knockout cells exhibit a crescent shape morphology associated with the microtubule-organizing center. Furthermore, this crescent shape ameliorates upon treatment with MT drugs, Nocodazole or Taxol. Expression of progerin, in LA knockout cells also rescues the crescent shape, although the response to Nocodazole or Taxol treatment is altered in comparison to cells expressing LA. Together these results describe a collaborative effort between LA and the MT network to maintain nuclear morphology.

  11. EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

    Science.gov (United States)

    Wang, Yi-Long; Chen, Hui; Zhan, Yi-Qun; Yin, Rong-Hua; Li, Chang-Yan; Ge, Chang-Hui; Yu, Miao; Yang, Xiao-Ming

    2016-08-17

    EWSR1, participating in transcription and splicing, has been identified as a translocation partner for various transcription factors, resulting in translocation, which in turn plays crucial roles in tumorigenesis. Recent studies have investigated the role of EWSR1 in mitosis. However, the effect of EWSR1 on mitosis is poorly understood. Here, we observed that depletion of EWSR1 resulted in cell cycle arrest in the mitotic phase, mainly due to an increase in the time from nuclear envelope breakdown to metaphase, resulting in a high percentage of unaligned chromosomes and multipolar spindles. We also demonstrated that EWSR1 is a spindle-associated protein that interacts with α-tubulin during mitosis. EWSR1 depletion increased the cold-sensitivity of spindle microtubules, and decreased the rate of spindle assembly. EWSR1 regulated the level of microtubule acetylation in the mitotic spindle; microtubule acetylation was rescued in EWSR1-depleted mitotic cells following suppression of HDAC6 activity by its specific inhibitor or siRNA treatment. In summary, these results suggest that EWSR1 regulates the acetylation of microtubules in a cell cycle-dependent manner through its dynamic location on spindle MTs, and may be a novel regulator for mitosis progress independent of its translocation.

  12. Anatomical evidence for direct fiber projections from the cerebellar nucleus interpositus to rubrospinal neurons. A quantitative EM study in the rat combining anterograde and retrograde intra-axonal tracing methods

    International Nuclear Information System (INIS)

    Dekker, J.J.

    1981-01-01

    A quantitative electron microscopic (EM) study combining the anterograde intra-axonal transport of radioactive amino acids and the retrograde intra-axonal transport of the enzyme horseradish peroxidase (HRP) was performed in the magnocellular red nucleus of the rat to obtain anatomical evidence as to whether there is a direct projection from the cerebellar nucleus interpositus to the cells in the red nucleus that give rise to the rubrospinal tract. Large asymmetrical synaptic terminals were radioactively labeled in the magnocellular red nucleus following injections of [ 3 H]leucine into the cerebellar nucleus interpositus. In these same animals, the postsynaptic target neurons were labeled with HRP granules after injection of this substance in the rubrospinal tract. A quantitative analysis showed that more than 85% of the large and giant neurons in the magnocellular red nucleus were labeled with HRP granules and also received synaptic contacts from radioactively-labeled terminals. Thus, it can be concluded that in the rat, afferents from the cerebellar nucleus interpositus establish asymmetrical synaptic contacts with large and giant rubrospinal neurons, thus confirming and extending the previous physiological evidence of such direct monosynaptic connections. (Auth.)

  13. EB1 recognizes the nucleotide state of tubulin in the microtubule lattice.

    Directory of Open Access Journals (Sweden)

    Marija Zanic

    Full Text Available Plus-end-tracking proteins (+TIPs are localized at the fast-growing, or plus end, of microtubules, and link microtubule ends to cellular structures. One of the best studied +TIPs is EB1, which forms comet-like structures at the tips of growing microtubules. The molecular mechanisms by which EB1 recognizes and tracks growing microtubule ends are largely unknown. However, one clue is that EB1 can bind directly to a microtubule end in the absence of other proteins. Here we use an in vitro assay for dynamic microtubule growth with two-color total-internal-reflection-fluorescence imaging to investigate binding of mammalian EB1 to both stabilized and dynamic microtubules. We find that under conditions of microtubule growth, EB1 not only tip tracks, as previously shown, but also preferentially recognizes the GMPCPP microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount of salt in solution. The ability to distinguish different nucleotide states of tubulin in microtubule lattice may contribute to the end-tracking mechanism of EB1.

  14. Heuristic consequences of a load of oxygen in microtubules.

    Science.gov (United States)

    Denis, Pierre A

    2014-04-01

    The current cell oxygen paradigm shows some major gaps that have not yet been resolved. Something seems to be lacking for the comprehensive statement of the oxygen distribution in the cell, especially the low cytoplasmic oxygen level. The entrapment of oxygen in microtubules (MTs) resolves the latter observation, as well as the occurrence of an extensive cytoplasmic foam formation. It leads to a novel oxygen paradigm for cells. During the steady-state treadmilling, the mobile cavity would absorb oxygenated cytoplasm forward, entrap gas nuclei and concentrate them. A fluorescence method is described to confirm the in vitro load of oxygen in MTs during their periodic growths and shrinkages. The latter operating mechanism is called the gas dynamic instability (GDI) of MTs. Several known biosystems could rest on the GDI. (1) The GTP-cap is linked with the gas meniscus encountered in a tube filled with gas. The GTP hydrolysis is linked to the conformational change of the GTPase domain according to the bubble pressure, and to the shaking of protofilaments with gas particles (soliton-like waves). (2) The GDI provides a free energy water pump because water molecules have to escape from MT pores when foam concentrates within the MT. Beside ATP hydrolysis in motor proteins, the GDI provides an additional driving force in intracellular transport of cargo. The water streams flowing from the MT through slits organize themselves as water layers between the cargo and the MT surface, and break ionic bridges. It makes the cargo glide over a water rail. (3) The GDI provides a universal motor for chromosome segregation because the depolymerization of kinetochorial MTs is expected to generate a strong cytoplasmic foam. Chromosomes are sucked up according to the pressure difference (or density difference) applied to opposite sides of the kinetochore, which is in agreement with Archimedes' principle of buoyancy. Non-kinetochorial MTs reabsorb foam during GDI. Last, the mitotic spindle

  15. Cell edges accumulate gamma tubulin complex components and nucleate microtubules following cytokinesis in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Chris Ambrose

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

  16. Hooks and comets: The story of microtubule polarity orientation in the neuron.

    Science.gov (United States)

    Baas, Peter W; Lin, Shen

    2011-06-01

    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. Recent studies on insect neurons suggest that it is the minus-end-distal microtubules that are the critical feature of the dendritic microtubule array, whether or not they are accompanied by plus-end-distal microtubules. 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. Copyright © 2010 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2012-11-16

    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.

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

    Directory of Open Access Journals (Sweden)

    Lei eLei

    2014-03-01

    Full Text Available 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 CSI1, 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.

  19. Molecular quantum robotics: particle and wave solutions, illustrated by "leg-over-leg" walking along microtubules.

    Science.gov (United States)

    Levi, Paul

    2015-01-01

    Remarkable biological examples of molecular robots are the proteins kinesin-1 and dynein, which move and transport cargo down microtubule "highways," e.g., of the axon, to final nerve nodes or along dendrites. They convert the energy of ATP hydrolysis into mechanical forces and can thereby push them forwards or backwards step by step. Such mechano-chemical cycles that generate conformal changes are essential for transport on all different types of substrate lanes. The step length of an individual molecular robot is a matter of nanometers but the dynamics of each individual step cannot be predicted with certainty (as it is a random process). Hence, our proposal is to involve the methods of quantum field theory (QFT) to describe an overall reliable, multi-robot system that is composed of a huge set of unreliable, local elements. The methods of QFT deliver techniques that are also computationally demanding to synchronize the motion of these molecular robots on one substrate lane as well as across lanes. Three different challenging types of solutions are elaborated. The impact solution reflects the particle point of view; the two remaining solutions are wave based. The second solution outlines coherent robot motions on different lanes. The third solution describes running waves. Experimental investigations are needed to clarify under which biological conditions such different solutions occur. Moreover, such a nano-chemical system can be stimulated by external signals, and this opens a new, hybrid approach to analyze and control the combined system of robots and microtubules externally. Such a method offers the chance to detect mal-functions of the biological system.

  20. Molecular Quantum Robotics: Particle and Wave Solutions, illustrated by "Leg-over-Leg" Walking along Microtubules

    Directory of Open Access Journals (Sweden)

    Paul eLevi

    2015-05-01

    Full Text Available Remarkable biological examples of molecular robots are the proteins kinesin-1 and dynein, which move and transport cargo down microtubule highways, e.g. of the axon, to final nerve nodes or along dendrites. They convert the energy of ATP hydrolysis into mechanical forces and can thereby push them forwards or backwards step by step. Such mechano-chemical cycles that generate conformal changes are essential for transport on all different types of substrate lanes. The step length of an individual molecular robot is a matter of nanometers but the dynamics of each individual step cannot be predicted with certainty (as it is a random process. Hence, our proposal is to involve the methods of quantum field theory (QFT to describe an overall reliable, multi–robot system that is composed of a huge set of unreliable, local elements. The methods of QFT deliver techniques that are also computationally demanding to synchronize the motion of these molecular robots on one substrate lane as well as across lanes.Three different challenging types of solutions are elaborated. The impact solution reflects the particle point of view; the two remaining solutions are wave based. The second solution outlines coherent robot motions on different lanes. The third solution describes running waves. Experimental investigations are needed to clarify under which biological conditions such different solutions occur.Moreover, such a nano-chemical system can be stimulated by external signals, and this opens a new, hybrid approach to analyze and control the combined system of robots and microtubules externally. Such a method offers the chance to detect mal-functions of the biological system. In our framework, such defects can be characterized by the distortion of typical features of dynamic systems like attractive fixed points, limit cycles, etc. However, such additional details would overload this presentation and obscure the essentials that we wish to point out.

  1. TgICMAP1 is a novel microtubule binding protein in Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Aoife T Heaslip

    Full Text Available The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction.

  2. The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

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    Kyle D Grode

    Full Text Available Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

  3. Microtubule-Organizing Centers: Towards a Minimal Parts List.

    Science.gov (United States)

    Paz, Joel; Lüders, Jens

    2018-03-01

    Despite decades of molecular analysis of the centrosome, an important microtubule-organizing center (MTOC) of animal cells, the molecular basis of microtubule organization remains obscure. A major challenge is the sheer complexity of the interplay of the hundreds of proteins that constitute the centrosome. However, this complexity owes not only to the centrosome's role as a MTOC but also to the requirements of its duplication cycle and to various other functions such as the formation of cilia, the integration of various signaling pathways, and the organization of actin filaments. Thus, rather than using the parts lists to reconstruct the centrosome, we propose to identify the subset of proteins minimally needed to assemble a MTOC and to study this process at non-centrosomal sites. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Vibrations of microtubules: Physics that has not met biology yet

    Czech Academy of Sciences Publication Activity Database

    Kučera, Ondřej; Havelka, Daniel; Cifra, Michal

    2017-01-01

    Roč. 72, 1 July (2017), s. 13-22 ISSN 0165-2125 R&D Projects: GA ČR(CZ) GA15-17102S Grant - others:AV ČR(CZ) SAV-15-22 Program:Bilaterální spolupráce Institutional support: RVO:67985882 Keywords : Models * Vibrations * Microtubules Subject RIV: BO - Biophysics OBOR OECD: Biophysics Impact factor: 1.575, year: 2016

  5. Human herpes virus-6 encephalitis causing severe anterograde amnesia associated with rituximab, azathioprine and prednisolone combination therapy for dermatomyositis.

    Science.gov (United States)

    Baumer, Thomas; Fry, Charlie; Luppe, Sebastian; Gunawardena, Harsha; Sieradzan, Kasia

    2017-06-01

    Human herpes virus-6 (HHV-6) reactivation is a well-recognised complication following haematological stem cell transplantation, but it is novel in the context of combination immunomodulatory therapy for autoimmune disease. We report a case of severe anterograde amnesia caused by HHV-6 encephalitis in a young female patient on rituximab, azathioprine and prednisolone for dermatomyositis (DM). The use of targeted biologic treatments for systemic autoimmune connective tissue diseases (CTDs) is increasing, particularly when refractory to conventional management. The anti-CD20 B cell depleting monoclonal antibody, rituximab is now increasingly used, often in combination with conventional immunomodulatory treatments, in certain autoimmune neurological conditions and systemic CTDs including DM. Physicians should be aware of the possibility of HHV-6 in those who develop encephalitis while CD20 B cell deplete, especially in the presence of additional immunomodulatory therapies. Prompt diagnosis and treatment of HHV-6 encephalitis with evidence-based anti-viral therapy may help reduce the extent of irreversible morbidity such as amnesia.

  6. Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics.

    Science.gov (United States)

    Carranza, Gerardo; Castaño, Raquel; Fanarraga, Mónica L; Villegas, Juan Carlos; Gonçalves, João; Soares, Helena; Avila, Jesus; Marenchino, Marco; Campos-Olivas, Ramón; Montoya, Guillermo; Zabala, Juan Carlos

    2013-01-01

    Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO(-) present in TBCB, which is similar to the EEY/F-COO(-) element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE-TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation.

  7. GIT1 enhances neurite outgrowth by stimulating microtubule assembly

    Directory of Open Access Journals (Sweden)

    Yi-sheng Li

    2016-01-01

    Full Text Available GIT1, a G-protein-coupled receptor kinase interacting protein, has been reported to be involved in neurite outgrowth. However, the neurobiological functions of the protein remain unclear. In this study, we found that GIT1 was highly expressed in the nervous system, and its expression was maintained throughout all stages of neuritogenesis in the brain. In primary cultured mouse hippocampal neurons from GIT1 knockout mice, there was a significant reduction in total neurite length per neuron, as well as in the average length of axon-like structures, which could not be prevented by nerve growth factor treatment. Overexpression of GIT1 significantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice. The GIT1 N terminal region, including the ADP ribosylation factor-GTPase activating protein domain, the ankyrin domains and the Spa2 homology domain, were sufficient to enhance axonal extension. Importantly, GIT1 bound to many tubulin proteins and microtubule-associated proteins, and it accelerated microtubule assembly in vitro. Collectively, our findings suggest that GIT1 promotes neurite outgrowth, at least partially by stimulating microtubule assembly. This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neurological diseases.

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

    Science.gov (United States)

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

    2013-01-01

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

  9. The Drosophila microtubule-associated protein mars stabilizes mitotic spindles by crosslinking microtubules through its N-terminal region.

    Science.gov (United States)

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

    2013-01-01

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

  10. The Drosophila microtubule-associated protein mars stabilizes mitotic spindles by crosslinking microtubules through its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Gang Zhang

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

  11. Mimicking cellular transport mechanism in stem cells through endosomal escape of new peptide-coated quantum dots.

    Science.gov (United States)

    Narayanan, Karthikeyan; Yen, Swee Kuan; Dou, Qingqing; Padmanabhan, Parasuraman; Sudhaharan, Thankiah; Ahmed, Sohail; Ying, Jackie Y; Selvan, Subramanian Tamil

    2013-01-01

    Protein transport is an important phenomenon in biological systems. Proteins are transported via several mechanisms to reach their destined compartment of cell for its complete function. One such mechanism is the microtubule mediated protein transport. Up to now, there are no reports on synthetic systems mimicking the biological protein transport mechanism. Here we report a highly efficient method of mimicking the microtubule mediated protein transport using newly designed biotinylated peptides encompassing a microtubule-associated sequence (MTAS) and a nuclear localization signaling (NLS) sequence, and their final conjugation with streptavidin-coated CdSe/ZnS quantum dots (QDs). Our results demonstrate that these novel bio-conjugated QDs enhance the endosomal escape and promote targeted delivery into the nucleus of human mesenchymal stem cells via microtubules. Mimicking the cellular transport mechanism in stem cells is highly desirable for diagnostics, targeting and therapeutic applications, opening up new avenues in the area of drug delivery.

  12. Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation.

    Science.gov (United States)

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

    2003-07-01

    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. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.

  13. The actin-binding protein capulet genetically interacts with the microtubule motor kinesin to maintain neuronal dendrite homeostasis.

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    Paul M B Medina

    Full Text Available BACKGROUND: Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGS: From a Drosophila forward genetic screen, we identified a mutation in capulet--encoding a conserved actin-binding protein--that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer's models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other. CONCLUSIONS/SIGNIFICANCE: The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer's and Parkinson's cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.

  14. Intrinsic synergistic-topological mechanism versus synergistic-topological matrix in microtubule self-organization

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    Buljan Vlado A

    2014-12-01

    Overall our data indicate that under crowded conditions in vitro, the self-organization of a microtubule fiber is governed by an intrinsic synergistic-topological mechanism, which in conjunction with the topological changes, GTP-tubulin depletion, and cooperative motion of fiber constituting microtubules, may generate and maintain a ‘synergistic-topological matrix’. Failure of the mechanism to form biologically feasible microtubule synergistic-topological matrix may, per se, precondition tumorigenesis.

  15. Dynamic instabilities in the kinetics of growth and disassembly of microtubules

    OpenAIRE

    Katrukha, Eugene

    2016-01-01

    Dynamic instability of microtubules is considered using frameworks of non-linear thermodynamics and non-equilibrium reaction-diffusion systems. Stochastic assembly/disassembly phases in the polymerization dynamics of microtubules are treated as a result of collective clusterization of microdefects (holes in structure). The model explains experimentally observed power law dependence of catastrophe frequency from the microtubule growth rate. Additional reaction-diffusion-precipitation model is ...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  18. Combing and self-assembly phenomena in dry films of Taxol-stabilized microtubules

    Directory of Open Access Journals (Sweden)

    Rose Franck

    2007-01-01

    Full Text Available AbstractMicrotubules are filamentous proteins that act as a substrate for the translocation of motor proteins. As such, they may be envisioned as a scaffold for the self-assembly of functional materials and devices. Physisorption, self-assembly and combing are here investigated as a potential prelude to microtubule-templated self-assembly. Dense films of self-assembled microtubules were successfully produced, as well as patterns of both dendritic and non-dendritic bundles of microtubules. They are presented in the present paper and the mechanism of their formation is discussed.

  19. Msd1/SSX2IP-dependent microtubule anchorage ensures spindle orientation and primary cilia formation

    OpenAIRE

    Hori, Akiko; Ikebe, Chiho; Tada, Masazumi; Toda, Takashi

    2014-01-01

    Anchoring microtubules to the centrosome is critical for cell geometry and polarity, yet the molecular mechanism remains unknown. Here we show that the conserved human Msd1/SSX2IP is required for microtubule anchoring. hMsd1/SSX2IP is delivered to the centrosome in a centriolar satellite-dependent manner and binds the microtubule-nucleator ?-tubulin complex. hMsd1/SSX2IP depletion leads to disorganised interphase microtubules and misoriented mitotic spindles with reduced length and intensity....

  20. The prostate-derived sterile 20-like kinase (PSK) regulates microtubule organization and stability.

    Science.gov (United States)

    Mitsopoulos, Costas; Zihni, Ceniz; Garg, Ritu; Ridley, Anne J; Morris, Jonathan D H

    2003-05-16

    Sterile 20 (STE20) protein kinases, which include germinal center kinases and p21-activated protein kinases, are known to activate mitogen-activated protein kinase pathways (c-Jun NH(2)-terminal kinase, p38, or extracellular signal-regulated kinase), leading to changes in gene transcription. Some STE20s can also regulate the cytoskeleton, and we have shown that the germinal center kinase-like kinase prostate-derived STE20-like kinase (PSK) affects actin cytoskeletal organization. Here, we demonstrate that PSK colocalizes with microtubules; and that this localization is disrupted by the microtubule depolymerizing agent nocodazole. The association of PSK with microtubules results in the production of stabilized perinuclear microtubule cables that are nocodazole-resistant and contain increased levels of acetylated alpha-tubulin. Kinase-defective PSK (K57A) or the C terminus of PSK (amino acids 745-1235) lacking the kinase domain are sufficient for microtubule binding and stabilization, demonstrating that the catalytic activity of the protein is not required. The localization of PSK to microtubules occurs via its C terminus, and PSK binds and phosphorylates alpha- and beta-tubulin in vitro. The N terminus of PSK (1-940) is unable to bind or stabilize microtubules, demonstrating that PSK must associate with microtubules for their reorganization to occur. These results demonstrate that PSK interacts with microtubules and affects their organization and stability independently of PSK kinase activity.

  1. Polyamine sharing between tubulin dimers favours microtubule nucleation and elongation via facilitated diffusion.

    Directory of Open Access Journals (Sweden)

    Alain Mechulam

    2009-01-01

    Full Text Available We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends. Facilitated diffusion can promote microtubule assembly, because, upon encountering a growing nucleus or the microtubule wall, random GTP-tubulin sliding on their surfaces will increase the probability of association to the target sites (nucleation sites or MT ends. This is an original explanation for understanding the apparent discrepancy between the high rate of microtubule elongation and the low rate of tubulin association at the microtubule ends in the viscous cytoplasm. The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions. Natural polyamines (putrescine, spermidine, and spermine are present in all living cells and are potent agents to trigger tubulin self-attraction. By using an analytical model, we analyze the implication of facilitated diffusion mediated by polyamines on nucleation and elongation of microtubules. In vitro experiments using pure tubulin indicate that the promotion of microtubule assembly by polyamines is typical of facilitated diffusion. The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics.

  2. Cep169, a Novel Microtubule Plus-End-Tracking Centrosomal Protein, Binds to CDK5RAP2 and Regulates Microtubule Stability.

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    Yusuke Mori

    Full Text Available The centrosomal protein, CDK5RAP2, is a microcephaly protein that regulates centrosomal maturation by recruitment of a γ-tubulin ring complex (γ-TuRC onto centrosomes. In this report, we identified a novel human centrosomal protein, Cep169, as a binding partner of CDK5RAP2, a member of microtubule plus-end-tracking proteins (+TIPs. Cep169 interacts directly with CDK5RAP2 through CM1, an evolutionarily conserved domain, and colocalizes at the pericentriolar matrix (PCM around centrioles with CDK5RAP2. In addition, Cep169 interacts with EB1 through SxIP-motif responsible for EB1 binding, and colocalizes with CDK5RAP2 at the microtubule plus-end. EB1-binding-deficient Cep169 abolishes EB1 interaction and microtubule plus-end attachment, indicating Cep169 as a novel member of +TIPs. We further show that ectopic expression of either Cep169 or CDK5RAP2 induces microtubule bundling and acetylation in U2OS cells, and depletion of Cep169 induces microtubule depolymerization in HeLa cells, although Cep169 is not required for assembly of γ-tubulin onto centrosome by CDK5RAP2. These results show that Cep169 targets microtubule tips and regulates stability of microtubules with CDK5RAP2.

  3. Distinct brainstem and forebrain circuits receiving tracheal sensory neuron inputs revealed using a novel conditional anterograde transsynaptic viral tracing system.

    Science.gov (United States)

    McGovern, Alice E; Driessen, Alexandria K; Simmons, David G; Powell, Joseph; Davis-Poynter, Nicholas; Farrell, Michael J; Mazzone, Stuart B

    2015-05-06

    Sensory nerves innervating the mucosa of the airways monitor the local environment for the presence of irritant stimuli and, when activated, provide input to the nucleus of the solitary tract (Sol) and paratrigeminal nucleus (Pa5) in the medulla to drive a variety of protective behaviors. Accompanying these behaviors are perceivable sensations that, particularly for stimuli in the proximal end of the airways, can be discrete and localizable. Airway sensations likely reflect the ascending airway sensory circuitry relayed via the Sol and Pa5, which terminates broadly throughout the CNS. However, the relative contribution of the Sol and Pa5 to these ascending pathways is not known. In the present study, we developed and characterized a novel conditional anterograde transneuronal viral tracing system based on the H129 strain of herpes simplex virus 1 and used this system in rats along with conventional neuroanatomical tracing with cholera toxin B to identify subcircuits in the brainstem and forebrain that are in receipt of relayed airway sensory inputs via the Sol and Pa5. We show that both the Pa5 and proximal airways disproportionately receive afferent terminals arising from the jugular (rather than nodose) vagal ganglia and the output of the Pa5 is predominately directed toward the ventrobasal thalamus. We propose the existence of a somatosensory-like pathway from the proximal airways involving jugular ganglia afferents, the Pa5, and the somatosensory thalamus and suggest that this pathway forms the anatomical framework for sensations arising from the proximal airway mucosa. Copyright © 2015 the authors 0270-6474/15/357041-15$15.00/0.

  4. Brainstem projections of neurons located in various subdivisions of the dorsolateral hypothalamic area – an anterograde tract-tracing study

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    Rege Sugárka Papp

    2014-05-01

    Full Text Available The projections from the dorsolateral hypothalamic area (DLH to the lower brainstem have been investigated by using biotinylated dextran amine (BDA, an anterograde tracer in rats. The DLH can be divided into 3 areas (dorsomedial hypothalamus, perifornical area, lateral hypothalamic area, and further subdivided into 8 subdivisions. After unilateral stereotaxic injections of BDA into individual DLH subdivisions, the correct sites of injections were controlled histologically, and the distribution patterns of BDA-positive fibers were mapped on serial sections between the hypothalamus and spinal cord in 22 rats. BDA-labeled fibers were observable over 100 different brainstem areas, nuclei or subdivisions. Injections into the 8 DLH subdivisions established distinct topographical patterns. In general, the density of labeled fibers was low in the lower brainstem. High density of fibers was seen only 4 of the 116 areas: in the lateral and ventrolateral parts of the periaqueductal gray, the Barrington’s and the pedunculopontine tegmental nuclei. All of the biogenic amine cell groups in the lower brainstem (9 noradrenaline, 3 adrenaline and 9 serotonin cell groups received labeled fibers, some of them from all, or at least 7 DLH subdivisions, mainly from perifornical and ventral lateral hypothalamic neurons. Some of the tegmental nuclei and nuclei of the reticular formation were widely innervated, although the density of the BDA-labeled fibers was generally low. No definitive descending BDA-positive pathway, but long-run solitaire BDA-labeled fibers were seen in the lower brainstem. These descending fibers joined some of the large tracts or fasciculi in the brainstem. The distribution pattern of BDA-positive fibers of DLH origin throughout the lower brainstem was comparable to patterns of previously published orexin- or melanin-concentrating hormone-immunoreactive fibers with somewhat differences.

  5. Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking.

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    Alice A Royal

    Full Text Available The slow delayed-rectifier potassium current (IKs is crucial for human cardiac action potential repolarization. The formation of IKs requires co-assembly of the KCNQ1 α-subunit and KCNE1 β-subunit, and mutations in either of these subunits can lead to hereditary long QT syndrome types 1 and 5, respectively. It is widely recognised that the KCNQ1/KCNE1 (Q1/E1 channel requires phosphatidylinositol-4,5-bisphosphate (PIP2 binding for function. We previously identified a cluster of basic residues in the proximal C-terminus of KCNQ1 that form a PIP2/phosphoinositide binding site. Upon charge neutralisation of these residues we found that the channel became more retained in the endoplasmic reticulum, which raised the possibility that channel-phosphoinositide interactions could play a role in channel trafficking. To explore this further we used a chemically induced dimerization (CID system to selectively deplete PIP2 and/or phosphatidylinositol-4-phosphate (PI(4P at the plasma membrane (PM or Golgi, and we subsequently monitored the effects on both channel trafficking and function. The depletion of PIP2 and/or PI(4P at either the PM or Golgi did not alter channel cell-surface expression levels. However, channel function was extremely sensitive to the depletion of PIP2 at the PM, which is in contrast to the response of other cardiac potassium channels tested (Kir2.1 and Kv11.1. Surprisingly, when using the CID system IKs was dramatically reduced even before dimerization was induced, highlighting limitations regarding the utility of this system when studying processes highly sensitive to PIP2 depletion. In conclusion, we identify that the Q1/E1 channel does not require PIP2 or PI(4P for anterograde trafficking, but is heavily reliant on PIP2 for channel function once at the PM.

  6. Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking.

    Science.gov (United States)

    Royal, Alice A; Tinker, Andrew; Harmer, Stephen C

    2017-01-01

    The slow delayed-rectifier potassium current (IKs) is crucial for human cardiac action potential repolarization. The formation of IKs requires co-assembly of the KCNQ1 α-subunit and KCNE1 β-subunit, and mutations in either of these subunits can lead to hereditary long QT syndrome types 1 and 5, respectively. It is widely recognised that the KCNQ1/KCNE1 (Q1/E1) channel requires phosphatidylinositol-4,5-bisphosphate (PIP2) binding for function. We previously identified a cluster of basic residues in the proximal C-terminus of KCNQ1 that form a PIP2/phosphoinositide binding site. Upon charge neutralisation of these residues we found that the channel became more retained in the endoplasmic reticulum, which raised the possibility that channel-phosphoinositide interactions could play a role in channel trafficking. To explore this further we used a chemically induced dimerization (CID) system to selectively deplete PIP2 and/or phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane (PM) or Golgi, and we subsequently monitored the effects on both channel trafficking and function. The depletion of PIP2 and/or PI(4)P at either the PM or Golgi did not alter channel cell-surface expression levels. However, channel function was extremely sensitive to the depletion of PIP2 at the PM, which is in contrast to the response of other cardiac potassium channels tested (Kir2.1 and Kv11.1). Surprisingly, when using the CID system IKs was dramatically reduced even before dimerization was induced, highlighting limitations regarding the utility of this system when studying processes highly sensitive to PIP2 depletion. In conclusion, we identify that the Q1/E1 channel does not require PIP2 or PI(4)P for anterograde trafficking, but is heavily reliant on PIP2 for channel function once at the PM.

  7. Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo

    Directory of Open Access Journals (Sweden)

    Kevin T Beier

    2013-02-01

    Full Text Available Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV can be endowed with anterograde or retrograde synaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of VSV with the rabies virus glycoprotein (RABV-G, and provide examples of its activity relative to the anterograde tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to a given area. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer.

  8. Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo.

    Science.gov (United States)

    Beier, Kevin T; Saunders, Arpiar B; Oldenburg, Ian A; Sabatini, Bernardo L; Cepko, Constance L

    2013-01-01

    Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer.

  9. Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Jessica M Esparza

    Full Text Available 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 (ε-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.

  10. Genetic analysis of a Drosophila microtubule-associated protein

    OpenAIRE

    1992-01-01

    The 205-kD microtubule-associated protein (205K MAP) is one of the principal MAPs in Drosophila. 205K MAP is similar to the HeLa 210K/MAP4 family of MAPs since it shares the following biochemical properties: it is present in several isoforms, has a molecular mass of approximately 200 kD, and is thermostable. Furthermore, immuno-crossreactivity has been observed between mouse MAP4, HeLa 210K, and Drosophila 205K MAP. Currently, there is little information concerning the biological function of ...

  11. Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

    Science.gov (United States)

    Melmed, RN; Karanian, PJ; Berlin, RD

    1981-01-01

    We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J

  12. Analysis of Soybean Microtubule Persistence Length; New Evidence on the Correlation between Structural Composition and Mechanical Properties

    Science.gov (United States)

    Shojania Feizabadi, Mitra; Winton, Carly; Barrientos, Jimmy

    2012-02-01

    Recent studies on microtubules composed of different β tubulin isotypes indicate their different functionality in terms of their dynamical behavior or the mechanism of their interaction with chemotherapeutic drugs. Along these lines, the result of our recent study measuring the rigidity of neural and non-neural samples of microtubules with different β tubulin isotype compositions suggests that the distinguished mechanical properties of microtubules, such as rigidity, may also be associated with the different distribution of their β tubulin isotypes. In our current study, we have reported the persistence length of a single soybean microtubule. This plant microtubule has a structural composition different from that of mammalian microtubules. Under the same experimental methods of measurement, the soybean microtubules showed a different persistence length as compared to the value of the persistence length that we estimated in the study of both single Bovine Brain and MCF7 microtubules.

  13. Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart.

    Science.gov (United States)

    Braun, Marcus; Lansky, Zdenek; Fink, Gero; Ruhnow, Felix; Diez, Stefan; Janson, Marcel E

    2011-09-04

    Short regions of overlap between ends of antiparallel microtubules are central elements within bipolar microtubule arrays. Although their formation requires motors, recent in vitro studies demonstrated that stable overlaps cannot be generated by molecular motors alone. Motors either slide microtubules along each other until complete separation or, in the presence of opposing motors, generate oscillatory movements. Here, we show that Ase1, a member of the conserved MAP65/PRC1 family of microtubule-bundling proteins, enables the formation of stable antiparallel overlaps through adaptive braking of Kinesin-14-driven microtubule-microtubule sliding. As overlapping microtubules start to slide apart, Ase1 molecules become compacted in the shrinking overlap and the sliding velocity gradually decreases in a dose-dependent manner. Compaction is driven by moving microtubule ends that act as barriers to Ase1 diffusion. Quantitative modelling showed that the molecular off-rate of Ase1 is sufficiently low to enable persistent overlap stabilization over tens of minutes. The finding of adaptive braking demonstrates that sliding can be slowed down locally to stabilize overlaps at the centre of bipolar arrays, whereas sliding proceeds elsewhere to enable network self-organization.

  14. A ROP2-RIC1 pathway fine-tunes microtubule reorganization for salt tolerance in Arabidopsis.

    Science.gov (United States)

    Li, Changjiang; Lu, Hanmei; Li, Wei; Yuan, Ming; Fu, Ying

    2017-07-01

    The reorganization of microtubules induced by salt stress is required for Arabidopsis survival under high salinity conditions. RIC1 is an effector of Rho-related GTPase from plants (ROPs) and a known microtubule-associated protein. In this study, we demonstrated that RIC1 expression decreased with long-term NaCl treatment, and ric1-1 seedlings exhibited a higher survival rate under salt stress. We found that RIC1 reduced the frequency of microtubule transition from shortening to growing status and knockout of RIC1 improved the reassembly of depolymerized microtubules caused by either oryzalin treatment or salt stress. Further investigation showed that constitutively active ROP2 promoted the reassembly of microtubules and the survival of seedlings under salt stress. A rop2-1 ric1-1 double mutant rescued the salt-sensitive phenotype of rop2-1, indicating that ROP2 functions in salt tolerance through RIC1. Although ROP2 did not regulate RIC1 expression upon salt stress, a quick but mild increase of ROP2 activity was induced, led to reduction of RIC1 on microtubules. Collectively, our study reveals an ROP2-RIC1 pathway that fine-tunes microtubule dynamics in response to salt stress in Arabidopsis. This finding not only reveals a new regulatory mechanism for microtubule reorganization under salt stress but also the importance of ROP signalling for salinity tolerance. © 2017 John Wiley & Sons Ltd.

  15. Microtubules in legume root hairs: cell polarity and response to rhizobial signal molecules

    NARCIS (Netherlands)

    Sieberer, B.

    2005-01-01

    Microtubules, which occur as hollow protein tubes with a diameter of 25 nanometers, are an important compound of the cytoskeleton and occur in plant cells as a highly organized and dynamic array, which actual arrangement will depend on its tasks during the cell cycle. Microtubules play a key-role in

  16. Four-stranded mini microtubules formed byProsthecobacterBtubAB show dynamic instability.

    Science.gov (United States)

    Deng, Xian; Fink, Gero; Bharat, Tanmay A M; He, Shaoda; Kureisaite-Ciziene, Danguole; Löwe, Jan

    2017-07-18

    Microtubules, the dynamic, yet stiff hollow tubes built from αβ-tubulin protein heterodimers, are thought to be present only in eukaryotic cells. Here, we report a 3.6-Å helical reconstruction electron cryomicroscopy structure of four-stranded mini microtubules formed by bacterial tubulin-like Prosthecobacter dejongeii BtubAB proteins. Despite their much smaller diameter, mini microtubules share many key structural features with eukaryotic microtubules, such as an M-loop, alternating subunits, and a seam that breaks overall helical symmetry. Using in vitro total internal reflection fluorescence microscopy, we show that bacterial mini microtubules treadmill and display dynamic instability, another hallmark of eukaryotic microtubules. The third protein in the btub gene cluster, BtubC, previously known as "bacterial kinesin light chain," binds along protofilaments every 8 nm, inhibits BtubAB mini microtubule catastrophe, and increases rescue. Our work reveals that some bacteria contain regulated and dynamic cytomotive microtubule systems that were once thought to be only useful in much larger and sophisticated eukaryotic cells.

  17. Synthesis and biological evaluation of structurally simplified noscapine analogues as microtubule binding agents

    Czech Academy of Sciences Publication Activity Database

    Ghaly, P.E.; Churchill, C.D.M.; Abou El-Magd, R.M.; Hájková, Zuzana; Dráber, Pavel; West, F.G.; Tuszyński, J.A.

    2017-01-01

    Roč. 95, č. 6 (2017), s. 649-655 ISSN 0008-4042 R&D Projects: GA ČR GA15-22194S Institutional support: RVO:68378050 Keywords : noscapine * microtubule * tubulin * cytotoxicity * microtubule dynamics * docking Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 1.080, year: 2016

  18. Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcement

    NARCIS (Netherlands)

    Brangwynne, C. P.; Mac Kintosh, F.C.; Kumar, S.B.; Geisse, N. A.; Talbot, J.; Mahadevan, L.; Parker, K.; Ingber, D. E.; Weitz, D. A.

    2006-01-01

    Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of

  19. The XMAP215-family protein DdCP224 is required for cortical interactions of microtubules

    Directory of Open Access Journals (Sweden)

    Hestermann Andrea

    2004-06-01

    Full Text Available Abstract Background Interactions of peripheral microtubule tips with the cell cortex are of crucial importance for nuclear migration, spindle orientation, centrosome positioning and directional cell movement. Microtubule plus end binding proteins are thought to mediate interactions of microtubule tips with cortical actin and membrane proteins in a dynein-dependent manner. XMAP215-family proteins are main regulators of microtubule plus end dynamics but so far they have not been implicated in the interactions of microtubule tips with the cell cortex. Results Here we show that overexpression of an N-terminal fragment of DdCP224, the Dictyostelium XMAP215 homologue, caused a collapse of the radial microtubule cytoskeleton, whereby microtubules lost contact with the cell cortex and were dragged behind like a comet tail of an unusually motile centrosome. This phenotype was indistinguishable from mutants overexpressing fragments of the dynein heavy chain or intermediate chain. Moreover, it was accompanied by dispersal of the Golgi apparatus and reduced cortical localization of the dynein heavy chain indicating a disrupted dynein/dynactin interaction. The interference of DdCP224 with cortical dynein function is strongly supported by the observations that DdCP224 and its N-terminal fragment colocalize with dynein and coimmunoprecipitate with dynein and dynactin. Conclusions Our data show that XMAP215-like proteins are required for the interaction of microtubule plus ends with the cell cortex in interphase cells and strongly suggest that this function is mediated by dynein.

  20. Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture.

    Science.gov (United States)

    Ganguly, Sujoy; Williams, Lucy S; Palacios, Isabel M; Goldstein, Raymond E

    2012-09-18

    Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA.

  1. Expansion and Polarity Sorting in Microtubule-Dynein Bundles(WHAT IS LIFE? THE NEXT 100 YEARS OF YUKAWA'S DREAM)

    OpenAIRE

    Assaf, ZEMEL; Alex, MOGILNER; Department of Neurobiology, Physiology and Behavior, University of California; Department of Neurobiology, Physiology and Behavior, University of California

    2008-01-01

    Interactions of multiple molecular motors with dynamic polymers, such as actin and microtubules, form the basis for many processes in the cell cytoskeleton. One example is the active 'sorting' of microtubule bundles by dynein molecular motors into aster-like arrays of microtubules; in these bundles dynein motors cross-link and slide neighboring microtubules apart. A number of models have been suggested to quantify the active dynamics of cross-linked bundles of polar filaments. In the case of ...

  2. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

    Science.gov (United States)

    Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

    2017-04-01

    Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

  3. Microtubules restrict plastid sedimentation in protonemata of the moss Ceratodon

    Science.gov (United States)

    Schwuchow, J.; Sack, F. D.

    1994-01-01

    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.

  4. Kindlin1 regulates microtubule function to ensure normal mitosis.

    Science.gov (United States)

    Patel, Hitesh; Stavrou, Ifigeneia; Shrestha, Roshan L; Draviam, Viji; Frame, Margaret C; Brunton, Valerie G

    2016-08-01

    Loss of Kindlin 1 (Kin1) results in the skin blistering disorder Kindler Syndrome (KS), whose symptoms also include skin atrophy and reduced keratinocyte proliferation. Kin1 binds to integrins to modulate their activation and more recently it has been shown to regulate mitotic spindles and cell survival in a Plk1-dependent manner. Here we report that short-term Kin1 deletion in mouse skin results in impaired mitosis, which is associated with reduced acetylated tubulin (ac-tub) levels and cell proliferation. In cells, impaired mitosis and reduced ac-tub levels are also accompanied by reduced microtubule stability, all of which are rescued by HDAC6 inhibition. The ability of Kin1 to regulate HDAC6-dependent cellular ac-tub levels is dependent on its phosphorylation by Plk1. Taken together, these data define a novel role for Kin1 in microtubule acetylation and stability and offer a mechanistic insight into how certain KS phenotypes, such as skin atrophy and reduced cell proliferation, arise. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.

  5. The evolution and diversification of plant microtubule-associated proteins.

    Science.gov (United States)

    Gardiner, John

    2013-07-01

    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. © 2013 The Author The Plant Journal © 2013 John Wiley & Sons Ltd.

  6. Localization of a microtubule organizing center by kinesin motors

    Science.gov (United States)

    Arita, Chikashi; Bosche, Jonas; Lück, Alexander; Santen, Ludger

    2017-12-01

    Molecular motors are proteins which bind to a polarized cytoskeletal filament and move steadily along it. Molecular motors of the kinesin family move along microtubules (MTs), which are a component of the cytoskeleton. A very processive kinesin motor Kip3p, is known to promote catastrophes and pausing of MT, in particular on cortical contact. These properties play an important role in positioning the mitotic spindle in budding yeast. We present a theoretical approach to positioning of MT networks under confinement. In order to explore a localization mechanism of a microtubule organizing center (MTOC), we introduce an idealized system of two MTs connected by a MTOC. The dynamics of Kip3p is modeled by interacting stochastic particles, which allows us to study the effects of motor-induced depolymerization in a finite volume. We find that localization in the middle of the cavity is realized in a parameter regime where the motor densities on the MTs are increasing with the distance from the MTOC. Localization at an asymmetric position is also possible by tuning model parameters.

  7. Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells.

    Science.gov (United States)

    Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka; Stearns, Tim

    2017-09-14

    Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

  8. Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex.

    Science.gov (United States)

    Ciferri, Claudio; Pasqualato, Sebastiano; Screpanti, Emanuela; Varetti, Gianluca; Santaguida, Stefano; Dos Reis, Gabriel; Maiolica, Alessio; Polka, Jessica; De Luca, Jennifer G; De Wulf, Peter; Salek, Mogjiborahman; Rappsilber, Juri; Moores, Carolyn A; Salmon, Edward D; Musacchio, Andrea

    2008-05-02

    Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.

  9. Microtubules Nonlinear Models Dynamics Investigations through the exp(−Φ(ξ-Expansion Method Implementation

    Directory of Open Access Journals (Sweden)

    Nur Alam

    2016-02-01

    Full Text Available In this research article, we present exact solutions with parameters for two nonlinear model partial differential equations(PDEs describing microtubules, by implementing the exp(−Φ(ξ-Expansion Method. The considered models, describing highly nonlinear dynamics of microtubules, can be reduced to nonlinear ordinary differential equations. While the first PDE describes the longitudinal model of nonlinear dynamics of microtubules, the second one describes the nonlinear model of dynamics of radial dislocations in microtubules. The acquired solutions are then graphically presented, and their distinct properties are enumerated in respect to the corresponding dynamic behavior of the microtubules they model. Various patterns, including but not limited to regular, singular kink-like, as well as periodicity exhibiting ones, are detected. Being the method of choice herein, the exp(−Φ(ξ-Expansion Method not disappointing in the least, is found and declared highly efficient.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Iku [Tokyo Denki University School of Information Environment 2-1200, Muzai-Gakuendai, Inzai, Chiba 270-1382 (Japan)]. E-mail: nemoto@sie.dendai.ac.jp; Kawamura, Kazuhisa [Tokyo Denki University School of Science and Engineering Hatoyama, Saitama 350-0394 (Japan)

    2005-05-15

    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.

  11. Interactive domains in the molecular chaperone human alphaB crystallin modulate microtubule assembly and disassembly.

    Directory of Open Access Journals (Sweden)

    Joy G Ghosh

    2007-06-01

    Full Text Available Small heat shock proteins regulate microtubule assembly during cell proliferation and in response to stress through interactions that are poorly understood.Novel functions for five interactive sequences in the small heat shock protein and molecular chaperone, human alphaB crystallin, were investigated in the assembly/disassembly of microtubules and aggregation of tubulin using synthetic peptides and mutants of human alphaB crystallin.The interactive sequence (113FISREFHR(120 exposed on the surface of alphaB crystallin decreased microtubule assembly by approximately 45%. In contrast, the interactive sequences, (131LTITSSLSSDGV(142 and (156ERTIPITRE(164, corresponding to the beta8 strand and the C-terminal extension respectively, which are involved in complex formation, increased microtubule assembly by approximately 34-45%. The alphaB crystallin peptides, (113FISREFHR(120 and (156ERTIPITRE(164, inhibited microtubule disassembly by approximately 26-36%, and the peptides (113FISREFHR(120 and (131LTITSSLSSDGV(142 decreased the thermal aggregation of tubulin by approximately 42-44%. The (131LTITSSLSSDGV(142 and (156ERTIPITRE(164 peptides were more effective than the widely used anti-cancer drug, Paclitaxel, in modulating tubulinmicrotubule dynamics. Mutagenesis of these interactive sequences in wt human alphaB crystallin confirmed the effects of the alphaB crystallin peptides on microtubule assembly/disassembly and tubulin aggregation. The regulation of microtubule assembly by alphaB crystallin varied over a narrow range of concentrations. The assembly of microtubules was maximal at alphaB crystallin to tubulin molar ratios between 1:4 and 2:1, while molar ratios >2:1 inhibited microtubule assembly.Interactive sequences on the surface of human alphaB crystallin collectively modulate microtubule assembly through a dynamic subunit exchange mechanism that depends on the concentration and ratio of alphaB crystallin to tubulin. These are the first

  12. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    Science.gov (United States)

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  13. Increased acetylation of microtubules rescues human tau-induced microtubule defects and neuromuscular junction abnormalities in Drosophila

    Directory of Open Access Journals (Sweden)

    Chuan-Xi Mao

    2017-10-01

    Full Text Available Tau normally associates with and stabilizes microtubules (MTs, but is hyperphosphorylated and aggregated into neurofibrillary tangles in Alzheimer's disease and related neurodegenerative diseases, which are collectively known as tauopathies. MTs are regulated by different forms of post-translational modification, including acetylation; acetylated MTs represent a more stable microtubule population. In our previous study, we showed that inhibition of histone deacetylase 6 (HDAC6, which deacetylates tubulin at lysine 40, rescues defects in MTs and in neuromuscular junction growth caused by tau overexpression. However, HDAC6 also acts on other proteins that are involved in distinct biological processes unrelated to tubulins. In order to examine directly the role of increased tubulin acetylation against tau toxicity, we generated a site-directed α-tubulinK40Q mutation by CRISPR/Cas9 technology to mimic the acetylated MTs and found that acetylation-mimicking α-tubulin rescued tau-induced MT defects and neuromuscular junction developmental abnormalities. We also showed that late administration of ACY-1215 and tubastatin A, two potent and selective inhibitors of HDAC6, rescued the tau-induced MT defects after the abnormalities had already become apparent. Overall, our results indicate that increasing MT acetylation by either genetic manipulations or drugs might be used as potential strategies for intervention in tauopathies.

  14. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

    NARCIS (Netherlands)

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A. Michael; Mansson, Alf; Kocer, Armagan

    2013-01-01

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies.

  15. Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling

    Directory of Open Access Journals (Sweden)

    Shi Xianglin

    2009-01-01

    Full Text Available Abstract Background Engineered iron nanoparticles are being explored for the development of biomedical applications and many other industry purposes. However, to date little is known concerning the precise mechanisms of translocation of iron nanoparticles into targeted tissues and organs from blood circulation, as well as the underlying implications of potential harmful health effects in human. Results The confocal microscopy imaging analysis demonstrates that exposure to engineered iron nanoparticles induces an increase in cell permeability in human microvascular endothelial cells. Our studies further reveal iron nanoparticles enhance the permeability through the production of reactive oxygen species (ROS and the stabilization of microtubules. We also showed Akt/GSK-3β signaling pathways are involved in iron nanoparticle-induced cell permeability. The inhibition of ROS demonstrate ROS play a major role in regulating Akt/GSK-3β – mediated cell permeability upon iron nanoparticle exposure. These results provide new insights into the bioreactivity of engineered iron nanoparticles which can inform potential applications in medical imaging or drug delivery. Conclusion Our results indicate that exposure to iron nanoparticles induces an increase in endothelial cell permeability through ROS oxidative stress-modulated microtubule remodeling. The findings from this study provide new understandings on the effects of nanoparticles on vascular transport of macromolecules and drugs.

  16. Altered nucleotide-microtubule coupling and increased mechanical output by a kinesin mutant.

    Directory of Open Access Journals (Sweden)

    Hong-Lei Liu

    Full Text Available Kinesin motors hydrolyze ATP to produce force and do work in the cell--how the motors do this is not fully understood, but is thought to depend on the coupling of ATP hydrolysis to microtubule binding by the motor. Transmittal of conformational changes from the microtubule- to the nucleotide-binding site has been proposed to involve the central β-sheet, which could undergo large structural changes important for force production. We show here that mutation of an invariant residue in loop L7 of the central β-sheet of the Drosophila kinesin-14 Ncd motor alters both nucleotide and microtubule binding, although the mutated residue is not present in either site. Mutants show weak-ADP/tight-microtubule binding, instead of tight-ADP/weak-microtubule binding like wild type--they hydrolyze ATP faster than wild type, move faster in motility assays, and assemble long spindles with greatly elongated poles, which are also produced by simulations of assembly with tighter microtubule binding and faster sliding. The mutated residue acts like a mechanochemical coupling element--it transmits changes between the microtubule-binding and active sites, and can switch the state of the motor, increasing mechanical output by the motor. One possibility, based on our findings, is that movements by the residue and the loop that contains it could bend or distort the central β-sheet, mediating free energy changes that lead to force production.

  17. Long astral microtubules and RACK-1 stabilize polarity domains during maintenance phase in Caenorhabditis elegans embryos.

    Directory of Open Access Journals (Sweden)

    Erkang Ai

    2011-04-01

    Full Text Available Cell polarity is a very well conserved process important for cell differentiation, cell migration, and embryonic development. After the establishment of distinct cortical domains, polarity cues have to be stabilized and maintained within a fluid and dynamic membrane to achieve proper cell asymmetry. Microtubules have long been thought to deliver the signals required to polarize a cell. While previous studies suggest that microtubules play a key role in the establishment of polarity, the requirement of microtubules during maintenance phase remains unclear. In this study, we show that depletion of Caenorhabditis elegans RACK-1, which leads to short astral microtubules during prometaphase, specifically affects maintenance of cortical PAR domains and Dynamin localization. We then investigated the consequence of knocking down other factors that also abolish astral microtubule elongation during polarity maintenance phase. We found a correlation between short astral microtubules and the instability of PAR-6 and PAR-2 domains during maintenance phase. Our data support a necessary role for astral microtubules in the maintenance phase of cell polarity.

  18. Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria.

    Directory of Open Access Journals (Sweden)

    Carsten Schwan

    2009-10-01

    Full Text Available Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase, which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 microm microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host-pathogen interactions.

  19. Probing a self-assembled fd virus membrane with a microtubule.

    Science.gov (United States)

    Xie, Sheng; Pelcovits, Robert A; Hagan, Michael F

    2016-06-01

    The self-assembly of highly anisotropic colloidal particles leads to a rich variety of morphologies whose properties are just beginning to be understood. This article uses computer simulations to probe a particle-scale perturbation of a commonly studied colloidal assembly, a monolayer membrane composed of rodlike fd viruses in the presence of a polymer depletant. Motivated by experiments currently in progress, we simulate the interaction between a microtubule and a monolayer membrane as the microtubule "pokes" and penetrates the membrane face-on. Both the viruses and the microtubule are modeled as hard spherocylinders of the same diameter, while the depletant is modeled using ghost spheres. We find that the force exerted on the microtubule by the membrane is zero either when the microtubule is completely outside the membrane or when it has fully penetrated the membrane. The microtubule is initially repelled by the membrane as it begins to penetrate but experiences an attractive force as it penetrates further. We assess the roles played by translational and rotational fluctuations of the viruses and the osmotic pressure of the polymer depletant. We find that rotational fluctuations play a more important role than the translational ones. The dependence on the osmotic pressure of the depletant of the width and height of the repulsive barrier and the depth of the attractive potential well is consistent with the assumed depletion-induced attractive interaction between the microtubule and viruses. We discuss the relevance of these studies to the experimental investigations.

  20. Structural differences between yeast and mammalian microtubules revealed by cryo-EM

    Energy Technology Data Exchange (ETDEWEB)

    Howes, Stuart C. [Univ. of California, Berkeley, CA (United States). Biophysics Graduate Group; Geyer, Elisabeth A. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; LaFrance, Benjamin [Univ. of California, Berkeley, CA (United States). Molecular and Cell Biology Graduate Program; Zhang, Rui [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Kellogg, Elizabeth H. [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Westermann, Stefan [Univ. of Duisburg-Essen, Essen (Germany). Dept. of Molecular Genetics, Center for Medical Biotechnology; Rice, Luke M. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; Nogales, Eva [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Univ. of California, Berkeley, CA (United States). Dept. of Molecular Biology and California Inst. for Quantitative Biosciences; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division

    2017-06-26

    Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. In conclusion, our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.

  1. Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

    Science.gov (United States)

    2014-01-01

    Toxoplasma gondii is an obligate intracellular parasite that causes serious opportunistic infections, birth defects, and blindness in humans. Microtubules are critically important components of diverse structures that are used throughout the Toxoplasma life cycle. As in other eukaryotes, spindle microtubules are required for chromosome segregation during replication. Additionally, a set of membrane-associated microtubules is essential for the elongated shape of invasive “zoites,” and motility follows a spiral trajectory that reflects the path of these microtubules. Toxoplasma zoites also construct an intricate, tubulin-based apical structure, termed the conoid, which is important for host cell invasion and associates with proteins typically found in the flagellar apparatus. Last, microgametes specifically construct a microtubule-containing flagellar axoneme in order to fertilize macrogametes, permitting genetic recombination. The specialized roles of these microtubule populations are mediated by distinct sets of associated proteins. This review summarizes our current understanding of the role of tubulin, microtubule populations, and associated proteins in Toxoplasma; these components are used for both novel and broadly conserved processes that are essential for parasite survival. PMID:25380753

  2. Variational Principles for Buckling of Microtubules Modeled as Nonlocal Orthotropic Shells

    Directory of Open Access Journals (Sweden)

    Sarp Adali

    2014-01-01

    Full Text Available 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.

  3. Buckling analysis of orthotropic protein microtubules under axial and radial compression based on couple stress theory.

    Science.gov (United States)

    Beni, Yaghoub Tadi; Zeverdejani, M Karimi; Mehralian, Fahimeh

    2017-10-01

    Protein microtubules (MTs) are one of the important intercellular components and have a vital role in the stability and strength of the cells. Due to applied external loads, protein microtubules may be involved buckling phenomenon. Due to impact of protein microtubules in cell reactions, it is important to determine their critical buckling load. Considering nature of protein microtubules, various parameters are effective on microtubules buckling. The small size of microtubules and also lack of uniformity of MTs properties in different directions caused the necessity of accuracy in the analysis of these bio-structure. In fact, microtubules must be considered as a size dependent cylinder, which behave as an orthotropic material. Hence, in the present work using first-order shear deformation model (FSDT), the buckling equations of anisotropic MTs are derived based on new modified couple stress theory (NMCST). After solving the stability equations, the influences of various parameters are measured on the MTs critical buckling load. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Microtubules CLASP to Adherens Junctions in epidermal progenitor cells

    DEFF Research Database (Denmark)

    Shahbazi, Marta N; Perez-Moreno, Mirna

    2014-01-01

    Cadherin-mediated cell adhesion at Adherens Junctions (AJs) and its dynamic connections with the microtubule (MT) cytoskeleton are important regulators of cellular architecture. However, the functional relevance of these interactions and the molecular players involved in different cellular contexts...... and cellular compartments are still not completely understood. Here, we comment on our recent findings showing that the MT plus-end binding protein CLASP2 interacts with the AJ component p120-catenin (p120) specifically in progenitor epidermal cells. Absence of either protein leads to alterations in MT...... dynamics and AJ functionality. These findings represent a novel mechanism of MT targeting to AJs that may be relevant for the maintenance of proper epidermal progenitor cell homeostasis. We also discuss the potential implication of other MT binding proteins previously associated to AJs in the wider context...

  5. The Role of Microtubule End Binding (EB) Proteins in Ciliogenesis

    DEFF Research Database (Denmark)

    Schrøder, Jacob Morville

    centrosomal MT array and abnormally long centriole-associated rootlet filaments. Cells lacking EB1 also had stumpy cilia and a disorganized centrosomal MT array, but rootlet filaments appeared normal. Further, live imaging revealed increased release frequency of MTs from the centrosome upon EB1 or EB3......EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends. EB1 plays a role in regulating MT dynamics, localizing other MT-associated proteins to the plus end, and in regulating interactions of MTs with the cell cortex, mitotic kinetochores and different......, are required for assembly of primary cilia in cultured human cells. The EB3 - siRNA ciliary phenotype could be rescued by GFP-EB1 expression, and GFP-EB3 over expression resulted in elongated cilia. Transmission electron microscopy (TEM) revealed that EB3-depleted cells possess stumpy cilia, a disorganized...

  6. STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

    Czech Academy of Sciences Publication Activity Database

    Hájková, Zuzana; Bugajev, Viktor; Dráberová, Eduarda; Vinopal, Stanislav; Dráberová, Lubica; Janáček, Jiří; Dráber, Petr; Dráber, Pavel

    2011-01-01

    Roč. 186, č. 2 (2011), s. 913-923 ISSN 0022-1767 R&D Projects: GA ČR(CZ) GD204/09/H084; GA ČR GA204/09/1777; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA MŠk LC545; GA MŠk(CZ) LC06063; GA AV ČR KAN200520701 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50110509 Keywords : STIM1 * bonemarrow-derived mast cells * microtubules Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.788, year: 2011

  7. STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

    Czech Academy of Sciences Publication Activity Database

    Hájková, Zuzana; Bugajev, Viktor; Dráberová, Eduarda; Vinopal, Stanislav; Dráberová, Lubica; Janáček, Jiří; Dráber, Petr; Dráber, Pavel

    2011-01-01

    Roč. 186, č. 2 (2011), s. 913-923 ISSN 0022-1767 R&D Projects: GA ČR(CZ) GD204/09/H084; GA ČR GA204/09/1777; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA MŠk LC545; GA MŠk(CZ) LC06063; GA AV ČR KAN200520701 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50110509 Keywords : STIM1 * bone marrow-derived mast cells * microtubules Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.788, year: 2011

  8. An ELMO2-RhoG-ILK network modulates microtubule dynamics.

    Science.gov (United States)

    Jackson, Bradley C; Ivanova, Iordanka A; Dagnino, Lina

    2015-07-15

    ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin-dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca(2+)-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes. © 2015 Jackson et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  9. Alteramide B is a microtubule antagonist of inhibiting Candida albicans.

    Science.gov (United States)

    Ding, Yanjiao; Li, Yaoyao; Li, Zhenyu; Zhang, Juanli; Lu, Chunhua; Wang, Haoxin; Shen, Yuemao; Du, Liangcheng

    2016-10-01

    Alteramide B (ATB), isolated from Lysobacter enzymogenes C3, was a new polycyclic tetramate macrolactam (PTM). ATB exhibited potent inhibitory activity against several yeasts, particularly Candida albicans SC5314, but its antifungal mechanism is unknown. The structure of ATB was established by extensive spectroscopic analyses, including high-resolution mass spectrometry, 1D- and 2D-NMR, and CD spectra. Flow cytometry, fluorescence microscope, transmission electron microscope, molecular modeling, overexpression and site-directed mutation studies were employed to delineate the anti-Candida molecular mechanism of ATB. ATB induced apoptosis in C. albicans through inducing reactive oxygen species (ROS) production by disrupting microtubules. Molecular dynamics studies revealed the binding patterns of ATB to the β-tubulin subunit. Overexpression of the wild type and site-directed mutants of the β-tubulin gene (TUBB) changed the sensitivity of C. albicans to ATB, confirming the binding of ATB to β-tubulin, and indicating that the binding sites are L215, L217, L273, L274 and R282. In vivo, ATB significantly improved the survival of the candidiasis mice and reduced fungal burden. The molecular mechanism underlying the ATB-induced apoptosis in C. albicans is through inhibiting tubulin polymerization that leads to cell cycle arrest at the G2/M phase. The identification of ATB and the study of its activity provide novel mechanistic insights into the mode of action of PTMs against the human pathogen. This study shows that ATB is a new microtubule inhibitor and a promising anti-Candida lead compound. The results also support β-tubulin as a potential target for anti-Candida drug discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Functional role of ɛ-tubulin in the assembly of the centriolar microtubule scaffold

    Science.gov (United States)

    Dupuis-Williams, Pascale; Fleury-Aubusson, Anne; de Loubresse, Nicole Garreau; Geoffroy, Hélène; Vayssié, Laurence; Galvani, Angélique; Espigat, Aude; Rossier, Jean

    2002-01-01

    Centrioles and basal bodies fascinate by their spectacular architecture, featuring an arrangement of nine microtubule triplets into an axial symmetry, whose biogenesis relies on yet elusive mechanisms. However, the recent discovery of new tubulins, such as δ-, ɛ-, or η-tubulin, could constitute a breakthrough for deciphering the assembly steps of this unconventional microtubule scaffold. Here, we report the functional analysis in vivo of ɛ-tubulin, based on gene silencing in Paramecium, which demonstrates that this protein, which localizes at the basal bodies, is essential for the assembly and anchorage of the centriolar microtubules. PMID:12356863

  11. The Kinesin Adaptor Calsyntenin-1 Organizes Microtubule Polarity and Regulates Dynamics during Sensory Axon Arbor Development

    Directory of Open Access Journals (Sweden)

    Mary C. Halloran

    2017-04-01

    Full Text Available Axon growth and branching, and development of neuronal polarity are critically dependent on proper organization and dynamics of the microtubule (MT cytoskeleton. MTs must organize with correct polarity for delivery of diverse cargos to appropriate subcellular locations, yet the molecular mechanisms regulating MT polarity remain poorly understood. Moreover, how an actively branching axon reorganizes MTs to direct their plus ends distally at branch points is unknown. We used high-speed, in vivo imaging of polymerizing MT plus ends to characterize MT dynamics in developing sensory axon arbors in zebrafish embryos. We find that axonal MTs are highly dynamic throughout development, and that the peripheral and central axons of sensory neurons show differences in MT behaviors. Furthermore, we show that Calsyntenin-1 (Clstn-1, a kinesin adaptor required for sensory axon branching, also regulates MT polarity in developing axon arbors. In wild type neurons the vast majority of MTs are directed in the correct plus-end-distal orientation from early stages of development. Loss of Clstn-1 causes an increase in MTs polymerizing in the retrograde direction. These misoriented MTs most often are found near growth cones and branch points, suggesting Clstn-1 is particularly important for organizing MT polarity at these locations. Together, our results suggest that Clstn-1, in addition to regulating kinesin-mediated cargo transport, also organizes the underlying MT highway during axon arbor development.

  12. Protein friction limits diffusive and directed movements of kinesin motors on microtubules.

    Science.gov (United States)

    Bormuth, Volker; Varga, Vladimir; Howard, Jonathon; Schäffer, Erik

    2009-08-14

    Friction limits the operation of macroscopic engines and is critical to the performance of micromechanical devices. We report measurements of friction in a biological nanomachine. Using optical tweezers, we characterized the frictional drag force of individual kinesin-8 motor proteins interacting with their microtubule tracks. At low speeds and with no energy source, the frictional drag was related to the diffusion coefficient by the Einstein relation. At higher speeds, the frictional drag force increased nonlinearly, consistent with the motor jumping 8 nanometers between adjacent tubulin dimers along the microtubule, and was asymmetric, reflecting the structural polarity of the microtubule. We argue that these frictional forces arise from breaking bonds between the motor domains and the microtubule, and they limit the speed and efficiency of kinesin.

  13. Katanin: A Sword Cutting Microtubules for Cellular, Developmental, and Physiological Purposes

    Directory of Open Access Journals (Sweden)

    Ivan Luptovčiak

    2017-11-01

    Full Text Available KATANIN is a well-studied microtubule severing protein affecting microtubule organization and dynamic properties in higher plants. By regulating mitotic and cytokinetic and cortical microtubule arrays it is involved in the progression of cell division and cell division plane orientation. KATANIN is also involved in cell elongation and morphogenesis during plant growth. In this way KATANIN plays critical roles in diverse plant developmental processes including the development of pollen, embryo, seed, meristem, root, hypocotyl, cotyledon, leaf, shoot, and silique. KATANIN-dependent microtubule regulation seems to be under the control of plant hormones. This minireview provides an overview on available KATANIN mutants and discusses advances in our understanding of KATANIN biological roles in plants.

  14. Proper microtubule structure is vital for timely progression through meiosis in fission yeast.

    Directory of Open Access Journals (Sweden)

    Akira Yamashita

    Full Text Available Cells of the fission yeast Schizosaccharomyces pombe normally reproduce by mitotic division in the haploid state. When subjected to nutrient starvation, two haploid cells fuse and undergo karyogamy, forming a diploid cell that initiates meiosis to form four haploid spores. Here, we show that deletion of the mal3 gene, which encodes a homolog of microtubule regulator EB1, produces aberrant asci carrying more than four spores. The mal3 deletion mutant cells have a disordered cytoplasmic microtubule structure during karyogamy and initiate meiosis before completion of karyogamy, resulting in twin haploid meiosis in the zygote. Treatment with anti-microtubule drugs mimics this phenotype. Mutants defective in karyogamy or mutants prone to initiate haploid meiosis exaggerate the phenotype of the mal3 deletion mutant. Our results indicate that proper microtubule structure is required for ordered progression through the meiotic cycle. Furthermore, the results of our study suggest that fission yeast do not monitor ploidy during meiosis.

  15. Non-linear dynamics in biological microtubules: solitons and dissipation-free energy transfer

    Science.gov (United States)

    Mavromatos, Nick E.

    2017-08-01

    I review some recent developments concerning soliton solutions in biological microtubules and their significance in transferring energy without dissipation. I discuss various types of soliton solutions, as well as ‘spikes’, of the associated non-linear Lagrange equations describing the dynamics of a ‘pseudo-spin non-linear σ-model’ that models the dynamics of a microtubule system with dipole-dipole interactions. These results will hopefully contribute to a better understanding of the functional properties of microtubules, including the motor protein dynamics and the information transfer processes. With regards to the latter we also speculate on the use of microtubules as ‘logical’ gates. Our considerations are classical, but the soliton solutions may have a microscopic quantum origin, which we briefly touch upon.

  16. Survivin counteracts the therapeutic effect of microtubule de-stabilizers by stabilizing tubulin polymers

    Directory of Open Access Journals (Sweden)

    Hsieh Hsing-Pang

    2009-07-01

    Full Text Available Abstract Background Survivin is a dual function protein. It inhibits the apoptosis of cells by inhibiting caspases, and also promotes cell growth by stabilizing microtubules during mitosis. Over-expression of survivin has been demonstrated to induce drug-resistance to various chemo-therapeutic agents such as cisplatin (DNA damaging agent and paclitaxel (microtubule stabilizer in cancers. However, survivin-induced resistance to microtubule de-stabilizers such as Vinca alkaloids and Combretastatin A-4 (CA-4-related compounds were seldom demonstrated in the past. Furthermore, the question remains as to whether survivin plays a dominant role in processing cytokinesis or inhibiting caspases activity in cells treated with anti-mitotic compounds. The purpose of this study is to evaluate the effect of survivin on the resistance and susceptibility of human cancer cells to microtubule de-stabilizer-induced cell death. Results BPR0L075 is a CA-4 analog that induces microtubule de-polymerization and subsequent caspase-dependent apoptosis. To study the relationship between the expression of survivin and the resistance to microtubule de-stabilizers, a KB-derived BPR0L075-resistant cancer cell line, KB-L30, was generated for this study. Here, we found that survivin was over-expressed in the KB-L30 cells. Down-regulation of survivin by siRNA induced hyper-sensitivity to BPR0L075 in KB cells and partially re-stored sensitivity to BPR0L075 in KB-L30 cells. Western blot analysis revealed that down-regulation of survivin induced microtubule de-stabilization in both KB and KB-L30 cells. However, the same treatment did not enhance the down-stream caspase-3/-7 activities in BPR0L075-treated KB cells. Translocation of a caspase-independent apoptosis-related molecule, apoptosis-inducing factor (AIF, from cytoplasm to the nucleus was observed in survivin-targeted KB cells under BPR0L075 treatment. Conclusion In this study, survivin plays an important role in the

  17. Dynamics of mitochondrial transport in axons

    Directory of Open Access Journals (Sweden)

    Robert Francis Niescier

    2016-05-01

    Full Text Available The polarized structure and long neurites of neurons pose a unique challenge for proper mitochondrial distribution. It is widely accepted that mitochondria move from the cell body to axon ends and vice versa; however, we have found that mitochondria originating from the axon ends moving in the retrograde direction never reach to the cell body, and only a limited number of mitochondria moving in the anterograde direction from the cell body arrive at the axon ends of mouse hippocampal neurons. Furthermore, we have derived a mathematical formula using the Fokker-Planck equation to characterize features of mitochondrial transport, and the equation could determine altered mitochondrial transport in axons overexpressing parkin. Our analysis will provide new insights into the dynamics of mitochondrial transport in axons of normal and unhealthy neurons.

  18. Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus.

    Science.gov (United States)

    Huang, Jialing; Lazear, Helen M; Friedman, Harvey M

    2011-01-05

    The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

    International Nuclear Information System (INIS)

    Huang Jialing; Lazear, Helen M.; Friedman, Harvey M.

    2011-01-01

    The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.

  20. Axonal Actin Transport Driven By Metastable Actin Filaments

    Science.gov (United States)

    Chakrabarty, Nilaj; Ganguly, Archan; Roy, Subhojit; Jung, Peter

    Actin is one of the key constituents of the neuronal cytoskeleton and is responsible for driving important cellular processes like axon elongation. Axonal actin is synthesized in the cell body and transported at rates of 0.25 - 3 mm/day, as shown by in-vivo pulse-chase radiolabelling studies. However, the underlying transport mechanisms are unknown. Recent experiments in cultured neurons have revealed a dynamic network of metastable actin filaments (actin trails). Actin trails seem to originate from focal actin hotspots which colocalize with stationary endosomes. Interestingly, the number of actin trails extending anterogradely is higher than the ones extending retrogradely. We hypothesize that the bulk axonal transport of actin originates from this directional asymmetry of the number of actin trails. To test this, we constructed a computational model of actin trail growth and simulated the pulse-chase experiment. In our model, local, metastable trails, which grow with their barbed ends anchored to the hotspots, drive the bulk anterograde transport. Our results indicate that the observed bias of the nucleation probabilities and the elongation rate of actin trails are sufficient to drive the bulk transport of actin at rates that agree with in-vivo pulse chase experiments.

  1. Kar3Vik1 uses a minus-end directed powerstroke for movement along microtubules.

    Directory of Open Access Journals (Sweden)

    Julia Cope

    Full Text Available We have used cryo-electron microscopy (cryo-EM and helical averaging to examine the 3-D structure of the heterodimeric kinesin-14 Kar3Vik1 complexed to microtubules at a resolution of 2.5 nm. 3-D maps were obtained at key points in Kar3Vik1's nucleotide hydrolysis cycle to gain insight into the mechanism that this motor uses for retrograde motility. In all states where Kar3Vik1 maintained a strong interaction with the microtubule, we found, as observed by cryo-EM, that the motor bound with one head domain while the second head extended outwards. 3-D reconstructions of Kar3Vik1-microtubule complexes revealed that in the nucleotide-free state, the motor's coiled-coil stalk points toward the plus-end of the microtubule. In the ATP-state, the outer head is shown to undergo a large rotation that reorients the stalk ∼75° to point toward the microtubule minus-end. To determine which of the two heads binds to tubulin in each nucleotide state, we employed specific Nanogold®-labeling of Vik1. The resulting maps confirmed that in the nucleotide-free, ATP and ADP+Pi states, Kar3 maintains contact with the microtubule surface, while Vik1 extends away from the microtubule and tracks with the coiled-coil as it rotates towards the microtubule minus-end. While many previous investigations have focused on the mechanisms of homodimeric kinesins, this work presents the first comprehensive study of the powerstroke of a heterodimeric kinesin. The stalk rotation shown here for Kar3Vik1 is highly reminiscent of that reported for the homodimeric kinesin-14 Ncd, emphasizing the conservation of a mechanism for minus-end directed motility.

  2. Enhanced stability of microtubules contributes in the development of colchicine resistance in MCF-7 cells.

    Science.gov (United States)

    Rai, Ankit; Kapoor, Sonia; Naaz, Afsana; Kumar Santra, Manas; Panda, Dulal

    2017-05-15

    Understanding the mechanism of resistance to tubulin-targeted anticancer drugs is important for improved chemotherapy. In this work, a colchicine-resistant MCF-7 cell line (MCF-7 Col30 ) was generated by the gradual increment of colchicine treatment and the MCF-7 Col30 showed ∼8-fold resistance towards colchicine. MCF-7 Col30 cells showed ∼2.5-fold resistance against microtubule depolymerizing agents, vinblastine, and nocodazole. In contrast, it displayed more sensitivity towards paclitaxel, a microtubule-polymerizing agent. MCF-7 and MCF-7 Col30 cells showed similar sensitivity towards cisplatin. Further, the level of P-glycoprotein did not increase in MCF-7 Col30 cells. MCF-7 Col30 cells resisted the microtubule depolymerizing effects of colchicine. The time-lapse imaging of individual microtubules in live cells showed that the dynamics of microtubules in MCF-7 Col30 cells was suppressed as compared to the parent MCF-7 cells. The levels of tubulin acetylation and glutamylation increased in MCF-7 Col30 cells than the parent MCF-7 cells suggesting that microtubules are stabilized in MCF-7 Col30 cells. Interestingly, the level of βIII tubulin was increased by 2.3 folds whereas that of βII and βIV tubulin was decreased by 55 and 150%, respectively in MCF-7 Col30 cells. The results suggested that the changes in the level of β-tubulin isoforms and the post-translational modifications of microtubules altered the stability and dynamics of microtubules and contributed to the development of colchicine-resistance in MCF-7 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Solution structure of the microtubule-targeting COS domain of MID1.

    Science.gov (United States)

    Wright, Katharine M; Du, Haijuan; Dagnachew, Mesgana; Massiah, Michael A

    2016-08-01

    The human MID1 protein is required for the proper development during embryogenesis. Mutations of MID1 are associated with X-linked Opitz G syndrome, characterized by midline anomalies. MID1 associates with the microtubules and functions as an ubiquitin E3 ligase, targeting protein phosphatase 2A for ubiquitin-mediated regulation. The mechanism of microtubule association is not known. Recently, a 60-amino acid region termed the C-terminal subgroup One Signature (COS) box/domain was identified at the C-terminal end of the coiled-coil (CC) domain that facilitates microtubule localization. Insertion of the MID1 COS domain at the C-terminal end of the CC domain of a nonmicrotubule-associated TRIM protein confers microtubule localization. Here, we report the solution structure of the COS domain of MID1. The domain adopts a helix-loop-helix structure in which the N- and C-terminal ends are in close proximity. Hydrophobic residues stabilizing the interaction of the two α-helices form a central hydrophobic core. The loop separating the α-helices is structured, with two of its hydrophobic residues making contact with the central core. On the outer surface, positively charged residues form a distinct basic patch near the termini that we postulate is important for microtubule binding. A model of the structure of the preceding coiled-coil and COS domains (CC-COS) show that the COS domain forms a helical bundle at the C-terminal end of the CC domain similar to the spectrin-like fold observed with some known microtubule-binding proteins. Interestingly, the CC-COS domains bind to microtubules, demonstrating for the first time that MID1 can directly associate with the microtubules. Structural data are available in PDB database under the accession number 5IM8. © 2016 Federation of European Biochemical Societies.

  4. Vimentin intermediate filaments template microtubule networks to enhance persistence in cell polarity and directed migration

    OpenAIRE

    Gan, Zhuo; Ding, Liya; Burckhardt, Christoph J.; Lowery, Jason; Zaritsky, Assaf; Sitterley, Karlyndsay; Mota, Andressa; Costigliola, Nancy; Starker, Colby G.; Voytas, Daniel F.; Tytell, Jessica; Goldman, Robert D.; Danuser, Gaudenz

    2016-01-01

    Increased expression of vimentin intermediate filaments (VIF) enhances directed cell migration, but the mechanism behind VIF’s effect on motility is not understood. VIF interact with microtubules, whose organization contributes to polarity maintenance in migrating cells. Here we characterize the dynamic coordination of VIF and microtubule networks in wounded monolayers of Retinal Pigment Epithelial cells. By genome editing we fluorescently labelled endogenous vimentin and α-...

  5. Effects of tertiary amine local anesthetics on the assembly and disassembly of brain microtubules in vitro.

    Science.gov (United States)

    Genna, J M; Coffe, G; Pudles, J

    1980-09-01

    From kinetic and electron microscopy studies on the effects of procaine, tetracaine and dibucaine on the polymerization and depolymerization of the microtubules isolated from pig and rat brains the following results were obtained. 1. Procaine or tetracaine, at the concentration range of 0.5--20 mM and of 0.5--5 mM respectively, increases the rate of tubulin polymerization (24 degrees C or 37 degrees C) and of microtubule depolymerization (4 degrees C) as a linear function of the concentration of the anesthetics, while identical amounts of microtubules are formed. In the absence of microtubule-associated proteins the polymerization of tubulin is not induced by 10 mM procaine, furthermore, the critical concentration of microtubule proteins necessary for assembly into microtubules is not affected at this concentration level of the anesthetic. This suggests that procaine affects not the nucleation, but rather the elongation process. 2. Dibucaine, from 0.5 mM to 3 mM increases the lag time of the polymerization reaction, while from 0.5 mM to 2 mM it linearly decreases both tubulin polymerization (24 degrees C) and microtubule depolymerization (4 degrees C) rates. Dibucaine, up to mM concentration, does not affect the extent of tubulin polymerization; however, above this concentration it induces the formation of amorphous aggregates. 3. Procaine or tetracaine enhances the depolymerizing effect of calcium on microtubules. The half-maximal values for the depolymerizing effect of calcium were 0.96, 0.71 and 0.51 mM for the control, in the presence of 10 mM procaine and 5 mM tetracaine respectively.

  6. Identification of a TPX2-like microtubule-associated protein in Drosophila.

    Directory of Open Access Journals (Sweden)

    Gohta Goshima

    Full Text Available Chromosome segregation during mitosis and meiosis relies on the spindle and the functions of numerous microtubule-associated proteins (MAPs. One of the best-studied spindle MAPs is the highly conserved TPX2, which has been reported to have characteristic intracellular dynamics and molecular activities, such as nuclear localisation in interphase, poleward movement in the metaphase spindle, microtubule nucleation, microtubule stabilisation, microtubule bundling, Aurora A kinase activation, kinesin-5 binding, and kinesin-12 recruitment. This protein has been shown to be essential for spindle formation in every cell type analysed so far. However, as yet, TPX2 homologues have not been found in the Drosophila genome. In this study, I found that the Drosophila protein Ssp1/Mei-38 has significant homology to TPX2. Sequence conservation was limited to the putative spindle microtubule-associated region of TPX2, and intriguingly, D-TPX2 (Ssp1/Mei-38 lacks Aurora A- and kinesin-5-binding domains, which are highly conserved in other animal and plant species, including many insects such as ants and bees. D-TPX2 uniformly localised to kinetochore microtubule-enriched regions of the metaphase spindle in the S2 cell line, and it had microtubule binding and bundling activities in vitro. In comparison with other systems, the contribution of D-TPX2 to cell division seems to be minor; live cell imaging of microtubules and chromosomes after RNAi knockdown identified significant delay in chromosome congression in only 18% of the cells. Thus, while this conserved spindle protein is present in Drosophila, other mechanisms may largely compensate for its spindle assembly and chromosome segregation functions.

  7. Dynamic release of nuclear RanGTP triggers TPX2-dependent microtubule assembly during the apoptotic execution phase.

    Science.gov (United States)

    Moss, David K; Wilde, Andrew; Lane, Jon D

    2009-03-01

    During apoptosis, the interphase microtubule network is dismantled then later replaced by a novel, non-centrosomal microtubule array. These microtubules assist in the peripheral redistribution of nuclear fragments in the apoptotic cell; however, the regulation of apoptotic microtubule assembly is not understood. Here, we demonstrate that microtubule assembly depends upon the release of nuclear RanGTP into the apoptotic cytoplasm because this process is blocked in apoptotic cells overexpressing dominant-negative GDP-locked Ran (T24N). Actin-myosin-II contractility provides the impetus for Ran release and, consequently, microtubule assembly is blocked in blebbistatin- and Y27632-treated apoptotic cells. Importantly, the spindle-assembly factor TPX2 (targeting protein for Xklp2), colocalises with apoptotic microtubules, and siRNA silencing of TPX2, but not of the microtubule motors Mklp1 and Kid, abrogates apoptotic microtubule assembly. These data provide a molecular explanation for the assembly of the apoptotic microtubule network, and suggest important similarities with the process of RanGTP- and TPX2-mediated mitotic spindle formation.

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

    Science.gov (United States)

    Delphin, Christian; Bouvier, Denis; Seggio, Maxime; Couriol, Emilie; Saoudi, Yasmina; Denarier, Eric; Bosc, Christophe; Valiron, Odile; Bisbal, Mariano; Arnal, Isabelle; Andrieux, Annie

    2012-10-12

    Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.

  9. A macroscopic model of traffic jams in axons.

    Science.gov (United States)

    Kuznetsov, A V; Avramenko, A A

    2009-04-01

    The purpose of this paper is to develop a minimal macroscopic model capable of explaining the formation of traffic jams in fast axonal transport. The model accounts for the decrease of the number density of positively (and negatively) oriented microtubules near the location of the traffic jam due to formation of microtubule swirls; the model also accounts for the reduction of the effective velocity of organelle transport in the traffic jam region due to organelles falling off microtubule tracks more often in the swirl region. The model is based on molecular-motor-assisted transport equations and the hydrodynamic model of traffic jams in highway traffic. Parametric analyses of the model's predictions for various values of viscosity of the traffic flow, variance of the velocity distribution, diffusivity of microtubule-bound and free organelles, rate constants for binding to and detachment from microtubules, relaxation time, and average motor velocities of the retrograde and anterograde transport, are carried out.

  10. Tubulin cofactor B regulates microtubule densities during microglia transition to the reactive states

    International Nuclear Information System (INIS)

    Fanarraga, M.L.; Villegas, J.C.; Carranza, G.; Castano, R.; Zabala, J.C.

    2009-01-01

    Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulated in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes

  11. Emerging roles for microtubules in angiosperm pollen tube growth highlight new research cues

    Directory of Open Access Journals (Sweden)

    Alessandra eMoscatelli

    2015-02-01

    Full Text Available In plants, actin filaments have an important role in organelle movement and cytoplasmic streaming. Otherwise microtubules have a role in restricting organelles to specific areas of the cell and in maintaining organelle morphology. In somatic plant cells, microtubules also participate in cell division and morphogenesis, allowing cells to take their definitive shape in order to perform specific functions. In the latter case, microtubules influence assembly of the cell wall, controlling the delivery of enzymes involved in cellulose synthesis and of wall modulation material to the proper sites.In angiosperm pollen tubes, organelle movement is generally attributed to the acto-myosin system, the main role of which is in distributing organelles in the cytoplasm and in carrying secretory vesicles to the apex for polarized growth. Recent data on membrane trafficking suggests a role of microtubules in fine delivery and repositioning of vesicles to sustain pollen tube growth. This review examines the role of microtubules in secretion and endocytosis, highlighting new research cues regarding cell wall construction and pollen tube-pistil crosstalk, that help unravel the role of microtubules in polarized growth.

  12. Actin and microtubule networks contribute differently to cell response for small and large strains

    Science.gov (United States)

    Kubitschke, H.; Schnauss, J.; Nnetu, K. D.; Warmt, E.; Stange, R.; Kaes, J.

    2017-09-01

    Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

  13. Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

    Science.gov (United States)

    Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

    2015-08-10

    Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Hypothesis: NDL Proteins Function in Stress Responses by Regulating Microtubule Organization

    Directory of Open Access Journals (Sweden)

    Nisha eKhatri

    2015-10-01

    Full Text Available N-MYC DOWNREGULATED-LIKE proteins (NDL, members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart NDRG suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein (MAP which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants.

  15. Interplay between microtubule bundling and sorting factors ensures acentriolar spindle stability during C. elegans oocyte meiosis.

    Directory of Open Access Journals (Sweden)

    Timothy J Mullen

    2017-09-01

    Full Text Available In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1 bundles microtubules and KLP-18 (kinesin-12 likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.

  16. An active matter analysis of intracellular Active Transport

    Science.gov (United States)

    Wang, Bo; Chen, Kejia; Bae, Sung Chul; Granick, Steve

    2012-02-01

    Tens of thousands of fluorescence-based trajectories at nm resolution have been analyzed, regarding active transport along microtubules in living cells. The following picture emerges. Directed motion to pre-determined locations is certainly an attractive idea, but cannot be pre-programmed as to do so would sacrifice adaptability. The polarity of microtubules is inadequate to identify these directions in cells, and no other mechanism is currently known. We conclude that molecular motors carry cargo through disordered intracellular microtubule networks in a statistical way, with loud cellular ``noise'' both in directionality and speed. Programmed random walks describe how local 1D active transport traverses crowded cellular space efficiently, rapidly, minimizing the energy waste that would result from redundant activity. The mechanism of statistical regulation is not yet understood, however.

  17. Microtubule length dependence of motor traffic in cells.

    Science.gov (United States)

    Zhang, Yunxin

    2012-10-01

    Motor proteins in living cells, such as kinesin and dynein, can move processively along the microtubule (MT), and can also detach from or attach to MT stochastically. Experiments found that the traffic of motors along MT may be jammed; thus various theoretical models were designed to understand this process. However, previous studies mainly focused on motor attachment/detachment rate dependent properties. Leduc et al. recently found that the traffic jam of motor protein Kip3 depends on MT length (Proc. Natl. Acad. Sci. U.S.A. 109, 6100 (2012)). Therefore, this study discusses the MT length-dependent properties of motor traffic. The results showed that MT length has one critical value N(c); a traffic jam occurs only when MT length N > N(c). The jammed MT length increases with total MT length N , whereas the non-jammed MT length may not change monotonically with N . The critical value N(c) increases with motor detachment rate from MT, but decreases with motor attachment rate to MT. Therefore, the traffic of motors will be more likely to be jammed when the MT is long, motor detachment rate is high, and motor detachment rate is low.

  18. Augmented stress fiber arrays after cytopharmacologic disassembly of microtubules

    Energy Technology Data Exchange (ETDEWEB)

    Godman, G.C.; Tannenbaum, J.; Brett, J.B.

    1986-03-01

    Disruption of microtubules (mt) of bovine aortic endothelial (BAE) cells, and normal and transformed fibroblasts, by exposure to 2.5 ..mu..M colchicine; 12 ..mu..M vinblastine; or 1 ..mu..M nocodazole, for 5 or 20 hrs results in aggregation of vimentin-intermediate filament (IF) and the development of markedly augmented stress fiber (SF) arrays. After disassembly of mt, confluent BAE, with circumferential marginal microfilament bands and few central SF, develop dense ribbon-like SF arrays, and spontaneously transformed fibroblasts (tHmf-e), which before treatment are apolar or epithelioid and have few or no SF, acquire extensive organized SF arrays. The axially oriented SF span the entire cell length and terminate in vinculin-containing adhesion plaques, polarizing these cells. The visible increase in SF associated actin is not accompanied by an increase either in actin synthesis (determined from electropherograms after pulse labeling with (/sup 35/S)methionine), or content (DNAse I assay for total cell actin). The reorganization of actin into SF and the development of vinculin adhesion plaques is independent of protein synthesis and occurs in the presence of cycloheximide (10 ..mu..g/ml). These results suggest a role for mt and IF in the regulation of the organizational state of the actin-based cytoskeleton.

  19. Effects of microtubule mechanics on hydrolysis and catastrophes

    International Nuclear Information System (INIS)

    Müller, N; Kierfeld, J

    2014-01-01

    We introduce a model for microtubule (MT) 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 ∘ to 22 ∘ by hydrolysis of a dimer. This also affects the lateral interaction and bending energies and, thus, the mechanical equilibrium state of the MT. 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 MT 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. (papers)

  20. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

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

    1992-12-31

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 {mu}M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 {mu}M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  1. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

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

    1992-01-01

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 [mu]M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  2. MTBindingSim: simulate protein binding to microtubules.

    Science.gov (United States)

    Philip, Julia T; Pence, Charles H; Goodson, Holly V

    2012-02-01

    Many protein-protein interactions are more complex than can be accounted for by 1:1 binding models. However, biochemists have few tools available to help them recognize and predict the behaviors of these more complicated systems, making it difficult to design experiments that distinguish between possible binding models. MTBindingSim provides researchers with an environment in which they can rapidly compare different models of binding for a given scenario. It is written specifically with microtubule polymers in mind, but many of its models apply equally well to any polymer or any protein-protein interaction. MTBindingSim can thus both help in training intuition about binding models and with experimental design. MTBindingSim is implemented in MATLAB and runs either within MATLAB (on Windows, Mac or Linux) or as a binary without MATLAB (on Windows or Mac). The source code (licensed under the GNU General Public License) and binaries are freely available at http://mtbindingsim.googlecode.com. jphilip@nd.edu; cpence@nd.edu.

  3. Microtubule modification influences cellular response to amyloid-β exposure

    Directory of Open Access Journals (Sweden)

    Nicole Shamitko-Klingensmith

    2016-05-01

    Full Text Available During the normal aging process, cytoskeletal changes such as a reduction in density or disruption of cytoskeletal components occur that can affect neuronal function. As aging is the biggest risk factor for Alzheimer's disease (AD, this study sought to determine how microtubule (MT modification influences cellular response upon exposure to β-amyloid1-42 (Aβ1-42, which is implicated in AD. The MT networks of hypothalamic GT1-7 neurons were modified by common disrupting or stabilizing drugs, and then the physical and mechanical properties of the modified neurons were determined. The MT modified neurons were then exposed to Aβ1-42 and the ability of the neurons to cope with this exposure was determined by a variety of biochemical assays. Flow cytometry studies indicated that MT disruption reduced the binding of Aβ1-42 to the plasma membrane by 45% per cell compared to neurons with stabilized or unaltered MTs. Although the cells with disrupted MTs experienced less peptide-membrane binding, they experienced similar or increased levels of cytotoxicity caused by the Aβ1-42 exposure. In contrast, MT stabilization delayed toxicity caused by Aβ1-42. These results demonstrate that MT modification significantly influences the ability of neurons to cope with toxicity induced by Aβ1-42.

  4. Buckling of Microtubules on a 2D Elastic Medium.

    Science.gov (United States)

    Kabir, Arif Md Rashedul; Inoue, Daisuke; Afrin, Tanjina; Mayama, Hiroyuki; Sada, Kazuki; Kakugo, Akira

    2015-11-24

    We have demonstrated compression stress induced mechanical deformation of microtubules (MTs) on a two-dimensional elastic medium and investigated the role of compression strain, strain rate, and a MT-associated protein in the deformation of MTs. We show that MTs, supported on a two-dimensional substrate by a MT-associated protein kinesin, undergo buckling when they are subjected to compression stress. Compression strain strongly affects the extent of buckling, although compression rate has no substantial effect on the buckling of MTs. Most importantly, the density of kinesin is found to play the key role in determining the buckling mode of MTs. We have made a comparison between our experimental results and the 'elastic foundation model' that theoretically predicts the buckling behavior of MTs and its connection to MT-associated proteins. Taking into consideration the role of kinesin in altering the mechanical property of MTs, we are able to explain the buckling behavior of MTs by the elastic foundation model. This work will help understand the buckling mechanism of MTs and its connection to MT-associated proteins or surrounding medium, and consequently will aid in obtaining a meticulous scenario of the compression stress induced deformation of MTs in cells.

  5. Microtubule patterning in the presence of moving motor proteins.

    Science.gov (United States)

    White, D; de Vries, G; Martin, J; Dawes, A

    2015-10-07

    Cytoskeletal polymers such as microtubules (MTs) interact with motor proteins to form higher-order structures. In vitro experiments have shown that MT patterns such as asters, bundles, and vortices can form under the influence of a single type of dynamic motor protein. MTs also can form anti-parallel bundles, similar to bundles that form the mitotic spindle during cell division, under the influence of two types of moving motors with opposite directionality. Despite the importance of MT structures, their mechanism of formation is not yet understood. We develop an integro-partial differential equation model to describe the dynamic interactions between MTs and moving motor proteins. Our model takes into account motor protein speed, processivity, density, and directionality, as well as MT treadmilling and reorganization due to interactions with motors. Simulation results show that plus-end directed motor proteins can form vortex patterns at low motor density, while minus-end directed motor proteins form aster patterns at similar densities. Also, motor proteins with opposite directionality are able to organize MTs into anti-parallel bundles. Our model is able to provide a quantitative and qualitative description of MT patterning, providing insights into possible mechanisms of spindle formation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Microtubule-Targeting Agents Enter the Central Nervous System (CNS): Double-edged Swords for Treating CNS Injury and Disease.

    Science.gov (United States)

    Hur, Eun-Mi; Lee, Byoung Dae

    2014-12-01

    Microtubules have been among the most successful targets in anticancer therapy and a large number of microtubule-targeting agents (MTAs) are in various stages of clinical development for the treatment of several malignancies. Given that injury and diseases in the central nervous system (CNS) are accompanied by acute or chronic disruption of the structural integrity of neurons and that microtubules provide structural support for the nervous system at cellular and intracellular levels, microtubules are emerging as potential therapeutic targets for treating CNS disorders. It has been postulated that exogenous application of MTAs might prevent the breakdown or degradation of microtubules after injury or during neurodegeneration, which will thereby aid in preserving the structural integrity and function of the nervous system. Here we review recent evidence that supports this notion and also discuss potential risks of targeting microtubules as a therapy for treating nerve injury and neurodegenerative diseases.

  7. Role of the microtubule-targeting drug vinflunine on cell-cell adhesions in bladder epithelial tumour cells

    OpenAIRE

    Aparicio, Luis A; Castosa, Raquel; Haz-Conde, Mar; Rodríguez, Marta; Blanco, Moisés; Valladares, Manuel; Figueroa, Angélica

    2014-01-01

    Background Vinflunine (VFL) is a microtubule-targeting drug that suppresses microtubule dynamics, showing anti-metastatic properties both in vitro and in living cancer cells. An increasing body of evidence underlines the influence of the microtubules dynamics on the cadherin-dependent cell-cell adhesions. E-cadherin is a marker of epithelial-to-mesenchymal transition (EMT) and a tumour suppressor; its reduced levels in carcinoma are associated with poor prognosis. In this report, we investiga...

  8. Aβ-mediated spine changes in the hippocampus are microtubule-dependent and can be reversed by a subnanomolar concentration of the microtubule-stabilizing agent epothilone D

    Science.gov (United States)

    Penazzi, Lorène; Tackenberg, Christian; Ghori, Adnan; Golovyashkina, Nataliya; Niewidok, Benedikt; Selle, Karolin; Ballatore, Carlo; Smith, Amos B.; Bakota, Lidia; Brandt, Roland

    2016-01-01

    Dendritic spines represent the major postsynaptic input of excitatory synapses. Loss of spines and changes in their morphology correlate with cognitive impairment in Alzheimer’s disease (AD) and are thought to occur early during pathology. Therapeutic intervention at a preclinical stage of AD to modify spine changes might thus be warranted. To follow the development and to potentially interfere with spine changes over time, we established a long term ex vivo model from organotypic cultures of the hippocampus from APP transgenic and control mice. The cultures exhibit spine loss in principal hippocampal neurons, which closely resembles the changes occurring in vivo, and spine morphology progressively changes from mushroom-shaped to stubby. We demonstrate that spine changes are completely reversed within few days after blocking amyloid-β (Aβ) production with the gamma-secretase inhibitor DAPT. We show that the microtubule disrupting drug nocodazole leads to spine loss similar to Aβ expressing cultures and suppresses DAPT-mediated spine recovery in slices from APP transgenic mice. Finally, we report that epothilone D (EpoD) at a subnanomolar concentration, which slightly stabilizes microtubules in model neurons, completely reverses Aβ-induced spine loss and increases thin spine density. Taken together the data indicate that Aβ causes spine changes by microtubule destabilization and that spine recovery requires microtubule polymerization. Moreover, our results suggest that a low, subtoxic concentration of EpoD is sufficient to reduce spine loss during the preclinical stage of AD. PMID:26772969

  9. The plant formin AtFH4 interacts with both actin and microtubules, and contains a newly identified microtubule-binding domain

    Czech Academy of Sciences Publication Activity Database

    Deeks, M.J.; Fendrych, Matyáš; Smertenko, A.; Bell, K.S.; Oparka, K.; Cvrčková, F.; Žárský, Viktor; Hussey, P.J.

    2010-01-01

    Roč. 123, č. 8 (2010), s. 1209-1215 ISSN 0021-9533 R&D Projects: GA MŠk(CZ) LC06004; GA ČR GAP305/10/0433 Institutional research plan: CEZ:AV0Z50380511 Keywords : Actin regulating proteins * Membrane * Microtubule Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.290, year: 2010

  10. Localized buckling of a microtubule surrounded by randomly distributed cross linkers.

    Science.gov (United States)

    Jin, M Z; Ru, C Q

    2013-07-01

    Microtubules supported by surrounding cross linkers in eukaryotic cells can bear a much higher compressive force than free-standing microtubules. Different from some previous studies, which treated the surroundings as a continuum elastic foundation or elastic medium, the present paper develops a micromechanics numerical model to examine the role of randomly distributed discrete cross linkers in the buckling of compressed microtubules. First, the proposed numerical approach is validated by reproducing the uniform multiwave buckling mode predicted by the existing elastic-foundation model. For more realistic buckling of microtubules surrounded by randomly distributed cross linkers, the present numerical model predicts that the buckling mode is localized at one end in agreement with some known experimental observations. In particular, the critical force for localized buckling, predicted by the present model, is insensitive to microtubule length and can be about 1 order of magnitude lower than those given by the elastic-foundation model, which suggests that the elastic-foundation model may have overestimated the critical force for buckling of microtubules in vivo. In addition, unlike the elastic-foundation model, the present model can capture the effect of end conditions on the critical force and wavelength of localized buckling. Based on the known data of spacing and elastic constants of cross linkers available in literature, the critical force and wavelength of the localized buckling mode, predicted by the present model, are compared to some experimental data with reasonable agreement. Finally, two empirical formulas are proposed for the critical force and wavelength of the localized buckling of microtubules surrounded by cross linkers.

  11. Taking directions: the role of microtubule-bound nucleation in the self-organization of the plant cortical array

    International Nuclear Information System (INIS)

    Deinum, Eva E; Tindemans, Simon H; Mulder, Bela M

    2011-01-01

    The highly aligned cortical microtubule array of interphase plant cells is a key regulator of anisotropic cell expansion. Recent computational and analytical work has shown that the non-equilibrium self-organization of this structure can be understood on the basis of experimentally observed collisional interactions between dynamic microtubules attached to the plasma membrane. Most of these approaches assumed that new microtubules are homogeneously and isotropically nucleated on the cortical surface. Experimental evidence, however, shows that nucleation mostly occurs from other microtubules and under specific relative angles. Here, we investigate the impact of directed microtubule-bound nucleations on the alignment process using computer simulations. The results show that microtubule-bound nucleations can increase the degree of alignment achieved, decrease the timescale of the ordering process and widen the regime of dynamic parameters for which the system can self-organize. We establish that the major determinant of this effect is the degree of co-alignment of the nucleations with the parent microtubule. The specific role of sideways branching nucleations appears to allow stronger alignment while maintaining a measure of overall spatial homogeneity. Finally, we investigate the suggestion that observed persistent rotation of microtubule domains can be explained through a handedness bias in microtubule-bound nucleations, showing that this is possible only for an extreme bias and over a limited range of parameters

  12. Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function.

    Science.gov (United States)

    Otero, Maria G; Alloatti, Matías; Cromberg, Lucas E; Almenar-Queralt, Angels; Encalada, Sandra E; Pozo Devoto, Victorio M; Bruno, Luciana; Goldstein, Lawrence S B; Falzone, Tomás L

    2014-04-01

    Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo 'hitch-hiking' and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.

  13. Novel microtubule-targeting agents – the epothilones

    Directory of Open Access Journals (Sweden)

    Daniel R Budman

    2008-10-01

    Full Text Available Kit L Cheng, Thomas Bradley, Daniel R Budman1Monter Cancer Center, North Shore – LIJ Health Systems, Lake Success, New York, USAAbstract: Epothilones are a new class of antimicrotubule agents currently in clinical trials. Their chemical structures are distinct from taxanes and are more amenable to synthetic modification. Six epothilones have been studied in preclinical and clinical trials: patupilone (epothilone B, ixabepilone (BMS247550, BMS 310705, sagopilone (ZK-EPO, KOS-862 (epothilone D, and KOS-1584. In vitro data have shown increased potency in taxane-sensitive and taxane-resistant cancer cell lines. This enhanced cytotoxic effect has been attributed to epothilone being a poor substrate for p-glycoprotein drug resistance protein and having high affinity to the various β tubulin isoforms. Phase I clinical data have shown different dose-limiting toxicities for each of the epothilones. These effects are drug specific, dose specific, and schedule of administration specific. While diarrhea and myelosuppression are the dose-limiting toxicities for patupilone and BMS 310705, respectively, neurologic toxicity, as seen with taxanes, is the dose-limiting toxicity of ixabepilone, sagopilone, and KOS-862. In an effort to decrease neurologic toxicity, investigators have modified dosing schedules with limited success. Ixabepilone has the most mature clinical results with published phase II and III data, and regulatory approval for clinical use in the treatment of breast cancer. Ixabepilone has also been combined with other anticancer agents and has regulatory approval in combination with capecitabine for heavily treated breast cancer.Keywords: microtubule-targeting agents, epothilones, taxanes, ixabepilone

  14. Multiscale modeling of the nanomechanics of microtubule protofilaments.

    Science.gov (United States)

    Theisen, Kelly E; Zhmurov, Artem; Newberry, Maycee E; Barsegov, Valeri; Dima, Ruxandra I

    2012-07-26

    Large-size biomolecular systems that spontaneously assemble, disassemble, and self-repair by controlled inputs play fundamental roles in biology. Microtubules (MTs), which play important roles in cell adhesion and cell division, are a prime example. MTs serve as ″tracks″ for molecular motors, and their biomechanical functions depend on dynamic instability-a stochastic switching between periods of rapid growing and shrinking. This process is controlled by many cellular factors so that growth and shrinkage periods are correlated with the life cycle of a cell. Resolving the molecular basis for the action of these factors is of paramount importance for understanding the diverse functions of MTs. We employed a multiscale modeling approach to study the force-induced MT depolymerization by analyzing the mechanical response of a MT protofilament to external forces. We carried out self-organized polymer (SOP) model based simulations accelerated on Graphics Processing Units (GPUs). This approach enabled us to follow the mechanical behavior of the molecule on experimental time scales using experimental force loads. We resolved the structural details and determined the physical parameters that characterize the stretching and bending modes of motion of a MT protofilament. The central result is that the severing action of proteins, such as katanin and kinesin, can be understood in terms of their mechanical coupling to a protofilament. For example, the extraction of tubulin dimers from MT caps by katanin can be achieved by pushing the protofilament toward the axis of the MT cylinder, while the removal of large protofilaments curved into ″ram's horn″ structures by kinesin is the result of the outward bending of the protofilament. We showed that, at the molecular level, these types of deformations are due to the anisotropic, but homogeneous, micromechanical properties of MT protofilaments.

  15. Quantum mechanical aspects of cell microtubules: science fiction or realistic possibility?

    Science.gov (United States)

    Mavromatos, Nick E.

    2011-07-01

    Recent experimental research with marine algae points towards quantum entanglement at ambient temperature, with correlations between essential biological units separated by distances as long as 20 Angströms. The associated decoherence times, due to environmental influences, are found to be of order 400 fs. This prompted some authors to connect such findings with the possibility of some kind of quantum computation taking place in these biological entities: within the decoherence time scales, the cell "quantum calculates" the optimal "path" along which energy and signal would be transported more efficiently. Prompted by these experimental results, in this talk I remind the audience of a related topic proposed several years ago in connection with the possible rôle of quantum mechanics and/or field theory on dissipation-free energy transfer in microtubules (MT), which constitute fundamental cell substructures. The basic assumption was to view the cell MT as quantum electrodynamical cavities, providing sufficient isolation in vivo to enable the formation of electric-dipole quantum coherent solitonic states across the tubulin dimer walls. Crucial to this, were argued to be the electromagnetic interactions of the dipole moments of the tubulin dimers with the dipole quanta in the ordered water interiors of the MT, that play the rôle of quantum coherent cavity modes. Quantum entanglement between tubulin dimers was argued to be possible, provided there exists sufficient isolation from other environmental cell effects. The model was based on certain ferroelectric aspects of MT. Subsequent experiments in vitro could not confirm ferroelectricity at room temperatures, however they provided experimental measurements of the induced electric dipole moments of the MT under the influence of external electric fields. Nevertheless, this does not demonstrate that in vivo MT are not ferroelectric materials. More refined experiments should be done. In the talk I review the model and

  16. Quantum mechanical aspects of cell microtubules: science fiction or realistic possibility?

    Energy Technology Data Exchange (ETDEWEB)

    Mavromatos, Nick E, E-mail: nikolaos.mavromatos@kcl.ac.uk [CERN - Theory Division, CH-1211 Geneva 23, (Switzerland)

    2011-07-08

    Recent experimental research with marine algae points towards quantum entanglement at ambient temperature, with correlations between essential biological units separated by distances as long as 20 Angstroems. The associated decoherence times, due to environmental influences, are found to be of order 400 fs. This prompted some authors to connect such findings with the possibility of some kind of quantum computation taking place in these biological entities: within the decoherence time scales, the cell 'quantum calculates' the optimal 'path' along which energy and signal would be transported more efficiently. Prompted by these experimental results, in this talk I remind the audience of a related topic proposed several years ago in connection with the possible role of quantum mechanics and/or field theory on dissipation-free energy transfer in microtubules (MT), which constitute fundamental cell substructures. The basic assumption was to view the cell MT as quantum electrodynamical cavities, providing sufficient isolation in vivo to enable the formation of electric-dipole quantum coherent solitonic states across the tubulin dimer walls. Crucial to this, were argued to be the electromagnetic interactions of the dipole moments of the tubulin dimers with the dipole quanta in the ordered water interiors of the MT, that play the role of quantum coherent cavity modes. Quantum entanglement between tubulin dimers was argued to be possible, provided there exists sufficient isolation from other environmental cell effects. The model was based on certain ferroelectric aspects of MT. Subsequent experiments in vitro could not confirm ferroelectricity at room temperatures, however they provided experimental measurements of the induced electric dipole moments of the MT under the influence of external electric fields. Nevertheless, this does not demonstrate that in vivo MT are not ferroelectric materials. More refined experiments should be done. In the talk I

  17. Fission yeast mitochondria are distributed by dynamic microtubules in a motor-independent manner

    Science.gov (United States)

    Li, Tianpeng; Zheng, Fan; Cheung, Martin; Wang, Fengsong; Fu, Chuanhai

    2015-01-01

    The cytoskeleton plays a critical role in regulating mitochondria distribution. Similar to axonal mitochondria, the fission yeast mitochondria are distributed by the microtubule cytoskeleton, but this is regulated by a motor-independent mechanism depending on the microtubule associated protein mmb1p as the absence of mmb1p causes mitochondria aggregation. In this study, using a series of chimeric proteins to control the subcellular localization and motility of mitochondria, we show that a chimeric molecule containing a microtubule binding domain and the mitochondria outer membrane protein tom22p can restore the normal interconnected mitochondria network in mmb1-deletion (mmb1∆) cells. In contrast, increasing the motility of mitochondria by using a chimeric molecule containing a kinesin motor domain and tom22p cannot rescue mitochondria aggregation defects in mmb1∆ cells. Intriguingly a chimeric molecule carrying an actin binding domain and tom22p results in mitochondria associated with actin filaments at the actomyosin ring during mitosis, leading to cytokinesis defects. These findings suggest that the passive motor-independent microtubule-based mechanism is the major contributor to mitochondria distribution in wild type fission yeast cells. Hence, we establish that attachment to microtubules, but not kinesin-dependent movement and the actin cytoskeleton, is required and crucial for proper mitochondria distribution in fission yeast. PMID:26046468

  18. TIPsy tour guides: How microtubule plus-end tracking proteins (+TIPs facilitate axon guidance

    Directory of Open Access Journals (Sweden)

    Elizabeth A Bearce

    2015-06-01

    Full Text Available The growth cone is a dynamic cytoskeletal vehicle, which drives the end of a developing axon. It serves to interpret and navigate through the complex landscape and guidance cues of the early nervous system. The growth cone’s distinctive cytoskeletal organization offers a fascinating platform to study how extracellular cues can be translated into mechanical outgrowth and turning behaviors. While many studies of cell motility highlight the importance of actin networks in signaling, adhesion, and propulsion, both seminal and emerging works in the field have highlighted a unique and necessary role for microtubules in growth cone navigation. Here, we focus on the role of singular pioneer microtubules, which extend into the growth cone periphery and are regulated by a diverse family of microtubule plus-end tracking proteins (+TIPs. These +TIPs accumulate at the dynamic ends of microtubules, where they are well-positioned to encounter and respond to key signaling events downstream of guidance receptors, catalyzing immediate changes in microtubule stability and actin cross-talk, that facilitate both axonal outgrowth and turning events.

  19. Disruption of microtubule network rescues aberrant actin comets in dynamin2-depleted cells.

    Directory of Open Access Journals (Sweden)

    Yuji Henmi

    Full Text Available A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.

  20. The spectraplakin Short stop is an essential microtubule regulator involved in epithelial closure in Drosophila

    Science.gov (United States)

    Takács, Zsanett; Vilmos, Péter; Lénárt, Péter; Röper, Katja; Erdélyi, Miklós

    2017-01-01

    ABSTRACT Dorsal closure of the Drosophila embryonic epithelium provides an excellent model system for the in vivo analysis of molecular mechanisms regulating cytoskeletal rearrangements. In this study, we investigated the function of the Drosophila spectraplakin Short stop (Shot), a conserved cytoskeletal structural protein, during closure of the dorsal embryonic epithelium. We show that Shot is essential for the efficient final zippering of the opposing epithelial margins. By using isoform-specific mutant alleles and genetic rescue experiments with truncated Shot variants, we demonstrate that Shot functions as an actin–microtubule cross-linker in mediating zippering. At the leading edge of epithelial cells, Shot regulates protrusion dynamics by promoting filopodia formation. Fluorescence recovery after photobleaching (FRAP) analysis and in vivo imaging of microtubule growth revealed that Shot stabilizes dynamic microtubules. The actin- and microtubule-binding activities of Shot are simultaneously required in the same molecule, indicating that Shot is engaged as a physical crosslinker in this process. We propose that Shot-mediated interactions between microtubules and actin filaments facilitate filopodia formation, which promotes zippering by initiating contact between opposing epithelial cells. PMID:28062848

  1. Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK.

    Science.gov (United States)

    Cross, Marie K; Powers, Maureen A

    2011-03-01

    During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere-associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.

  2. Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen; Cosgrove, Daniel J.; Anderson, Charles T.

    2015-11-02

    Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.

  3. Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation.

    Directory of Open Access Journals (Sweden)

    Sumio Ishijima

    Full Text Available It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.

  4. Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

    Science.gov (United States)

    Borek, Weronika E.; Groocock, Lynda M.; Samejima, Itaru; Zou, Juan; de Lima Alves, Flavia; Rappsilber, Juri; Sawin, Kenneth E.

    2015-01-01

    Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation. PMID:26243668

  5. Disruption of microtubule network rescues aberrant actin comets in dynamin2-depleted cells.

    Science.gov (United States)

    Henmi, Yuji; Tanabe, Kenji; Takei, Kohji

    2011-01-01

    A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.

  6. Fission yeast cells undergo nuclear division in the absence of spindle microtubules.

    Directory of Open Access Journals (Sweden)

    Stefania Castagnetti

    2010-10-01

    Full Text Available Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.

  7. An essential role for katanin p80 and microtubule severing in male gamete production.

    Directory of Open Access Journals (Sweden)

    Liza O'Donnell

    Full Text Available Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line, we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development.

  8. TBCD links centriologenesis, spindle microtubule dynamics, and midbody abscission in human cells.

    Directory of Open Access Journals (Sweden)

    Mónica López Fanarraga

    2010-01-01

    Full Text Available Microtubule-organizing centers recruit alpha- and beta-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs A-E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into "centriolar rosettes". These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways.

  9. Identification and characterization of SSE15206, a microtubule depolymerizing agent that overcomes multidrug resistance

    KAUST Repository

    Manzoor, Safia

    2018-02-13

    Microtubules are highly dynamic structures that form spindle fibres during mitosis and are one of the most validated cancer targets. The success of drugs targeting microtubules, however, is often limited by the development of multidrug resistance. Here we describe the discovery and characterization of SSE15206, a pyrazolinethioamide derivative [3-phenyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide] that has potent antiproliferative activities in cancer cell lines of different origins and overcomes resistance to microtubule-targeting agents. Treatment of cells with SSE15206 causes aberrant mitosis resulting in G2/M arrest due to incomplete spindle formation, a phenotype often associated with drugs that interfere with microtubule dynamics. SSE15206 inhibits microtubule polymerization both in biochemical and cellular assays by binding to colchicine site in tubulin as shown by docking and competition studies. Prolonged treatment of cells with the compound results in apoptotic cell death [increased Poly (ADP-ribose) polymerase cleavage and Annexin V/PI staining] accompanied by p53 induction. More importantly, we demonstrate that SSE15206 is able to overcome resistance to chemotherapeutic drugs in different cancer cell lines including multidrug-resistant KB-V1 and A2780-Pac-Res cell lines overexpressing MDR-1, making it a promising hit for the lead optimization studies to target multidrug resistance.

  10. Amyloid-Beta Induced Changes in Vesicular Transport of BDNF in Hippocampal Neurons

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    Bianca Seifert

    2016-01-01

    Full Text Available The neurotrophin brain derived neurotrophic factor (BDNF is an important growth factor in the CNS. Deficits in transport of this secretory protein could underlie neurodegenerative diseases. Investigation of disease-related changes in BDNF transport might provide insights into the cellular mechanism underlying, for example, Alzheimer’s disease (AD. To analyze the role of BDNF transport in AD, live cell imaging of fluorescently labeled BDNF was performed in hippocampal neurons of different AD model systems. BDNF and APP colocalized with low incidence in vesicular structures. Anterograde as well as retrograde transport of BDNF vesicles was reduced and these effects were mediated by factors released from hippocampal neurons into the extracellular medium. Transport of BDNF was altered at a very early time point after onset of human APP expression or after acute amyloid-beta(1-42 treatment, while the activity-dependent release of BDNF remained unaffected. Taken together, extracellular cleavage products of APP induced rapid changes in anterograde and retrograde transport of BDNF-containing vesicles while release of BDNF was unaffected by transgenic expression of mutated APP. These early transport deficits might lead to permanently impaired brain functions in the adult brain.

  11. Regulation of kinetochore-microtubule attachments through homeostatic control during mitosis.

    Science.gov (United States)

    Godek, Kristina M; Kabeche, Lilian; Compton, Duane A

    2015-01-01

    Faithful chromosome segregation during mitosis is essential for genome integrity and is mediated by the bi-oriented attachment of replicated chromosomes to spindle microtubules through kinetochores. Errors in kinetochore-microtubule (k-MT) attachment that could cause chromosome mis-segregation are frequent and are corrected by the dynamic turnover of k-MT attachments. Thus, regulating the rate of spindle microtubule attachment and detachment to kinetochores is crucial for mitotic fidelity and is frequently disrupted in cancer cells displaying chromosomal instability. A model based on homeostatic principles involving receptors, a core control network, effectors and feedback control may explain the precise regulation of k-MT attachment stability during mitotic progression to ensure error-free mitosis.

  12. Microtubule dynamic instability: A new model with coupled GTP hydrolysis and multistep catastrophe

    Science.gov (United States)

    Bowne-Anderson, Hugo; Zanic, Marija; Kauer, Monika; Howard, Jonathon

    2013-01-01

    A key question in understanding microtubule dynamics is how GTP hydrolysis leads to catastrophe, the switch from slow growth to rapid shrinkage. We first provide a review of the experimental and modeling literature, and then present a new model of microtubule dynamics. We demonstrate that vectorial, random, and coupled hydrolysis mechanisms are not consistent with the dependence of catastrophe on tubulin concentration and show that, although single-protofilament models can explain many features of dynamics, they do not describe catastrophe as a multistep process. Finally, we present a new combined (coupled plus random hydrolysis) multiple-protofilament model that is a simple, analytically solvable generalization of a single-protofilament model. This model accounts for the observed lifetimes of growing microtubules, the delay to catastrophe following dilution and describes catastrophe as a multistep process. PMID:23532586

  13. Structural Basis of Microtubule Destabilization by Potent Auristatin Anti-Mitotics.

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    Andrew B Waight

    Full Text Available The auristatin class of microtubule destabilizers are highly potent cytotoxic agents against several cancer cell types when delivered as antibody drug conjugates. Here we describe the high resolution structures of tubulin in complex with both monomethyl auristatin E and F and unambiguously define the trans-configuration of both ligands at the Val-Dil amide bond in their tubulin bound state. Moreover, we illustrate how peptidic vinca-site agents carrying terminal carboxylate residues may exploit an observed extended hydrogen bond network with the M-loop Arg278 to greatly improve the affinity of the corresponding analogs and to maintain the M-loop in an incompatible conformation for productive lateral tubulin-tubulin contacts in microtubules. Our results highlight a potential, previously undescribed molecular mechanism by which peptidic vinca-site agents maintain unparalleled potency as microtubule-destabilizing agents.

  14. Quinolin-6-Yloxyacetamides Are Microtubule Destabilizing Agents That Bind to the Colchicine Site of Tubulin

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    Ashwani Sharma

    2017-06-01

    Full Text Available Quinolin-6-yloxyacetamides (QAs are a chemical class of tubulin polymerization inhibitors that were initially identified as fungicides. Here, we report that QAs are potent anti-proliferative agents against human cancer cells including ones that are drug-resistant. QAs act by disrupting the microtubule cytoskeleton and by causing severe mitotic defects. We further demonstrate that QAs inhibit tubulin polymerization in vitro. The high resolution crystal structure of the tubulin-QA complex revealed that QAs bind to the colchicine site on tubulin, which is targeted by microtubule-destabilizing agents such as colchicine and nocodazole. Together, our data establish QAs as colchicine-site ligands and explain the molecular mechanism of microtubule destabilization by this class of compounds. They further extend our structural knowledge on antitubulin agents and thus should aid in the development of new strategies for the rational design of ligands against multidrug-resistant cancer cells.

  15. Katanin spiral and ring structures shed light on power stroke for microtubule severing

    Energy Technology Data Exchange (ETDEWEB)

    Zehr, Elena; Szyk, Agnieszka; Piszczek, Grzegorz; Szczesna, Ewa; Zuo, Xiaobing; Roll-Mecak, Antonina

    2017-08-07

    Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases critical for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of 3D structures, their mechanism has remained poorly understood. We report the first X-ray structure of the monomeric AAA katanin module and cryo-EM reconstructions of the hexamer in two conformations. These reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to severing enzymes and critical for their function. Our reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes inter-protomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

  16. Reassessing the roles of PIN proteins and anticlinal microtubules during pavement cell morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Belteton, Samuel; Sawchuk, Megan G.; Donohoe, Bryon S.; Scarpella, Enrico; Szymanski, Daniel B.

    2017-11-30

    The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here we used Arabidopsis reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells, and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls.

  17. The Effect of the Crocus Sativus L. Carotenoid, Crocin, on the Polymerization of Microtubules, in Vitro

    Directory of Open Access Journals (Sweden)

    Hossein Zarei Jaliani

    2013-01-01

    Full Text Available Objective(s: Crocin, as the main carotenoid of saffron, has shown anti-tumor activity both in vitro and in vivo. Crocin might interact with cellular proteins and modulate their functions, but the exact target of this carotenoid and the other compounds of the saffron have not been discovered yet. Microtubular proteins, as one of the most important proteins inside the cells, have several functions in nearly all kinds of cellular processes. The aim of this study was to investigate whether crocin affects microtubule polymerization and tubulin structure. Materials and Methods: Microtubules were extracted from sheep brains after two cycles of temperature-dependant assembly-disassembly in the polymerization buffer (PMG. Then phosphocellulose P11 column was used to prepare MAP-free tubulin. Turbidimetric assay of microtubules was performed by incubation of tubulins at 37 ºC in PIPES buffer. To investigate the intrinsic fluorescence spectra of tubulins, the emission spectra of tryptophans was monitored. To test the interaction of crocin with tubulin in more details, ANS has been used. Results: Crocin extremely affected the tubulin polymerization and structure. Ultraviolet spectroscopy indicated that crocin increased polymerization of microtubules by nearly a factor of two. Fluorescence spectroscopic data also pointed to significant conformational changes of tubulin. Conclusion: We showed that crocin increased tubulin polymerization and microtubule nucleation rate and this effect was concentration dependant. After entering cell, crocin can modulate cellular proteins and their functions. Concerning the results of this study, crocin would be able to affect several cell processes through interaction with tubulin proteins or microtubules.

  18. Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein.

    Science.gov (United States)

    Sakato, Miho; King, Stephen M

    2003-10-31

    The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.

  19. A computational framework for cortical microtubule dynamics in realistically shaped plant cells

    KAUST Repository

    Chakrabortty, Bandan

    2018-02-02

    Plant morphogenesis is strongly dependent on the directional growth and the subsequent oriented division of individual cells. It has been shown that the plant cortical microtubule array plays a key role in controlling both these processes. This ordered structure emerges as the collective result of stochastic interactions between large numbers of dynamic microtubules. To elucidate this complex self-organization process a number of analytical and computational approaches to study the dynamics of cortical microtubules have been proposed. To date, however, these models have been restricted to two dimensional planes or geometrically simple surfaces in three dimensions, which strongly limits their applicability as plant cells display a wide variety of shapes. This limitation is even more acute, as both local as well as global geometrical features of cells are expected to influence the overall organization of the array. Here we describe a framework for efficiently simulating microtubule dynamics on triangulated approximations of arbitrary three dimensional surfaces. This allows the study of microtubule array organization on realistic cell surfaces obtained by segmentation of microscopic images. We validate the framework against expected or known results for the spherical and cubical geometry. We then use it to systematically study the individual contributions of global geometry, cell-edge induced catastrophes and cell-face induced stability to array organization in a cuboidal geometry. Finally, we apply our framework to analyze the highly non-trivial geometry of leaf pavement cells of Arabidopsis thaliana, Nicotiana benthamiana and Hedera helix. We show that our simulations can predict multiple features of the microtubule array structure in these cells, revealing, among others, strong constraints on the orientation of division planes.

  20. Interaction of epothilone B (patupilone) with microtubules as detected by two-dimensional solid-state NMR spectroscopy

    NARCIS (Netherlands)

    Kumar, A.; Heise, H.; Blommers, M. J. J.; Krastel, P.; Schmitt, E.; Petersen, F.; Jeganathan, S.; Mandelkow, E. -M; Carlomagno, T.; Griesinger, C.; Baldus, M.

    2010-01-01

    Solid evidence: Induction of the polymerization of β-tubulin dimers into microtubules by epothilones, such as patupilone, by an as yet unknown mechanism leads to the apoptosis of cancer cells. Solid-state NMR spectroscopy of patupilone bound to microtubules has now enabled the identification of

  1. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

    NARCIS (Netherlands)

    Gutierrez, R.; Lindeboom, J.J.; Paredez, A.R.; Emons, A.M.C.; Ehrhardt, D.W.

    2009-01-01

    Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is

  2. A Toll receptor–FoxO pathway represses Pavarotti/MKLP1 to promote microtubule dynamics in motoneurons

    Science.gov (United States)

    Liu, Nan

    2016-01-01

    FoxO proteins are evolutionarily conserved regulators of neuronal structure and function, yet the neuron-specific pathways within which they act are poorly understood. To elucidate neuronal FoxO function in Drosophila melanogaster, we first screened for FoxO’s upstream regulators and downstream effectors. On the upstream side, we present genetic and molecular pathway analyses indicating that the Toll-6 receptor, the Toll/interleukin-1 receptor domain adaptor dSARM, and FoxO function in a linear pathway. On the downstream side, we find that Toll-6–FoxO signaling represses the mitotic kinesin Pavarotti/MKLP1 (Pav-KLP), which itself attenuates microtubule (MT) dynamics. We next probed in vivo functions for this novel pathway and found that it is essential for axon transport and structural plasticity in motoneurons. We demonstrate that elevated expression of Pav-KLP underlies transport and plasticity phenotypes in pathway mutants, indicating that Toll-6–FoxO signaling promotes MT dynamics by limiting Pav-KLP expression. In addition to uncovering a novel molecular pathway, our work reveals an unexpected function for dynamic MTs in enabling rapid activity-dependent structural plasticity. PMID:27502486

  3. Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC, Microtubules and Lysosomal/Proteasomal Degradation

    Science.gov (United States)

    McHarg, Selina; Hopkins, Gemma; Lim, Lusiana; Garrod, David

    2014-01-01

    Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds. PMID:25291180

  4. CELL DIVISION CYCLE. Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80C.

    Science.gov (United States)

    Ji, Zhejian; Gao, Haishan; Yu, Hongtao

    2015-06-12

    The spindle checkpoint of the cell division cycle senses kinetochores that are not attached to microtubules and prevents precocious onset of anaphase, which can lead to aneuploidy. The nuclear division cycle 80 complex (Ndc80C) is a major microtubule receptor at the kinetochore. Ndc80C also mediates the kinetochore recruitment of checkpoint proteins. We found that the checkpoint protein kinase monopolar spindle 1 (Mps1) directly bound to Ndc80C through two independent interactions. Both interactions involved the microtubule-binding surfaces of Ndc80C and were directly inhibited in the presence of microtubules. Elimination of one such interaction in human cells caused checkpoint defects expected from a failure to detect unattached kinetochores. Competition between Mps1 and microtubules for Ndc80C binding thus constitutes a direct mechanism for the detection of unattached kinetochores. Copyright © 2015, American Association for the Advancement of Science.

  5. Evidence for multiple sensory circuits in the brain arising from the respiratory system: an anterograde viral tract tracing study in rodents.

    Science.gov (United States)

    McGovern, Alice E; Davis-Poynter, Nicholas; Yang, Seung-Kwon; Simmons, David G; Farrell, Michael J; Mazzone, Stuart B

    2015-11-01

    Complex sensations accompany the activation of sensory neurons within the respiratory system, yet little is known about the organization of sensory pathways in the brain that mediate these sensations. In the present study, we employ anterograde viral neuroanatomical tract tracing with isogenic self-reporting recombinants of HSV-1 strain H129 to map the higher brain regions in receipt of vagal sensory neurons arising from the trachea versus the lungs, and single-cell PCR to characterize the phenotype of sensory neurons arising from these two divisions of the respiratory tree. The results suggest that the upper and lower airways are predominantly innervated by sensory neurons derived from the somatic jugular and visceral nodose cranial ganglia, respectively. This coincides with central circuitry that is predominately somatic-like, arising from the trachea, and visceral-like, arising from the lungs. Although some convergence of sensory pathways was noted in preautonomic cell groups, this was notably absent in thalamic and cortical regions. These data support the notion that distinct afferent subtypes, via distinct central circuits, subserve sensations arising from the upper versus lower airways. The findings may explain why sensations arising from different levels of the respiratory tree are qualitatively and quantitatively unique.

  6. The kinesin-13 KLP10A motor regulates oocyte spindle length and affects EB1 binding without altering microtubule growth rates

    Directory of Open Access Journals (Sweden)

    Kevin K. Do

    2014-06-01

    Full Text Available Kinesin-13 motors are unusual in that they do not walk along microtubules, but instead diffuse to the ends, where they remove tubulin dimers, regulating microtubule dynamics. Here we show that Drosophila kinesin-13 klp10A regulates oocyte meiosis I spindle length and is haplo-insufficient – KLP10A, reduced by RNAi or a loss-of-function P element insertion mutant, results in elongated and mispositioned oocyte spindles, and abnormal cortical microtubule asters and aggregates. KLP10A knockdown by RNAi does not significantly affect microtubule growth rates in oocyte spindles, but, unexpectedly, EB1 binding and unbinding are slowed, suggesting a previously unobserved role for kinesin-13 in mediating EB1 binding interactions with microtubules. Kinesin-13 may regulate spindle length both by disassembling subunits from microtubule ends and facilitating EB1 binding to plus ends. We also observe an increased number of paused microtubules in klp10A RNAi knockdown spindles, consistent with a reduced frequency of microtubule catastrophes. Overall, our findings indicate that reduced kinesin-13 decreases microtubule disassembly rates and affects EB1 interactions with microtubules, rather than altering microtubule growth rates, causing spindles to elongate and abnormal cortical microtubule asters and aggregates to form.

  7. Exploring the effect of end-binding proteins and microtubule targeting chemotherapy drugs on microtubule dynamic instability.

    Science.gov (United States)

    White, Diana; Honoré, Stéphane; Hubert, Florence

    2017-09-21

    Microtubules (MTs) play a key role in normal cell development and are a primary target for many cancer chemotherapy MT targeting agents (MTAs). As such, understanding MT dynamics in the presence of such agents, as well as other proteins that alter MT dynamics, is extremely important. In general, MTs grow relatively slowly and shorten very fast (almost instantaneously), an event referred to as a catastrophe. These dynamics, referred to as dynamic instability, have been studied in both experimental and theoretical settings. In the presence of MTAs, it is well known that such agents work by suppressing MT dynamics, either by promoting MT polymerization or promoting MT depolymerization. However, recent in vitro experiments show that in the presence of end-binding proteins (EBs), low doses of MTAs can increase MT dynamic instability, rather than suppress it. Here, we develop a novel mathematical model, to describe MT and EB dynamics, something which has not been done in a theoretical setting. Our MT model is based on previous modeling efforts, and consists of a pair of partial differential equations to describe length distributions for growing and shortening MT populations, and an ordinary differential equation (ODE) system to describe the time evolution for concentrations of GTP- and GDP-bound tubulin. A new extension of our approach is the use of an integral term, rather than an advection term, to describe very fast MT shortening events. Further, we introduce an ODE system to describe the binding and unbinding of EBs with MTs. To compare simulation results with experiment, we define novel mathematical expressions for time- and distance-based catastrophe frequencies. These quantities help to define MT dynamics in in vivo and in vitro settings. Simulation results show that increasing concentrations of EBs work to increase time-based catastrophe while distance-based catastrophe is less affected by changes in EB concentration, a result that is consistent with experiment

  8. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    Science.gov (United States)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  9. Vesicular Axonal Transport is Modified In Vivo by Tau Deletion or Overexpression in Drosophila

    Directory of Open Access Journals (Sweden)

    Yasmina Talmat-Amar

    2018-03-01

    Full Text Available Structural microtubule associated protein Tau is found in high amount in axons and is involved in several neurodegenerative diseases. Although many studies have highlighted the toxicity of an excess of Tau in neurons, the in vivo understanding of the endogenous role of Tau in axon morphology and physiology is poor. Indeed, knock-out mice display no strong cytoskeleton or axonal transport phenotype, probably because of some important functional redundancy with other microtubule-associated proteins (MAPs. Here, we took advantage of the model organism Drosophila, which genome contains only one homologue of the Tau/MAP2/MAP4 family to decipher (endogenous Tau functions. We found that Tau depletion leads to a decrease in microtubule number and microtubule density within axons, while Tau excess leads to the opposite phenotypes. Analysis of vesicular transport in tau mutants showed altered mobility of vesicles, but no change in the total amount of putatively mobile vesicles, whereas both aspects were affected when Tau was overexpressed. In conclusion, we show that loss of Tau in tau mutants not only leads to a decrease in axonal microtubule density, but also impairs axonal vesicular transport, albeit to a lesser extent compared to the effects of an excess of Tau.

  10. XMAP215, XKCM1, NuMA, and cytoplasmic dynein are required for the assembly and organization of the transient microtubule array during the maturation of Xenopus oocytes.

    Science.gov (United States)

    Becker, Bret E; Romney, S Joshua; Gard, David L

    2003-09-15

    During the maturation of Xenopus oocytes, a transient microtubule array (TMA) is nucleated from a novel MTOC near the base of the germinal vesicle. The MTOC-TMA transports the meiotic chromosomes to the animal cortex, where it serves as the precursor to the first meiotic spindle. To understand more fully the assembly of the MTOC-TMA, we used confocal immunofluorescence microscopy to examine the localization and function of XMAP215, XKCM1, NuMA, and cytoplasmic dynein during oocyte maturation. XMAP215, XKCM1, and NuMA were all localized to the base of the MTOC-TMA and the meiotic spindle. Microinjection of anti-XMAP215 inhibited microtubule (MT) assembly during oocyte maturation, disrupting assembly of the MTOC-TMA and subsequent assembly of the first meiotic spindle. In contrast, microinjection of anti-XKCM1 promoted MT assembly throughout the cytoplasm, disrupting organization of the MTOC-TMA and meiotic spindle. Finally, microinjection of anti-dynein or anti-NuMA disrupted the organization of the MTOC-TMA and subsequent assembly of the meiotic spindles. These results suggest that XMAP215 and XKCM1 act antagonistically to regulate MT assembly and organization during maturation of Xenopus oocytes, and that dynein and NuMA are required for organization of the MTOC-TMA.

  11. Measuring Pushing and Braking Forces Generated by Ensembles of Kinesin-5 Crosslinking Two Microtubules.

    Science.gov (United States)

    Shimamoto, Yuta; Forth, Scott; Kapoor, Tarun M

    2015-09-28

    The proper organization of the microtubule-based mitotic spindle is proposed to depend on nanometer-sized motor proteins generating forces that scale with a micron-sized geometric feature, such as microtubule overlap length. However, it is unclear whether such regulation can be achieved by any mitotic motor protein. Here, we employ an optical-trap- and total internal reflection fluorescence (TIRF)-based assay to show that ensembles of kinesin-5, a conserved mitotic motor protein, can push apart overlapping antiparallel microtubules to generate a force whose magnitude scales with filament overlap length. We also find that kinesin-5 can produce overlap-length-dependent "brake-like" resistance against relative microtubule sliding in both parallel and antiparallel geometries, an activity that has been suggested by cell biological studies but had not been directly measured. Together, these findings, along with numerical simulations, reveal how a motor protein can function as an analog converter, "reading" simple geometric and dynamic features in cytoskeletal networks to produce regulated force outputs. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. CYK4 promotes antiparallel microtubule bundling by optimizing MKLP1 neck conformation.

    Directory of Open Access Journals (Sweden)

    Tim Davies

    2015-04-01

    Full Text Available Centralspindlin, a constitutive 2:2 heterotetramer of MKLP1 (a kinesin-6 and the non-motor subunit CYK4, plays important roles in cytokinesis. It is crucial for the formation of central spindle microtubule bundle structure. Its accumulation at the central antiparallel overlap zone is key for recruitment and regulation of downstream cytokinesis factors and for stable anchoring of the plasma membrane at the midbody. Both MKLP1 and CYK4 are required for efficient microtubule bundling. However, the mechanism by which CYK4 contributes to this is unclear. Here we performed structural and functional analyses of centralspindlin using high-speed atomic force microscopy, Fӧrster resonance energy transfer analysis, and in vitro reconstitution. Our data reveal that CYK4 binds to a globular mass in the atypically long MKLP1 neck domain between the catalytic core and the coiled coil and thereby reconfigures the two motor domains in the MKLP1 dimer to be suitable for antiparallel microtubule bundling. Our work provides insights into the microtubule bundling during cytokinesis and into the working mechanisms of the kinesins with non-canonical neck structures.

  13. Small Molecules Revealed in a Screen Targeting Epithelial Scattering Are Inhibitors of Microtubule Polymerization.

    Science.gov (United States)

    Hoj, Taylor H; Robinson, Ryan J; Burton, Jason C; Densley-Ure, Rachel A; Olson, Tyler V; Williams, Logan K; Coward, Lori; Gorman, Greg; Hansen, Marc D H

    2016-08-01

    Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in the detachment of cell-cell junctions and initiation of cell migration. Instead of coordinating collective cell behavior within a tissue, cells become solitary and have few cell-cell interactions. Since epithelial scattering is recapitulated in cancer progression and since HGF signaling drives cancer metastasis in many cases, inhibitors of HGF signaling have been proposed to act as anticancer agents. We previously sought to better understand critical components required for HGF-induced epithelial scattering by performing a forward chemical genetics screen, which resulted in the identification of compounds with no previously reported biological activity that we report here. In efforts to determine the mechanism of these compounds, we find that many compounds have broad antiproliferative effects on cancer cell lines by arrest of cell division in G2/M with minimal induction of apoptosis. This effect is reminiscent of microtubule-targeting agents, and we find that several of these scaffolds directly inhibit microtubule polymerization. Compounds are assessed for their toxicity and pharmacokinetics in vivo. The identification of novel small-molecule inhibitors of microtubule polymerization highlights the role of the microtubule cytoskeleton in HGF-induced epithelial scattering. © 2016 Society for Laboratory Automation and Screening.

  14. Nod factors alter the microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair reorientation

    NARCIS (Netherlands)

    Sieberer, B.; Timmers, A.C.J.; Emons, A.M.C.

    2005-01-01

    The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the

  15. Binding of dihydroxynaphthyl aryl ketones to tubulin colchicine site inhibits microtubule assembly.

    Science.gov (United States)

    Gutierrez, Eunices; Benites, Julio; Valderrama, Jaime A; Calderon, Pedro Buc; Verrax, Julien; Nova, Esteban; Villanelo, Felipe; Maturana, Daniel; Escobar, Cristian; Lagos, Rosalba; Monasterio, Octavio

    2015-10-23

    Dihydroxynaphthyl aryl ketones 1-5 have been evaluated for their abilities to inhibit microtubule assembly and the binding to tubulin. Compounds 3, 4 and 5 displayed competitive inhibition against colchicine binding, and docking analysis showed that they bind to the tubulin colchicine-binding pocket inducing sheets instead of microtubules. Remarkable differences in biological activity observed among the assayed compounds seem to be related to the structure and position of the aryl substituent bonded to the carbonyl group. Compounds 2, 3 and 4, which contain a heterocyclic ring, presented higher affinity for tubulin compared to the carbocyclic analogue 5. Compound 4 showed the best affinity of the series, with an IC50 value of 2.1 μM for microtubule polymerization inhibition and a tubulin dissociation constant of 1.0 ± 0.2 μM, as determined by thermophoresis. Compound 4 was more efficacious in disrupting microtubule assembly in vitro than compound 5 although it contains the trimethoxyphenyl ring present in colchicine. Hydrogen bonds with Asn101 of α-tubulin seem to be responsible for the higher affinity of compound 4 respects to the others. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Deformation pattern in vibrating microtubule: Structural mechanics study based on an atomistic approach

    Czech Academy of Sciences Publication Activity Database

    Havelka, Daniel; Deriu, M.A.; Cifra, Michal; Kučera, Ondřej

    2017-01-01

    Roč. 7, č. 1 (2017), č. článku 4227. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA15-17102S Institutional support: RVO:67985882 Keywords : Continuum model * Protein microtubules * Molecular-dymamics Subject RIV: BO - Biophysics OBOR OECD: Biophysics Impact factor: 4.259, year: 2016

  17. Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing.

    Directory of Open Access Journals (Sweden)

    Tatsuroh Sugiyama

    Full Text Available Cells migrate by extending pseudopods such as lamellipodia and blebs. Although the signals leading to lamellipodia extension have been extensively investigated, those for bleb extension remain unclear. Here, we investigated signals for blebbing in Dictyostelium cells using a newly developed assay to induce blebbing. When cells were cut into two pieces with a microneedle, the anucleate fragments vigorously extended blebs. This assay enabled us to induce blebbing reproducibly, and analyses of knockout mutants and specific inhibitors identified candidate molecules that regulate blebbing. Blebs were also induced in anucleate fragments of leukocytes, indicating that this assay is generally applicable to animal cells. After cutting, microtubules in the anucleate fragments promptly depolymerized, followed by the extension of blebs. Furthermore, when intact cells were treated with a microtubule inhibitor, they frequently extended blebs. The depolymerization of microtubules induced the delocalization of inositol lipid phosphatidylinositol 3,4,5-trisphosphate from the cell membrane. PI3 kinase-null cells frequently extended blebs, whereas PTEN-null cells extended fewer blebs. From these observations, we propose a model in which microtubules play a critical role in bleb regulation via inositol lipid metabolism.

  18. NuMA distribution and microtubule configuration in rabbit oocytes and cloned embryos.

    Science.gov (United States)

    Yan, Li-Ying; Huang, Jun-Cheng; Zhu, Zi-Yu; Lei, Zi-Li; Shi, Li-Hong; Nan, Chang-Long; Zhao, Zhen-Jun; Ouyang, Ying-Chun; Song, Xiang-Fen; Sun, Qing-Yuan; Chen, Da-Yuan

    2006-12-01

    The assembly of microtubules and the distribution of NuMA were analyzed in rabbit oocytes and early cloned embryos. Alpha-tubulin was localized around the periphery of the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), multi-arrayed microtubules were found tightly associated with the condensed chromosomes and assembled into spindles. After the enucleated oocyte was fused with a fibroblast, microtubules were observed around the introduced nucleus in most reconstructed embryos and formed a transient spindle 2-4 h post-fusion (hpf). A mass of microtubules surrounded the swollen pseudo-pronucleus 5 hpf and a normal spindle was formed 13 hpf in cloned embryos. NuMAwas detected in the nucleus in germinal vesicle-stage oocytes, and it was concentrated at the spindle poles in both meiotic and mitotic metaphase. In both donor cell nucleus and enucleated oocyte cytoplasm, NuMA was not detected, while NuMA reappeared in pseudo-pronucleus as reconstructed embryo development proceeded. However, no evident NuMA staining was observed in the poles of transient spindle and first mitotic spindle in nuclear transfer eggs. These results indicate that NuMA localization and its spindle pole tethering function are different during rabbit oocyte meiosis and cloned embryo mitosis.

  19. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    International Nuclear Information System (INIS)

    Swanson, J.; Bushnell, A.; Silverstein, S.C.

    1987-01-01

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of ≅ 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4 0 C or in medium containing 5 μM colchicine or nocodazole at 37 0 C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37 0 C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures

  20. Aluminum ions inhibit phospholipase D in a microtubule-dependent manner

    Czech Academy of Sciences Publication Activity Database

    Pejchar, Přemysl; Pleskot, R.; Schwarzerová, K.; Martinec, Jan; Valentová, O.; Novotná, Z.

    2008-01-01

    Roč. 32, č. 5 (2008), s. 554-556 ISSN 1065-6995 R&D Projects: GA ČR GA522/05/0340 Institutional research plan: CEZ:AV0Z50380511 Keywords : Aluminum toxicity * Phospholipase D * Microtubules Subject RIV: ED - Physiology Impact factor: 1.619, year: 2008

  1. Emission of mitochondrial biophotons and their effect on electrical activity of membrane via microtubules

    Czech Academy of Sciences Publication Activity Database

    Rahmana, M.; Tuszynski, J. A.; Bokkon, I.; Cifra, Michal; Sardar, P.; Salari, V.

    2011-01-01

    Roč. 10, č. 1 (2011), 65-88 ISSN 0219-6352 R&D Projects: GA ČR GPP102/10/P454 Institutional research plan: CEZ:AV0Z20670512 Keywords : bioelectric phenomena * cellular biophysics * microtubules Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 0.761, year: 2011

  2. Activation of tubulin assembly into microtubules upon a series of repeated femtosecond laser impulses

    NARCIS (Netherlands)

    Tulub, AA; Stefanov, VE

    2004-01-01

    Tubulin, a globular protein, mostly distributed in nature in the dimeric alpha, beta form, can polymerize in vivo and in vitro into microtubules-longitudinal dynamic assemblies, involved in numerous cellular functions, including cell division and signaling. Tubulin polymerization starts upon binding

  3. Are coiled-coils of dimeric kinesins unwound during their walking on microtubule?

    Science.gov (United States)

    Duan, Zhao-Wen; Xie, Ping; Li, Wei; Wang, Peng-Ye

    2012-01-01

    Dimeric kinesin motor proteins such as homodimeric kinesin-1, homodimeric Ncd and heterodimeric Kar3/Vik1are composed of two head domains which are connected together by a rod-shaped, coiled-coil stalk. Despite the extensive and intensive studies on structures, kinetics, dynamics and walking mechanism of the dimers, whether their coiled-coils are unwound or not during their walking on the microtubule is still an unclear issue. Here, we try to clarify this issue by using molecular dynamics simulations. Our simulation results showed that, for Ncd, a large change in potential of mean force is required to unwind the coiled-coil by only several pairs of residues. For both Ncd and kinesin-1, the force required to initiate the coiled-coil unwinding is larger than that required for unfolding of the single [Formula: see text]-helix that forms the coiled-coil or is larger than that required to unwind the DNA duplex, which is higher than the unbinding force of the kinesin head from the microtubule in strong microtubule-binding states. Based on these results and the comparison of the sequence between the coiled-coil of Kar3/Vik1 and those of Ncd and kinesin-1, it was deduced that the coiled-coil of the Kar3/Vik1 should also be very stable. Thus, we concluded that the coiled-coils of kinesin-1, Ncd and Kar3/Vik1 are almost impossible to unwind during their walking on the microtubule.

  4. Microtubule Reorganization during Mitosis and Cytokinesis: Lessons Learned from Developing Microgametophytes in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bo eLiu

    2011-07-01

    Full Text Available In angiosperms, mitosis and cytokinesis take place in the absence of structurally defined microtubule-organizing centers and the underlying mechanisms are largely unknown. In the spindle and phragmoplast, microtubule reorganization depends on microtubule-interacting factors like the -tubulin complex. Because of their critical functions in cell division, loss-of-function mutations in the corresponding genes are often homozygous or sporophytic lethal. However, a number of mutations like gem1, gcp2, and nedd1 can be maintained in heterozygous mutants in Arabidopsis thaliana. When mutant microspores produced by a heterozygous parent undergo pollen mitosis I, they are amenable for phenotypic characterization by fluorescence microscopy. The results would allow us to pinpoint at specific functions of particular proteins in microtubule reorganization that are characteristic to specific stages of mitosis and cytokinesis. Conclusions made in the developing microgametophytes can be extrapolated to somatic cells regarding mechanisms that regulate nuclear migration, spindle pole formation, phragmoplast assembly, and cell division plane determination.

  5. Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo.

    Directory of Open Access Journals (Sweden)

    Janina Baumbach

    2015-05-01

    Full Text Available Acentriolar microtubule organizing centers (aMTOCs are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2--the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems. We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

  6. Phospholipase D family interactions with the cytoskeleton: isoform delta promotes plasma membrane anchoring of cortical microtubules

    Czech Academy of Sciences Publication Activity Database

    Andreeva, Z.; Ho, A. Y. Y.; Barthet, M. M.; Potocký, Martin; Bezvoda, R.; Žárský, Viktor; Marc, J.

    2009-01-01

    Roč. 36, č. 7 (2009), s. 600-612 ISSN 1445-4408 R&D Projects: GA AV ČR IAA601110916 Institutional research plan: CEZ:AV0Z50380511 Keywords : Allium * Arabidopsis * F-actin-microtubule interactions Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.678, year: 2009

  7. Effects of ultraviolet radiation on microtubule organisation and morphogenesis in plants

    International Nuclear Information System (INIS)

    Staxen, I.

    1994-09-01

    The involvement of the cytoskeleton in the development of somatic embryos was studied in Larix x eurolepis. Protoplasts were isolated from both somatic embryo-regenerating and non-generating cultures and fractionated on a discontinuous Percoll density gradient, whereby a highly embryogenic protoplast fraction could be enriched. Protoplasts of two cell lines of Larix eurolepis, one with regenerating potential and one lacking this potential, were compared. In contrast to the non-regenerating line were a protoplast-like organisation of the cortical microtubules was maintained, re-organisation of this microtubular network occurred in the regenerable line after only three days of culture, indicating that organised growth was occurring. However, this early organisation of cortical microtubules may not always be a valid marker for regenerable and non-regenerable material. In order to investigate the effect of ultraviolet-B (UV-B, 280-320 nm) radiation on the microtubule cytoskeleton, protoplasts were isolated from leaves of Petunia hybrida and subjected to four different doses of UV-B radiation. The organisation of the microtubules and the progression of the cells through the cell cycle was observed at 0, 24, 48 and 72 h after irradiation. UV-B induced breaks in the cortical microtubules resulting in shorter fragments with increasing amounts of radiation. Also, the division of the protoplasts was delayed, which was related to the absence of an microtubule network. Whole Petunia plants were grown in growth chambers in the presence and absence of UV-B. The plants responded to UV-B with increased rates of CO 2 assimilation, a 60% increase in UV-screening compounds and the changes in the morphology of the leaves that were reflected in a 70-100% increase in leaf area and 20% decrease in leaf thickness. The microtubules of the epidermal cells was not affected by UV-B, nor was the number of epidermal cells (per unit area). The increase in leaf area in the UV-treated plants

  8. Colchicine Depolymerizes Microtubules, Increases Junctophilin-2, and Improves Right Ventricular Function in Experimental Pulmonary Arterial Hypertension.

    Science.gov (United States)

    Prins, Kurt W; Tian, Lian; Wu, Danchen; Thenappan, Thenappan; Metzger, Joseph M; Archer, Stephen L

    2017-05-31

    Pulmonary arterial hypertension (PAH) is a lethal disease characterized by obstructive pulmonary vascular remodeling and right ventricular (RV) dysfunction. Although RV function predicts outcomes in PAH, mechanisms of RV dysfunction are poorly understood, and RV-targeted therapies are lacking. We hypothesized that in PAH, abnormal microtubular structure in RV cardiomyocytes impairs RV function by reducing junctophilin-2 (JPH2) expression, resulting in t-tubule derangements. Conversely, we assessed whether colchicine, a microtubule-depolymerizing agent, could increase JPH2 expression and enhance RV function in monocrotaline-induced PAH. Immunoblots, confocal microscopy, echocardiography, cardiac catheterization, and treadmill testing were used to examine colchicine's (0.5 mg/kg 3 times/week) effects on pulmonary hemodynamics, RV function, and functional capacity. Rats were treated with saline (n=28) or colchicine (n=24) for 3 weeks, beginning 1 week after monocrotaline (60 mg/kg, subcutaneous). In the monocrotaline RV, but not the left ventricle, microtubule density is increased, and JPH2 expression is reduced, with loss of t-tubule localization and t-tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves t-tubule morphology in RV cardiomyocytes. Colchicine therapy diminishes RV hypertrophy, improves RV function, and enhances RV-pulmonary artery coupling. Colchicine reduces small pulmonary arteriolar thickness and improves pulmonary hemodynamics. Finally, colchicine increases exercise capacity. Monocrotaline-induced PAH causes RV-specific derangement of microtubules marked by reduction in JPH2 and t-tubule disarray. Colchicine reduces microtubule density, increases JPH2 expression, and improves both t-tubule architecture and RV function. Colchicine also reduces adverse pulmonary vascular remodeling. These results provide biological plausibility for a clinical trial to repurpose colchicine as a RV-directed therapy for PAH

  9. Biochemical and structural insights into microtubule perturbation by CopN from Chlamydia pneumoniae.

    Science.gov (United States)

    Nawrotek, Agata; Guimarães, Beatriz G; Velours, Christophe; Subtil, Agathe; Knossow, Marcel; Gigant, Benoît

    2014-09-05

    Although the actin network is commonly hijacked by pathogens, there are few reports of parasites targeting microtubules. The proposed member of the LcrE protein family from some Chlamydia species (e.g. pCopN from C. pneumoniae) binds tubulin and inhibits microtubule assembly in vitro. From the pCopN structure and its similarity with that of MxiC from Shigella, we definitively confirm CopN as the Chlamydia homolog of the LcrE family of bacterial proteins involved in the regulation of type III secretion. We have also investigated the molecular basis for the pCopN effect on microtubules. We show that pCopN delays microtubule nucleation and acts as a pure tubulin-sequestering protein at steady state. It targets the β subunit interface involved in the tubulin longitudinal self-association in a way that inhibits nucleotide exchange. pCopN contains three repetitions of a helical motif flanked by disordered N- and C-terminal extensions. We have identified the pCopN minimal tubulin-binding region within the second and third repeats. Together with the intriguing observation that C. trachomatis CopN does not bind tubulin, our data support the notion that, in addition to the shared function of type III secretion regulation, these proteins have evolved different functions in the host cytosol. Our results provide a mechanistic framework for understanding the C. pneumoniae CopN-specific inhibition of microtubule assembly. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

    Science.gov (United States)

    Kudalkar, Emily M.; Scarborough, Emily A.; Umbreit, Neil T.; Zelter, Alex; Gestaut, Daniel R.; Riffle, Michael; Johnson, Richard S.; MacCoss, Michael J.; Asbury, Charles L.; Davis, Trisha N.

    2015-01-01

    Multiple protein subcomplexes of the kinetochore cooperate as a cohesive molecular unit that forms load-bearing microtubule attachments that drive mitotic chromosome movements. There is intriguing evidence suggesting that central kinetochore components influence kinetochore–microtubule attachment, but the mechanism remains unclear. Here, we find that the conserved Mis12/MIND (Mtw1, Nsl1, Nnf1, Dsn1) and Ndc80 (Ndc80, Nuf2, Spc24, Spc25) complexes are connected by an extensive network of contacts, each essential for viability in cells, and collectively able to withstand substantial tensile load. Using a single-molecule approach, we demonstrate that an individual MIND complex enhances the microtubule-binding affinity of a single Ndc80 complex by fourfold. MIND itself does not bind microtubules. Instead, MIND binds Ndc80 complex far from the microtubule-binding domain and confers increased microtubule interaction of the complex. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a folded conformation. Together, our results suggest a previously unidentified mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore complex. PMID:26430240

  11. Influence of carbon nanotubes on the buckling of microtubule bundles in viscoelastic cytoplasm using nonlocal strain gradient theory

    Directory of Open Access Journals (Sweden)

    A. Farajpour

    Full Text Available Carbon nanotubes are a new class of microtubule-stabilizing agents since they interact with protein microtubules in living cells, interfering with cell division and inducing apoptosis. In the present work, a modified beam model is developed to investigate the effect of carbon nanotubes on the buckling of microtubule bundles in living cell. A realistic interaction model is employed using recent experimental data on the carbon nanotube-stabilized microtubules. Small scale and surface effects are taken into account applying the nonlocal strain gradient theory and surface elasticity theory. Pasternak model is used to describe the normal and shearing effects of enclosing filament matrix on the buckling behavior of the system. An exact solution is obtained for the buckling growth rates of the mixed bundle in viscoelastic surrounding cytoplasm. The present results are compared with those reported in the open literature for single microtubules and an excellent agreement is found. Finally, the effects of different parameters such as the size, chirality, position and surface energy of carbon nanotubes on the buckling growth rates of microtubule bundles are studied. It is found that the buckling growth rate may increase or decrease by adding carbon nanotubes, depending on the diameter and chirality of carbon nanotubes. Keywords: Microtubules, Carbon nanotubes, Buckling, Size effects

  12. State of the Art Review: Information Processing in Microtubules

    Science.gov (United States)

    2009-10-06

    the lecture. Some of these included Dr Robert Ewing, Chrissy Grabinski, Nikki Schaeublin, Dr Laura Stolle , Carol Garrett, David Ellis, Janice Speshock...been several proposal for intelligent control of machines [patent ref. 8] basic issues of signal transport from one neuron to another eventually is...for Quantum Boltzman Machine neuron devices, Jpn JAP, 38, 439 (1999) [non-patent ref. 7] Supriyo Datta, Magnus Paulsson, Ferdows Zahid, Electrical

  13. Nup62, associated with spindle microtubule rather than spindle matrix, is involved in chromosome alignment and spindle assembly during mitosis.

    Science.gov (United States)

    Wu, Zhige; Jin, Zhihua; Zhang, Xinhong; Shen, Na; Wang, Jinbo; Zhao, Yingxian; Mei, Lehe

    2016-09-01

    An increasing number of the active mitotic functions of nucleoporins in the distinct steps of mitosis have been assigned over the past few years. As one of FG-repeats containing nucleoporins, Nup62 has been found to be involved in nuclear transport, cell migration, virus infection, and cell cycle regulation. However, the role and mechanism of Nup62 in mitotic regulation have not been fully revealed. In this paper, it was revealed that a fraction of Nup62 was associated with mitotic spindle microtubule instead of spindle matrix, and the localization of Nup62 in the mitotic spindle depended on its three coiled-coil domains rather than Crm1, although Nup62 strongly interacted with Crm1 during mitosis. Moreover, depletion of Nup62 by small interference of RNA seriously induced the defects of chromosome alignment and spindle assembly although the bipolar spindle was observed in most of the Nup62 knock-down cells. Notably, congression of polar chromosome defect was observed in more than 30% of Nup62 knock-down cells. These findings revealed that Nup62 was a novel mitotic spindle associated nucleoporin and involved in chromosome alignment and spindle assembly. © 2016 International Federation for Cell Biology.

  14. The NAP motif of activity-dependent neuroprotective protein (ADNP) regulates dendritic spines through microtubule end binding proteins.

    Science.gov (United States)

    Oz, S; Kapitansky, O; Ivashco-Pachima, Y; Malishkevich, A; Giladi, E; Skalka, N; Rosin-Arbesfeld, R; Mittelman, L; Segev, O; Hirsch, J A; Gozes, I

    2014-10-01

    The NAP motif of activity-dependent neuroprotective protein (ADNP) enhanced memory scores in patients suffering from mild cognitive impairment and protected activities of daily living in schizophrenia patients, while fortifying microtubule (MT)-dependent axonal transport, in mice and flies. The question is how does NAP fortify MTs? Our sequence analysis identified the MT end-binding protein (EB1)-interacting motif SxIP (SIP, Ser-Ile-Pro) in ADNP/NAP and showed specific SxIP binding sites in all members of the EB protein family (EB1-3). Others found that EB1 enhancement of neurite outgrowth is attenuated by EB2, while EB3 interacts with postsynaptic density protein 95 (PSD-95) to modulate dendritic plasticity. Here, NAP increased PSD-95 expression in dendritic spines, which was inhibited by EB3 silencing. EB1 or EB3, but not EB2 silencing inhibited NAP-mediated cell protection, which reflected NAP binding specificity. NAPVSKIPQ (SxIP=SKIP), but not NAPVAAAAQ mimicked NAP activity. ADNP, essential for neuronal differentiation and brain formation in mouse, a member of the SWI/SNF chromatin remodeling complex and a major protein mutated in autism and deregulated in schizophrenia in men, showed similar EB interactions, which were enhanced by NAP treatment. The newly identified shared MT target of NAP/ADNP is directly implicated in synaptic plasticity, explaining the breadth and efficiency of neuroprotective/neurotrophic capacities.

  15. The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

    Directory of Open Access Journals (Sweden)

    Jennifer Pöhlmann

    2014-09-01

    Full Text Available Microtubules (MTs are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

  16. Structure-based simulations of kinesin motor domain for the study and characterization of its different microtubule and ligand-binding states

    Science.gov (United States)

    Chakraborty, Srirupa; Zheng, Wenjun

    2014-03-01

    Kinesins are molecular motors acting as enzyme-based nanomachines that transport intracellular cargo along microtubules (MT). To obtain a detailed structural and energetic picture of the various conformations of the kinesin motor domain, we built atomistic models using available crystal structures, homology modeling and flexible fitting into cryo-electron microscopy (EM) maps. These models depict the various biochemical states of the kinesin head, such as - with the neck-linker docked and undocked in the MT-free state, and the different nucleotide (ADP, ATP and APO) bound states in the kinesin-MT complex. Here we perform molecular dynamics simulation techniques and large-scale computational probing of differences in these states, by an exhaustive search of interactions that differ between them, identify key residues in the active site and binding interface, and investigate the binding free-energy between kinesin and MT, and kinesin and ligand to compare with experimentally obtained results.

  17. Direct visualization of microtubules using a genetic tool to analyse radial progenitor-astrocyte continuum in mice

    OpenAIRE

    Eom, Tae-Yeon; Stanco, Amelia; Weimer, Jill; Stabingas, Kristen; Sibrack, Elizabeth; Gukassyan, Vladimir; Cheng, JrGang; Anton, E. S.

    2011-01-01

    Microtubule cytoskeletal dynamics of cortical progenitors and astroglial cells play critical roles in the emergence of normal functional organization of cerebral cortex and in disease processes such as tumorigenesis. However, tools to efficiently visualize these events are lacking. Here we describe a mouse genetic model to efficiently visualize and analyze radial progenitors, their astroglial progeny, and the microtubule cytoskeleton of these cells in the developing and adult brain. Using thi...

  18. Dissecting the nanoscale distributions and functions of microtubule-end-binding proteins EB1 and ch-TOG in interphase HeLa cells.

    Directory of Open Access Journals (Sweden)

    Satoko Nakamura

    Full Text Available Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other's localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.

  19. Microtubule-Dependent Mitochondria Alignment Regulates Calcium Release in Response to Nanomechanical Stimulus in Heart Myocytes

    Directory of Open Access Journals (Sweden)

    Michele Miragoli

    2016-01-01

    Full Text Available Arrhythmogenesis during heart failure is a major clinical problem. Regional electrical gradients produce arrhythmias, and cellular ionic transmembrane gradients are its originators. We investigated whether the nanoscale mechanosensitive properties of cardiomyocytes from failing hearts have a bearing upon the initiation of abnormal electrical activity. Hydrojets through a nanopipette indent specific locations on the sarcolemma and initiate intracellular calcium release in both healthy and heart failure cardiomyocytes, as well as in human failing cardiomyocytes. In healthy cells, calcium is locally confined, whereas in failing cardiomyocytes, calcium propagates. Heart failure progressively stiffens the membrane and displaces sub-sarcolemmal mitochondria. Colchicine in healthy cells mimics the failing condition by stiffening the cells, disrupting microtubules, shifting mitochondria, and causing calcium release. Uncoupling the mitochondrial proton gradient abolished calcium initiation in both failing and colchicine-treated cells. We propose the disruption of microtubule-dependent mitochondrial mechanosensor microdomains as a mechanism for abnormal calcium release in failing heart.

  20. RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization

    Science.gov (United States)

    Havin, Leora; Git, Anna; Elisha, Zichrini; Oberman, Froma; Yaniv, Karina; Schwartz, Sigal Pressman; Standart, Nancy; Yisraeli, Joel K.

    1998-01-01

    Vg1 mRNA translocation to the vegetal cortex of Xenopus oocytes requires intact microtubules, and a 3′ UTR cis-acting element (termed VLE), which also mediates sequence-specific binding of several proteins. One protein, the 69-kD Vg1 RBP, associates Vg1 RNA to microtubules in vitro. Here we show that Vg1 RBP-binding sites correlate with vegetal localization. Purification and cloning of Vg1 RBP revealed five RNA-binding motifs: four KH and one RRM domains. Surprisingly, Vg1 RBP is highly homologous to the zipcode binding protein implicated in the microfilament-mediated localization of β actin mRNA in fibroblasts. These data support Vg1 RBP’s direct role in vegetal localization and suggest the existence of a general, evolutionarily conserved mechanism for mRNA targeting. PMID:9620847

  1. RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization.

    Science.gov (United States)

    Havin, L; Git, A; Elisha, Z; Oberman, F; Yaniv, K; Schwartz, S P; Standart, N; Yisraeli, J K

    1998-06-01

    Vg1 mRNA translocation to the vegetal cortex of Xenopus oocytes requires intact microtubules, and a 3' UTR cis-acting element (termed VLE), which also mediates sequence-specific binding of several proteins. One protein, the 69-kD Vg1 RBP, associates Vg1 RNA to microtubules in vitro. Here we show that Vg1 RBP-binding sites correlate with vegetal localization. Purification and cloning of Vg1 RBP revealed five RNA-binding motifs: four KH and one RRM domains. Surprisingly, Vg1 RBP is highly homologous to the zipcode binding protein implicated in the microfilament-mediated localization of beta actin mRNA in fibroblasts. These data support Vg1 RBP's direct role in vegetal localization and suggest the existence of a general, evolutionarily conserved mechanism for mRNA targeting.

  2. Proteomics of cancer cell lines resistant to microtubule-stabilizing agents

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Angeletti, Ruth H; Horwitz, Susan Band

    2014-01-01

    was compared with two drug-resistant daughter cell lines, an EpoB-resistant cell line (EpoB8) and an ixabepilone-resistant cell line (Ixab80). All 2D DIGE results were validated by Western blot analyses. A variety of cytoskeletal and cytoskeleton-associated proteins were differentially expressed in drug......Despite the clinical success of microtubule-interacting agents (MIA), a significant challenge for oncologists is the inability to predict the response of individual patients with cancer to these drugs. In the present study, six cell lines were compared by 2D DIGE proteomics to investigate cellular...... resistance to the class of MIAs known as microtubule-stabilizing agents (MSA). The human lung cancer cell line A549 was compared with two drug-resistant daughter cell lines, a taxol-resistant cell line (AT12) and an epothilone B (EpoB)-resistant cell line (EpoB40). The ovarian cancer cell line Hey...

  3. The organization of the Golgi complex and microtubules in skeletal muscle is fiber type-dependent

    DEFF Research Database (Denmark)

    Ralston, E; Lu, Z; Ploug, Thorkil

    1999-01-01

    and experimentally denervated. The total number of GC elements, small polarized stacks of cisternae, is quite similar in all fibers, but their intracellular distribution is fiber type-dependent. Thus, in slow-twitch, type I fibers, approximately 75% of all GC elements are located within 1 micrometer from the plasma...... membrane, and each nucleus is surrounded by a belt of GC elements. In contrast, in the fast-twitch type IIB fibers, most GC elements are in the fiber core, and most nuclei only have GC elements at their poles. Intermediate, type IIA fibers also have an intermediate distribution of GC elements...... of the hindlimb muscles, GC elements as well as microtubules converge toward a common pattern, that of the slow-twitch fibers, in all fibers. Our data suggest that innervation regulates the distribution of microtubules, which in turn organize the Golgi complex according to muscle fiber type....

  4. Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging.

    Science.gov (United States)

    Celler, Katherine; Fujita, Miki; Kawamura, Eiko; Ambrose, Chris; Herburger, Klaus; Holzinger, Andreas; Wasteneys, Geoffrey O

    2016-01-01

    Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.

  5. Stimulation of Vesicular Stomatitis Virus in vitro RNA Synthesis by Microtubule-Associated Proteins

    Science.gov (United States)

    Hill, Virginia M.; Harmon, Shirley A.; Summers, Donald F.

    1986-08-01

    Microtubule-associated proteins purified from bovine brains stimulated the in vitro transcription and replication reactions of vesicular stomatitis virus. The products of these reactions were intact messenger or genome-sized RNA species. A preparation from HeLa cells containing tubulin and microtubule-associated proteins also stimulated vesicular stomatitis virus transcription in vitro. This observation is in accord with previous studies, which suggested that a host cell factor was involved with the function of the vesicular stomatitis virus RNA polymerase, and others that indicated that several animal viruses displayed an association with host cell cytoskeletal elements during their replication cycles. We show evidence in this report of a host cell protein that seems to have a functional role in interacting with the virion polymerase.

  6. Microtubule and cortical forces determine platelet size during vascular platelet production.

    Science.gov (United States)

    Thon, Jonathan N; Macleod, Hannah; Begonja, Antonija Jurak; Zhu, Jie; Lee, Kun-Chun; Mogilner, Alex; Hartwig, John H; Italiano, Joseph E

    2012-05-22

    Megakaryocytes release large preplatelet intermediates into the sinusoidal blood vessels. Preplatelets convert into barbell-shaped proplatelets in vitro to undergo repeated abscissions that yield circulating platelets. These observations predict the presence of circular-preplatelets and barbell-proplatelets in blood, and two fundamental questions in platelet biology are what are the forces that determine barbell-proplatelet formation, and how is the final platelet size established. Here we provide insights into the terminal mechanisms of platelet production. We quantify circular-preplatelets and barbell-proplatelets in human blood in high-resolution fluorescence images, using a laser scanning cytometry assay. We demonstrate that force constraints resulting from cortical microtubule band diameter and thickness determine barbell-proplatelet formation. Finally, we provide a mathematical model for the preplatelet to barbell conversion. We conclude that platelet size is limited by microtubule bundling, elastic bending, and actin-myosin-spectrin cortex forces.

  7. Non-equilibrium assembly of microtubules: from molecules to autonomous chemical robots.

    Science.gov (United States)

    Hess, H; Ross, Jennifer L

    2017-09-18

    Biological systems have evolved to harness non-equilibrium processes from the molecular to the macro scale. It is currently a grand challenge of chemistry, materials science, and engineering to understand and mimic biological systems that have the ability to autonomously sense stimuli, process these inputs, and respond by performing mechanical work. New chemical systems are responding to the challenge and form the basis for future responsive, adaptive, and active materials. In this article, we describe a particular biochemical-biomechanical network based on the microtubule cytoskeletal filament - itself a non-equilibrium chemical system. We trace the non-equilibrium aspects of the system from molecules to networks and describe how the cell uses this system to perform active work in essential processes. Finally, we discuss how microtubule-based engineered systems can serve as testbeds for autonomous chemical robots composed of biological and synthetic components.

  8. Cyclin A2 modulates kinetochore–microtubule attachment in meiosis II

    Science.gov (United States)

    Yuen, Wai Shan; Adhikari, Deepak; FitzHarris, Greg; Conti, Marco; Sicinski, Piotr; Nabti, Ibtissem; Marangos, Petros

    2017-01-01

    Cyclin A2 is a crucial mitotic Cdk regulatory partner that coordinates entry into mitosis and is then destroyed in prometaphase within minutes of nuclear envelope breakdown. The role of cyclin A2 in female meiosis and its dynamics during the transition from meiosis I (MI) to meiosis II (MII) remain unclear. We found that cyclin A2 decreases in prometaphase I but recovers after the first meiotic division and persists, uniquely for metaphase, in MII-arrested oocytes. Conditional deletion of cyclin A2 from mouse oocytes has no discernible effect on MI but leads to disrupted MII spindles and increased merotelic attachments. On stimulation of exit from MII, there is a dramatic increase in lagging chromosomes and an inhibition of cytokinesis. These defects are associated with an increase in microtubule stability in MII spindles, suggesting that cyclin A2 mediates the fidelity of MII by maintaining microtubule dynamics during the rapid formation of the MII spindle. PMID:28819014

  9. GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

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    Massimilano Scolz

    Full Text Available The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.

  10. Randomization of cortical microtubules in root epidermal cells induces root hair initiation in lettuce (Lactuca sativa L.) seedlings.

    Science.gov (United States)

    Takahashi, Hidenori; Hirota, Kayoko; Kawahara, Aiko; Hayakawa, Erika; Inoue, Yasunori

    2003-03-01

    Root hair formation is induced when lettuce seedlings are transferred from liquid medium at pH 6.0 to fresh medium at pH 4.0. If seedlings are transferred to pH 6.0, no root hairs are formed. We investigated the role of microtubules in this low pH-induced root hair initiation in lettuce. At the hair-forming zone in root epidermal cells, microtubules were perpendicular to the longitudinal axis of the cell just after pre-culture. This arrangement became disordered as early as 5 min after transfer to pH 4.0, and became random by 30 min later. At pH 4.0, the randomization extended to the entire hair-forming zone of seedlings; at pH 6.0, however, randomization did not occur and transverse microtubules were maintained. When seedlings at pH 6.0 were treated with microtubule-depolymerizing drugs, root hairs were formed. In contrast, when a microtubule-stabilizing drug, taxol, was added to the medium, no root hairs formed, even at pH 4.0. These results suggest that the transverse cortical microtubules inhibit root hair formation, and that their destruction is necessary for initiation. Furthermore, the microfilament-disrupting drugs cytochalasin B and latrunculin B inhibited root hair initiation, suggesting that actin filaments are necessary for root hair initiation.

  11. The transcription factor Egr3 is a putative component of the microtubule organizing center in mouse oocytes.

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    Hyejin Shin

    Full Text Available The early growth response (Egr family of zinc finger transcription factors consists of 4 members. During an investigation of Egr factor localization in mouse ovaries, we noted that Egr3 exhibits a subcellular localization that overlaps with the meiotic spindle in oocytes. Using Egr3-specific antibodies, we establish that Egr3 co-localizes with the spindle and cytosolic microtubule organizing centers (MTOCs in oocytes during meiotic maturation. Notably, the Egr3 protein appears to accumulate around γ-tubulin in MTOCs. Nocodazole treatment, which induces microtubule depolymerization, resulted in the disruption of spindle formation and Egr3 localization, suggesting that Egr3 localization is dependent on the correct configuration of the spindle. Shortly after warming of vitrified oocytes, growing arrays of microtubules were observed near large clusters of Egr3. An in vitro microtubule interaction assay showed that Egr3 does not directly interact with polymerized microtubules. Egr3 localization on the spindle was sustained in early preimplantation mouse embryos, but this pattern did not persist until the blastocyst stage. Collectively, our result shows for the first time that the Egr3 a transcription factor may play a novel non-transcriptional function during microtubule organization in mouse oocytes.

  12. The transcription factor Egr3 is a putative component of the microtubule organizing center in mouse oocytes.

    Science.gov (United States)

    Shin, Hyejin; Kwon, Sojung; Song, Haengseok; Lim, Hyunjung Jade

    2014-01-01

    The early growth response (Egr) family of zinc finger transcription factors consists of 4 members. During an investigation of Egr factor localization in mouse ovaries, we noted that Egr3 exhibits a subcellular localization that overlaps with the meiotic spindle in oocytes. Using Egr3-specific antibodies, we establish that Egr3 co-localizes with the spindle and cytosolic microtubule organizing centers (MTOCs) in oocytes during meiotic maturation. Notably, the Egr3 protein appears to accumulate around γ-tubulin in MTOCs. Nocodazole treatment, which induces microtubule depolymerization, resulted in the disruption of spindle formation and Egr3 localization, suggesting that Egr3 localization is dependent on the correct configuration of the spindle. Shortly after warming of vitrified oocytes, growing arrays of microtubules were observed near large clusters of Egr3. An in vitro microtubule interaction assay showed that Egr3 does not directly interact with polymerized microtubules. Egr3 localization on the spindle was sustained in early preimplantation mouse embryos, but this pattern did not persist until the blastocyst stage. Collectively, our result shows for the first time that the Egr3 a transcription factor may play a novel non-transcriptional function during microtubule organization in mouse oocytes.

  13. Microtubule array observed in the posterior-vegetal cortex during cytoplasmic and cortical reorganization of the ascidian egg.

    Science.gov (United States)

    Ishii, Hirokazu; Goto, Toshiyuki; Nishikata, Takahito

    2017-10-01

    Body axis formation during embryogenesis results from asymmetric localization of maternal factors in the egg. Shortly before the first cleavage in ascidian eggs, cell polarity along the anteroposterior (A-P) axis is established and the cytoplasmic domain (myoplasm) relocates from the vegetal to the posterior region in a microtubule-dependent manner. Through immunostaining, tubulin accumulation during this reorganization is observable on the myoplasm cortex. However, more detailed morphological features of microtubules remain relatively unknown. In this study, we invented a new reagent that improves the immunostaining of cortical microtubules and successfully visualized a parallel array of thick microtubules. During reorganization, they covered nearly the entire myoplasm cortical region, beneath the posterior-vegetal cortex. We designated this microtubule array as CAMP (cortical array of microtubules in posterior vegetal region). During the late phase of reorganization, CAMP shrank and the myoplasm formed a crescent-like cytoplasmic domain. When the CAMP formation was inhibited by sodium azide, myoplasmic reorganization and A-P axis formation were both abolished, suggesting that CAMP is important for these two processes. © 2017 The Authors Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.

  14. TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana.

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    Liangliang Chen

    2016-10-01

    Full Text Available How cell shape is controlled is a fundamental question in developmental biology, but the genetic and molecular mechanisms that determine cell shape are largely unknown. Arabidopsis trichomes have been used as a good model system to investigate cell shape at the single-cell level. Here we describe the trichome cell shape 1 (tcs1 mutants with the reduced trichome branch number in Arabidopsis. TCS1 encodes a coiled-coil domain-containing protein. Pharmacological analyses and observations of microtubule dynamics show that TCS1 influences the stability of microtubules. Biochemical analyses and live-cell imaging indicate that TCS1 binds to microtubules and promotes the assembly of microtubules. Further results reveal that TCS1 physically associates with KCBP/ZWICHEL, a microtubule motor involved in the regulation of trichome branch number. Genetic analyses indicate that kcbp/zwi is epistatic to tcs1 with respect to trichome branch number. Thus, our findings define a novel genetic and molecular mechanism by which TCS1 interacts with KCBP to regulate trichome cell shape by influencing the stability of microtubules.

  15. Kinetic analysis of tubulin assembly in the presence of the microtubule-associated protein TOGp

    OpenAIRE

    Bonfils, Claude; Bec, Nicole; Lacroix, Benjamin; Harricane, Marie-Cécile; Larroque, Christian

    2006-01-01

    International audience; The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the clas...

  16. Microtubule-Associated Protein Mdp3 Promotes Breast Cancer Growth and Metastasis

    OpenAIRE

    Tala,; Xie, Songbo; Sun, Xiaodong; Sun, Xiaoou; Ran, Jie; Zhang, Linlin; Li, Dengwen; Liu, Min; Bao, Gang; Zhou, Jun

    2014-01-01

    Breast cancer is the most prevalent cancer in women worldwide with a high mortality rate, and the identification of new biomarkers and targets for this disease is greatly needed. Here we present evidence that microtubule-associated protein (MAP) 7 domain-containing protein 3 (Mdp3) is highly expressed in clinical samples and cell lines of breast cancer. The expression of Mdp3 correlates with clinicopathological parameters indicating breast cancer malignancy. In addition, Mdp3 promotes breast ...

  17. KIF7 Controls the Proliferation of Cells of the Respiratory Airway through Distinct Microtubule Dependent Mechanisms.

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    Garry L Coles

    2015-10-01

    Full Text Available The cell cycle must be tightly coordinated for proper control of embryonic development and for the long-term maintenance of organs such as the lung. There is emerging evidence that Kinesin family member 7 (Kif7 promotes Hedgehog (Hh signaling during embryonic development, and its misregulation contributes to diseases such as ciliopathies and cancer. Kif7 encodes a microtubule interacting protein that controls Hh signaling through regulation of microtubule dynamics within the primary cilium. However, whether Kif7 has a function in nonciliated cells remains largely unknown. The role Kif7 plays in basic cell biological processes like cell proliferation or cell cycle progression also remains to be elucidated. Here, we show that Kif7 is required for coordination of the cell cycle, and inactivation of this gene leads to increased cell proliferation in vivo and in vitro. Immunostaining and transmission electron microscopy experiments show that Kif7dda/dda mutant lungs are hyperproliferative and exhibit reduced alveolar epithelial cell differentiation. KIF7 depleted C3H10T1/2 fibroblasts and Kif7dda/dda mutant mouse embryonic fibroblasts have increased growth rates at high cellular densities, suggesting that Kif7 may function as a general regulator of cellular proliferation. We ascertained that in G1, Kif7 and microtubule dynamics regulate the expression and activity of several components of the cell cycle machinery known to control entry into S phase. Our data suggest that Kif7 may function to regulate the maintenance of the respiratory airway architecture by controlling cellular density, cell proliferation, and cycle exit through its role as a microtubule associated protein.

  18. Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches.

    Science.gov (United States)

    Canales, Angeles; Nieto, Lidia; Rodríguez-Salarichs, Javier; Sánchez-Murcia, Pedro A; Coderch, Claire; Cortés-Cabrera, Alvaro; Paterson, Ian; Carlomagno, Teresa; Gago, Federico; Andreu, José M; Altmann, Karl-Heinz; Jiménez-Barbero, Jesús; Díaz, J Fernando

    2014-04-18

    The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.

  19. Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties

    Energy Technology Data Exchange (ETDEWEB)

    Sloman, David L.; Noucti, Njamkou; Altman, Michael D.; Chen, Dapeng; Mislak, Andrea C.; Szewczak, Alexander; Hayashi, Mansuo; Warren, Lee; Dellovade, Tammy; Wu, Zhenhua; Marcus, Jacob; Walker, Deborah; Su, Hua-Poo; Edavettal, Suzanne C.; Munshi, Sanjeev; Hutton, Michael; Nuthall, Hugh; Stanton, Matthew G. (Merck)

    2016-09-01

    Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer’s disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

  20. The centrosomal linker and microtubules provide dual levels of spatial coordination of centrosomes.

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    Marko Panic

    2015-05-01

    Full Text Available The centrosome is the principal microtubule organizing center in most animal cells. It consists of a pair of centrioles surrounded by pericentriolar material. The centrosome, like DNA, duplicates exactly once per cell cycle. During interphase duplicated centrosomes remain closely linked by a proteinaceous linker. This centrosomal linker is composed of rootletin filaments that are anchored to the centrioles via the protein C-Nap1. At the onset of mitosis the linker is dissolved by Nek2A kinase to support the formation of the bipolar mitotic spindle. The importance of the centrosomal linker for cell function during interphase awaits characterization. Here we assessed the phenotype of human RPE1 C-Nap1 knockout (KO cells. The absence of the linker led to a modest increase in the average centrosome separation from 1 to 2.5 μm. This small impact on the degree of separation is indicative of a second level of spatial organization of centrosomes. Microtubule depolymerisation or stabilization in C-Nap1 KO cells dramatically increased the inter-centrosomal separation (> 8 μm. Thus, microtubules position centrosomes relatively close to one another in the absence of linker function. C-Nap1 KO cells had a Golgi organization defect with a two-fold expansion of the area occupied by the Golgi. When the centrosomes of C-Nap1 KO cells showed considerable separation, two spatially distinct Golgi stacks could be observed. Furthermore, migration of C-Nap1 KO cells was slower than their wild type RPE1 counterparts. These data show that the spatial organization of centrosomes is modulated by a combination of centrosomal cohesion and microtubule forces. Furthermore a modest increase in centrosome separation has major impact on Golgi organization and cell migration.

  1. Opposing microtubule motors drive robust nuclear dynamics in developing muscle cells.

    Science.gov (United States)

    Wilson, Meredith H; Holzbaur, Erika L F

    2012-09-01

    Dynamic interactions with the cytoskeleton drive the movement and positioning of nuclei in many cell types. During muscle cell development, myoblasts fuse to form syncytial myofibers with nuclei positioned regularly along the length of the cell. Nuclear translocation in developing myotubes requires microtubules, but the mechanisms involved have not been elucidated. We find that as nuclei actively translocate through the cell, they rotate in three dimensions. The nuclear envelope, nucleoli and chromocenters within the nucleus rotate together as a unit. Both translocation and rotation require an intact microtubule cytoskeleton, which forms a dynamic bipolar network around nuclei. The plus- and minus-end-directed microtubule motor proteins, kinesin-1 and dynein, localize to the nuclear envelope in myotubes. Kinesin-1 localization is mediated at least in part by interaction with klarsicht/ANC-1/Syne homology (KASH) proteins. Depletion of kinesin-1 abolishes nuclear rotation and significantly inhibits nuclear translocation, resulting in the abnormal aggregation of nuclei at the midline of the myotube. Dynein depletion also inhibits nuclear dynamics, but to a lesser extent, leading to altered spacing between adjacent nuclei. Thus, oppositely directed motors acting from the surface of the nucleus drive nuclear motility in myotubes. The variable dynamics observed for individual nuclei within a single myotube are likely to result from the stochastic activity of competing motors interacting with a complex bipolar microtubule cytoskeleton that is also continuously remodeled as the nuclei move. The three-dimensional rotation of myotube nuclei may facilitate their motility through the complex and crowded cellular environment of the developing muscle cell, allowing for proper myonuclear positioning.

  2. Cargo distributions differentiate pathological axonal transport impairments.

    Science.gov (United States)

    Mitchell, Cassie S; Lee, Robert H

    2012-05-07

    Axonal transport is an essential process in neurons, analogous to shipping goods, by which energetic and cellular building supplies are carried downstream (anterogradely) and wastes are carried upstream (retrogradely) by molecular motors, which act as cargo porters. Impairments in axonal transport have been linked to devastating and often lethal neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis, Huntington's, and Alzheimer's. Axonal transport impairment types include a decrease in available motors for cargo transport (motor depletion), the presence of defective or non-functional motors (motor dilution), and the presence of increased or larger cargos (protein aggregation). An impediment to potential treatment identification has been the inability to determine what type(s) of axonal transport impairment candidates that could be present in a given disease. In this study, we utilize a computational model and common axonal transport experimental metrics to reveal the axonal transport impairment general characteristics or "signatures" that result from three general defect types of motor depletion, motor dilution, and protein aggregation. Our results not only provide a means to discern these general impairments types, they also reveal key dynamic and emergent features of axonal transport, which potentially underlie multiple impairment types. The identified characteristics, as well as the analytical method, can be used to help elucidate the axonal transport impairments observed in experimental and clinical data. For example, using the model-predicted defect signatures, we identify the defect candidates, which are most likely to be responsible for the axonal transport impairments in the G93A SOD1 mouse model of ALS. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Structural Model for Tubulin Recognition and Deformation by Kinesin-13 Microtubule Depolymerases

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    Ana B. Asenjo

    2013-03-01

    Full Text Available To elucidate the structural basis of the mechanism of microtubule depolymerization by kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10A by electron microscopy (EM and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear between tubulin subunits in addition to curvature. In this configuration, the kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.

  4. Prostaglandins from a zoanthid: paclitaxel-like neurite-degenerating and microtubule-stabilizating activities.

    Science.gov (United States)

    Han, Chunguang; Qi, Jianhua; Shi, Xiaojin; Sakagami, Youji; Shibata, Takahiro; Uchida, Koji; Ojika, Makoto

    2006-03-01

    Two prostaglandins, PGA2 and PGB2, were isolated from the Okinawan zoanthid, Palythoa kochii, during a search for paclitaxel-like neurite-degenerating compounds from natural sources using a cell-based assay method. In the presence of PGA2 at 30 microM, the neuronal processes induced in PC12 cells by the nerve growth factor (NGF) degenerated over 24 h, whereas PGB2 had no effect on the neuronal processes of PC12 cells. This activity of PGA2 was similar to that of the microtubule-stabilizing agents, paclitaxel (Taxol) and epothilone A, unlike the microtubule-depolymerizing agent, colchicine, which brought about quick neurite degeneration within 3 h. PGA2 stimulated tubulin polymerization, although less potently than paclitaxel. An examination of structure-activity relationships across several PGs suggests that the cyclopentenone ring structure and the orientation of its dipolar moment played an important role in the paclitaxel-like neurite-degenerating activity. These results suggest that the cyclopentenone-type PGs can interact with microtubules to inhibit their function like paclitaxel.

  5. Curcumin alters the cytoskeleton and microtubule organization on trophozoites of Giardia lamblia.

    Science.gov (United States)

    Gutiérrez-Gutiérrez, Filiberto; Palomo-Ligas, Lissethe; Hernández-Hernández, José Manuel; Pérez-Rangel, Armando; Aguayo-Ortiz, Rodrigo; Hernández-Campos, Alicia; Castillo, Rafael; González-Pozos, Sirenia; Cortés-Zárate, Rafael; Ramírez-Herrera, Mario Alberto; Mendoza-Magaña, María Luisa; Castillo-Romero, Araceli

    2017-08-01

    Giardia lamblia is a worldwide protozoan responsible for a significant number of intestinal infections. There are several drugs for the treatment of giardiasis, but they often cause side effects. Curcumin, a component of turmeric, has antigiardial activity; however, the molecular target and mechanism of antiproliferative activity are not clear. The effects of curcumin on cellular microtubules have been widely investigated. Since tubulin is the most abundant protein in the cytoskeleton of Giardia, to elucidate whether curcumin has activity against the microtubules of this parasite, we treated trophozoites with curcumin and the cells were analyzed by scanning electron microscopy and confocal microscopy. Curcumin inhibited Giardia proliferation and adhesion in a time-concentration-dependent mode. The higher inhibitory concentrations of curcumin (3 and 15μM) disrupted the cytoskeletal structures of trophozoites; the damage was evident on the ventral disk, flagella and in the caudal region, also the membrane was affected. The immunofluorescence images showed altered distribution of tubulin staining on ventral disk and flagella. Additionally, we found that curcumin caused a clear reduction of tubulin expression. By docking analysis and molecular dynamics we showed that curcumin has a high probability to bind at the interface of the tubulin dimer close to the vinblastine binding site. All the data presented indicate that curcumin may inhibit Giardia proliferation by perturbing microtubules. Copyright © 2017. Published by Elsevier B.V.

  6. Microtubule dynamics and the neurodegenerative triad of Alzheimer's disease: The hidden connection.

    Science.gov (United States)

    Brandt, Roland; Bakota, Lidia

    2017-11-01

    Alzheimer's disease (AD) is the most common neurodegenerative disorder and is, on a histopathological level, characterized by the presence of extracellular amyloid plaques composed of the protein fragment Aβ, and intracellular neurofibrillary tangles, which contain the microtubule-associated protein tau in a hyperphosphorylated state. In AD defects in microtubule (MT) assembly and organization have also been reported; however, it is unclear whether MT abnormalities have a causal and early role in the disease process or represent a common end point downstream of the neurodegenerative cascade. Recent evidence indicates that microtubule-stabilizing drugs prevent axonopathy in animal models of tauopathies and reverse Aβ-induced loss of synaptic connectivity in an ex vivo model of amyloidosis. This could suggest that MT dysfunction connects some of the degenerative events and provides a useful target to simultaneously prevent several neurodegenerative processes in AD. Here, we describe how changes in the structure and dynamics of MTs are involved in the different aspects of the neurodegenerative triad of AD. We discuss evidence that MTs are affected both by tau-dependent and tau-independent mechanisms but appear to be regulated in a distinct way in different neuronal compartments. We argue that modulation of MT dynamics could be of potential benefit but needs to be precisely controlled in a cell and compartment-specific manner to avoid harmful side effects. This article is part of the series "Beyond Amyloid". © 2017 International Society for Neurochemistry.

  7. SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture.

    Science.gov (United States)

    Hilbert, Manuel; Noga, Akira; Frey, Daniel; Hamel, Virginie; Guichard, Paul; Kraatz, Sebastian H W; Pfreundschuh, Moritz; Hosner, Sarah; Flückiger, Isabelle; Jaussi, Rolf; Wieser, Mara M; Thieltges, Katherine M; Deupi, Xavier; Müller, Daniel J; Kammerer, Richard A; Gönczy, Pierre; Hirono, Masafumi; Steinmetz, Michel O

    2016-04-01

    Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

  8. Phosphatase PP2A and microtubule-mediated pulling forces disassemble centrosomes during mitotic exit

    Directory of Open Access Journals (Sweden)

    Stephen J. Enos

    2018-01-01

    Full Text Available Centrosomes are microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2ASUR-6, together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, ∼25% of PCM persisted from one cell cycle into the next. Purified PP2ASUR-6 could dephosphorylate the major PCM scaffold protein SPD-5 in vitro. Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and force-driven fragmentation of the PCM scaffold.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Haixin; Downing, Kenneth H.

    2006-05-22

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

  10. Hippocampal hyperexcitability is modulated by microtubule-active agent: evidence from in vivo and in vitro epilepsy models in the rat.

    OpenAIRE

    Fabio eCarletti; Pierangelo eSardo; Giuditta eGambino; Xin-An eLiu; Giuseppe eFerraro; Valerio eRizzo; Valerio eRizzo

    2016-01-01

    The involvement of microtubule dynamics on bioelectric activity of neurons and neurotransmission represents a fascinating target of research in the context of neural excitability. It has been reported that alteration of microtubule cytoskeleton can lead to profound modifications of neural functioning, with a putative impact on hyperexcitability phenomena. Altogether, in the present study we pointed at exploring the outcomes of modulating the degree of microtubule polymerization in two electro...

  11. Axonal transport of enzymes and labeled proteins in experimental axonopathy induced by p-bromophenylacetylurea

    International Nuclear Information System (INIS)

    Jakobsen, J.; Brimijoin, S.

    1981-01-01

    Axonal transport was studied by several techniques in the sciatic nerves of adult male Sprague-Dawley rats with neuropathy induced by treatment with p-bromophenylacetylurea (BPAU) in dimethylsulfoxide solution. Control rats were treated with solvent alone. BPAU, 200 mg/kg, induced severe muscle weakness in the hindlimbs, beginning after a latent period of 1 week and progressing to near total paralysis by 2 weeks. Axonal transport of the endogenous transmitter enzymes, acetylcholinesterase, dopamine-β-hydroxylase and choline acetyltransferase, was normal at both 2 and 15 days after administration of BPAU, as judged by the accumulation of enzyme activity above and below a set of double ligatures on the sciatic nerve. The velocity of fast anterograde transport of [ 35 S]methionine labeled protein was also unaffected by BPAU. However, 4 abnormalities of transport were detected in BPAU treated rats. These abnormalities are discussed. (Auth.)

  12. Nuclear transport factors: global regulation of mitosis.

    Science.gov (United States)

    Forbes, Douglass J; Travesa, Anna; Nord, Matthew S; Bernis, Cyril

    2015-08-01

    The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator - the γ-TuRC complex - and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Random intermittent search and the tug-of-war model of motor-driven transport

    KAUST Repository

    Newby, Jay

    2010-04-16

    We formulate the \\'tug-of-war\\' model of microtubule cargo transport by multiple molecular motors as an intermittent random search for a hidden target. A motor complex consisting of multiple molecular motors with opposing directional preference is modeled using a discrete Markov process. The motors randomly pull each other off of the microtubule so that the state of the motor complex is determined by the number of bound motors. The tug-of-war model prescribes the state transition rates and corresponding cargo velocities in terms of experimentally measured physical parameters. We add space to the resulting Chapman-Kolmogorov (CK) equation so that we can consider delivery of the cargo to a hidden target at an unknown location along the microtubule track. The target represents some subcellular compartment such as a synapse in a neuron\\'s dendrites, and target delivery is modeled as a simple absorption process. Using a quasi-steady-state (QSS) reduction technique we calculate analytical approximations of the mean first passage time (MFPT) to find the target. We show that there exists an optimal adenosine triphosphate (ATP) concentration that minimizes the MFPT for two different cases: (i) the motor complex is composed of equal numbers of kinesin motors bound to two different microtubules (symmetric tug-of-war model) and (ii) the motor complex is composed of different numbers of kinesin and dynein motors bound to a single microtubule (asymmetric tug-of-war model). © 2010 IOP Publishing Ltd.

  14. Three-dimensional nanometry of vesicle transport in living cells using dual-focus imaging optics

    International Nuclear Information System (INIS)

    Watanabe, Tomonobu M.; Sato, Takashi; Gonda, Kohsuke; Higuchi, Hideo

    2007-01-01

    Dual-focus imaging optics for three-dimensional tracking of individual quantum dots has been developed to study the molecular mechanisms of motor proteins in cells. The new system has a high spatial and temporal precision, 2 nm in the x-y sample plane and 5 nm along the z-axis at a frame time of 2 ms. Three-dimensional positions of the vesicles labeled with quantum dots were detected in living cells. Vesicles were transported on the microtubules using 8-nm steps towards the nucleus. The steps had fluctuation of ∼20 nm which were perpendicular to the axis of the microtubule but with the constant distance from the microtubule. The most of perpendicular movement was not synchronized with the 8-nm steps, indicating that dynein moved on microtubules without changing the protofilaments. When the vesicles changed their direction of movement toward the cell membrane, they moved perpendicular with the constant distance from the microtubule. The present method is powerful tool to investigate three dimensional movement of molecules in cells with nanometer and millisecond accuracy

  15. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    Science.gov (United States)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

  16. How molecular motors are arranged on a cargo is important for vesicular transport.

    Directory of Open Access Journals (Sweden)

    Robert P Erickson

    2011-05-01

    Full Text Available The spatial organization of the cell depends upon intracellular trafficking of cargos hauled along microtubules and actin filaments by the molecular motor proteins kinesin, dynein, and myosin. Although much is known about how single motors function, there is significant evidence that cargos in vivo are carried by multiple motors. While some aspects of multiple motor function have received attention, how the cargo itself--and motor organization on the cargo--affects transport has not been considered. To address this, we have developed a three-dimensional Monte Carlo simulation of motors transporting a spherical cargo, subject to thermal fluctuations that produce both rotational and translational diffusion. We found that these fluctuations could exert a load on the motor(s, significantly decreasing the mean travel distance and velocity of large cargos, especially at large viscosities. In addition, the presence of the cargo could dramatically help the motor to bind productively to the microtubule: the relatively slow translational and rotational diffusion of moderately sized cargos gave the motors ample opportunity to bind to a microtubule before the motor/cargo ensemble diffuses out of range of that microtubule. For rapidly diffusing cargos, the probability of their binding to a microtubule was high if there were nearby microtubules that they could easily reach by translational diffusion. Our simulations found that one reason why motors may be approximately 100 nm long is to improve their 'on' rates when attached to comparably sized cargos. Finally, our results suggested that to efficiently regulate the number of active motors, motors should be clustered together rather than spread randomly over the surface of the cargo. While our simulation uses the specific parameters for kinesin, these effects result from generic properties of the motors, cargos, and filaments, so they should apply to other motors as well.

  17. Computational Predictions of Volatile Anesthetic Interactions with the Microtubule Cytoskeleton: Implications for Side Effects of General Anesthesia

    Science.gov (United States)

    Craddock, Travis J. A.; St. George, Marc; Freedman, Holly; Barakat, Khaled H.; Damaraju, Sambasivarao; Hameroff, Stuart; Tuszynski, Jack A.

    2012-01-01

    The cytoskeleton is essential to cell morphology, cargo trafficking, and cell division. As the neuronal cytoskeleton is extremely complex, it is no wonder that a startling number of neurodegenerative disorders (including but not limited to Alzheimer’s disease, Parkinson’s disease and Huntington’s disease) share the common feature of a dysfunctional neuronal cytoskeleton. Recently, concern has been raised about a possible link between anesthesia, post-operative cognitive dysfunction, and the exacerbation of neurodegenerative disorders. Experimental investigations suggest that anesthetics bind to and affect cytoskeletal microtubules, and that anesthesia-related cognitive dysfunction involves microtubule instability, hyper-phosphorylation of the microtubule-associated protein tau, and tau separation from microtubules. However, exact mechanisms are yet to be identified. In this paper the interaction of anesthetics with the microtubule subunit protein tubulin is investigated using computer-modeling methods. Homology modeling, molecular dynamics simulations and surface geometry techniques were used to determine putative binding sites for volatile anesthetics on tubulin. This was followed by free energy based docking calculations for halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the tubulin body, and C-terminal regions for specific tubulin isotypes. Locations of the putative binding sites, halothane binding energies and the relation to cytoskeleton function are reported in this paper. PMID:22761654

  18. Computational predictions of volatile anesthetic interactions with the microtubule cytoskeleton: implications for side effects of general anesthesia.

    Directory of Open Access Journals (Sweden)

    Travis J A Craddock

    Full Text Available The cytoskeleton is essential to cell morphology, cargo trafficking, and cell division. As the neuronal cytoskeleton is extremely complex, it is no wonder that a startling number of neurodegenerative disorders (including but not limited to Alzheimer's disease, Parkinson's disease and Huntington's disease share the common feature of a dysfunctional neuronal cytoskeleton. Recently, concern has been raised about a possible link between anesthesia, post-operative cognitive dysfunction, and the exacerbation of neurodegenerative disorders. Experimental investigations suggest that anesthetics bind to and affect cytoskeletal microtubules, and that anesthesia-related cognitive dysfunction involves microtubule instability, hyper-phosphorylation of the microtubule-associated protein tau, and tau separation from microtubules. However, exact mechanisms are yet to be identified. In this paper the interaction of anesthetics with the microtubule subunit protein tubulin is investigated using computer-modeling methods. Homology modeling, molecular dynamics simulations and surface geometry techniques were used to determine putative binding sites for volatile anesthetics on tubulin. This was followed by free energy based docking calculations for halothane (2-bromo-2-chloro-1,1,1-trifluoroethane on the tubulin body, and C-terminal regions for specific tubulin isotypes. Locations of the putative binding sites, halothane binding energies and the relation to cytoskeleton function are reported in this paper.

  19. Auxin-dependent microtubule responses and seedling development are affected in a rice mutant resistant to EPC

    International Nuclear Information System (INIS)

    Nick, P.; Yatou, O.; Furuya, M.; Lambert, A.M.

    1994-01-01

    Mutants in rice (Oryza sativa L. cv. japonica) were used to study the role of the cytoskeleton in signal-dependent morphogenesis. Mutants obtained by gamma ray irradiation were selected that failed to show inhibition of coleoptile elongation by the anti microtubular drug ethyl-N-phenylcarbamate (EPC). The mutation EPC-Resistant 31 (ER31), isolated from such a screen, caused lethality in putatively homozygous embryos. Heterozygotes exhibited drug resistance, impaired development of crown roots, and characteristic changes in the pattern of cell elongation: cell elongation was enhanced in mesocotyls and leaf sheaths, but inhibited in coleoptiles. The orientation of cortical microtubules changed correspondingly: for etiolated seedlings, compared with the wild-type, they were more transverse with respect to the long cell axis in mesocotyls and leaf sheaths, but more longitudinal in coleoptiles. In mutant coleoptiles, in contrast to wild-type, microtubules did not reorient in response to auxin, and their response to microtubule-eliminating and microtubule-stabilizing drugs was conspicuously reduced. In contrast, they responded normally to other stimuli such as gibberellins or red light. Auxin sensitivity as assayed by the dose-response for callus induction did not show any significant differences between wild-type and mutant. The mutant phenotype is interpreted in terms of an interrupted link between auxin-triggered signal transduction and microtubule reorientation. (author)

  20. Effect of axonal micro-tubules on the morphology of retinal nerve fibers studied by second-harmonic generation

    Science.gov (United States)

    Lim, Hyungsik; Danias, John

    2012-11-01

    Many studies suggest that the degradation of microtubules in the retinal ganglion cells may be an early event in the progression of glaucoma. Because reflectance and birefringence of the retinal nerve fibers arise primarily from microtubules, the optical properties have been intensively studied for early detection of the disease. We previously reported a novel nonlinear optical signal from axonal microtubules for visualizing the retinal nerve fibers, namely second-harmonic generation (SHG). We demonstrate the use of axonal SHG to investigate the effect of microtubules on the morphology of the retinal nerve fiber bundles. Time-lapse SHG imaging of ex vivo rat retinal flat mounts was performed during pharmacological treatment of nocodazole, and the intensity of axonal SHG and the changes in nerve fiber bundle morphology were monitored. We found that the microtubule disruption does not lead to immediate modification in the morphology of the nerve fibers. Our results indicate that microtubular SHG may provide a useful means for sensitive detection of axonal injuries. Since the intrinsic radiation depends on the regular architecture of the cytoskeleton element as maintained by active cellular regulations, the intensity of signal reflects the health of the retinal ganglion cell axons.

  1. Metabolic amplification of insulin secretion by glucose is independent of β-cell microtubules.

    Science.gov (United States)

    Mourad, Nizar I; Nenquin, Myriam; Henquin, Jean-Claude

    2011-03-01

    Glucose-induced insulin secretion (IS) by β-cells is controlled by two pathways. The triggering pathway involves ATP-sensitive potassium (K(ATP)) channel-dependent depolarization, Ca(2+) influx, and rise in the cytosolic Ca(2+) concentration ([Ca(2+)](c)), which triggers exocytosis of insulin granules. The metabolic amplifying pathway augments IS without further increasing [Ca(2+)](c). After exclusion of the contribution of actin microfilaments, we here tested whether amplification implicates microtubule-dependent granule mobilization. Mouse islets were treated with nocodazole or taxol, which completely depolymerized and polymerized tubulin. They were then perifused to measure [Ca(2+)](c) and IS. Metabolic amplification was studied during imposed steady elevation of [Ca(2+)](c) by tolbutamide or KCl or by comparing [Ca(2+)](c) and IS responses to glucose and tolbutamide. Nocodazole did not alter [Ca(2+)](c) or IS changes induced by the three secretagogues, whereas taxol caused a small inhibition of IS that is partly ascribed to a decrease in [Ca(2+)](c). When [Ca(2+)](c) was elevated and controlled by KCl or tolbutamide, the amplifying action of glucose was unaffected by microtubule disruption or stabilization. Both phases of IS were larger in response to glucose than tolbutamide, although triggering [Ca(2+)](c) was lower. This difference, due to amplification, persisted in nocodazole- or taxol-treated islets, even when IS was augmented fourfold by microfilament disruption with cytochalasin B or latrunculin B. In conclusion, metabolic amplification rapidly augments first and second phases of IS independently of insulin granule translocation along microtubules. We therefore extend our previous proposal that it does not implicate the cytoskeleton but corresponds to acceleration of the priming process conferring release competence to insulin granules.

  2. Polygamain, a New Microtubule Depolymerizing Agent That Occupies a Unique Pharmacophore in the Colchicine Site

    Science.gov (United States)

    Hartley, R. M.; Peng, J.; Fest, G. A.; Dakshanamurthy, S.; Frantz, D. E.; Brown, M. L.

    2012-01-01

    Bioassay-guided fractionation was used to isolate the lignan polygamain as the microtubule-active constituent in the crude extract of the Mountain torchwood, Amyris madrensis. Similar to the effects of the crude plant extract, polygamain caused dose-dependent loss of cellular microtubules and the formation of aberrant mitotic spindles that led to G2/M arrest. Polygamain has potent antiproliferative activities against a wide range of cancer cell lines, with an average IC50 of 52.7 nM. Clonogenic studies indicate that polygamain effectively inhibits PC-3 colony formation and has excellent cellular persistence after washout. In addition, polygamain is able to circumvent two clinically relevant mechanisms of drug resistance, the expression of P-glycoprotein and the βIII isotype of tubulin. Studies with purified tubulin show that polygamain inhibits the rate and extent of purified tubulin assembly and displaces colchicine, indicating a direct interaction of polygamain within the colchicine binding site on tubulin. Polygamain has structural similarities to podophyllotoxin, and molecular modeling simulations were conducted to identify the potential orientations of these compounds within the colchicine binding site. These studies suggest that the benzodioxole group of polygamain occupies space similar to the trimethoxyphenyl group of podophyllotoxin but with distinct interactions within the hydrophobic pocket. Our results identify polygamain as a new microtubule destabilizer that seems to occupy a unique pharmacophore within the colchicine site of tubulin. This new pharmacophore will be used to design new colchicine site compounds that might provide advantages over the current agents. PMID:22169850

  3. Stabilization of microtubules by taxane diterpenoids: insight from docking and MD simulations.

    Science.gov (United States)

    Yadava, Umesh; Gupta, Hariom; Roychoudhury, Mihir

    2015-03-01

    Microtubules are formed from the molecules of tubulin, whose dynamics is important for many functions in a cell, the most dramatic of which is mitosis. Taxol is known to interact within a specific site on tubulin and also believed to block cell-cycle progression during mitosis by binding to and stabilizing microtubules. Along with the tremendous potential that taxol has shown as an anticancer drug, clinical problems exist with solubility, toxicity, and development of drug resistance. The crystal structure of taxane diterpenoids, namely, 10, 13-deacetyl-abeo-baccatin-IV (I), 5-acetyl-2-deacetoxydecinnamoyl-taxinine-0.29hydrate (II), 7, 9-dideacetyltaxayuntin (III), and Taxawallin-K (IV), are very similar to the taxol molecule. Considerable attention has been given to such molecules whose archetype is taxol but do not posses long aliphatic chains, to be developed as a substitute for taxol with fewer side effects. In the present work, the molecular docking of these taxane diterpenoids has been carried out with the tubulin alpha-beta dimer (1TUB) and refined microtubule structure (1JFF) using Glide-XP, in order to assess the potential of tubulin binding of these cytotoxic agents. Results show that all the ligands dock into the classical taxol binding site of tubulin. Taxol shows the best binding capabilities. On the basis of docking energy and interactions, apart from taxol, molecule II has a better tendency of binding with 1TUB while molecule I shows better binding capability with bovine tubulin 1JFF. To validate the binding capabilities, molecular dynamics (MD) simulations of the best docked complexes of ligands with 1JFF have been carried out for 15.0 ns using DESMOND. Average RMSD variations and time line study of interactions and contacts indicate that these complexes remain stable during the course of the dynamics. However, taxol and molecule II prevail over other taxoids.

  4. The effect of a 94 GHz electromagnetic field on neuronal microtubules.

    Science.gov (United States)

    Samsonov, Andrey; Popov, Sergey V

    2013-02-01

    Hardware that generates electromagnetic waves with wavelengths from 1 to 10 mm (millimeter waves, "MMW") is being used in a variety of applications, including high-speed data communication and medical devices. This raises both practical and fundamental issues concerning the interaction of MMW electromagnetic fields (EMF) with biological tissues. A 94 GHz EMF is of particular interest because a number of applications, such as active denial systems, rely on this specific frequency. Most of the energy associated with MMW radiation is absorbed in the skin and, for a 94 GHz field, the power penetration depth is shallow (≈0.4 mm). At sufficiently high energies, skin heating is expected to activate thermal pain receptors, leading to the perception of pain. In addition to this "thermal" mechanism of action, a number of "non-thermal" effects of MMW fields have been previously reported. Here, we investigated the influence of a 94 GHz EMF on the assembly/disassembly of neuronal microtubules in Xenopus spinal cord neurons. We reasoned that since microtubule array is regulated by a large number of intracellular signaling cascades, it may serve as an exquisitely sensitive reporter for the biochemical status of neuronal cytoplasm. We found that exposure to 94 GHz radiation increases the rate of microtubule assembly and that this effect can be entirely accounted for by the rapid EMF-elicited temperature jump. Our data are consistent with the notion that the cellular effects of a 94 GHz EMF are mediated entirely by cell heating. Copyright © 2012 Wiley Periodicals, Inc.

  5. Simultaneous Manipulation and Super-Resolution Fluorescence Imaging of Individual Kinetochores Coupled to Microtubule Tips.

    Science.gov (United States)

    Deng, Yi; Asbury, Charles L

    2017-01-01

    Kinetochores are large multiprotein complexes that drive mitotic chromosome movements by mechanically coupling them to the growing and shortening tips of spindle microtubules. Kinetochores are also regulatory hubs, somehow sensing when they are erroneously attached and, in response, releasing their incorrect attachments and generating diffusible wait signals to delay anaphase until proper attachments can form. The remarkable ability of a kinetochore to sense and respond to its attachment status might stem from attachment- or tension-dependent changes in the structural arrangement of its core subcomplexes. However, direct tests of the relationship between attachment, tension, and core kinetochore structure have not previously been possible because of the difficulties of applying well-controlled forces and determining unambiguously the attachment status of individual kinetochores in vivo. The recent purification of native yeast kinetochores has enabled in vitro optical trapping-based assays of kinetochore tip-coupling and, in separate experiments, fluorescence imaging of single kinetochore particles. Here we introduce a dual instrument, combining optical trapping with multicolor total internal reflection fluorescence (TIRF) imaging, to allow kinetochore structure to be monitored directly with nanometer precision while mechanical tension is simultaneously applied. Our instrument incorporates differential interference contrast (DIC) imaging as well, to minimize the photo-bleaching of fluorescent tags during preparative bead and microtubule manipulations. A simple modification also allows the trapping laser to be easily converted into a real-time focus detection and correction system. Using this combined instrument, the distance between specific subcomplexes within a single kinetochore particle can be measured with 2-nm precision after 50 s observation time, or with 11-nm precision at 1 s temporal resolution. While our instrument was constructed specifically for

  6. The role of actin and microtubule networks in plasmid DNA intracellular trafficking

    Czech Academy of Sciences Publication Activity Database

    Ondřej, Vladan; Lukášová, Emilie; Falk, Martin; Kozubek, Stanislav

    2007-01-01

    Roč. 54, č. 3 (2007), s. 657-663 ISSN 0001-527X R&D Projects: GA ČR(CZ) GA202/02/0804; GA ČR(CZ) GA202/04/0907; GA AV ČR(CZ) 1QS500040508; GA MŠk(CZ) LC535 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : plasmid DNA * actin filaments * microtubules Subject RIV: BO - Biophysics Impact factor: 1.261, year: 2007

  7. Colchitaxel, a coupled compound made from microtubule inhibitors colchicine and paclitaxel

    Directory of Open Access Journals (Sweden)

    Uppal Sonal O

    2006-06-01

    Full Text Available Abstract Background Tumor promoters enhance tumor yield in experimental animals without directly affecting the DNA of the cell. Promoters may play a role in the development of cancer, as humans are exposed to them in the environment. In work based on computer-assisted microscopy and sophisticated classification methods, we showed that cells could be classified by reference to a database of known normal and cancerous cell phenotypes. Promoters caused loss of properties specific to normal cells and gain of properties of cancer cells. Other compounds, including colchicine, had a similar effect. Colchicine given together with paclitaxel, however, caused cells to adopt properties of normal cells. This provided a rationale for tests of microtubule inhibitor combinations in cancer patients. The combination of a depolymerizing and a stabilizing agent is a superior anti-tumor treatment. The biological basis of the effect is not understood. Results A single compound containing both colchicine and paclitaxel structures was synthesized. Colchicine is an alkaloid with a trimethoxyphenyl ring (ring A, a ring with an acetamide linkage (ring B, and a tropolone ring (ring C. Although rings A and C are important for tubulin-binding activity, the acetamide linkage on ring B could be replaced by an amide containing a glutamate linker. Alteration of the C-7 site on paclitaxel similarly had little or no inhibitory effect on its biological activity. The linker was attached to this position. The coupled compound, colchitaxel (1, had some of the same effects on microtubules as the combination of starting compounds. It also caused shortening and fragmentation of the + end protein cap. Conclusion Since microtubule inhibitor combinations give results unlike those obtained with either inhibitor alone, it is important to determine how such combinations affect cell shape and growth. Colchitaxel shows a subset of the effects of the inhibitor combination. Thus, it may be able

  8. XTACC3-XMAP215 association reveals an asymmetric interaction promoting microtubule elongation

    DEFF Research Database (Denmark)

    Mortuza, Gulnahar B; Cavazza, Tommaso; Garcia-Mayoral, Maria Flor

    2014-01-01

    215 (chTOG), dissecting the mechanism by which their interaction promotes microtubule elongation during spindle assembly. Using SAXS, we show that the TACC domain (TD) is an elongated structure that mediates the interaction with the C terminus of XMAP215. Our data suggest that one TD and two XMAP215...... molecules associate to form a four-helix coiled-coil complex. A hybrid methods approach was used to define the precise regions of the TACC heptad repeat and the XMAP215 C terminus required for assembly and functioning of the complex. We show that XTACC3 can induce the recruitment of larger amounts of XMAP...

  9. Asymmetric Microtubule Function Is an Essential Requirement for Polarized Organization of the Drosophila Bristle▿ †

    OpenAIRE

    Bitan, Amir; Guild, Gregory M.; Bar-Dubin, Dikla; Abdu, Uri

    2009-01-01

    While previous studies have shown that microtubules (MTs) are essential for maintaining the highly biased axial growth of the Drosophila bristle, the mechanism for this process has remained vague. We report that the MT minus-end marker, Nod-KHC, accumulates at the bristle tip, suggesting that the MT network in the bristle is organized minus end out. Potential markers for studying the importance of properly polarized MTs to bristle axial growth are Ik2 and Spindle-F (Spn-F), since mutations in...

  10. Normal mammary epithelial cells promote carcinoma basement membrane invasion by inducing microtubule-rich protrusions.

    Science.gov (United States)

    Lee, Meng-Horng; Wu, Pei-Hsun; Gilkes, Daniele; Aifuwa, Ivie; Wirtz, Denis

    2015-10-20

    Recent work suggests that the dissemination of tumor cells may occur in parallel with, and even preceed, tumor growth. The mechanism for this early invasion is largely unknown. Here, we find that mammary epithelial cells (MECs) induce neighboring breast carcinoma cells (BCCs) to cross the basement membrane by secreting soluble laminin. Laminin continuously produced by MECs induce long membrane cellular protrusions in BCCs that promote their contractility and invasion into the surrounding matrix. These protrusions depend on microtubule bundles assembled de novo through laminin-integrin β1 signaling. These results describe how non-cancerous MECs can actively participate in the invasive process of BCCs.

  11. Phosphorylation of the yeast γ-tubulin Tub4 regulates microtubule function

    DEFF Research Database (Denmark)

    Lin, Tien-chen; Gombos, Linda; Neuner, Annett

    2011-01-01

    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...... 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...

  12. GIT1/beta PIX signaling proteins and PAK1 kinase regulate microtubule nucleation

    Czech Academy of Sciences Publication Activity Database

    Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

    2016-01-01

    Roč. 1863, č. 6 (2016), s. 1282-1297 ISSN 0167-4889 R&D Projects: GA ČR GAP302/12/1673; GA ČR GA15-22194S; GA MŠk LH12050; GA MZd NT14467; GA ČR GA16-23702S Institutional support: RVO:68378050 Keywords : Centrosome * Microtubule nucleation * gamma-tubulin * GIT1/beta PIX signaling proteins * PAK1 kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.521, year: 2016

  13. Microtubule Regulation of Kv7 Channels Orchestrates cAMP-Mediated Vasorelaxations in Rat Arterial Smooth Muscle

    DEFF Research Database (Denmark)

    Lindman, Johanna; Khammy, Makhala M; Lundegaard, Pia R

    2018-01-01

    of this pathway in vascular smooth muscle cells, we investigated the role of microtubule stability on β-adrenoceptor signaling in rat renal and mesenteric arteries. In isometric tension experiments, incubation with the microtubule inhibitors colchicine and nocodazole enhanced isoprenaline-mediated relaxations...... of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented. Sharp microelectrode experiments showed that colchicine treatment caused increased hyperpolarization of mesenteric artery segments in response to isoprenaline. Application of the Kv7 channel blocker, XE991, attenuated...... the effect of colchicine on isoprenaline relaxations, whereas iberiotoxin-a BKCa channel blocker-had no effect. In addition, colchicine improved the relaxations to the Kv7.2 to 7.5 activator, S-1, in both renal and mesenteric artery segments compared with dimethyl sulfoxide incubation. We determined...

  14. CYLD regulates spindle orientation by stabilizing astral microtubules and promoting dishevelled-NuMA-dynein/dynactin complex formation.

    Science.gov (United States)

    Yang, Yunfan; Liu, Min; Li, Dengwen; Ran, Jie; Gao, Jinmin; Suo, Shaojun; Sun, Shao-Cong; Zhou, Jun

    2014-02-11

    Oriented cell division is critical for cell fate specification, tissue organization, and tissue homeostasis, and relies on proper orientation of the mitotic spindle. The molecular mechanisms underlying the regulation of spindle orientation remain largely unknown. Herein, we identify a critical role for cylindromatosis (CYLD), a deubiquitinase and regulator of microtubule dynamics, in the control of spindle orientation. CYLD is highly expressed in mitosis and promotes spindle orientation by stabilizing astral microtubules and deubiquitinating the cortical polarity protein dishevelled. The deubiquitination of dishevelled enhances its interaction with nuclear mitotic apparatus, stimulating the cortical localization of nuclear mitotic apparatus and the dynein/dynactin motor complex, a requirement for generating pulling forces on astral microtubules. These findings uncover CYLD as an important player in the orientation of the mitotic spindle and cell division and have important implications in health and disease.

  15. Three-dimensional tracking of plus-tips by lattice light-sheet microscopy permits the quantification of microtubule growth trajectories within the mitotic apparatus

    Science.gov (United States)

    Yamashita, Norio; Morita, Masahiko; Legant, Wesley R.; Chen, Bi-Chang; Betzig, Eric; Yokota, Hideo; Mimori-Kiyosue, Yuko

    2015-10-01

    Mitotic apparatus, which comprises hundreds of microtubules, plays an essential role in cell division, ensuring the correct segregation of chromosomes into each daughter cell. To gain insight into its regulatory mechanisms, it is essential to detect and analyze the behavior of individual microtubule filaments. However, the discrimination of discrete microtubule filaments within the mitotic apparatus is beyond the capabilities of conventional light microscopic technologies. Recently, we detected three-dimensional (3-D) microtubule growth dynamics within the cellular cytoplasmic space using lattice light-sheet microscopy in conjunction with microtubule growth marker protein end-binding 1, a microtubule plus-end-tracking protein, which was fused to green fluorescent protein (EB1-GFP). This technique enables high-resolution 3-D imaging at subsecond intervals. We adapted mathematical computing and geometric representation techniques to analyze spatial variations in microtubule growth dynamics within the mitotic spindle apparatus. Our analytical approach enabled the different dynamic properties of individual microtubules to be determined, including the direction and speed of their growth, and their growth duration within a 3-D spatial map. Our analysis framework provides an important step toward a more comprehensive understanding of the mechanisms driving cellular machinery at the whole-cell level.

  16. An antitubulin agent BCFMT inhibits proliferation of cancer cells and induces cell death by inhibiting microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Ankit Rai

    Full Text Available Using cell based screening assay, we identified a novel anti-tubulin agent (Z-5-((5-(4-bromo-3-chlorophenylfuran-2-ylmethylene-2-thioxothiazolidin-4-one (BCFMT that inhibited proliferation of human cervical carcinoma (HeLa (IC(50, 7.2 ± 1.8 µM, human breast adenocarcinoma (MCF-7 (IC(50, 10.0 ± 0.5 µM, highly metastatic breast adenocarcinoma (MDA-MB-231 (IC(50, 6.0 ± 1 µM, cisplatin-resistant human ovarian carcinoma (A2780-cis (IC(50, 5.8 ± 0.3 µM and multi-drug resistant mouse mammary tumor (EMT6/AR1 (IC(50, 6.5 ± 1 µM cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 µM, BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably state by 135% and reduced the dynamicity (dimer exchange per unit time of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3 ± 1.8 µM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K(i of 5.2 ± 1.5 µM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2 at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug

  17. [Wide spectrum of arrhythmias, with inductions and terminations of atrio-ventricular replica-nodal tachycardia by automaticity during successful radiofrequency ablation of long anterogradely slow conducting atrio-fasciculo-ventricular accessory pathway].

    Science.gov (United States)

    Bodalski, Robert; Szumowski, Lukasz; Walczak, Franciszek

    2012-01-01

    Mahaim syndrome is still a challenge. The specific characteristics of the pathway, the fact that it seldom occurs, and ambiguities about the pathophysiology and nomenclature make it, for electrophysiologists, still difficult to ablate. We present a case of a patient with anterogradely slow conducting accessory pathway, which has been effectively ablated although M potential was not observed nor was a mechanical conducting block of the accessory pathway. The accessory pathway showed a wide spectrum of electrophysiological characteristics of the "AV node physiology" including inductions and terminations of atrio-ventricular replica-nodal tachycardia by local automaticity, not only during electrophysiological study, but during successful application. The accessory pathway presented signs of two types of pseudo Mahaim fibers: atrio-fascicular and slow conducting atrio-ventricular connection.

  18. ATX-2, the C. elegans Ortholog of Human Ataxin-2, Regulates Centrosome Size and Microtubule Dynamics.

    Science.gov (United States)

    Stubenvoll, Michael D; Medley, Jeffrey C; Irwin, Miranda; Song, Mi Hye

    2016-09-01

    Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT) to form mitotic bipolar spindles that ensure proper chromosome segregation. We report a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2, in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, γ-Tubulin), increasing MT nucleating activity but impeding MT growth. We show that ATX-2 influences MT behavior through γ-Tubulin at the centrosome. Our data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics.

  19. Kinetic theory of pattern formation in mixtures of microtubules and molecular motors

    Science.gov (United States)

    Maryshev, Ivan; Marenduzzo, Davide; Goryachev, Andrew B.; Morozov, Alexander

    2018-02-01

    In this study we formulate a theoretical approach, based on a Boltzmann-like kinetic equation, to describe pattern formation in two-dimensional mixtures of microtubular filaments and molecular motors. Following the previous work by Aranson and Tsimring [Phys. Rev. E 74, 031915 (2006), 10.1103/PhysRevE.74.031915] we model the motor-induced reorientation of microtubules as collision rules, and devise a semianalytical method to calculate the corresponding interaction integrals. This procedure yields an infinite hierarchy of kinetic equations that we terminate by employing a well-established closure strategy, developed in the pattern-formation community and based on a power-counting argument. We thus arrive at a closed set of coupled equations for slowly varying local density and orientation of the microtubules, and study its behavior by performing a linear stability analysis and direct numerical simulations. By comparing our method with the work of Aranson and Tsimring, we assess the validity of the assumptions required to derive their and our theories. We demonstrate that our approximation-free evaluation of the interaction integrals and our choice of a systematic closure strategy result in a rather different dynamical behavior than was previously reported. Based on our theory, we discuss the ensuing phase diagram and the patterns observed.

  20. Integrins Regulate Apical Constriction via Microtubule Stabilization in the Drosophila Eye Disc Epithelium

    Directory of Open Access Journals (Sweden)

    Vilaiwan M. Fernandes

    2014-12-01

    Full Text Available During morphogenesis, extracellular signals trigger actomyosin contractility in subpopulations of cells to coordinate changes in cell shape. To illuminate the link between signaling-mediated tissue patterning and cytoskeletal remodeling, we study the progression of the morphogenetic furrow (MF, the wave of apical constriction that traverses the Drosophila eye imaginal disc preceding photoreceptor neurogenesis. Apical constriction depends on actomyosin contractility downstream of the Hedgehog (Hh and bone morphogenetic protein (BMP pathways. We identify a role for integrin adhesion receptors in MF progression. We show that Hh and BMP regulate integrin expression, the loss of which disrupts apical constriction and slows furrow progression; conversely, elevated integrins accelerate furrow progression. We present evidence that integrins regulate MF progression by promoting microtubule stabilization, since reducing microtubule stability rescues integrin-mediated furrow acceleration. Thus, integrins act as a genetic link between tissue-level signaling events and morphological change at the cellular level, leading to morphogenesis and neurogenesis in the eye.

  1. Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules

    International Nuclear Information System (INIS)

    Kolobova, Elena; Efimov, Andrey; Kaverina, Irina; Rishi, Arun K.; Schrader, John W.; Ham, Amy-Joan; Larocca, M. Cecilia; Goldenring, James R.

    2009-01-01

    Recent investigations have highlighted the importance of subcellular localization of mRNAs to cell function. While AKAP350A, a multifunctional scaffolding protein, localizes to the Golgi apparatus and centrosomes, we have now identified a cytosolic pool of AKAP350A. Analysis of AKAP350A scaffolded complexes revealed two novel interacting proteins, CCAR1 and caprin-1. CCAR1, caprin-1 and AKAP350A along with G3BP, a stress granule marker, relocate to RNA stress granules after arsenite treatment. Stress also caused loss of AKAP350 from the Golgi and fragmentation of the Golgi apparatus. Disruption of microtubules with nocodazole altered stress granule formation and changed their morphology by preventing fusion of stress granules. In the presence of nocodazole, arsenite induced smaller granules with the vast majority of AKAP350A and CCAR1 separated from G3BP-containing granules. Similar to nocodazole treatment, reduction of AKAP350A or CCAR1 expression also altered the size and number of G3BP-containing stress granules induced by arsenite treatment. A limited set of 69 mRNA transcripts was immunoisolated with AKAP350A even in the absence of stress, suggesting the association of AKAP350A with mRNA transcripts. These results provide the first evidence for the microtubule dependent association of AKAP350A and CCAR1 with RNA stress granules

  2. Saccharomyces cerevisiae gene ISW2 encodes a microtubule-interacting protein required for premeiotic DNA replication.

    Science.gov (United States)

    Trachtulcová, P; Janatová, I; Kohlwein, S D; Hasek, J

    2000-01-15

    A molecular genetic characterization of the ORF YOR304W (ISW2), identified in a screen of a yeast lambdagt11 library using a monoclonal antibody that reacts with a 210 kDa mammalian microtubule-interacting protein, is presented in this paper. The protein encoded by the ORF YOR304W is 50% identical to the Drosophila nucleosome remodelling factor ISWI and is therefore a new member of the SNF2 protein family and has been recently entered into SDG as ISW2. Although not essential for vegetative growth, we found that the ISW2 gene is required for early stages in sporulation. The isw2 homozygous deletant diploid strain was blocked in the G(1) phase of the cell cycle, unable to execute the premeiotic DNA replication and progress through the nuclear meiotic division cycle. ISW2 expression from a multicopy plasmid had the same effect as deletion, but ISW2 expression from a centromeric plasmid rescued the deletion phenotype. In vegetatively growing diploid cells, the Isw2 protein was preferentially found in the cytoplasm, co-localizing with microtubules. An accumulation of the Isw2 protein within the nucleus was observed in cells entering sporulation. Together with data published very recently by Tsukiyama et al. (1999), we propose a role for the Isw2 protein in facilitating chromatin accessibility for transcriptional factor(s) that positively regulate meiosis/sporulation-specific genes. Copyright 2000 John Wiley & Sons, Ltd.

  3. Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix

    Directory of Open Access Journals (Sweden)

    Ramírez G.

    1999-01-01

    Full Text Available As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.

  4. Interaction between RB protein and NuMA is required for proper alignment of spindle microtubules.

    Science.gov (United States)

    Uchida, Chiharu; Hattori, Takayuki; Takahashi, Hirotaka; Yamamoto, Naoki; Kitagawa, Masatoshi; Taya, Yoichi

    2014-02-01

    Retinoblastoma protein (pRB) controls cell cycle progression and cell cycle exit through interactions with cellular proteins. Many pRB-binding proteins, which function in gene transcription or modulation of chromatin structure, harbor LXCXE motifs in their binding domain for pRB. In this study, we found that nuclear mitotic apparatus protein (NuMA), a mitotic spindle organizer, interacts with pRB through LSCEE sequences located in its C-terminal region. siRNA-mediated down-regulation of pRB caused aberrant distribution of NuMA and alignment of spindle microtubules in mitotic cells. Abnormal organization of spindle microtubules was also accompanied by misalignment of an over-expressed NuMA mutant (mut-NuMA) with a defect in pRB binding caused by an LSGEK mutation. The mut-NuMA-over-expressing cells showed lower potency for survival than wild-type NuMA (wt-NuMA)-over-expressing cells during 2 weeks of culture. Interestingly, knockdown of pRB reduced the population of wt-NuMA-over-expressing cells to the same level as mut-NuMA cells after 2 weeks. Taken together, pRB may have a novel function in regulating the mitotic function of NuMA and spindle organization, which are required for proper cell cycle progression. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  5. A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells

    Science.gov (United States)

    Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.

    2017-09-01

    The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

  6. ATX-2, the C. elegans Ortholog of Human Ataxin-2, Regulates Centrosome Size and Microtubule Dynamics.

    Directory of Open Access Journals (Sweden)

    Michael D Stubenvoll

    2016-09-01

    Full Text Available Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT to form mitotic bipolar spindles that ensure proper chromosome segregation. We report a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2, in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, γ-Tubulin, increasing MT nucleating activity but impeding MT growth. We show that ATX-2 influences MT behavior through γ-Tubulin at the centrosome. Our data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics.

  7. Microtubule affinity-regulating kinases are potential druggable targets for Alzheimer's disease.

    Science.gov (United States)

    Annadurai, Narendran; Agrawal, Khushboo; Džubák, Petr; Hajdúch, Marián; Das, Viswanath

    2017-11-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects normal functions of the brain. Currently, AD is one of the leading causes of death in developed countries and the only one of the top ten diseases without a means to prevent, cure, or significantly slow down its progression. Therefore, newer therapeutic concepts are urgently needed to improve survival and the quality of life of AD patients. Microtubule affinity-regulating kinases (MARKs) regulate tau-microtubule binding and play a crucial role in neurons. However, their role in hyperphosphorylation of tau makes them potential druggable target for AD therapy. Despite the relevance of MARKs in AD pathogenesis, only a few small molecules are known to have anti-MARK activity and not much has been done to progress these compounds into therapeutic candidates. But given the diverse role of MARKs, the specificity of novel inhibitors is imperative for their successful translation from bench to bedside. In this regard, a recent co-crystal structure of MARK4 in association with a pyrazolopyrimidine-based inhibitor offers a potential scaffold for the development of more specific MARK inhibitors. In this manuscript, we review the biological role of MARKs in health and disease, and draw attention to the largely unexplored area of MARK inhibitors for AD.

  8. Induction of microtubule damage in Allium cepa meristematic cells by pharmaceutical formulations of thiabendazole and griseofulvin.

    Science.gov (United States)

    Andrioli, Nancy B; Soloneski, Sonia; Larramendy, Marcelo L; Mudry, Marta D

    2014-09-15

    Microtubules (MT) are formed by the assembly of α- and β-tubulins and MT-associated proteins. We characterized the effects of pharmaceutical formulations containing the microtubule disruptors thiabendazole (TBZ) and griseofulvin (GF) on the mitotic machinery of plant (A. cepa) meristematic cells. GF concentrations between 10 and 250 μg/ml were tested. GF induced mitotic index inhibition and genotoxic effects, including chromosome fragments, bridges, lagged chromosomes, C-metaphases, tripolar cell division, disorganized anaphases and nuclear abnormalities in interphase cells. Efects on the mitotic machinery were studied by direct immunofluorescence with β-tubulin labeling and by DNA counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Exposure of meristematic root cells to TBZ or GF, 100 μg/ml, caused microtubular damage which led to abnormal MT arrays. Our results suggest that GF induces abnormalities in spindle symmetry/polarity, while TBZ causes chromosome missegregation, polyploidy, and lack of cytokinesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Ivermectin binds to Haemonchus contortus tubulins and promotes stability of microtubules.

    Science.gov (United States)

    Ashraf, Shoaib; Beech, Robin N; Hancock, Mark A; Prichard, Roger K

    2015-08-01

    Haemonchus contortus is a nematode of livestock that can cause severe disease and mortality. Ivermectin, an anti-parasitic drug that targets glutamate-gated chloride channels, is widely used in humans, livestock, companion animals and agriculture. Although an association between genetic changes to β-tubulin and exposure to ivermectin has been previously reported, direct binding between ivermectin and tubulin has not been demonstrated to date. Tubulin/microtubules are key targets for many anti-mitotic drugs used in anti-parasite and cancer therapies. We now report that ivermectin exposure increased the rate and extent of polymerisation of H. contortus recombinant α- and β-tubulin, and protected the parasitic α- and β-tubulins from limited trypsin proteolysis. Direct binding between ivermectin and the tubulin monomers exhibited low micromolar affinities, as determined using surface plasmon resonance. Subsequent equilibrium dialysis indicated that ivermectin and Taxol compete for binding to tubulin, supporting our molecular modelling that predicts ivermectin interacts with the Taxol binding pocket of both parasitic and mammalian tubulins. Collectively, our data indicate that ivermectin can bind to and stabilise microtubules (i.e., alter the tubulin polymerisation equilibrium) and this can then lead to mitotic arrest. This work extends the range of known pharmacological effects of ivermectin, and reveals its potential as an anti-mitotic agent. Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  10. Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction

    Science.gov (United States)

    Xiao, Ping-Jie; Mitchell, Angela M.; Huang, Lu; Li, Chengwen; Samulski, R. Jude

    2016-01-01

    Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion. PMID:26942476

  11. Synthesis and biological evaluation of indolyl-pyridinyl-propenones having either methuosis or microtubule disruption activity.

    Science.gov (United States)

    Trabbic, Christopher J; Overmeyer, Jean H; Alexander, Evan M; Crissman, Emily J; Kvale, Heather M; Smith, Marcie A; Erhardt, Paul W; Maltese, William A

    2015-03-12

    Methuosis is a form of nonapoptotic cell death characterized by an accumulation of macropinosome-derived vacuoles with eventual loss of membrane integrity. Small molecules inducing methuosis could offer significant advantages compared to more traditional anticancer drug therapies that typically rely on apoptosis. Herein we further define the effects of chemical substitutions at the 2- and 5-indolyl positions on our lead compound 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP). We have identified a number of compounds that induce methuosis at similar potencies, including an interesting analogue having a hydroxypropyl substituent at the 2-position. In addition, we have discovered that certain substitutions on the 2-indolyl position redirect the mode of cytotoxicity from methuosis to microtubule disruption. This switch in activity is associated with an increase in potency as large as 2 orders of magnitude. These compounds appear to represent a new class of potent microtubule-active anticancer agents.

  12. Cyclin A2 modulates kinetochore-microtubule attachment in meiosis II.

    Science.gov (United States)

    Zhang, Qing-Hua; Yuen, Wai Shan; Adhikari, Deepak; Flegg, Jennifer A; FitzHarris, Greg; Conti, Marco; Sicinski, Piotr; Nabti, Ibtissem; Marangos, Petros; Carroll, John

    2017-10-02

    Cyclin A2 is a crucial mitotic Cdk regulatory partner that coordinates entry into mitosis and is then destroyed in prometaphase within minutes of nuclear envelope breakdown. The role of cyclin A2 in female meiosis and its dynamics during the transition from meiosis I (MI) to meiosis II (MII) remain unclear. We found that cyclin A2 decreases in prometaphase I but recovers after the first meiotic division and persists, uniquely for metaphase, in MII-arrested oocytes. Conditional deletion of cyclin A2 from mouse oocytes has no discernible effect on MI but leads to disrupted MII spindles and increased merotelic attachments. On stimulation of exit from MII, there is a dramatic increase in lagging chromosomes and an inhibition of cytokinesis. These defects are associated with an increase in microtubule stability in MII spindles, suggesting that cyclin A2 mediates the fidelity of MII by maintaining microtubule dynamics during the rapid formation of the MII spindle. © 2017 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.

  13. Intracellular transport: from physics to ... biology.

    Science.gov (United States)

    Roux, Aurélien; Cuvelier, Damien; Bassereau, Patricia; Goud, Bruno

    2008-03-01

    Considerable effort over the past three decades has allowed the identification of the protein families that control the cellular machinery responsible for intracellular transport within eukaryotic cells. These proteins are estimated to represent about 10-20% of the human "proteome." The complexity of intracellular transport makes useful the development of model membranes. We describe here experimental systems based on lipid giant unilamellar vesicles (GUVs), which are attached to kinesin molecules. These systems give rise to thin membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. This type of assay, which mimics key events occurring during intracellular transport, allows physicists and biologists to understand how the unique mechanical properties of lipid membranes could be involved in the budding process, the sorting of cargo proteins and lipids, and the separation of the buds from a donor membrane.

  14. The Microtubule Plus-End Tracking Protein CLASP2 Is Required for Hematopoiesis and Hematopoietic Stem Cell Maintenance

    Directory of Open Access Journals (Sweden)

    Ksenija Drabek

    2012-10-01

    Full Text Available Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

  15. The microtubule-associated protein 1A (MAP1A) is an early molecular target of soluble Aβ-peptide

    DEFF Research Database (Denmark)

    Clemmensen, C; Aznar, S; Knudsen, G M

    2012-01-01

    ) protein, proposing that microtubule perturbations might be central for the Aβ-induced neuronal dysfunctions as PSD-95 plays a key role in synaptic plasticity. In conclusion, this study suggests that disruption of MAP1A could be a very early manifestation of Aβ-mediated synaptic dysfunction...

  16. The plant microtubule-associated protein AtMAP65-3/PLE is essential for cytokinetic phragmoplast function.

    Science.gov (United States)

    Müller, Sabine; Smertenko, Andrei; Wagner, Vera; Heinrich, Maria; Hussey, Patrick J; Hauser, Marie-Theres

    2004-03-09

    Directional cell expansion in interphase and nuclear and cell division in M-phase are mediated by four microtubule arrays, three of which are unique to plants: the interphase array, the preprophase band, and the phragmoplast. The plant microtubule-associated protein MAP65 has been identified as a key structural component in these arrays. The Arabidopsis genome has nine MAP65 genes, and here we show that one, AtMAP65-3/PLE, locates only to the mitotic arrays and is essential for cytokinesis. The Arabidopsis pleiade (ple) alleles are single recessive mutations, and we show that these mutations are in the AtMAP65-3 gene. Moreover, these mutations cause C-terminal truncations that abolish microtubule binding. In the ple mutants the anaphase spindle is normal, and the cytokinetic phragmoplast can form but is distorted; not only is it wider, but the midline, the region where oppositely oriented microtubules overlap, is unusually expanded. Here we present data that demonstrate an essential role for AtMAP65-3/PLE in cytokinesis in plant cells.

  17. Microtubule-associated protein 6 mediates neuronal connectivity through Semaphorin 3E-dependent signalling for axonal growth

    Science.gov (United States)

    Deloulme, Jean-Christophe; Gory-Fauré, Sylvie; Mauconduit, Franck; Chauvet, Sophie; Jonckheere, Julie; Boulan, Benoit; Mire, Erik; Xue, Jing; Jany, Marion; Maucler, Caroline; Deparis, Agathe A.; Montigon, Olivier; Daoust, Alexia; Barbier, Emmanuel L.; Bosc, Christophe; Deglon, Nicole; Brocard, Jacques; Denarier, Eric; Le Brun, Isabelle; Pernet-Gallay, Karin; Vilgrain, Isabelle; Robinson, Phillip J.; Lahrech, Hana; Mann, Fanny; Andrieux, Annie

    2015-01-01

    Structural microtubule associated proteins (MAPs) stabilize microtubules, a property that was thought to be essential for development, maintenance and function of neuronal circuits. However, deletion of the structural MAPs in mice does not lead to major neurodevelopment defects. Here we demonstrate a role for MAP6 in brain wiring that is independent of microtubule binding. We find that MAP6 deletion disrupts brain connectivity and is associated with a lack of post-commissural fornix fibres. MAP6 contributes to fornix development by regulating axonal elongation induced by Semaphorin 3E. We show that MAP6 acts downstream of receptor activation through a mechanism that requires a proline-rich domain distinct from its microtubule-stabilizing domains. We also show that MAP6 directly binds to SH3 domain proteins known to be involved in neurite extension and semaphorin function. We conclude that MAP6 is critical to interface guidance molecules with intracellular signalling effectors during the development of cerebral axon tracts. PMID:26037503

  18. Control of the initiation and termination of kinesin-1-driven transport by myosin-Ic and nonmuscle tropomyosin.

    Science.gov (United States)

    McIntosh, Betsy B; Holzbaur, Erika L F; Ostap, E Michael

    2015-02-16

    Intracellular transport is largely driven by processive microtubule- and actin-based molecular motors. Nonprocessive motors have also been localized to trafficking cargos, but their roles are not well understood. Myosin-Ic (Myo1c), a nonprocessive actin motor, functions in a variety of exocytic events, although the underlying mechanisms are not yet clear. To investigate the interplay between myosin-I and the canonical long-distance transport motor kinesin-1, we attached both motor types to lipid membrane-coated bead cargo, using an attachment strategy that allows motors to actively reorganize within the membrane in response to the local cytoskeletal environment. We compared the motility of kinesin-1-driven cargos in the absence and presence of Myo1c at engineered actin/microtubule intersections. We found that Myo1c significantly increases the frequency of kinesin-1-driven microtubule-based runs that begin at actin/microtubule intersections. Myo1c also regulates the termination of processive runs. Beads with both motors bound have a significantly higher probability of pausing at actin/microtubule intersections, remaining tethered for an average of 20 s, with some pauses lasting longer than 200 s. The actin-binding protein nonmuscle tropomyosin (Tm) provides spatially specific regulation of interactions between myosin motors and actin filaments in vivo; in the crossed-filament in vitro assay, we found that Tm2-actin abolishes Myo1c-specific effects on both run initiation and run termination. Together, these observations suggest Myo1c is important for the selective initiation and termination of kinesin-1-driven runs along microtubules at specific actin filament populations within the cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Kinetics of cadmium accumulation and its effects on microtubule integrity and cell viability in the seagrass Cymodocea nodosa

    Energy Technology Data Exchange (ETDEWEB)

    Malea, Paraskevi, E-mail: malea@bio.auth.gr [Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki (Greece); Adamakis, Ioannis-Dimosthenis S. [Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki (Greece); Kevrekidis, Theodoros [Laboratory of Environmental Research and Education, Democritus University of Thrace, Nea Hili, GR-68100 Alexandroupolis (Greece)

    2013-11-15

    Highlights: •Cd effect on microtubules and viability of seagrass leaf cells was assessed. •The Michaelis–Menten equation satisfactorily dercribed the kinetics of Cd uptake. •Cd depolymerized MTs after 3–9 d of exposure, cell death occurred at later time. •Toxicity appeared to depend on Cd uptake rate rather than on tissue Cd content. •MTs can be used as biomarker of Cd stress and uptake rate for predicting effects. -- Abstract: The kinetics of cadmium accumulation and its effects on microtubule cytoskeleton and cell viability in leaf blades of the seagrass Cymodocea nodosa were investigated under laboratory conditions in exposure concentrations ranging from 0.5 to 40 mg L{sup −1}. An initial rapid accumulation of cadmium was followed by a steady state. The Michaelis–Menten model adequately described metal accumulation; equilibrium concentration and uptake velocity tended to increase, whereas bioconcentration factor at equilibrium to decrease, as the exposure concentration increased. Cadmium depolymerized microtubules after 3–9 d of exposure, depending on trace metal concentration, indicating that microtubules could be used as an early biomarker of cadmium stress; cell death, occurring at later time than microtubule disturbance, was also observed. Microtubule depolymerization expressed as percentage of reduction of fluorescence intensity and cell mortality expressed as percentage of live cells increased with time. The lowest experimental tissue concentration associated with the onset of microtubule depolymerization and cell death (98.5–128.9 μg g{sup −1} dry wt, 0.5 mg L{sup −1} treatment, 7th and 9th d) was within the wide range of reported cadmium concentrations in leaves of seagrass species from various geographical areas. This lowest tissue concentration was exceeded up to the 3rd d at higher exposure concentrations, but toxic effects were generally detected at later time. The time periods required for the onset of depolymerization and

  20. Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine

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    Balaji Petety V

    2010-05-01

    Full Text Available Abstract Background Griseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug. Methods The effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied in vitro by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1. Results Griseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the αβ intra-dimer interface. In combination studies

  1. Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine.

    Science.gov (United States)

    Rathinasamy, Krishnan; Jindal, Bhavya; Asthana, Jayant; Singh, Parminder; Balaji, Petety V; Panda, Dulal

    2010-05-19

    Griseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug. The effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied in vitro by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1. Griseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the alphabeta intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic

  2. Kinetics of cadmium accumulation and its effects on microtubule integrity and cell viability in the seagrass Cymodocea nodosa

    International Nuclear Information System (INIS)

    Malea, Paraskevi; Adamakis, Ioannis-Dimosthenis S.; Kevrekidis, Theodoros

    2013-01-01

    Highlights: •Cd effect on microtubules and viability of seagrass leaf cells was assessed. •The Michaelis–Menten equation satisfactorily dercribed the kinetics of Cd uptake. •Cd depolymerized MTs after 3–9 d of exposure, cell death occurred at later time. •Toxicity appeared to depend on Cd uptake rate rather than on tissue Cd content. •MTs can be used as biomarker of Cd stress and uptake rate for predicting effects. -- Abstract: The kinetics of cadmium accumulation and its effects on microtubule cytoskeleton and cell viability in leaf blades of the seagrass Cymodocea nodosa were investigated under laboratory conditions in exposure concentrations ranging from 0.5 to 40 mg L −1 . An initial rapid accumulation of cadmium was followed by a steady state. The Michaelis–Menten model adequately described metal accumulation; equilibrium concentration and uptake velocity tended to increase, whereas bioconcentration factor at equilibrium to decrease, as the exposure concentration increased. Cadmium depolymerized microtubules after 3–9 d of exposure, depending on trace metal concentration, indicating that microtubules could be used as an early biomarker of cadmium stress; cell death, occurring at later time than microtubule disturbance, was also observed. Microtubule depolymerization expressed as percentage of reduction of fluorescence intensity and cell mortality expressed as percentage of live cells increased with time. The lowest experimental tissue concentration associated with the onset of microtubule depolymerization and cell death (98.5–128.9 μg g −1 dry wt, 0.5 mg L −1 treatment, 7th and 9th d) was within the wide range of reported cadmium concentrations in leaves of seagrass species from various geographical areas. This lowest tissue concentration was exceeded up to the 3rd d at higher exposure concentrations, but toxic effects were generally detected at later time. The time periods required for the onset of depolymerization and for 10 and 50

  3. Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro and in Xenopus egg extract spindles.

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    Julie Cahu

    Full Text Available Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1 during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro.We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation.These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.

  4. Spindle pole body-anchored Kar3 drives the nucleus along microtubules from another nucleus in preparation for nuclear fusion during yeast karyogamy.

    Science.gov (United States)

    Gibeaux, Romain; Politi, Antonio Z; Nédélec, François; Antony, Claude; Knop, Michael

    2013-02-01

    Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule-microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion.

  5. Vertebrate Fidgetin Restrains Axonal Growth by Severing Labile Domains of Microtubules

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    Lanfranco Leo

    2015-09-01

    Full Text Available Individual microtubules (MTs in the axon consist of a stable domain that is highly acetylated and a labile domain that is not. Traditional MT-severing proteins preferentially cut the MT in the stable domain. In Drosophila, fidgetin behaves in this fashion, with targeted knockdown resulting in neurons with a higher fraction of acetylated (stable MT mass in their axons. Conversely, in a fidgetin knockout mouse, the fraction of MT mass that is acetylated is lower than in the control animal. When fidgetin is depleted from cultured rodent neurons, there is a 62% increase in axonal MT mass, all of which is labile. Concomitantly, there are more minor processes and a longer axon. Together with experimental data showing that vertebrate fidgetin targets unacetylated tubulin, these results indicate that vertebrate fidgetin (unlike its fly ortholog regulates neuronal development by tamping back the expansion of the labile domains of MTs.

  6. Podoverine A--a novel microtubule destabilizing natural product from the Podophyllum species.

    Science.gov (United States)

    Tran, Tuyen Thi Ngoc; Gerding-Reimers, Claas; Schölermann, Beate; Stanitzki, Bettina; Henkel, Thomas; Waldmann, Herbert; Ziegler, Slava

    2014-09-15

    Natural products represent compound classes with high chemical and structural diversity and various biological activities. Libraries based