A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

DEFF Research Database (Denmark)

Bach, Anders*; Clausen, Bettina H; Møller, Magda

2012-01-01

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are ...... of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke....

Curcumin alters the cytoskeleton and microtubule organization on trophozoites of Giardia lamblia.

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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.

UCCB01-125, a dimeric inhibitor of PSD-95, reduces inflammatory pain without disrupting cognitive or motor performance: Comparison with the NMDA receptor antagonist MK-801

DEFF Research Database (Denmark)

Andreasen, Jesper T.; Bach, Anders; Gynther, Mikko

2013-01-01

Excessive N-Methyl-d-aspartate receptor (NMDAR)-dependent production of nitric oxide (NO) is involved in the development and maintenance of chronic pain states, and is mediated by postsynaptic density protein-95 (PSD-95). By binding to both the NMDAR and neuronal NO synthase (nNOS), PSD-95 mediates...... a specific coupling between NMDAR activation and NO production. NMDAR antagonism shows anti-nociceptive action in humans and animal models of chronic pain but is associated with severe disturbances of cognitive and motor functions. An alternative approach to modulate the NMDAR-related activity is to perturb......'s adjuvant (CFA) model of inflammatory pain. To examine side-effect profiles we also compared the effects of UCCB01-125 and MK-801 in tests of attention, long-term memory, and motor performance. When administered concurrently with CFA, both MK-801 and UCCB01-125 prevented the development of CFA...

Manipulation and quantification of microtubule lattice integrity

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Taylor A. Reid

2017-08-01

Full Text Available Microtubules are structural polymers that participate in a wide range of cellular functions. The addition and loss of tubulin subunits allows the microtubule to grow and shorten, as well as to develop and repair defects and gaps in its cylindrical lattice. These lattice defects act to modulate the interactions of microtubules with molecular motors and other microtubule-associated proteins. Therefore, tools to control and measure microtubule lattice structure will be invaluable for developing a quantitative understanding of how the structural state of the microtubule lattice may regulate its interactions with other proteins. In this work, we manipulated the lattice integrity of in vitro microtubules to create pools of microtubules with common nucleotide states, but with variations in structural states. We then developed a series of novel semi-automated analysis tools for both fluorescence and electron microscopy experiments to quantify the type and severity of alterations in microtubule lattice integrity. These techniques will enable new investigations that explore the role of microtubule lattice structure in interactions with microtubule-associated proteins.

Microtubule nucleation and organization in dendrites

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Delandre, Caroline; Amikura, Reiko; Moore, Adrian W.

2016-01-01

ABSTRACT Dendrite branching is an essential process for building complex nervous systems. It determines the number, distribution and integration of inputs into a neuron, and is regulated to create the diverse dendrite arbor branching patterns characteristic of different neuron types. The microtubule cytoskeleton is critical to provide structure and exert force during dendrite branching. It also supports the functional requirements of dendrites, reflected by differential microtubule architectural organization between neuron types, illustrated here for sensory neurons. Both anterograde and retrograde microtubule polymerization occur within growing dendrites, and recent studies indicate that branching is enhanced by anterograde microtubule polymerization events in nascent branches. The polarities of microtubule polymerization events are regulated by the position and orientation of microtubule nucleation events in the dendrite arbor. Golgi outposts are a primary microtubule nucleation center in dendrites and share common nucleation machinery with the centrosome. In addition, pre-existing dendrite microtubules may act as nucleation sites. We discuss how balancing the activities of distinct nucleation machineries within the growing dendrite can alter microtubule polymerization polarity and dendrite branching, and how regulating this balance can generate neuron type-specific morphologies. PMID:27097122

Effects of the dimeric PSD-95 inhibitor UCCB01-144 in mouse models of pain, cognition and motor function

DEFF Research Database (Denmark)

Andreasen, Jesper T; Nasser, Arafat; Caballero-Puntiverio, Maitane

2016-01-01

NOS interaction has therefore been proposed as an alternative analgesic mechanism. We recently reported that UCCB01-125, a dimeric PSD-95 inhibitor with limited blood-brain-barrier permeability, reduced mechanical hypersensitivity in the complete Freund's adjuvant (CFA) inflammatory pain model, without disrupting...... of neuropathic pain. Potential cognitive effects of UCCB01-144 were examined using the social transmission of food preference (STFP) test and the V-maze test, and motor coordination was assessed with the rotarod test. UCCB01-144 (10mg/kg) reversed CFA-induced mechanical hypersensitivity after 1h, and completely...

CDKL5 ensures excitatory synapse stability by reinforcing NGL-1-PSD95 interaction in the postsynaptic compartment and is impaired in patient iPSC-derived neurons.

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Ricciardi, Sara; Ungaro, Federica; Hambrock, Melanie; Rademacher, Nils; Stefanelli, Gilda; Brambilla, Dario; Sessa, Alessandro; Magagnotti, Cinzia; Bachi, Angela; Giarda, Elisa; Verpelli, Chiara; Kilstrup-Nielsen, Charlotte; Sala, Carlo; Kalscheuer, Vera M; Broccoli, Vania

2012-09-01

Mutations of the cyclin-dependent kinase-like 5 (CDKL5) and netrin-G1 (NTNG1) genes cause a severe neurodevelopmental disorder with clinical features that are closely related to Rett syndrome, including intellectual disability, early-onset intractable epilepsy and autism. We report here that CDKL5 is localized at excitatory synapses and contributes to correct dendritic spine structure and synapse activity. To exert this role, CDKL5 binds and phosphorylates the cell adhesion molecule NGL-1. This phosphorylation event ensures a stable association between NGL-1 and PSD95. Accordingly, phospho-mutant NGL-1 is unable to induce synaptic contacts whereas its phospho-mimetic form binds PSD95 more efficiently and partially rescues the CDKL5-specific spine defects. Interestingly, similarly to rodent neurons, iPSC-derived neurons from patients with CDKL5 mutations exhibit aberrant dendritic spines, thus suggesting a common function of CDKL5 in mice and humans.

Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma.

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Landen, Jaren W; Lang, Roland; McMahon, Steve J; Rusan, Nasser M; Yvon, Anne-Marie; Adams, Ashley W; Sorcinelli, Mia D; Campbell, Ross; Bonaccorsi, Paola; Ansel, John C; Archer, David R; Wadsworth, Patricia; Armstrong, Cheryl A; Joshi, Harish C

2002-07-15

Cellular microtubules, polymers of tubulin, alternate relentlessly between phases of growth and shortening. We now show that noscapine, a tubulin-binding agent, increases the time that cellular microtubules spend idle in a paused state. As a result, most mammalian cell types observed arrest in mitosis in the presence of noscapine. We demonstrate that noscapine-treated murine melanoma B16LS9 cells do not arrest in mitosis but rather become polyploid followed by cell death, whereas primary melanocytes reversibly arrest in mitosis and resume a normal cell cycle after noscapine removal. Furthermore, in a syngeneic murine model of established s.c. melanoma, noscapine treatment resulted in an 85% inhibition of tumor volume on day 17 when delivered by gavage compared with untreated animals (P alterations in microtubule dynamics, with no detected toxicity to the host. Consequently, noscapine could be a valuable chemotherapeutic agent, alone or in combination, for the treatment of advanced melanoma.

Microtubules as mechanical force sensors.

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Karafyllidis, Ioannis G; Lagoudas, Dimitris C

2007-03-01

Microtubules are polymers of tubulin subunits (dimers) arranged on a hexagonal lattice. Each tubulin dimer comprises two monomers, the alpha-tubulin and beta-tubulin, and can be found in two states. In the first state a mobile negative charge is located into the alpha-tubulin monomer and in the second into the beta-tubulin monomer. Each tubulin dimer is modeled as an electrical dipole coupled to its neighbors by electrostatic forces. The location of the mobile charge in each dimer depends on the location of the charges in the dimer's neighborhood. Mechanical forces that act on the microtubule affect the distances between the dimers and alter the electrostatic potential. Changes in this potential affect the mobile negative charge location in each dimer and the charge distribution in the microtubule. The net effect is that mechanical forces affect the charge distribution in microtubules. We propose to exploit this effect and use microtubules as mechanical force sensors. We model each dimer as a two-state quantum system and, following the quantum computation paradigm, we use discrete quantum random walk on the hexagonal microtubule lattice to determine the charge distribution. Different forces applied on the microtubule are modeled as different coin biases leading to different probability distributions of the quantum walker location, which are directly connected to different charge distributions. Simulation results show that there is a strong indication that microtubules can be used as mechanical force sensors and that they can also detect the force directions and magnitudes.

Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models

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Ba M

2014-12-01

administration twice daily for 22 days to parkinsonian rats shortened the rotational duration and increased the peak turning responses. The altered rotational responses were attenuated by PSD-95 ASO pretreatment. Meanwhile, PSD-95 ASO pretreatment decreased the level of PSD-95 protein expression and reduced both the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B triggered during the levodopa administration in the lesioned striatum of PD rats. However, the protein levels of Fyn and NR2B showed no difference under the above conditions. Conclusion: The data demonstrate that the inhibition of PSD-95 protein expression suppressed the interactions of Fyn with NR2B and NR2B tyrosine phosphorylation and subsequently downregulated NMDA receptor overactivation, thus providing benefit for the therapy of LID. Therefore, PSD-95 is important for overactivity of NMDA receptor function due to facilitating NR2B tyrosine phosphorylation dependent on Fyn kinase by regulating interactions of Fyn with NR2B under the pathological conditions of LID. Keywords: PSD-95 ASO, NMDA, rotational response

GDP-tubulin incorporation into growing microtubules modulates polymer stability.

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

2010-06-04

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

Ligand binding to the PDZ domains of postsynaptic density protein 95

DEFF Research Database (Denmark)

Toto, Angelo; Pedersen, Søren W; Karlsson, O Andreas

2016-01-01

Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 ...

Psd1 Effects on Candida albicans Planktonic Cells and Biofilms

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Sónia Gonçalves

2017-06-01

Full Text Available Candida albicans is an important human pathogen, causing opportunistic infections. The adhesion of planktonic cells to a substrate is the first step for biofilm development. The antimicrobial peptide (AMP Psd1 is a defensin isolated from Pisum sativum seeds. We tested the effects of this AMP on C. albicans biofilms and planktonic cells, comparing its activity with amphotericin B and fluconazole. Three C. albicans variants were studied, one of them a mutant deficient in glucosylceramide synthase, conferring resistance to Psd1 antifungal action. Atomic force microscopy (AFM was used to assess morphological and biomechanical changes on fungal cells. Surface alterations, with membrane disruption and leakage of cellular contents, were observed. Cytometry assays and confocal microscopy imaging showed that Psd1 causes cell death, in a time and concentration-dependent manner. These results demonstrate Psd1 pleiotropic action against a relevant fungal human pathogen, suggesting its use as natural antimycotic agent.

2-Methyl-6-(phenylethynyl pyridine (MPEP reverses maze learning and PSD-95 deficits in Fmr1 knock-out mice.

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Réno Michelle Gandhi

2014-03-01

Full Text Available Fragile X syndrome (FXS is caused by the lack of expression of the fragile X mental retardation protein (FMRP, which results in intellectual disability and other debilitating symptoms including impairment of visual-spatial functioning. FXS is the only single-gene disorder that is highly co-morbid with autism spectrum disorder and can therefore provide insight into its pathophysiology. Lack of FMRP results in altered group I metabotropic glutamate receptor (mGluR signalling, which is a target for putative treatments. The Hebb-Williams (H-W mazes are a set of increasingly complex spatial navigation problems that depend on intact hippocampal and thus mGluR-5 functioning. In the present investigation, we examined whether an antagonist of mGluR-5 would reverse previously described behavioural deficits in Fmr1 KO mice. Mice were trained on a subset of the H-W mazes and then treated with either 20 mg/kg of an mGluR-5 antagonist, 2-Methyl-6-(phenylethynyl pyridine (MPEP; n = 11 or an equivalent dose of saline (n = 11 prior to running test mazes. Latency and errors were dependent variables recorded during the test phase. Immediately after completing each test, marble-burying behavior was assessed which confirmed that the drug treatment was pharmacologically active during maze learning. Although latency was not statistically different between the groups, MPEP treated Fmr1 KO mice made significantly fewer errors on mazes deemed more difficult suggesting a reversal of the behavioural deficit. MPEP treated mice were also less perseverative and impulsive when navigating mazes. Furthermore, MPEP treatment reversed PSD-95 protein deficits in Fmr1 KO treated mice, whereas levels of a control protein (β-tubulin remained unchanged. These data further validate MPEP as a potentially beneficial treatment for FXS. Our findings also suggest that adapted H-W mazes may be a useful tool to document alterations in behavioural functioning following pharmacological

Postsynaptic density protein 95 in the striosome and matrix compartments of the human neostriatum.

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Ryoma eMorigaki

2015-11-01

Full Text Available The human neostriatum consists of two functional subdivisions referred to as the striosome (patch and matrix compartments. The striosome-matrix dopamine systems play a central role in cortico-thalamo-basal ganglia circuits, and their involvement is thought to underlie the genesis of multiple movement and behavioral disorders, and of drug addiction. Human neuropathology also has shown that striosomes and matrix have differential vulnerability patterns in several striatal neurodegenerative diseases. Postsynaptic density protein 95 (PSD-95, also known as DLG4, is a major scaffolding protein in the postsynaptic densities of dendritic spines. PSD-95 is now known to negatively regulate not only N-methyl-D-aspartate glutamate signaling, but also dopamine D1 signals at sites of postsynaptic transmission. Accordingly, a neuroprotective role for PSD-95 against dopamine D1 receptor (D1R-mediated neurotoxicity in striatal neurodegeneration also has been suggested. Here, we used a highly sensitive immunohistochemistry technique to show that in the human neostriatum, PSD-95 is differentially concentrated in the striosome and matrix compartments, with a higher density of PSD-95 labeling in the matrix compartment than in the striosomes. This compartment-specific distribution of PSD-95 was strikingly complementary to that of D1R. In addition to the possible involvement of PSD-95-mediated synaptic function in compartment-specific dopamine signals, we suggest that the striosomes might be more susceptible to D1R-mediated neurotoxicity than the matrix compartment. This notion may provide new insight into the compartment-specific vulnerability of MSNs in striatal neurodegeneration.

Interaction partners of PSD-93 studied by X-ray crystallography and fluorescence polarization spectroscopy

DEFF Research Database (Denmark)

Fiorentini, Monica; Bach, Anders; Strømgaard, Kristian

2013-01-01

PSD-93 (chapsyn-110, DLG2) is a member of the family of membrane-associated guanylate kinase (MAGUK) proteins. The MAGUK proteins are involved in receptor localization and signalling pathways. The best characterized MAGUK protein, PSD-95, is known to be involved in NMDA receptor signalling via it...

Shaping the tracks : Regulation of microtubule dynamics by kinesins KIF21A and KIF21B

NARCIS (Netherlands)

van Riel, W.E.|info:eu-repo/dai/nl/338772634

2016-01-01

Control of microtubule dynamics is important for cell morphogenesis. Kinesins, motor proteins known to function in cargo transport, were recently also implicated in altering the microtubule network. Several kinesins are described to cause microtubule network reorganization or stabilization, either

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

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Kwiatkowska, Maria; Stępiński, Dariusz; Polit, Justyna T; Popłońska, Katarzyna; Wojtczak, Agnieszka

2011-01-01

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

Microtubule-Organizing Centers.

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Wu, Jingchao; Akhmanova, Anna

2017-10-06

The organization of microtubule networks is crucial for controlling chromosome segregation during cell division, for positioning and transport of different organelles, and for cell polarity and morphogenesis. The geometry of microtubule arrays strongly depends on the localization and activity of the sites where microtubules are nucleated and where their minus ends are anchored. Such sites are often clustered into structures known as microtubule-organizing centers, which include the centrosomes in animals and spindle pole bodies in fungi. In addition, other microtubules, as well as membrane compartments such as the cell nucleus, the Golgi apparatus, and the cell cortex, can nucleate, stabilize, and tether microtubule minus ends. These activities depend on microtubule-nucleating factors, such as γ-tubulin-containing complexes and their activators and receptors, and microtubule minus end-stabilizing proteins with their binding partners. Here, we provide an overview of the current knowledge on how such factors work together to control microtubule organization in different systems.

In vitro and in vivo effects of a novel dimeric inhibitor of PSD-95 on excitotoxicity and functional recovery after experimental traumatic brain injury

DEFF Research Database (Denmark)

Sommer, Jens Bak; Bach, Anders; Rytter, Hana Malá

2017-01-01

weeks post-trauma, spatial learning and memory were assessed in a water maze, and at 3 months, brains were removed for estimation of lesion volumes. Overall, neither treatment with UCCB01-147 nor MK-801 resulted in significant improvements of cognition and histopathology after CCI. Although MK-801......PSD-95 inhibitors have been shown to be neuroprotective in stroke, but have only to a very limited extent been evaluated in the treatment of traumatic brain injury (TBI) that has pathophysiological mechanisms in common with stroke. The aims of the current study were to assess the effects of a novel...

Centrosome and microtubule instability in aging Drosophila cells

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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.

Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy.

Science.gov (United States)

Flex, Elisabetta; Niceta, Marcello; Cecchetti, Serena; Thiffault, Isabelle; Au, Margaret G; Capuano, Alessandro; Piermarini, Emanuela; Ivanova, Anna A; Francis, Joshua W; Chillemi, Giovanni; Chandramouli, Balasubramanian; Carpentieri, Giovanna; Haaxma, Charlotte A; Ciolfi, Andrea; Pizzi, Simone; Douglas, Ganka V; Levine, Kara; Sferra, Antonella; Dentici, Maria Lisa; Pfundt, Rolph R; Le Pichon, Jean-Baptiste; Farrow, Emily; Baas, Frank; Piemonte, Fiorella; Dallapiccola, Bruno; Graham, John M; Saunders, Carol J; Bertini, Enrico; Kahn, Richard A; Koolen, David A; Tartaglia, Marco

2016-10-06

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αβ-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective β-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/β-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with

Microtubule catastrophe and rescue.

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

2013-02-01

Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed 'dynamic instability'. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule 'catastrophe'), and then by switching back from shrinkage to growth (called microtubule 'rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Effect of Dexmedetomidine on Cognitive Function and Protein Expression of Aβ, p-Tau, and PSD95 after Extracorporeal Circulation Operation in Aged Rats

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Ying Zhang

2018-01-01

Full Text Available Postoperative cognitive dysfunction (POCD is a kind of serious neurologic complications and dexmedetomidine has a certain effect on POCD. However, functional mechanism of dexmedetomidine on POCD still remains unclear, so the research mainly studied the effect of dexmedetomidine on cognitive function and protein expression in hippocampus and prefrontal cortex cerebrospinal fluid after extracorporeal circulation operation in aged rats. We Found that, compared with POCD group, the cognitive function was improved in POCD + Dex group. We speculate that dexmedetomidine could improve the cognitive function after extracorporeal circulation operation in aged rats and Aβ, p-Tau, and PSD95 protein might have contributed to this favorable outcome.

Biochemical investigations of the mechanism of action of small molecules ZL006 and IC87201 as potential inhibitors of the nNOS-PDZ/PSD-95-PDZ interactions

DEFF Research Database (Denmark)

Bach, Anders

2015-01-01

ZL006 and IC87201 have been presented as efficient inhibitors of the nNOS/PSD-95 protein-protein interaction and shown great promise in cellular experiments and animal models of ischemic stroke and pain. Here, we investigate the proposed mechanism of action of ZL006 and IC87201 using biochemical...... by interacting with the β-finger of nNOS-PDZ. Our findings have implications for further medicinal chemistry efforts of ZL006, IC87201 and analogues, and challenge the general and widespread view on their mechanism of action....

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

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

2014-01-01

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

PKA Inhibitor H89 (N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulfonamide Attenuates Synaptic Dysfunction and Neuronal Cell Death following Ischemic Injury

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Juhyun Song

2015-01-01

Full Text Available The cyclic AMP-dependent protein kinase (PKA, which activates prosurvival signaling proteins, has been implicated in the expression of long-term potentiation and hippocampal long-term memory. It has come to light that H89 commonly known as the PKA inhibitor have diverse roles in the nervous system that are unrelated to its role as a PKA inhibitor. We have investigated the role of H89 in ischemic and reperfusion injury. First, we examined the expression of postsynaptic density protein 95 (PSD95, microtubule-associated protein 2 (MAP2, and synaptophysin in mouse brain after middle cerebral artery occlusion injury. Next, we examined the role of H89 pretreatment on the expression of brain-derived neurotrophic factor (BDNF, PSD95, MAP2, and the apoptosis regulators Bcl2 and cleaved caspase-3 in cultured neuroblastoma cells exposed to hypoxia and reperfusion injury. In addition, we investigated the alteration of AKT activation in H89 pretreated neuroblastoma cells under hypoxia and reperfusion injury. The data suggest that H89 may contribute to brain recovery after ischemic stroke by regulating neuronal death and proteins related to synaptic plasticity.

Oscillatory fluid flow influences primary cilia and microtubule mechanics.

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Espinha, Lina C; Hoey, David A; Fernandes, Paulo R; Rodrigues, Hélder C; Jacobs, Christopher R

2014-07-01

Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues, such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity. Copyright © 2014 Wiley Periodicals, Inc.

The effects of 60Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes

International Nuclear Information System (INIS)

Li Qianqian; Mao Zijun; Yin Zhiwei; Hu Yumin

1989-05-01

The effects of 60 Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes were investigated by immunofluorescence microscopy and scanning electron microscope. The results indicated. (1) microtubule organization of the irradiated cells remarkably differed from that of the control since the doses over 4 Gy; (2) 144 hours after irradiation the alterations of microtubules have been shown to be basically r epaired ; (3) the cytoplasmic microtubules and centrioles disappeared under transmission electron microscope, the membranes irradiated and microvilli showed changes under scanning electron microscope too. From these observations and those of other workers who studied the radiation effect on extracted microtubule proteins in vitro, the authors support that 60 Co γ-ray irradiation can inhabits cytoplasmic microtubule assembling

Puffy skin disease (PSD) in rainbow trout, Oncorhynchus mykiss (Walbaum): a case definition.

Science.gov (United States)

Maddocks, C E; Nolan, E T; Feist, S W; Crumlish, M; Richards, R H; Williams, C F

2015-07-01

Puffy skin disease (PSD) is a disease that causes skin pathology in rainbow trout, Oncorhynchus mykiss (Walbaum). Incidence of PSD in UK fish farms and fisheries has increased sharply in the last decade, with growing concern from both industry sectors. This paper provides the first comprehensive case definition of PSD, combining clinical and pathological observations of diseased rainbow trout from both fish farms and fisheries. The defining features of PSD, as summarized in the case definition, were focal lateral flank skin lesions that appeared as cutaneous swelling with pigment loss and petechiae. These were associated with lethargy, poor body condition, inappetance and low level mortality. Epidermal hyperplasia and spongiosis, oedema of the dermis stratum spongiosum and a mild diffuse inflammatory cellularity were typical in histopathology of skin. A specific pathogen or aetiology was not identified. Prevalence and severity of skin lesions was greatest during late summer and autumn, with the highest prevalence being 95%. Atypical lesions seen in winter and spring were suggestive of clinical resolution. PSD holds important implications for both trout aquaculture and still water trout fisheries. This case definition will aid future diagnosis, help avoid confusion with other skin conditions and promote prompt and consistent reporting. © 2014 John Wiley & Sons Ltd.

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

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

Stabilizing versus Destabilizing the Microtubules: A Double-Edge Sword for an Effective Cancer Treatment Option?

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Daniele Fanale

2015-01-01

Full Text Available Microtubules are dynamic and structural cellular components involved in several cell functions, including cell shape, motility, and intracellular trafficking. In proliferating cells, they are essential components in the division process through the formation of the mitotic spindle. As a result of these functions, tubulin and microtubules are targets for anticancer agents. Microtubule-targeting agents can be divided into two groups: microtubule-stabilizing, and microtubule-destabilizing agents. The former bind to the tubulin polymer and stabilize microtubules, while the latter bind to the tubulin dimers and destabilize microtubules. Alteration of tubulin-microtubule equilibrium determines the disruption of the mitotic spindle, halting the cell cycle at the metaphase-anaphase transition and, eventually, resulting in cell death. Clinical application of earlier microtubule inhibitors, however, unfortunately showed several limits, such as neurological and bone marrow toxicity and the emergence of drug-resistant tumor cells. Here we review several natural and synthetic microtubule-targeting agents, which showed antitumor activity and increased efficacy in comparison to traditional drugs in various preclinical and clinical studies. Cryptophycins, combretastatins, ombrabulin, soblidotin, D-24851, epothilones and discodermolide were used in clinical trials. Some of them showed antiangiogenic and antivascular activity and others showed the ability to overcome multidrug resistance, supporting their possible use in chemotherapy.

Microtubule bundling plays a role in ethylene-mediated cortical microtubule reorientation in etiolated Arabidopsis hypocotyls.

Science.gov (United States)

Ma, Qianqian; Sun, Jingbo; Mao, Tonglin

2016-05-15

The gaseous hormone ethylene is known to regulate plant growth under etiolated conditions (the 'triple response'). Although organization of cortical microtubules is essential for cell elongation, the underlying mechanisms that regulate microtubule organization by hormone signaling, including ethylene, are ambiguous. In the present study, we demonstrate that ethylene signaling participates in regulation of cortical microtubule reorientation. In particular, regulation of microtubule bundling is important for this process in etiolated hypocotyls. Time-lapse analysis indicated that selective stabilization of microtubule-bundling structures formed in various arrays is related to ethylene-mediated microtubule orientation. Bundling events and bundle growth lifetimes were significantly increased in oblique and longitudinal arrays, but decreased in transverse arrays in wild-type cells in response to ethylene. However, the effects of ethylene on microtubule bundling were partially suppressed in a microtubule-bundling protein WDL5 knockout mutant (wdl5-1). This study suggests that modulation of microtubule bundles that have formed in certain orientations plays a role in reorienting microtubule arrays in response to ethylene-mediated etiolated hypocotyl cell elongation. © 2016. Published by The Company of Biologists Ltd.

Microtubule Catastrophe and Rescue

OpenAIRE

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

2012-01-01

Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends...

Cellular effects of curcumin on Plasmodium falciparum include disruption of microtubules.

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Rimi Chakrabarti

Full Text Available Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC50 of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.

Exogenous Alpha-Synuclein Alters Pre- and Post-Synaptic Activity by Fragmenting Lipid Rafts

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Marco Emanuele

2016-05-01

Full Text Available Alpha-synuclein (αSyn interferes with multiple steps of synaptic activity at pre-and post-synaptic terminals, however the mechanism/s by which αSyn alters neurotransmitter release and synaptic potentiation is unclear. By atomic force microscopy we show that human αSyn, when incubated with reconstituted membrane bilayer, induces lipid rafts' fragmentation. As a consequence, ion channels and receptors are displaced from lipid rafts with consequent changes in their activity. The enhanced calcium entry leads to acute mobilization of synaptic vesicles, and exhaustion of neurotransmission at later stages. At the post-synaptic terminal, an acute increase in glutamatergic transmission, with increased density of PSD-95 puncta, is followed by disruption of the interaction between N-methyl-d-aspartate receptor (NMDAR and PSD-95 with ensuing decrease of long term potentiation. While cholesterol loading prevents the acute effect of αSyn at the presynapse; inhibition of casein kinase 2, which appears activated by reduction of cholesterol, restores the correct localization and clustering of NMDARs.

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

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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.

Feeding cells induced by phytoparasitic nematodes require γ-tubulin ring complex for microtubule reorganization.

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Mohamed Youssef Banora

2011-12-01

Full Text Available Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound γ-tubulin ring complexes. Here we investigate the requirement of γ-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that γ-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis γ-tubulin (TUBG1 and TUBG2 and two γ-tubulin complex proteins genes (GCP3 and GCP4 are upregulated in galls. Electron microscopy demonstrates association of GCP3 and γ-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both γ-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the γ-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts.

Cellular effects of the microtubule-targeting agent peloruside A in hypoxia-conditioned colorectal carcinoma cells.

Science.gov (United States)

Řehulka, Jiří; Annadurai, Narendran; Frydrych, Ivo; Znojek, Pawel; Džubák, Petr; Northcote, Peter; Miller, John H; Hajdúch, Marián; Das, Viswanath

2017-07-01

Hypoxia is a prominent feature of solid tumors, dramatically remodeling microtubule structures and cellular pathways and contributing to paclitaxel resistance. Peloruside A (PLA), a microtubule-targeting agent, has shown promising anti-tumor effects in preclinical studies. Although it has a similar mode of action to paclitaxel, it binds to a distinct site on β-tubulin that differs from the classical taxane site. In this study, we examined the unexplored effects of PLA in hypoxia-conditioned colorectal HCT116 cancer cells. Cytotoxicity of PLA was determined by cell proliferation assay. The effects of a pre-exposure to hypoxia on PLA-induced cell cycle alterations and apoptosis were examined by flow cytometry, time-lapse imaging, and western blot analysis of selected markers. The hypoxia effect on stabilization of microtubules by PLA was monitored by an intracellular tubulin polymerization assay. Our findings show that the cytotoxicity of PLA is not altered in hypoxia-conditioned cells compared to paclitaxel and vincristine. Furthermore, hypoxia does not alter PLA-induced microtubule stabilization nor the multinucleation of cells. PLA causes cyclin B1 and G2/M accumulation followed by apoptosis. The cellular and molecular effects of PLA have been determined in normoxic conditions, but there are no reports of PLA effects in hypoxic cells. Our findings reveal that hypoxia preconditioning does not alter the sensitivity of HCT116 to PLA. These data report on the cellular and molecular effects of PLA in hypoxia-conditioned cells for the first time, and will encourage further exploration of PLA as a promising anti-tumor agent. Copyright © 2017 Elsevier B.V. All rights reserved.

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

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Baconnais Sonia

2008-09-01

Full Text Available Abstract Background YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. Results We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. Conclusion These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.

Large eddy simulation of cooling flows in underground subway station according to different PSD operating conditions

Energy Technology Data Exchange (ETDEWEB)

Jang, Yong Jun; Kim, Jin Ho; Park, Sung Huk; Koo, Dong Hoe [Korea Railroad Research Institute, Uiwang (Korea, Republic of)

2015-11-15

Large eddy simulation (LES) method is applied to systematically investigate the cooling fluid flow and the temperature distribution under the operating of air conditioning in the deeply underground subway station. The Shin-Gum-Ho subway station in Seoul which is the 8{sup th} floor and 43.6 m deep is selected for this analysis. The entire station is covered for simulation. The ventilation mode for air conditioning is kept as ordinary state. Different operating conditions for Platform screen door (PSD) are applied. First one is PSD is completely close and second one is PSD is regularly open and close which imitate the actual circumstances in the platform. The ventilation diffusers are modeled as 95 square shapes in the lobby and 222 squares in the platform. The temperature variations and flow behaviors are numerically simulated after operating of air conditioning for the whole station and the calculated results are compared with experimental data. LES method solves the momentum and thermal equations. Werner-Wengle wall law is applied to viscous sub layers for near wall resolution. The total grid numbers are 7.5 million and the whole domain is divided to 22 blocks. Multi blocks are computed in parallel using MPI. The results show the temperature difference in the platform between PSD-close and PSD-regularly open and close cases is 3-4 .deg. C.

Large eddy simulation of cooling flows in underground subway station according to different PSD operating conditions

International Nuclear Information System (INIS)

Jang, Yong Jun; Kim, Jin Ho; Park, Sung Huk; Koo, Dong Hoe

2015-01-01

Large eddy simulation (LES) method is applied to systematically investigate the cooling fluid flow and the temperature distribution under the operating of air conditioning in the deeply underground subway station. The Shin-Gum-Ho subway station in Seoul which is the 8"t"h floor and 43.6 m deep is selected for this analysis. The entire station is covered for simulation. The ventilation mode for air conditioning is kept as ordinary state. Different operating conditions for Platform screen door (PSD) are applied. First one is PSD is completely close and second one is PSD is regularly open and close which imitate the actual circumstances in the platform. The ventilation diffusers are modeled as 95 square shapes in the lobby and 222 squares in the platform. The temperature variations and flow behaviors are numerically simulated after operating of air conditioning for the whole station and the calculated results are compared with experimental data. LES method solves the momentum and thermal equations. Werner-Wengle wall law is applied to viscous sub layers for near wall resolution. The total grid numbers are 7.5 million and the whole domain is divided to 22 blocks. Multi blocks are computed in parallel using MPI. The results show the temperature difference in the platform between PSD-close and PSD-regularly open and close cases is 3-4 .deg. C

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

Science.gov (United States)

Atkinson, Samantha; Kirik, Angela; Kirik, Viktor

2014-01-01

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

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.

Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

International Nuclear Information System (INIS)

Hamel, E.; Batra, J.K.; Lin, C.M.

1986-01-01

Using highly purified calf brain tubulin bearing [8- 14 C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins, the authors have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg 2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly

Remote Laser Diffraction PSD Analyzer

International Nuclear Information System (INIS)

Batcheller, T.A.; Huestis, G.M.; Bolton, S.M.

2000-01-01

Particle size distribution (PSD) analysis of radioactive slurry samples were obtained using a modified off-the-shelf classical laser light scattering particle size analyzer. A Horiba Instruments Inc. Model La-300 PSD analyzer, which has a 0.1 to 600 micron measurement range, was modified for remote application in a hot cell (gamma radiation) environment. The general details of the modifications to this analyzer are presented in this paper. This technology provides rapid and simple PSD analysis, especially down in the fine and microscopic particle size regime. Particle size analysis of these radioactive slurries down in this smaller range was not achievable - making this technology far superior than the traditional methods used previously. Remote deployment and utilization of this technology is in an exploratory stage. The risk of malfunction in this radiation environment is countered by gaining of this tremendously useful fundamental engineering data. Successful acquisition of this data, in conjunction with other characterization analyses, provides important information that can be used in the myriad of potential radioactive waste management alternatives

Remote Laser Diffraction PSD Analyzer

International Nuclear Information System (INIS)

Batcheller, Thomas Aquinas; Huestis, Gary Michael; Bolton, Steven Michael

2000-01-01

Particle size distribution (PSD) analysis of radioactive slurry samples were obtained using a modified ''off-the-shelf'' classical laser light scattering particle size analyzer. A Horiba Instruments Inc. Model La-300 PSD analyzer, which has a 0.1 to 600 micron measurement range, was modified for remote application in a ''hot cell'' (gamma radiation) environment. The general details of the modifications to this analyzer are presented in this paper. This technology provides rapid and simple PSD analysis, especially down in the fine and microscopic particle size regime. Particle size analysis of these radioactive slurries down in this smaller range was not achievable--making this technology far superior than the traditional methods used previously. Remote deployment and utilization of this technology is in an exploratory stage. The risk of malfunction in this radiation environment is countered by gaining of this tremendously useful fundamental engineering data. Successful acquisition of this data, in conjunction with other characterization analyses, provides important information that can be used in the myriad of potential radioactive waste management alternatives

Cep192 controls the balance of centrosome and non-centrosomal microtubules during interphase.

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Brian P O'Rourke

Full Text Available Cep192 is a centrosomal protein that contributes to the formation and function of the mitotic spindle in mammalian cells. Cep192's mitotic activities stem largely from its role in the recruitment to the centrosome of numerous additional proteins such as gamma-tubulin and Pericentrin. Here, we examine Cep192's function in interphase cells. Our data indicate that, as in mitosis, Cep192 stimulates the nucleation of centrosomal microtubules thereby regulating the morphology of interphase microtubule arrays. Interestingly, however, cells lacking Cep192 remain capable of generating normal levels of MTs as the loss of centrosomal microtubules is augmented by MT nucleation from other sites, most notably the Golgi apparatus. The depletion of Cep192 results in a significant decrease in the level of centrosome-associated gamma-tubulin, likely explaining its impact on centrosome microtubule nucleation. However, in stark contrast to mitosis, Cep192 appears to maintain an antagonistic relationship with Pericentrin at interphase centrosomes. Interphase cells depleted of Cep192 display significantly higher levels of centrosome-associated Pericentrin while overexpression of Cep192 reduces the levels of centrosomal Pericentrin. Conversely, depletion of Pericentrin results in elevated levels of centrosomal Cep192 and enhances microtubule nucleation at centrosomes, at least during interphase. Finally, we show that depletion of Cep192 negatively impacts cell motility and alters normal cell polarization. Our current working hypothesis is that the microtubule nucleating capacity of the interphase centrosome is determined by an antagonistic balance of Cep192, which promotes nucleation, and Pericentrin, which inhibits nucleation. This in turn determines the relative abundance of centrosomal and non-centrosomal microtubules that tune cell movement and shape.

Synchronous Oscillations in Microtubule Polymerization

Science.gov (United States)

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

1987-08-01

Under conditions where microtubule nucleation and growth are fast (i.e., high magnesium ion and tubulin concentrations and absence of glycerol), microtubule assembly in vitro exhibits an oscillatory regime preceding the establishment of steady state. The amplitude of the oscillations can represent >50% of the maximum turbidity change and oscillations persist for up to 20 periods of 80 s each. Oscillations are accompanied by extensive length redistribution of microtubules. Preliminary work suggests that the oscillatory kinetics can be simulated using a model in which many microtubules undergo synchronous transitions between growing and rapidly depolymerizing phases, complicated by the kinetically limiting rate of nucleotide exchange on free tubulin.

Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization

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Jia Shen

2017-07-01

Full Text Available Background: The tubulin/microtubule system, which is an integral component of the cytoskeleton, plays an essential role in mitosis. Targeting mitotic progression by disturbing microtubule dynamics is a rational strategy for cancer treatment. Methods: Microtubule polymerization assay was performed to examine the effect of Magnolol (a novel natural phenolic compound isolated from Magnolia obovata on cellular microtubule polymerization in human non-small cell lung cancer (NSCLC cells. Cell cycle analysis, mitotic index assay, cell proliferation assay, colony formation assay, western blotting analysis of cell cycle regulators, Annexin V-FITC/PI staining, and live/dead viability staining were carried out to investigate the Magnolol’s inhibitory effect on proliferation and viability of NSCLS cells in vitro. Xenograft model of human A549 NSCLC tumor was used to determine the Magnolol’s efficacy in vivo. Results: Magnolol treatment effectively inhibited cell proliferation and colony formation of NSCLC cells. Further study proved that Magnolol induced the mitotic phase arrest and inhibited G2/M progression in a dose-dependent manner, which were mechanistically associated with expression alteration of a series of cell cycle regulators. Furthermore, Magnolol treatment disrupted the cellular microtubule organization via inhibiting the polymerization of microtubule. We also found treatment with NSCLC cells with Magnolol resulted in apoptosis activation through a p53-independent pathway, and autophgy induction via down-regulation of the Akt/mTOR pathway. Finally, Magnolol treatment significantly suppressed the NSCLC tumor growth in mouse xenograft model in vivo. Conclusion: These findings identify Magnolol as a promising candidate with anti-microtubule polymerization activity for NSCLC treatment.

Assembly and control of large microtubule complexes

Science.gov (United States)

Korolev, Kirill; Ishihara, Keisuke; Mitchison, Timothy

Motility, division, and other cellular processes require rapid assembly and disassembly of microtubule structures. We report a new mechanism for the formation of asters, radial microtubule complexes found in very large cells. The standard model of aster growth assumes elongation of a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we found evidence for microtubule nucleation away from centrosomes. By combining polymerization dynamics and auto-catalytic nucleation of microtubules, we developed a new biophysical model of aster growth. The model predicts an explosive transition from an aster with a steady-state radius to one that expands as a travelling wave. At the transition, microtubule density increases continuously, but aster growth rate discontinuously jumps to a nonzero value. We tested our model with biochemical perturbations in egg extract and confirmed main theoretical predictions including the jump in the growth rate. Our results show that asters can grow even though individual microtubules are short and unstable. The dynamic balance between microtubule collapse and nucleation could be a general framework for the assembly and control of large microtubule complexes. NIH GM39565; Simons Foundation 409704; Honjo International 486 Scholarship Foundation.

Microtubules self-repair in response to mechanical stress

Science.gov (United States)

Schaedel, Laura; John, Karin; Gaillard, Jérémie; Nachury, Maxence V.; Blanchoin, Laurent; Théry, Manuel

2015-11-01

Microtubules--which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport--can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules' adaptation to mechanical stresses.

Mechanics of microtubules: effects of protofilament orientation.

Science.gov (United States)

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

2010-09-08

Microtubules are hollow cylindrical polymers of the protein tubulin that play a number of important dynamic and structural roles in eukaryotic cells. Both in vivo and in vitro microtubules can exist in several possible configurations, differing in the number of protofilaments, helical rise of tubulin dimers, and protofilament skew angle with respect to the main tube axis. Here, finite element modeling is applied to examine the mechanical response of several known microtubule types when subjected to radial deformation. The data presented here provide an important insight into microtubule stiffness and reveal that protofilament orientation does not affect radial stiffness. Rather, stiffness is primarily dependent on the effective Young's modulus of the polymerized material and the effective radius of the microtubule. These results are also directly correlated to atomic force microscopy nanoindentation measurements to allow a more detailed interpretation of previous experiments. When combined with experimental data that show a significant difference between microtubules stabilized with a slowly hydrolyzable GTP analog and microtubules stabilized with paclitaxel, the finite element data suggest that paclitaxel increases the overall radial flexibility of the microtubule wall. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Sleep Deprivation Alters Rat Ventral Prostate Morphology, Leading to Glandular Atrophy: A Microscopic Study Contrasted with the Hormonal Assays

Directory of Open Access Journals (Sweden)

Daniel P. Venâncio

2012-01-01

Full Text Available We investigated the effect of 96 h paradoxical sleep deprivation (PSD and 21-day sleep restriction (SR on prostate morphology using stereological assays in male rats. After euthanasia, the rat ventral prostate was removed, weighed, and prepared for conventional light microscopy. Microscopic analysis of the prostate reveals that morphology of this gland was altered after 96 h of PSD and 21 days of SR, with the most important alterations occurring in the epithelium and stroma in the course of both procedures compared with the control group. Both 96 h PSD and 21-day SR rats showed lower serum testosterone and higher corticosterone levels than control rats. The significance of our result referring to the sleep deprivation was responsible for deep morphological alterations in ventral prostate tissue, like to castration microscopic modifications. This result is due to the marked alterations in hormonal status caused by PSD and SR.

Sleep Deprivation Alters Rat Ventral Prostate Morphology, Leading to Glandular Atrophy: A Microscopic Study Contrasted with the Hormonal Assays

Science.gov (United States)

Venâncio, Daniel P.; Andersen, Monica L.; Vilamaior, Patricia S. L.; Santos, Fernanda C.; Zager, Adriano; Tufik, Sérgio; Taboga, Sebastião R.; De Mello, Marco T.

2012-01-01

We investigated the effect of 96 h paradoxical sleep deprivation (PSD) and 21-day sleep restriction (SR) on prostate morphology using stereological assays in male rats. After euthanasia, the rat ventral prostate was removed, weighed, and prepared for conventional light microscopy. Microscopic analysis of the prostate reveals that morphology of this gland was altered after 96 h of PSD and 21 days of SR, with the most important alterations occurring in the epithelium and stroma in the course of both procedures compared with the control group. Both 96 h PSD and 21-day SR rats showed lower serum testosterone and higher corticosterone levels than control rats. The significance of our result referring to the sleep deprivation was responsible for deep morphological alterations in ventral prostate tissue, like to castration microscopic modifications. This result is due to the marked alterations in hormonal status caused by PSD and SR. PMID:22927719

A novel mechanism important for the alignment of microtubules.

Science.gov (United States)

Wightman, Raymond; Turner, Simon R

2008-04-01

Using a live-cell imaging approach to study individual micro-tubules, we have compared microtubule behavior between net-like and aligned cortical arrays. In contrast to previous studies, a steep angled collision between the growing end of a microtubule and a preexisting microtubule was found to favor crossover. Frequencies of microtubule crossovers, bundling and catastrophes are similar regardless of whether the cell exhibited a net-like or aligned microtubule array. In the predominantly aligned array of petiole cells, severing occurs at the sites of microtubule crossovers and serves to remove unaligned microtubules and to increase microtubule density. Severing was observed to be rare in net-like arrays. Microtubule severing is carried out by the katanin enzyme. In this addendum, we present new insights into the possible mechanism of crossing over and preliminary data looking at organization of the array in a katanin mutant.

Structure of the first PDZ domain of human PSD-93

DEFF Research Database (Denmark)

Fiorentini, Monica; Nielsen, Ann Kallehauge; Kristensen, Ole

2009-01-01

The crystal structure of the PDZ1 domain of human PSD-93 has been determined to 2.0 A resolution. The PDZ1 domain forms a crystallographic trimer that is also predicted to be stable in solution. The main contributions to the stabilization of the trimer seem to arise from interactions involving...... the PDZ1-PDZ2 linker region at the extreme C-terminus of PDZ1, implying that the oligomerization that is observed is not of biological significance in full-length PSD-93. Comparison of the structures of the binding cleft of PSD-93 PDZ1 with the previously reported structures of PSD-93 PDZ2 and PDZ3...

On-orbit Status and Light Attenuation Behavior of the DAMPE-PSD

Science.gov (United States)

Li, Y.; Zhang, Y. P.; Zhang, Y. J.; Sun, Z. Y.; Yu, Y. H.; Dong, T. K.; Ma, P. X.; Wang, Y. P.; Yuan, Q.

2017-11-01

The DArk Matter Particle Explorer (DAMPE) is a high-resolution multi-purpose space-borne device for detecting the high-energy cosmic-rays like e±, γ-rays, protons, and heavy-ions, which was launched on 2015 December 17th. The Plastic Scintillator Detector (PSD) is the top-most sub-detector of DAMPE. The PSD is designed to measure the charge of incident high-energy particles, and to serve as a veto detector for discriminating γ-rays from the charged particles. In this paper, the on-orbit status of the PSD after launching in terms of high voltage (HV) and temperature stabilities is presented. The temperature and the HV variations of the PSD are less than 1°C and 0.5%, respectively. By using the on-orbit data, the attenuation lengths of PSD bars are obtained according to an empirical formula. A preliminary charge spectrum reconstructed from the X-layer of the PSD is obtained.

Cell proliferation, cell shape, and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

NARCIS (Netherlands)

Sieberer, B.; Kieft, H.; Franssen-Verheijen, M.A.W.; Emons, A.M.C.; Vos, J.W.

2009-01-01

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g.

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.

Measurement of in vitro microtubule polymerization by turbidity and fluorescence.

Science.gov (United States)

Mirigian, Matthew; Mukherjee, Kamalika; Bane, Susan L; Sackett, Dan L

2013-01-01

Tubulin polymerization may be conveniently monitored by the increase in turbidity (optical density, or OD) or by the increase in fluorescence intensity of diamidino-phenylindole. The resulting data can be a quantitative measure of microtubule (MT) assembly, but some care is needed in interpretation, especially of OD data. Buffer formulations used for the assembly reaction significantly influence the polymerization, both by altering the critical concentration for polymerization and by altering the exact polymer produced-for example, by increasing the production of sheet polymers in addition to MT. Both the turbidity and the fluorescence methods are useful for demonstrating the effect of MT-stabilizing or -destabilizing additives. 2013 Published by Elsevier Inc.

A low cost PSD-based monocular motion capture system

Science.gov (United States)

Ryu, Young Kee; Oh, Choonsuk

2007-10-01

This paper describes a monocular PSD-based motion capture sensor to employ with commercial video game systems such as Microsoft's XBOX and Sony's Playstation II. The system is compact, low-cost, and only requires a one-time calibration at the factory. The system includes a PSD(Position Sensitive Detector) and active infrared (IR) LED markers that are placed on the object to be tracked. The PSD sensor is placed in the focal plane of a wide-angle lens. The micro-controller calculates the 3D position of the markers using only the measured intensity and the 2D position on the PSD. A series of experiments were performed to evaluate the performance of our prototype system. From the experimental results we see that the proposed system has the advantages of the compact size, the low cost, the easy installation, and the high frame rates to be suitable for high speed motion tracking in games.

Overexpression of cypin alters dendrite morphology, single neuron activity, and network properties via distinct mechanisms

Science.gov (United States)

Rodríguez, Ana R.; O'Neill, Kate M.; Swiatkowski, Przemyslaw; Patel, Mihir V.; Firestein, Bonnie L.

2018-02-01

Objective. This study investigates the effect that overexpression of cytosolic PSD-95 interactor (cypin), a regulator of synaptic PSD-95 protein localization and a core regulator of dendrite branching, exerts on the electrical activity of rat hippocampal neurons and networks. Approach. We cultured rat hippocampal neurons and used lipid-mediated transfection and lentiviral gene transfer to achieve high levels of cypin or cypin mutant (cypinΔPDZ PSD-95 non-binding) expression cellularly and network-wide, respectively. Main results. Our analysis revealed that although overexpression of cypin and cypinΔPDZ increase dendrite numbers and decrease spine density, cypin and cypinΔPDZ distinctly regulate neuronal activity. At the single cell level, cypin promotes decreases in bursting activity while cypinΔPDZ reduces sEPSC frequency and further decreases bursting compared to cypin. At the network level, by using the Fano factor as a measure of spike count variability, cypin overexpression results in an increase in variability of spike count, and this effect is abolished when cypin cannot bind PSD-95. This variability is also dependent on baseline activity levels and on mean spike rate over time. Finally, our spike sorting data show that overexpression of cypin results in a more complex distribution of spike waveforms and that binding to PSD-95 is essential for this complexity. Significance. Our data suggest that dendrite morphology does not play a major role in cypin action on electrical activity.

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

Microtubules become more dynamic but not shorter during preprophase band formation: A possible "search-and-capture" mechanism for microtubule translocation

NARCIS (Netherlands)

Vos, J.W.; Dogterom, M.; Emons, A.M.C.

2004-01-01

The dynamic behavior of the microtubule cytoskeleton plays a crucial role in cellular organization, but the physical mechanisms underlying microtubule (re)organization in plant cells are poorly understood. We investigated microtubule dynamics in tobacco BY-2 suspension cells during interphase and

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

Science.gov (United States)

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

Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process.

Science.gov (United States)

Hutchins, B Ian; Wray, Susan

2014-01-01

Microtubules are a critical part of neuronal polarity and leading process extension, thus microtubule movement plays an important role in neuronal migration. However, the dynamics of microtubules during the forward movement of the nucleus into the leading process (nucleokinesis) is unclear and may be dependent on the cell type and mode of migration used. In particular, little is known about cytoskeletal changes during axophilic migration, commonly used in anteroposterior neuronal migration. We recently showed that leading process actin flow in migrating GnRH neurons is controlled by a signaling cascade involving IP3 receptors, CaMKK, AMPK, and RhoA. In the present study, microtubule dynamics were examined in GnRH neurons. Failure of the migration of these cells leads to the neuroendocrine disorder Kallmann Syndrome. Microtubules translocated forward along the leading process shaft during migration, but reversed direction and moved toward the nucleus when migration stalled. Blocking calcium release through IP3 receptors halted migration and induced the same reversal of microtubule translocation, while blocking cortical actin flow prevented microtubules from translocating toward the distal leading process. Super-resolution imaging revealed that microtubule plus-end tips are captured at the actin cortex through calcium-dependent mechanisms. This work shows that cortical actin flow draws the microtubule network forward through calcium-dependent capture in order to promote nucleokinesis, revealing a novel mechanism engaged by migrating neurons to facilitate movement.

The IPEN/CNEN-SP PSD neutron diffractometer

International Nuclear Information System (INIS)

Parente, Carlos Benedicto Ramos; Mazzocchi, Vera Lucia; Mascarenhas, Yvonne Primerano

2004-01-01

Full text: A new IPEN-CNEN/SP neutron powder diffractometer is under construction at the 4 MW thermal IEA-R1m research reactor. It is an upgrading of the old IPEN-CNEN/SP multipurpose neutron diffractometer. The main modifications introduced in the old instrument are: installation of a position sensitive detector (PSD) and a bent perfect single crystal monochromator (a focusing Si monochromator). The PSD is formed by eleven linear detector elements, clamped together at each end to form a rigid plane. The PSD is installed in a detector shielding which is supported by two arms fixed in a large rotary table. This table provides the instrument with the 2θ angular movement. A smaller rotary table, placed underneath and concentric with the larger one, provides the ω(θ) movement. Both tables are driven by a computer controlled geared mechanism. The computer also makes the data acquisition. A rotating-oscillating collimator, placed at the entrance to the detector shielding, eliminates parasitic scattering from furnace or cryorefrigerator heat shields in the vicinity of the sample. The collimator also makes the PSD less sensitive to ambient background. The PSD spans an angular range of 20 deg of a diffraction pattern, resulting in a quite good resolution for the instrument. An extended powder diffraction pattern can be obtained by moving the detector and collecting the data in 20 deg segments. With a take-off angle of 84 deg, the monochromator can be positioned to produce 4 different wavelengths, namely 1.111, 1.399, 1.667 and 2.191 A. Other parts constructed for the new instrument are: a in-pile collimator, a monochromatic beam collimator and a neutron shield, large enough to accommodate the monochromator, a beam shutter and the monochromatic beam collimator. In comparison to the former instrument, the new diffractometer will have better resolution and will be ca. 600 times faster in data acquisition. At the present time, the new instrument is in the final steps of

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).

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

Nerve growth factor alters microtubule targeting agent-induced neurotransmitter release but not MTA-induced neurite retraction in sensory neurons.

Science.gov (United States)

Pittman, Sherry K; Gracias, Neilia G; Fehrenbacher, Jill C

2016-05-01

Peripheral neuropathy is a dose-limiting side effect of anticancer treatment with the microtubule-targeted agents (MTAs), paclitaxel and epothilone B (EpoB); however, the mechanisms by which the MTAs alter neuronal function and morphology are unknown. We previously demonstrated that paclitaxel alters neuronal sensitivity, in vitro, in the presence of nerve growth factor (NGF). Evidence in the literature suggests that NGF may modulate the neurotoxic effects of paclitaxel. Here, we examine whether NGF modulates changes in neuronal sensitivity and morphology induced by paclitaxel and EpoB. Neuronal sensitivity was assessed using the stimulated release of calcitonin gene-related peptide (CGRP), whereas morphology of established neurites was evaluated using a high content screening system. Dorsal root ganglion cultures, maintained in the absence or presence of NGF, were treated from day 7 to day 12 in culture with paclitaxel (300nM) or EpoB (30nM). Following treatment, the release of CGRP was stimulated using capsaicin or high extracellular potassium. In the presence of NGF, EpoB mimicked the effects of paclitaxel: capsaicin-stimulated release was attenuated, potassium-stimulated release was slightly enhanced and the total peptide content was unchanged. In the absence of NGF, both paclitaxel and EpoB decreased capsaicin- and potassium-stimulated release and the total peptide content, suggesting that NGF may reverse MTA-induced hyposensitivity. Paclitaxel and EpoB both decreased neurite length and branching, and this attenuation was unaffected by NGF in the growth media. These differential effects of NGF on neuronal sensitivity and morphology suggest that neurite retraction is not a causative factor to alter neuronal sensitivity. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Biological Information Processing in Single Microtubules

Science.gov (United States)

2014-03-05

generated by synchronized oscillations of microtubules, centrosomes and chromosomes regulate the dynamics of mitosis and meiosis, Yue Zhao and Qimin...of frequency by a single microtubule. Green arrows depict the peaks that appear in absorption and disappear in transmission. Purple arrows show

Practical Findings from Applying the PSD Model for Evaluating Software Design Specifications

Science.gov (United States)

Räisänen, Teppo; Lehto, Tuomas; Oinas-Kukkonen, Harri

This paper presents practical findings from applying the PSD model to evaluating the support for persuasive features in software design specifications for a mobile Internet device. On the one hand, our experiences suggest that the PSD model fits relatively well for evaluating design specifications. On the other hand, the model would benefit from more specific heuristics for evaluating each technique to avoid unnecessary subjectivity. Better distinction between the design principles in the social support category would also make the model easier to use. Practitioners who have no theoretical background can apply the PSD model to increase the persuasiveness of the systems they design. The greatest benefit of the PSD model for researchers designing new systems may be achieved when it is applied together with a sound theory, such as the Elaboration Likelihood Model. Using the ELM together with the PSD model, one may increase the chances for attitude change.

Maximum Likelihood PSD Estimation for Speech Enhancement in Reverberation and Noise

DEFF Research Database (Denmark)

Kuklasinski, Adam; Doclo, Simon; Jensen, Søren Holdt

2016-01-01

In this contribution we focus on the problem of power spectral density (PSD) estimation from multiple microphone signals in reverberant and noisy environments. The PSD estimation method proposed in this paper is based on the maximum likelihood (ML) methodology. In particular, we derive a novel ML...... instrumental measures and is shown to be higher than when the competing estimator is used. Moreover, we perform a speech intelligibility test where we demonstrate that both the proposed and the competing PSD estimators lead to similar intelligibility improvements......., it is shown numerically that the mean squared estimation error achieved by the proposed method is near the limit set by the corresponding Cram´er-Rao lower bound. The speech dereverberation performance of a multi-channel Wiener filter (MWF) based on the proposed PSD estimators is measured using several...

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

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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.

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.

VID22 is required for transcriptional activation of the PSD2 gene in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Miyata, Non; Miyoshi, Takuya; Yamaguchi, Takanori; Nakazono, Toshimitsu; Tani, Motohiro; Kuge, Osamu

2015-12-15

Phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae is synthesized through decarboxylation of phosphatidylserine (PS), catalysed by PS decarboxylase 1 (Psd1p) and 2 (Psd2p) and the cytidine 5'-diphosphate (CDP)-ethanolamine (CDP-Etn) pathway. PSD1 null (psd1Δ) and PSD2 null (psd2Δ) mutants are viable in a synthetic minimal medium, but a psd1Δ psd2Δ double mutant exhibits Etn auxotrophy, which is incorporated into PE through the CDP-Etn pathway. We have previously shown that psd1Δ is synthetic lethal with deletion of VID22 (vid22Δ) [Kuroda et al. (2011) Mol. Microbiol. 80: , 248-265]. In the present study, we found that vid22Δ mutant exhibits Etn auxotrophy under PSD1-depressed conditions. Deletion of VID22 in wild-type and PSD1-depressed cells caused partial defects in PE formation through decarboxylation of PS. The enzyme activity of PS decarboxylase in an extract of vid22Δ cells was ∼70% of that in wild-type cells and similar to that in psd2Δ cells and the PS decarboxylase activity remaining in the PSD1-depressed cells became almost negligible with deletion of VID22. Thus, the vid22Δ mutation was suggested to cause a defect in the Psd2p activity. Furthermore, vid22Δ cells were shown to be defective in expression of the PSD2 gene tagged with 6×HA, the defect being ameliorated by replacement of the native promoter of the PSD2 gene with a CYC1 promoter. In addition, an α-galactosidase reporter assay revealed that the activity of the promoter of the PSD2 gene in vid22Δ cells was ∼5% of that in wild-type cells. These results showed that VID22 is required for transcriptional activation of the PSD2 gene. © 2015 Authors; published by Portland Press Limited.

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...

Changes in microtubule-associated protein tau during peripheral nerve injury and regeneration

Directory of Open Access Journals (Sweden)

Guang-bin Zha

2016-01-01

Full Text Available Tau, a primary component of microtubule-associated protein, promotes microtubule assembly and/or disassembly and maintains the stability of the microtubule structure. Although the importance of tau in neurodegenerative diseases has been well demonstrated, whether tau is involved in peripheral nerve regeneration remains unknown. In the current study, we obtained sciatic nerve tissue from adult rats 0, 1, 4, 7, and 14 days after sciatic nerve crush and examined tau mRNA and protein expression levels and the location of tau in the sciatic nerve following peripheral nerve injury. The results from our quantitative reverse transcription polymerase chain reaction analysis showed that compared with the uninjured control sciatic nerve, mRNA expression levels for both tau and tau tubulin kinase 1, a serine/threonine kinase that regulates tau phosphorylation, were decreased following peripheral nerve injury. Our western blot assay results suggested that the protein expression levels of tau and phosphorylated tau initially decreased 1 day post nerve injury but then gradually increased. The results of our immunohistochemical labeling showed that the location of tau protein was not altered by nerve injury. Thus, these results showed that the expression of tau was changed following sciatic nerve crush, suggesting that tau may be involved in peripheral nerve repair and regeneration.

Taxol crystals can masquerade as stabilized microtubules.

Directory of Open Access Journals (Sweden)

Margit Foss

Full Text Available Taxol is a potent anti-mitotic drug used in chemotherapy, angioplastic stents, and cell biology research. By binding and stabilizing microtubules, Taxol inhibits their dynamics, crucial for cell division, motility, and survival. The drug has also been reported to induce formation of asters and bundles composed of stabilized microtubules. Surprisingly, at commonly used concentrations, Taxol forms crystals that rapidly bind fluorescent tubulin subunits, generating structures with an uncanny resemblance to microtubule asters and bundles. Kinetic and topological considerations suggest that tubulin subunits, rather than microtubules, bind the crystals. This sequestration of tubulin from the subunit pool would be expected to shift the equilibrium of free to polymerized tubulin to disfavor assembly. Our results imply that some previously reported Taxol-induced asters or bundles could include or be composed of tubulin-decorated Taxol crystals. Thus, reevaluation of certain morphological, chemical, and physical properties of Taxol-treated microtubules may be necessary. Moreover, our findings suggest a novel mechanism for chemotherapy-induced cytotoxicity in non-dividing cells, with far-reaching medical implications.

Effect of radiation on microtubule structure in cancer cells

International Nuclear Information System (INIS)

Tripath, Shambhoo Sharan; Panda, Dulal; Jayakumar, S.; Maikho, Thoh; Sandur, Santosh Kumar

2017-01-01

Microtubules (MT) are dynamic structural cellular components. In proliferating cells, they are essential components in cell division through the formation of the mitotic spindle. Radiotherapy is an integral part of cancer treatment for most of the solid cancers. Scanty data exists in the literature related to how ionizing radiation affects microtubule reorganization in tumor cells. In the present study, breast cancer cell line (MCF-7 cells) was exposed to different doses of radiation (2-10Gy). Cells were cultured for 24 h, fixed and stained with antitubulin antibody and subjected to immunofluorescence microscopy. In another experiment, cells were subjected to cold treatment for 5 min or 30 min for studying the disassembly of microtubules after 24 h of irradiation. Further, these cells were incubated at 37°C for 20 min for studying the reassembly of microtubules. Acetylation of microtubule was also examined after exposure of cells to radiation. Experiments were also performed by combining radiation with low concentration of CXI-Benzo 84 (MT destabilizing agent 1 and 2.5 uM). Exposure of MCF-7 cells to radiation lead to destabilization of microtubules. Interestingly, destabilization of microtubule was faster upon cold treatment in irradiated group as compared to control group. These cells failed to re-stabilize at 37°C. Radiation also reduced the acetylation level of microtubule. Combination treatment of CXI-Benzo 84 with radiation exhibited additive effect in terms of depolymerization of MT. Our results suggest that ionizing radiation indeed modulates microtubule dynamics. (author)

Microtubules are organized independently of the centrosome in Drosophila neurons

Directory of Open Access Journals (Sweden)

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.

PSD Determination - Baseline

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

PSD Increment Baseline

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

EML proteins in microtubule regulation and human disease.

Science.gov (United States)

Fry, Andrew M; O'Regan, Laura; Montgomery, Jessica; Adib, Rozita; Bayliss, Richard

2016-10-15

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

The investigation of fatigue load on a PSD using finite element method

Energy Technology Data Exchange (ETDEWEB)

Choi, Mood Duck; Cho, Chong Du; Choi, Won June [Inha Univ., Incheon (Korea, Republic of); Kim, Jung Yup [Korea Institute of Construction Technology, Goyang (Korea, Republic of)

2008-07-01

Subway railway systems are being increasingly adopted in metro cities to ease the passenger transportation. But there are some concerns related to the safety of the passengers. Nowadays, PSD(Platform Screen Doors) are commonly used to assure the safety of passengers. PSD is used to prevent the fire disasters, air turbulence, and dust which may pose a threat to the passenger's safety. Moreover the design of PSD itself has to take some parameters into consideration, crowd loading, wind pressure, etc. In our present study we perform a fatigue analysis considering these parameters. Commercial finite element software package ANSYS Workbench 11.0 has been used for the structural analysis. In correlation with this analysis, the structural safety of the testing PSD equipment was confirmed, and the critical load condition was found.

The investigation of fatigue load on a PSD using finite element method

International Nuclear Information System (INIS)

Choi, Mood Duck; Cho, Chong Du; Choi, Won June; Kim, Jung Yup

2008-01-01

Subway railway systems are being increasingly adopted in metro cities to ease the passenger transportation. But there are some concerns related to the safety of the passengers. Nowadays, PSD(Platform Screen Doors) are commonly used to assure the safety of passengers. PSD is used to prevent the fire disasters, air turbulence, and dust which may pose a threat to the passenger's safety. Moreover the design of PSD itself has to take some parameters into consideration, crowd loading, wind pressure, etc. In our present study we perform a fatigue analysis considering these parameters. Commercial finite element software package ANSYS Workbench 11.0 has been used for the structural analysis. In correlation with this analysis, the structural safety of the testing PSD equipment was confirmed, and the critical load condition was found

Data acquisition system for linear PSD based neutron diffractometer

International Nuclear Information System (INIS)

Pande, S.S.; Borkar, S.P.; Behere, Anita; Ghodgaonkar, M.D.

2001-01-01

Single or multi-PSD configurations are used in different neutron diffractometer setups. A data acquisition system is developed to serve the gross requirements of all the diffractometer setups. It is also customized to specific requirements of different setups. The hardware is developed as a Transputer based add-on card. Most of the hardware functionality is handled in the Transputer program thus improving throughput of the system. The card can handle 16 RDCs, a few motor controls and on/off controls. The software comprises of a front-end Windows98 application, a Transputer program and a device driver. The data acquisition system performs data acquisition, analysis, display and storage. Analysis includes converting raw data of linear PSD to equiangular format, merging and clubbing the data to make a continuous equiangular spectrum. Calibration of individual PSD is a crucial activity in correctly merging the data coming from PSDs. (author)

Microtubule dynamics: Caps, catastrophes, and coupled hydrolysis

DEFF Research Database (Denmark)

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

1996-01-01

An effective theory is formulated for the dynamics of the guanosine triphosphate (GTP) cap believed to stabilize growing microtubules. The theory provides a ''coarse-grained'' description of the cap's dynamics. ''Microscopic'' details, such as the microtubule lattice structure and the fate of its...

Nonlinear dynamics of C-terminal tails in cellular microtubules

Science.gov (United States)

Sekulic, Dalibor L.; Sataric, Bogdan M.; Zdravkovic, Slobodan; Bugay, Aleksandr N.; Sataric, Miljko V.

2016-07-01

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.

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2

Science.gov (United States)

Petry, Sabine; Groen, Aaron C.; Ishihara, Keisuke; Mitchison, Timothy J.; Vale, Ronald D.

2013-01-01

Summary The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence also has suggested that microtubules might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires gamma-tubulin and augmin and is stimulated by GTP-bound Ran and its effector TPX2, factors previously implicated in chromatin-stimulated nucleation. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance. PMID:23415226

Kinesin expands and stabilizes the GDP-microtubule lattice

Science.gov (United States)

Peet, Daniel R.; Burroughs, Nigel J.; Cross, Robert A.

2018-05-01

Kinesin-1 is a nanoscale molecular motor that walks towards the fast-growing (plus) ends of microtubules, hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organization of eukaryotic cells and shows great promise as a tool for nano-engineering1. Recent work hints that kinesin may also play a role in modulating the stability of its microtubule track, both in vitro2,3 and in vivo4, but the results are conflicting5-7 and the mechanisms are unclear. Here, we report a new dimension to the kinesin-microtubule interaction, whereby strong-binding state (adenosine triphosphate (ATP)-bound and apo) kinesin-1 motor domains inhibit the shrinkage of guanosine diphosphate (GDP) microtubules by up to two orders of magnitude and expand their lattice spacing by 1.6%. Our data reveal an unexpected mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, and so allow motile kinesins to influence the structure, stability and mechanics of their microtubule track.

Quantitative Analysis of Tau-Microtubule Interaction Using FRET

Directory of Open Access Journals (Sweden)

Isabelle L. Di Maïo

2014-08-01

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

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

2015-01-01

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.

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.

75 FR 70254 - PSD and Title V Permitting Guidance for Greenhouse Gases

Science.gov (United States)

2010-11-17

... Guidance for Greenhouse Gases AGENCY: Environmental Protection Agency (EPA). ACTION: Notice of availability..., ``PSD and Title V Permitting Guidance for Greenhouse Gases'' on its significant guidance Internet Web... guidance titled, ``PSD and Title V Permitting Guidance for Greenhouse Gases.'' This document has been...

Optical Tweezers-Based Measurements of Forces and Dynamics at Microtubule Ends.

Science.gov (United States)

Baclayon, Marian; Kalisch, Svenja-Marei; Hendel, Ed; Laan, Liedewij; Husson, Julien; Munteanu, E Laura; Dogterom, Marileen

2017-01-01

Microtubules are dynamic cytoskeletal polymers that polymerize and depolymerize while interacting with different proteins and structures within the cell. The highly regulated dynamic properties as well as the pushing and pulling forces generated by dynamic microtubule ends play important roles in processes such as in cell division. For instance, microtubule end-binding proteins are known to affect dramatically the dynamic properties of microtubules, and cortical dyneins are known to mediate pulling forces on microtubule ends. We discuss in this chapter our efforts to reconstitute these systems in vitro and mimic their interactions with structures within the cell using micro-fabricated barriers. Using an optical tweezers setup, we investigate the dynamics and forces of microtubules growing against functionalized barriers in the absence and presence of end-binding proteins and barrier-attached motor proteins. This setup allows high-speed as well as nanometer and piconewton resolution measurements on dynamic microtubules.

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.

Neutron-gamma discrimination via PSD plastic scintillator and SiPMs

Science.gov (United States)

Taggart, M. P.; Payne, C.; Sellin, P. J.

2016-10-01

The reduction in availability and inevitable increase in cost of traditional neutron detectors based on the 3He neutron capture reaction has resulted in a concerted effort to seek out new techniques and detection media to meet the needs of national nuclear security. Traditionally, the alternative has been provided through pulse shape discrimination (PSD) using liquid scintillators. However, these are not without their own inherent issues, primarily concerning user safety and ongoing maintenance. A potential system devised to separate neutron and gamma ray pulses utilising the PSD technique takes advantage of recent improvements in silicon photomultiplier (SiPM) technology and the development of plastic scintillators exhibiting the PSD phenomena. In this paper we present the current iteration of this ongoing work having achieved a Figure of Merit (FoM) of 1.39 at 1.5 MeVee.

Multi-beam synchronous measurement based on PSD phase detection using frequency-domain multiplexing

Science.gov (United States)

Duan, Ying; Qin, Lan; Xue, Lian; Xi, Feng; Mao, Jiubing

2013-10-01

According to the principle of centroid measurement, position-sensitive detectors (PSD) are commonly used for micro displacement detection. However, single-beam detection method cannot satisfy such tasks as multi-dimension position measurement, three dimension vision reconstruction, and robot precision positioning, which require synchronous measurement of multiple light beams. Consequently, we designed PSD phase detection method using frequency-domain multiplexing for synchronous detection of multiple modulated light beams. Compared to previous PSD amplitude detection method, the phase detection method using FDM has advantages of simplified measuring system, low cost, high capability of resistance to light interference as well as improved resolution. The feasibility of multi-beam synchronous measurement based on PSD phase detection using FDM was validated by multi-beam measuring experiments. The maximum non-linearity error of the multi-beam synchronous measurement is 6.62%.

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.

Model for the orientational ordering of the plant microtubule cortical array

Science.gov (United States)

Hawkins, Rhoda J.; Tindemans, Simon H.; Mulder, Bela M.

2010-07-01

The plant microtubule cortical array is a striking feature of all growing plant cells. It consists of a more or less homogeneously distributed array of highly aligned microtubules connected to the inner side of the plasma membrane and oriented transversely to the cell growth axis. Here, we formulate a continuum model to describe the origin of orientational order in such confined arrays of dynamical microtubules. The model is based on recent experimental observations that show that a growing cortical microtubule can interact through angle dependent collisions with pre-existing microtubules that can lead either to co-alignment of the growth, retraction through catastrophe induction or crossing over the encountered microtubule. We identify a single control parameter, which is fully determined by the nucleation rate and intrinsic dynamics of individual microtubules. We solve the model analytically in the stationary isotropic phase, discuss the limits of stability of this isotropic phase, and explicitly solve for the ordered stationary states in a simplified version of the model.

Adding rectifying/stripping section type heat integration to a pressure-swing distillation (PSD) process

International Nuclear Information System (INIS)

Huang Kejin; Shan Lan; Zhu Qunxiong; Qian Jixin

2008-01-01

This paper studies the economical effect of considering rectifying/stripping section type heat integration in a pressure-swing distillation (PSD) process separating a binary homogeneous pressure-sensitive azeotrope. The schemes for arranging heat integration between the rectifying section and the stripping section of the high- and low-pressure distillation columns, respectively, are derived and an effective procedure is devised for the conceptual process design of the heat-integrated PSD processes. In terms of the separation of a binary azeotropic mixture of acetonitrile and water, intensive comparisons are made between the conventional and heat-integrated PSD processes. It is demonstrated that breaking a pressure-sensitive azeotropic mixture can be made more economical than the current practice with the conventional PSD process. For boosting further the thermodynamic efficiency of a PSD process, it is strongly suggested to consider simultaneously the condenser/reboiler type heat integration with the rectifying/stripping section type heat integration in process synthesis and design

Hadron calorimeter (PSD) with new photo-detectors (MPPC) in NA61 experiment at CERN

Science.gov (United States)

Golubeva, M.; Guber, F.; Ivashkin, A.; Izvestnyy, A.; Kurepin, A.; Morozov, S.; Petukhov, O.; Selyuzhenkov, I.; Svintsov, I.; Taranenko, A.

2017-01-01

The Projectile Spectator Detector (PSD) is a segmented hadron calorimeter used in NA61 experiment (CERN) to determine a collision centrality as well as an event plane orientation in nucleus-nucleus collisions. The main goal of the experiment includes studying the onset of de-confinement and searching for the critical point of strongly interacting matter. It is of crucial importance to have a precise characterization of the event class with the PSD for the analysis of event-by-event observables. The PSD has been already used for centrality selection on trigger level in measurements of Be+Be and Ar+Sc reactions at beam energies 13 - 158 AGeV and Pb+Pb reaction at beam energy 30 AGeV. In 2016, the central modules of PSD have been equipped with new Hamamatsu MPPC silicon photo-detectors in order to extend dynamic range for studying Pb+Pb reaction at the full energy range 13 - 158 AGeV. Results of the PSD response on proton and lead beams are presented.

Adding rectifying/stripping section type heat integration to a pressure-swing distillation (PSD) process

Energy Technology Data Exchange (ETDEWEB)

Huang Kejin [School of Information Science and Technology, Beijing University of Chemical Technology, Chaoyang-qu, Beijing-shi, Beijing 100029 (China)], E-mail: huangkj@mail.buct.edu.cn; Shan Lan; Zhu Qunxiong [School of Information Science and Technology, Beijing University of Chemical Technology, Chaoyang-qu, Beijing-shi, Beijing 100029 (China); Qian Jixin [School of Information Science and Technology, Zhejiang University, Xihu-qu, Hangzhou-shi, Zhejiang 300027 (China)

2008-06-15

This paper studies the economical effect of considering rectifying/stripping section type heat integration in a pressure-swing distillation (PSD) process separating a binary homogeneous pressure-sensitive azeotrope. The schemes for arranging heat integration between the rectifying section and the stripping section of the high- and low-pressure distillation columns, respectively, are derived and an effective procedure is devised for the conceptual process design of the heat-integrated PSD processes. In terms of the separation of a binary azeotropic mixture of acetonitrile and water, intensive comparisons are made between the conventional and heat-integrated PSD processes. It is demonstrated that breaking a pressure-sensitive azeotropic mixture can be made more economical than the current practice with the conventional PSD process. For boosting further the thermodynamic efficiency of a PSD process, it is strongly suggested to consider simultaneously the condenser/reboiler type heat integration with the rectifying/stripping section type heat integration in process synthesis and design.

Fuzzy norm method for evaluating random vibration of airborne platform from limited PSD data

Directory of Open Access Journals (Sweden)

Wang Zhongyu

2014-12-01

Full Text Available For random vibration of airborne platform, the accurate evaluation is a key indicator to ensure normal operation of airborne equipment in flight. However, only limited power spectral density (PSD data can be obtained at the stage of flight test. Thus, those conventional evaluation methods cannot be employed when the distribution characteristics and priori information are unknown. In this paper, the fuzzy norm method (FNM is proposed which combines the advantages of fuzzy theory and norm theory. The proposed method can deeply dig system information from limited data, which probability distribution is not taken into account. Firstly, the FNM is employed to evaluate variable interval and expanded uncertainty from limited PSD data, and the performance of FNM is demonstrated by confidence level, reliability and computing accuracy of expanded uncertainty. In addition, the optimal fuzzy parameters are discussed to meet the requirements of aviation standards and metrological practice. Finally, computer simulation is used to prove the adaptability of FNM. Compared with statistical methods, FNM has superiority for evaluating expanded uncertainty from limited data. The results show that the reliability of calculation and evaluation is superior to 95%.

Four-stranded mini microtubules formed by Prosthecobacter BtubAB 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.

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.

Nonlinear dynamics of C–terminal tails in cellular microtubules

International Nuclear Information System (INIS)

Sekulic, Dalibor L.; Sataric, Bogdan M.; Sataric, Miljko V.; Zdravkovic, Slobodan; Bugay, Aleksandr N.

2016-01-01

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.

76 FR 7546 - Proposed Information Collection; Comment Request; Prohibited Species Donation (PSD) Program

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2011-02-10

... Collection; Comment Request; Prohibited Species Donation (PSD) Program AGENCY: National Oceanic and... species donation (PSD) program for Pacific salmon and Pacific halibut has effectively reduced regulatory... individuals through tax-exempt organizations. Vessels and processing plants participating in the donation...

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).

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.

PSD Increment Consumption Guidance

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This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

PSD Increment Consumption Question

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Microtubules move the nucleus to quiescence.

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Laporte, Damien; Sagot, Isabelle

2014-01-01

The nucleus is a cellular compartment that hosts several macro-molecular machines displaying a highly complex spatial organization. This tight architectural orchestration determines not only DNA replication and repair but also regulates gene expression. In budding yeast microtubules play a key role in structuring the nucleus since they condition the Rabl arrangement in G1 and chromosome partitioning during mitosis through their attachment to centromeres via the kinetochore proteins. Recently, we have shown that upon quiescence entry, intranuclear microtubules emanating from the spindle pole body elongate to form a highly stable bundle that spans the entire nucleus. Here, we examine some molecular mechanisms that may underlie the formation of this structure. As the intranuclear microtubule bundle causes a profound re-organization of the yeast nucleus and is required for cell survival during quiescence, we discuss the possibility that the assembly of such a structure participates in quiescence establishment.

Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells.

Science.gov (United States)

Akita, Kae; Higaki, Takumi; Kutsuna, Natsumaro; Hasezawa, Seiichiro

2015-01-01

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.

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.

Lysosomes are associated with microtubules and not with intermediate filaments in cultured fibroblasts.

OpenAIRE

Collot, M; Louvard, D; Singer, S J

1984-01-01

Double immunofluorescent labeling experiments for lysosomes and either microtubules or vimentin intermediate filaments in cultured well-spread fibroblasts show a remarkable degree of superposition of the lysosomes and the microtubules. Under two different sets of conditions where the microtubules and intermediate filaments are well segregated from one another, the lysosomes remain codistributed with the microtubules. It is suggested that this specific association of lysosomes with microtubule...

On the nature and shape of tubulin trails: implications on microtubule self-organization.

Science.gov (United States)

Glade, Nicolas

2012-06-01

Microtubules, major elements of the cell skeleton are, most of the time, well organized in vivo, but they can also show self-organizing behaviors in time and/or space in purified solutions in vitro. Theoretical studies and models based on the concepts of collective dynamics in complex systems, reaction-diffusion processes and emergent phenomena were proposed to explain some of these behaviors. In the particular case of microtubule spatial self-organization, it has been advanced that microtubules could behave like ants, self-organizing by 'talking to each other' by way of hypothetic (because never observed) concentrated chemical trails of tubulin that are expected to be released by their disassembling ends. Deterministic models based on this idea yielded indeed like-looking spatio-temporal self-organizing behaviors. Nevertheless the question remains of whether microscopic tubulin trails produced by individual or bundles of several microtubules are intense enough to allow microtubule self-organization at a macroscopic level. In the present work, by simulating the diffusion of tubulin in microtubule solutions at the microscopic scale, we measure the shape and intensity of tubulin trails and discuss about the assumption of microtubule self-organization due to the production of chemical trails by disassembling microtubules. We show that the tubulin trails produced by individual microtubules or small microtubule arrays are very weak and not elongated even at very high reactive rates. Although the variations of concentration due to such trails are not significant compared to natural fluctuations of the concentration of tubuline in the chemical environment, the study shows that heterogeneities of biochemical composition can form due to microtubule disassembly. They could become significant when produced by numerous microtubule ends located in the same place. Their possible formation could play a role in certain conditions of reaction. In particular, it gives a mesoscopic

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

Science.gov (United States)

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

2000-01-01

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

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...

Homer2 deletion alters dendritic spine morphology but not alcohol-associated adaptations in GluN2B-containing NMDA receptors in the nucleus accumbens

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Natalie S McGuier

2015-02-01

Full Text Available Repeated exposure to ethanol followed by withdrawal leads to the alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc in both clinical and preclinical models of ethanol exposure. Homer2 is a member of a family of postsynaptic density (PSD scaffolding proteins that functions in part to cluster NMDA signaling complexes in the PSD, and has been shown to be critically important for plasticity in multiple models of drug and alcohol abuse. Here we used Homer2 KO mice and a chronic intermittent intraperitoneal (IP ethanol injection model to investigate a potential role for the protein in ethanol-induced adaptations in dendritic spine morphology and PSD protein expression. While deletion of Homer2 was associated with increased density of long spines on medium spiny neurons of the NAc core of saline treated mice, ethanol exposure had no effect on dendritic spine morphology in either wild-type (WT or Homer2 KO mice. Western blot analysis of tissue samples from the NAc enriched for PSD proteins revealed a main effect of ethanol treatment on the expression of GluN2B, but there was no effect of genotype or treatment on the expression other glutamate receptor subunits or PSD95. These data indicate that the global deletion of Homer2 leads to aberrant regulation of dendritic spine morphology in the NAc core that is associated with an increased density of long, thin spines. Unexpectedly, intermittent IP ethanol did not affect spine morphology in either WT or KO mice. Together these data implicate Homer2 in the formation of long, thin spines and further supports its role in neuronal structure.

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

The nucleation of microtubules in Aspergillus nidulans germlings

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Cristina de Andrade-Monteiro

1999-09-01

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.Microtúbulos são filamentos compostos por dímeros das tubulinas a e b e têm uma variedade de funções nas células vivas. Em fungos, os corpúsculos polares dos fusos são geralmente considerados os centros organizadores dos microtúbulos. Com o objetivo de contribuir para uma melhor compreensão dos processos de nucleação dos microtúbulos no fungo filamentoso A. nidulans, nós utilizamos a droga antimicrotúbulo Benomil em experimentos de bloqueio e liberação para depolimerizar e repolimerizar os microtúbulos. Após 20 segundos de reincubação em meio sem Benomil, pequenos microtúbulos foram formados a partir de pontos distribuídos pela célula, sugerindo que os pontos de nucleação de microtúbulos s

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

Application of PSD for low level alpha counting using liquid scintillation counting

International Nuclear Information System (INIS)

Krishnamachari, G.; Vaze, P.K.; Iyer, M.R.

1989-01-01

In the liquid scintillator the light produced by alpha particles decays differently than those produced by electrons. Pulse shape discrimination (PSD) methods are employed to estimate low levels of alpha emitting radionuclides by reducing the background due to either beta or gamma events. An attempt is being made to develop a liquid scintillation counting sytem using a simple PSD circuit to achieve a background of 0.01 counts/min. The PSD circuit is based on measuring zero cross over points to differentiate particle types. The input signal is first differentiated by a delay line and subsequently by a RC circuit. The width of the initial part of the doubly differentiated pulse is different for alpha and beta pulses. This width is converted to amplitude by a time-to-amplitude converter (TAC). The higher amplitude pulses from the TAC are due to alpha particles and they are separated by an integral discriminator. The output from the integral discriminator opens a linear gate to record the pulse height spectrum. The figure of merit of the PSD circuit and background in the alpha energy channel have been worked out using different scintillator types. (author). 4 figs

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.

Thermodynamics of TMPC/PSd/Fullerene Nanocomposites: SANS Study

KAUST Repository

Chua, Yang-Choo; Chan, Alice; Wong, Him-Cheng; Higgins, Julia S.; Cabral, João T.

2010-01-01

) analysis demonstrate that 1-2 mass % of C60 fullerenes destabilizes a highly interacting mixture of poly(tetramethyl bisphenol A polycarbonate) and deuterated polystyrene (TMPC/PSd). We unequivocally corroborate these findings with time-resolved temperature

Transcriptional response to deletion of the phosphatidylserine decarboxylase Psd1p in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Gsell, Martina; Mascher, Gerald; Schuiki, Irmgard; Ploier, Birgit; Hrastnik, Claudia; Daum, Günther

2013-01-01

In the yeast, Saccharomyces cerevisiae, the synthesis of the essential phospholipid phosphatidylethanolamine (PE) is accomplished by a network of reactions which comprises four different pathways. The enzyme contributing most to PE formation is the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) which catalyzes conversion of phosphatidylserine (PS) to PE. To study the genome wide effect of an unbalanced cellular and mitochondrial PE level and in particular the contribution of Psd1p to this depletion we performed a DNA microarray analysis with a ∆psd1 deletion mutant. This approach revealed that 54 yeast genes were significantly up-regulated in the absence of PSD1 compared to wild type. Surprisingly, marked down-regulation of genes was not observed. A number of different cellular processes in different subcellular compartments were affected in a ∆psd1 mutant. Deletion mutants bearing defects in all 54 candidate genes, respectively, were analyzed for their growth phenotype and their phospholipid profile. Only three mutants, namely ∆gpm2, ∆gph1 and ∆rsb1, were affected in one of these parameters. The possible link of these mutations to PE deficiency and PSD1 deletion is discussed.

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).

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

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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

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

Novel Method of Detecting Movement of the Interference Fringes Using One-Dimensional PSD

Directory of Open Access Journals (Sweden)

Qi Wang

2015-06-01

Full Text Available In this paper, a method of using a one-dimensional position-sensitive detector (PSD by replacing charge-coupled device (CCD to measure the movement of the interference fringes is presented first, and its feasibility is demonstrated through an experimental setup based on the principle of centroid detection. Firstly, the centroid position of the interference fringes in a fiber Mach-Zehnder (M-Z interferometer is solved in theory, showing it has a higher resolution and sensitivity. According to the physical characteristics and principles of PSD, a simulation of the interference fringe’s phase difference in fiber M-Z interferometers and PSD output is carried out. Comparing the simulation results with the relationship between phase differences and centroid positions in fiber M-Z interferometers, the conclusion that the output of interference fringes by PSD is still the centroid position is obtained. Based on massive measurements, the best resolution of the system is achieved with 5.15, 625 μm. Finally, the detection system is evaluated through setup error analysis and an ultra-narrow-band filter structure. The filter structure is configured with a one-dimensional photonic crystal containing positive and negative refraction material, which can eliminate background light in the PSD detection experiment. This detection system has a simple structure, good stability, high precision and easily performs remote measurements, which makes it potentially useful in material small deformation tests, refractivity measurements of optical media and optical wave front detection.

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.

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.

Direct evidence for GTP and GDP-Pi intermediates in microtubule assembly

International Nuclear Information System (INIS)

Melki, R.; Carlier, M.F.; Pantaloni, D.

1990-01-01

Identification of the kinetic intermediates in GTP hydrolysis on microtubules and characterization of their assembly properties is essential in understanding microtubule dynamics. By using an improved glass filter assay that selectively traps microtubules with a dead time of 2 s and monitoring taxol-induced rapid assembly of microtubules from [γ- 32 P, 3 H]GTP-tubulin 1:1 complex, direct evidence has been obtained for GTP- and GDP-P i -microtubule transient states in the early stages of the polymerization process. A simple kinetic analysis of GTP hydrolysis on microtubules within two sequential pseudo-first-order processes led to apparent first-order rate constants of 0.065 s -1 for the cleavage of the γ-phosphate and 0.02 s -1 for the liberation of P i , assuming a simple random model. Apparent rate constants for GTP hydrolysis and P i release were independent of the composition of the buffer used to polymerize tubulin. The significance of these values with respect to those derived from previous studies from this and other laboratories and the possibility of a vectorial model for GTP hydrolysis are discussed

Mathematical Model and Calibration Procedure of a PSD Sensor Used in Local Positioning Systems.

Science.gov (United States)

Rodríguez-Navarro, David; Lázaro-Galilea, José Luis; Bravo-Muñoz, Ignacio; Gardel-Vicente, Alfredo; Domingo-Perez, Francisco; Tsirigotis, Georgios

2016-09-15

Here, we propose a mathematical model and a calibration procedure for a PSD (position sensitive device) sensor equipped with an optical system, to enable accurate measurement of the angle of arrival of one or more beams of light emitted by infrared (IR) transmitters located at distances of between 4 and 6 m. To achieve this objective, it was necessary to characterize the intrinsic parameters that model the system and obtain their values. This first approach was based on a pin-hole model, to which system nonlinearities were added, and this was used to model the points obtained with the nA currents provided by the PSD. In addition, we analyzed the main sources of error, including PSD sensor signal noise, gain factor imbalances and PSD sensor distortion. The results indicated that the proposed model and method provided satisfactory calibration and yielded precise parameter values, enabling accurate measurement of the angle of arrival with a low degree of error, as evidenced by the experimental results.

GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

Science.gov (United States)

Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

2016-06-01

Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Measuring and modeling polymer concentration profiles near spindle boundaries argues that spindle microtubules regulate their own nucleation

Science.gov (United States)

Kaye, Bryan; Stiehl, Olivia; Foster, Peter J.; Shelley, Michael J.; Needleman, Daniel J.; Fürthauer, Sebastian

2018-05-01

Spindles are self-organized microtubule-based structures that segregate chromosomes during cell division. The mass of the spindle is controlled by the balance between microtubule turnover and nucleation. The mechanisms that control the spatial regulation of microtubule nucleation remain poorly understood. While previous work found that microtubule nucleators bind to pre-existing microtubules in the spindle, it is still unclear whether this binding regulates the activity of those nucleators. Here we use a combination of experiments and mathematical modeling to investigate this issue. We measured the concentration of microtubules and soluble tubulin in and around the spindle. We found a very sharp decay in the concentration of microtubules at the spindle interface. This is inconsistent with a model in which the activity of nucleators is independent of their association with microtubules but consistent with a model in which microtubule nucleators are only active when bound to pre-existing microtubules. This argues that the activity of microtubule nucleators is greatly enhanced when bound to pre-existing microtubules. Thus, microtubule nucleators are both localized and activated by the microtubules they generate.

PSD Determination, Portland Cement Plant

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

United Salt Northeast PSD Determination

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

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.

Marshall Space Flight Center Propulsion Systems Department (PSD) Knowledge Management (KM) Initiative

Science.gov (United States)

Caraccioli, Paul; Varnedoe, Tom; Smith, Randy; McCarter, Mike; Wilson, Barry; Porter, Richard

2006-01-01

NASA Marshall Space Flight Center's Propulsion Systems Department (PSD) is four months into a fifteen month Knowledge Management (KM) initiative to support enhanced engineering decision making and analyses, faster resolution of anomalies (near-term) and effective, efficient knowledge infused engineering processes, reduced knowledge attrition, and reduced anomaly occurrences (long-term). The near-term objective of this initiative is developing a KM Pilot project, within the context of a 3-5 year KM strategy, to introduce and evaluate the use of KM within PSD. An internal NASA/MSFC PSD KM team was established early in project formulation to maintain a practitioner, user-centric focus throughout the conceptual development, planning and deployment of KM technologies and capabilities within the PSD. The PSD internal team is supported by the University of Alabama's Aging Infrastructure Systems Center of Excellence (AISCE), lntergraph Corporation, and The Knowledge Institute. The principle product of the initial four month effort has been strategic planning of PSD KNI implementation by first determining the "as is" state of KM capabilities and developing, planning and documenting the roadmap to achieve the desired "to be" state. Activities undertaken to suppoth e planning phase have included data gathering; cultural surveys, group work-sessions, interviews, documentation review, and independent research. Assessments and analyses have beon pedormed including industry benchmarking, related local and Agency initiatives, specific tools and techniques used and strategies for leveraging existing resources, people and technology to achieve common KM goals. Key findings captured in the PSD KM Strategic Plan include the system vision, purpose, stakeholders, prioritized strategic objectives mapped to the top ten practitioner needs and analysis of current resource usage. Opportunities identified from research, analyses, cultural1KM surveys and practitioner interviews include

Brassinosteroids regulate pavement cell growth by mediating BIN2-induced microtubule stabilization.

Science.gov (United States)

Liu, Xiaolei; Yang, Qin; Wang, Yuan; Wang, Linhai; Fu, Ying; Wang, Xuelu

2018-02-23

Brassinosteroids (BRs), a group of plant steroid hormones, play important roles in regulating plant development. The cytoskeleton also affects key developmental processes and a deficiency in BR biosynthesis or signaling leads to abnormal phenotypes similar to those of microtubule-defective mutants. However, how BRs regulate microtubule and cell morphology remains unknown. Here, using liquid chromatography-tandem mass spectrometry, we identified tubulin proteins that interact with Arabidopsis BRASSINOSTEROID INSENSITIVE2 (BIN2), a negative regulator of BR responses in plants. In vitro and in vivo pull-down assays confirmed that BIN2 interacts with tubulin proteins. High-speed co-sedimentation assays demonstrated that BIN2 also binds microtubules. The Arabidopsis genome also encodes two BIN2 homologs, BIN2-LIKE 1 (BIL1) and BIL2, which function redundantly with BIN2. In the bin2-3 bil1 bil2 triple mutant, cortical microtubules were more sensitive to treatment with the microtubule-disrupting drug oryzalin than in wild-type, whereas in the BIN2 gain-of-function mutant bin2-1, cortical microtubules were insensitive to oryzalin treatment. These results provide important insight into how BR regulates plant pavement cell and leaf growth by mediating the stabilization of microtubules by BIN2.

IB-11PSEUDO-PROGRESSION (PsdPg) IS A HARBINGER OF A MORE EFFECTIVE ANTI-TUMOR RESPONSE

Science.gov (United States)

Sturla, Lisa; Donahue, John; Machan, Jason; Delamonte, Suzanne; Jeyapalan, Suriya

2014-01-01

BACKGROUND: PsdPg is the increased contrast enhancement, high choline/creatine ratio and increased perfusion observed in the residual tumor bed of high-grade glioma patients after completion of temozolomide/radiation. It resolves within 3-6 months and incidence ranges from 10 - 31%. Though correlated with longer patient survival, its pathological basis is unclear. We used a cytokine/chemokine focused approach to compare the tumor microenvironment in pre- and post-treatment tumor tissue from patients with PsdPg to patients with true progression (TP). METHODS: We obtained pre-treatment formalin fixed paraffin embedded (FFPE) tissue from 35 GBM patients and post-treatment FFPE tissue from five patients with PsdPg and TP. A quantitative PCR array and custom Quantigene 2.0 multiplex was used to quantify gene expression corresponding to major cytokines/chemokines. An 18-gene signature was used to determine the macrophage polarization score (cumulative M2-associated cytokine expression - cumulative M1-associated cytokine expression). Immunohistochemistry (IHC) was used to confirm significantly different targets at the protein level. RESULTS: IHC revealed 7-fold higher B-cell infiltration in TP patients as compared to patients with PsdPg (p = 0.003). Macrophage and T-cell infiltration were not significantly different between the two groups. Nevertheless, the cytokines associated with macrophage polarization indicated pro-tumorigenic (M2) polarization in TP patients while PsdPg patients exhibited classical anti-tumorigenic (M1) polarization. TP patients had a 10-fold higher M2 score (p = 0.03) compared to PsdPg patients. The M1 score of tissue from PsdPg patients post-treatment was 25-fold higher than their pre-treatment tissue (p = 0.01). Analysis of a 7-gene signature associated with natural killer (NK) cell recruitment and activation showed a 8-fold higher expression in pre-treatment tissue from PsdPg patients compared to TP patients (p = 0.009) suggesting that NK cells

Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module.

Science.gov (United States)

Roostalu, Johanna; Cade, Nicholas I; Surrey, Thomas

2015-11-01

Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilizes growing microtubule ends and stimulates microtubule nucleation by stabilizing early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homologue) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking the interaction of TPX2 with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells.

Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation

Science.gov (United States)

Foe, Victoria E.; von Dassow, George

2008-01-01

The cytokinetic furrow arises from spatial and temporal regulation of cortical contractility. To test the role microtubules play in furrow specification, we studied myosin II activation in echinoderm zygotes by assessing serine19-phosphorylated regulatory light chain (pRLC) localization after precisely timed drug treatments. Cortical pRLC was globally depressed before cytokinesis, then elevated only at the equator. We implicated cell cycle biochemistry (not microtubules) in pRLC depression, and differential microtubule stability in localizing the subsequent myosin activation. With no microtubules, pRLC accumulation occurred globally instead of equatorially, and loss of just dynamic microtubules increased equatorial pRLC recruitment. Nocodazole treatment revealed a population of stable astral microtubules that formed during anaphase; among these, those aimed toward the equator grew longer, and their tips coincided with cortical pRLC accumulation. Shrinking the mitotic apparatus with colchicine revealed pRLC suppression near dynamic microtubule arrays. We conclude that opposite effects of stable versus dynamic microtubules focuses myosin activation to the cell equator during cytokinesis. PMID:18955555

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.

Short Linear Sequence Motif LxxPTPh Targets Diverse Proteins to Growing Microtubule Ends

NARCIS (Netherlands)

Kumar, Anil; Manatschal, Cristina; Rai, Ankit; Grigoriev, Ilya; Degen, Miriam Steiner; Jaussi, Rolf; Kretzschmar, Ines; Prota, Andrea E; Volkmer, Rudolf; Kammerer, Richard A.; Akhmanova, Anna; Steinmetz, Michel O.

2017-01-01

Microtubule plus-end tracking proteins (+TIPs) are involved in virtually all microtubule-based processes. End-binding (EB) proteins are considered master regulators of +TIP interaction networks, since they autonomously track growing microtubule ends and recruit a plethora of proteins to this

Data acquisition and processing software for linear PSD based neutron diffractometers

International Nuclear Information System (INIS)

Pande, S.S.; Borkar, S.P.; Ghodgaonkar, M.D.

2003-01-01

As a part of data acquisition system for various single and multi-PSD diffractometers software is developed to acquire the data and support the requirements of diffraction experiments. The software is a front-end Windows 98 application on PC and a transputer program on the MPSD card. The front-end application provides entire user interface required for data acquisition, control, presentation and system setup. Data is acquired and the diffraction spectra are generated in the transputer program. All the required hardware control is also implemented in the transputer program. The two programs communicate using a device driver named VTRANSPD. The software plays a vital role in customizing and integrating the data acquisition system for various diffractometer setups. Also the experiments are effectively automated in the software which has helped in making best use of available beam time. These and other features of the data acquisition and processing software are presented here. This software is being used along with the data acquisition system at a few single PSD and multi-PSD diffractometers. (author)

Template-free electrosynthesis of aligned poly(p-phenylene) microtubules

Institute of Scientific and Technical Information of China (English)

无

2003-01-01

Poly(p-phenylene) (PPP) microtubules with diameters of 0.2-0.8μm and lengths of～10 (m have been synthesized by direct oxidation of benzene in the mixed electrolyte of boron trifluoride diethyl etherate (BFEE) and trifluoroacetic acid (TFA) (BFEE:TFA= 2:1, by volume), containing a certain amount of sodium dodecylbenzene- sulfonate (SDBS) as surfactant. The microtubules were grown vertically on the working electrode surface. The tubular morphology has been confirmed by scanning and transmission electron microscopies and the chain structure of the skin of the tubules has been characterized by Raman spectroscopy. The electrode property, monomer/surfactant molar ratio and the value of applied potential have strong effects on the morphology of the microtubules.

Antihypertensive drug Valsartan promotes dendritic spine density by altering AMPA receptor trafficking

Science.gov (United States)

Sohn, Young In; Lee, Nathanael J.; Chung, Andrew; Saavedra, Juan M.; Turner, R. Scott; Pak, Daniel T. S.; Hoe, Hyang-Sook

2013-01-01

Recent studies demonstrated that the antihypertensive drug Valsartan improved spatial and episodic memory in mouse models of Alzheimer’s Disease (AD) and human subjects with hypertension. However, the molecular mechanism by which Valsartan can regulate cognitive function is still unknown. Here, we investigated the effect of Valsartan on dendritic spine formation in primary hippocampal neurons, which is correlated with learning and memory. Interestingly, we found that Valsartan promotes spinogenesis in developing and mature neurons. In addition, we found that Valsartan increases the puncta number of PSD-95 and trends toward an increase in the puncta number of synaptophysin. Moreover, Valsartan increased the cell surface levels of AMPA receptors and selectively altered the levels of spinogenesis-related proteins, including CaMKIIα and phospho-CDK5. These data suggest that Valsartan may promote spinogenesis by enhancing AMPA receptor trafficking and synaptic plasticity signaling. PMID:24012668

Lithium ameliorates sleep deprivation-induced mania-like behavior, hypothalamic-pituitary-adrenal (HPA) axis alterations, oxidative stress and elevations of cytokine concentrations in the brain and serum of mice.

Science.gov (United States)

Valvassori, Samira S; Resende, Wilson R; Dal-Pont, Gustavo; Sangaletti-Pereira, Heron; Gava, Fernanda F; Peterle, Bruna R; Carvalho, André F; Varela, Roger B; Dal-Pizzol, Felipe; Quevedo, João

2017-06-01

The goal of the present study was to investigate the effects of lithium administration on behavior, oxidative stress parameters and cytokine levels in the periphery and brain of mice subjected to an animal model of mania induced by paradoxical sleep deprivation (PSD). Male C57 mice were treated with saline or lithium for 7 days. The sleep deprivation protocol started on the 5th day during for the last 36 hours of the treatment period. Immediately after the sleep deprivation protocol, animals locomotor activity was evaluated and serum and brain samples was extracted to evaluation of corticosterone and adrenocorticotropic hormone circulating levels, oxidative stress parameters and citokynes levels. The results showed that PSD induced hyperactivity in mice, which is considered a mania-like behavior. PSD increased lipid peroxidation and oxidative damage to DNA, as well as causing alterations to antioxidant enzymes in the frontal cortex, hippocampus and serum of mice. In addition, PSD increased the levels of cytokines in the brains of mice. Treatment with lithium prevented the mania-like behavior, oxidative damage and cytokine alterations induced by PSD. Improving our understanding of oxidative damage in biomolecules, antioxidant mechanisms and the inflammatory system - alterations presented in the animal models of mania - is important in helping us to improve our knowledge concerning the pathophysiology of BD, and the mechanisms of action employed by mood stabilizers. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Heterotrimeric Kinesin II Is the Microtubule Motor Protein Responsible for Pigment Dispersion in Xenopus Melanophores

Science.gov (United States)

Tuma, M. Carolina; Zill, Andrew; Le Bot, Nathalie; Vernos, Isabelle; Gelfand, Vladimir

1998-01-01

Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720–3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles. PMID:9852150

Neutron beam applications; development of texture measuring technique using 1-dimensional PSD

Energy Technology Data Exchange (ETDEWEB)

Park, No Jin; Lee, Moon Kyu; Joung, Tae Won; Lee, In Sung [Kumoh National University of Technology, Kumi (Korea)

2002-03-01

The new developed materials have often a low crystal symmetry or/and multi-phase state. Because the diffraction patterns of those materials are very complex and some peaks are overlapped, the measured pole figures with a conventional detector (0-dimensional detector) are not sufficient to use for the texture analysis. And also the widely broaden diffraction patterns caused by sever deformation, can only measured with lots of measuring errors using 0-dimensional detector. In this study the 1-dimensional and 2-dimensional position sensitive detector(PSD) is used such pattern to analyse. With PSD the more accurate pole figures can be measured, and the texture analysis, the estimation of the properties are determined more precisely. The measurement using PSD needs special technique for the analysis of the measured pattern. In this study the measuring and analysing technique is developed and compared with the conventional detector. 11 refs., 92 figs., 21 tabs. (Author)

Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

Directory of Open Access Journals (Sweden)

Eiko Kitamura

Full Text Available Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS, an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

Science.gov (United States)

Kitamura, Eiko; Gribanova, Yekaterina E.; Farber, Debora B.

2011-01-01

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process. PMID:21738583

Maximum likelihood PSD estimation for speech enhancement in reverberant and noisy conditions

DEFF Research Database (Denmark)

Kuklasinski, Adam; Doclo, Simon; Jensen, Jesper

2016-01-01

of the estimator is in speech enhancement algorithms, such as the Multi-channel Wiener Filter (MWF) and the Minimum Variance Distortionless Response (MVDR) beamformer. We evaluate these two algorithms in a speech dereverberation task and compare the performance obtained using the proposed and a competing PSD...... estimator. Instrumental performance measures indicate an advantage of the proposed estimator over the competing one. In a speech intelligibility test all algorithms significantly improved the word intelligibility score. While the results suggest a minor advantage of using the proposed PSD estimator...

Simultaneous 3D tracking of passive tracers and microtubule bundles in an active gel

Science.gov (United States)

Fan, Yi; Breuer, Kenneth S.; Fluids Team

Kinesin-driven microtubule bundles generate a spontaneous flow in unconfined geometries. They exhibit properties of active matter, including the emergence of collective motion, reduction of apparent viscosity and consumption of local energy. Here we present results from 3D tracking of passive tracers (using Airy rings and 3D scanning) synchronized with 3D measurement of the microtubule bundles motion. This technique is applied to measure viscosity variation and collective flow in a confined geometry with particular attention paid to the self-pumping system recently reported by Wu et al. (2016). Results show that the viscosity in an equilibrium microtubule network is around half that of the isotropic unbundled microtubule solution. Cross-correlations of the active microtubule network and passive tracers define a neighborhood around microtubule bundles in which passive tracers are effectively transported. MRSEC NSF.

Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

Science.gov (United States)

Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

2010-07-13

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

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.

Heat shock gene expression and cytoskeletal alterations in mouse neuroblastoma cells

NARCIS (Netherlands)

Bergen en Henegouwen, P.M.P. van; Linnemans, W.A.M.

The cytoskeleton of neuroblastoma cells, clone Neuro 2A, is altered by two stress conditions: heat shock and arsenite treatment. Microtubules are reorganized, intermediate filaments are aggregated around the nucleus, and the number of stress fibers is reduced. Since both stress modalities induce

Potential mechanisms of resistance to microtubule inhibitors.

Science.gov (United States)

Kavallaris, Maria; Annereau, Jean-Philippe; Barret, Jean-Marc

2008-06-01

Antimitotic drugs targeting the microtubules, such as the taxanes and vinca alkaloids, are widely used in the treatment of neoplastic diseases. Development of drug resistance over time, however, limits the efficacy of these agents and poses a clinical challenge to long-term improvement of patient outcomes. Understanding the mechanism(s) of drug resistance becomes paramount to allowing for alternative, if not improved, therapeutic options that might circumvent this challenge. Vinflunine, a novel microtubule inhibitor, has shown superior preclinical antitumor activity, and displays a different pattern of resistance, compared with other agents in the vinca alkaloid class.

5-1 Beam Test of the PSD EQM at CERN

Institute of Scientific and Technical Information of China (English)

Zhou; Yong[1; Sun; Zhiyu[1; Yu; Yuhong[1; Fang; Fang[1; Zhang; Yongjie[1

2014-01-01

Dark Matter Particle Explorer (DAMPE) is a powerful space telescope for high energy cosmic rays detectionsuch as -ray, electron and heavy ions. The main motivation of DAMPE is to find the evidence of dark matterexistence, and the satellite is scheduled for launching before the end of 2015.The Plastic Scintillator Detector (PSD), which is oneof the key components of the DAMPE system, has twomajor functionalities: distinguish photons from chargedparticles by anti-coincidence and measure the charge ofheavy ions. The PSD is designed and fabricated by theinstitute of modern physics, CAS. It consists of 82 plasticscintillator strips, each of which is readout by PMTat both ends, and a double-dynode readout scheme forPMT is utilized in order to cover the large dynamicrange (from H to Ca).

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

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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.

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

Hypothesis: NDL proteins function in stress responses by regulating microtubule organization.

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Khatri, Nisha; Mudgil, Yashwanti

2015-01-01

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 N-MYC DOWNREGULATED GENE (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 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.

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

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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.

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.

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.

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

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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.

Regulation of developmental and environmental signaling by interaction between microtubules and membranes in plant cells

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Qun Zhang

2015-12-01

Full Text Available ABSTRACT Cell division and expansion require the ordered arrangement of microtubules, which are subject to spatial and temporal modifications by developmental and environmental factors. Understanding how signals translate to changes in cortical microtubule organization is of fundamental importance. A defining feature of the cortical microtubule array is its association with the plasma membrane; modules of the plasma membrane are thought to play important roles in the mediation of microtubule organization. In this review, we highlight advances in research on the regulation of cortical microtubule organization by membrane-associated and membrane-tethered proteins and lipids in response to phytohormones and stress. The transmembrane kinase receptor Rho-like guanosine triphosphatase, phospholipase D, phosphatidic acid, and phosphoinositides are discussed with a focus on their roles in microtubule organization.

CENTROSOMES AND MICROTUBULES DURING MEIOSIS IN THE MUSHROOM BOLETUS RUBINELLUS

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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

TBCD links centriologenesis, spindle microtubule dynamics, and midbody abscission in human cells.

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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.

Fission yeast cells undergo nuclear division in the absence of spindle microtubules.

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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.

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

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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

Microtubule-targeting drugs rescue axonal swellings in cortical neurons from spastin knockout mice

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Coralie Fassier

2013-01-01

Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP, a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.

PSD - Routine Maintenance Repair and Replacement

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This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Baseline Value for PSD Increment Consumption

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This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

International Nuclear Information System (INIS)

Nieznanski, Krzysztof; Podlubnaya, Zoya A.; Nieznanska, Hanna

2006-01-01

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

EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

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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.

Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells.

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

2015-10-28

Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies suggested that fisetin binds to β-tubulin with superior affinity compared to paclitaxel. Fisetin treatment of human prostate cancer cells resulted in robust up-regulation of microtubule associated proteins (MAP)-2 and -4. In addition, fisetin treated cells were enriched in α-tubulin acetylation, an indication of stabilization of microtubules. Fisetin significantly inhibited PCa cell proliferation, migration, and invasion. Nudc, a protein associated with microtubule motor dynein/dynactin complex that regulates microtubule dynamics, was inhibited with fisetin treatment. Further, fisetin treatment of a P-glycoprotein overexpressing multidrug-resistant cancer cell line NCI/ADR-RES inhibited the viability and colony formation. Our results offer in vitro proof-of-concept for fisetin as a microtubule targeting agent. We suggest that fisetin could be developed as an adjuvant for treatment of prostate and other cancer types. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

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

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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

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

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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.

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.

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

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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

Learning-induced and stathmin-dependent changes in microtubule stability are critical for memory and disrupted in ageing

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Uchida, Shusaku; Martel, Guillaume; Pavlowsky, Alice; Takizawa, Shuichi; Hevi, Charles; Watanabe, Yoshifumi; Kandel, Eric R.; Alarcon, Juan Marcos; Shumyatsky, Gleb P.

2014-01-01

Changes in the stability of microtubules regulate many biological processes, but their role in memory remains unclear. Here we show that learning causes biphasic changes in the microtubule-associated network in the hippocampus. In the early phase, stathmin is dephosphorylated, enhancing its microtubule-destabilizing activity by promoting stathmin-tubulin binding, whereas in the late phase these processes are reversed leading to an increase in microtubule/KIF5-mediated localization of the GluA2 subunit of AMPA receptors at synaptic sites. A microtubule stabilizer paclitaxel decreases or increases memory when applied at the early or late phases, respectively. Stathmin mutations disrupt changes in microtubule stability, GluA2 localization, synaptic plasticity and memory. Aged wild-type mice show impairments in stathmin levels, changes in microtubule stability, and GluA2 localization. Blocking GluA2 endocytosis rescues memory deficits in stathmin mutant and aged wild-type mice. These findings demonstrate a role for microtubules in memory in young adult and aged individuals. PMID:25007915

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

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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

Optical properties of template synthesized nanowalled ZnS microtubules

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Kumar, Rajesh; Chakarvarti, S. K.

2007-12-01

Electrodeposition is a versatile technique combining low processing cost with ambient conditions that can be used to prepare metallic, polymeric and semiconducting nano/micro structures. In the present work, track-etch membranes (TEMs) of makrofol (KG) have been used as templates for synthesis of ZnS nanowalled microtubules using electrodeposition technique. The morphology of the microtubules was characterized by scanning electron microscopy. Size effects on the band gap of tubules have also been studied by UV-visible spectrophotometer.

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

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.

Dinitroaniline herbicide resistance and the microtubule cytoskeleton.

Science.gov (United States)

Anthony; Hussey

1999-03-01

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

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

Prenatal chronic mild stress induces depression-like behavior and sex-specific changes in regional glutamate receptor expression patterns in adult rats.

Science.gov (United States)

Wang, Y; Ma, Y; Hu, J; Cheng, W; Jiang, H; Zhang, X; Li, M; Ren, J; Li, X

2015-08-20

Chronic stress during critical periods of human fetal brain development is associated with cognitive, behavioral, and mood disorders in later life. Altered glutamate receptor (GluR) expression has been implicated in the pathogenesis of stress-dependent disorders. To test whether prenatal chronic mild stress (PCMS) enhances offspring's vulnerability to stress-induced behavioral and neurobiological abnormalities and if this enhanced vulnerability is sex-dependent, we measured depression-like behavior in the forced swimming test (FST) and regional changes in GluR subunit expression in PCMS-exposed adult male and female rats. Both male and female PCMS-exposed rats exhibited stronger depression-like behavior than controls. Males and females exhibited unique regional changes in GluR expression in response to PCMS alone, FST alone (CON-FST), and PCMS with FST (PCMS-FST). In females, PCMS alone did not alter N-methyl-d-aspartate receptor (NMDAR) or metabotropic glutamate receptor (mGluR) expression, while in PCMS males, higher mGluR2/3, mGluR5, and NR1 expression levels were observed in the prefrontal cortex. In addition, PCMS altered the change in GluR expression induced by acute stress (the FST test), and this too was sex-specific. Male PCMS-FST rats expressed significantly lower mGluR5 levels in the hippocampus, lower mGluR5, NR1, postsynaptic density protein (PSD)95, and higher mGluR2/3 in the prefrontal cortex, and higher mGluR5 and PSD95 in the amygdala than male CON-FST rats. Female PCMS-FST rats expressed lower NR1 in the hippocampus, lower NR2B and PSD95 in the prefrontal cortex, lower mGluR2/3 in the amygdala, and higher PSD95 in the amygdala than female CON-FST rats. PCMS may increase the offspring's vulnerability to depression by altering sex-specific stress-induced changes in glutamatergic signaling. Copyright © 2015. Published by Elsevier Ltd.

Dictyoceratidan poisons: Defined mark on microtubule-tubulin dynamics.

Science.gov (United States)

Gnanambal K, Mary Elizabeth; Lakshmipathy, Shailaja Vommi

2016-03-01

Tubulin/microtubule assembly and disassembly is characterized as one of the chief processes during cell growth and division. Hence drugs those perturb these process are considered to be effective in killing fast multiplying cancer cells. There is a collection of natural compounds which disturb microtubule/tubulin dis/assemblage and there have been a lot of efforts concerted in the marine realm too, to surveying such killer molecules. Close to half the natural compounds shooting out from marine invertebrates are generally with no traceable definite mechanisms of action though may be tough anti-cancerous hits at nanogram levels, hence fatefully those discoveries conclude therein without a capacity of translation from laboratory to pharmacy. Astoundingly at least 50% of natural compounds which have definite mechanisms of action causing disorders in tubulin/microtubule kinetics have an isolation history from sponges belonging to the Phylum: Porifera. Poriferans have always been a wonder worker to treat cancers with a choice of, yet precise targets on cancerous tissues. There is a specific order: Dictyoceratida within this Phylum which has contributed to yielding at least 50% of effective compounds possessing this unique mechanism of action mentioned above. However, not much notice is driven to Dictyoceratidans alongside the order: Demospongiae thus dictating the need to know its select microtubule/tubulin irritants since the unearthing of avarol in the year 1974 till date. Hence this review selectively pinpoints all the compounds, noteworthy derivatives and analogs stemming from order: Dictyoceratida focusing on the past, present and future. Copyright © 2016 Elsevier Inc. All rights reserved.

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....

Centrosome structure and function is altered by chloral hydrate and diazepam during the first reproductive cell cycles in sea urchin eggs

Science.gov (United States)

Schatten, H.; Chakrabarti, A.

1998-01-01

This paper explores the mode of action of the tranquillizers chloral hydrate and diazepam during fertilization and mitosis of the first reproductive cell cycles in sea urchin eggs. Most striking effects of these drugs are the alteration of centrosomal material and the abnormal microtubule configurations during exposure and after recovery from the drugs. This finding is utilized to study the mechanisms of centrosome compaction and decompaction and the dynamic configurational changes of centrosomal material and its interactions with microtubules. When 0.1% chloral hydrate or 350-750 microM diazepam is applied at specific phases during the first cell cycle of sea urchin eggs, expanded centrosomal material compacts at distinct regions and super-compacts into dense spheres while microtubules disassemble. When eggs are treated before pronuclear fusion, centrosomal material aggregates around each of the two pronuclei while microtubules disappear. Upon recovery, atypical asters oftentimes with multiple foci are formed from centrosomal material surrounding the pronuclei which indicates that the drugs have affected centrosomal material and prevent it from functioning normally. Electron microscopy and immunofluorescence studies with antibodies that routinely stain centrosomes in sea urchin eggs (4D2; and Ah-6) depict centrosomal material that is altered when compared to control cells. This centrosomal material is not able to reform normal microtubule patterns upon recovery but will form multiple asters around the two pronuclei. When cells are treated with 0.1% chloral hydrate or 350-750 microM diazepam during mitosis, the bipolar centrosomal material becomes compacted and aggregates into multiple dense spheres while spindle and polar microtubules disassemble. With increased incubation time, the smaller dense centrosome particles aggregate into bigger and fewer spheres. Upon recovery, unusual irregular microtubule configurations are formed from centrosomes that have lost their

ATPase Cycle of the Nonmotile Kinesin NOD Allows Microtubule End Tracking and Drives Chromosome Movement

Energy Technology Data Exchange (ETDEWEB)

Cochran, J.; Sindelar, C; Mulko, N; Collins, K; Kong, S; Hawley, R; Kull, F

2009-01-01

Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile kinesin-10. We have determined the X-ray crystal structure of the NOD catalytic domain in the ADP- and AMPPNP-bound states. These structures reveal an alternate conformation of the microtubule binding region as well as a nucleotide-sensitive relay of hydrogen bonds at the active site. Additionally, a cryo-electron microscopy reconstruction of the nucleotide-free microtubule-NOD complex shows an atypical binding orientation. Thermodynamic studies show that NOD binds tightly to microtubules in the nucleotide-free state, yet other nucleotide states, including AMPPNP, are weakened. Our pre-steady-state kinetic analysis demonstrates that NOD interaction with microtubules occurs slowly with weak activation of ADP product release. Upon rapid substrate binding, NOD detaches from the microtubule prior to the rate-limiting step of ATP hydrolysis, which is also atypical for a kinesin. We propose a model for NOD's microtubule plus-end tracking that drives chromosome movement.

The dynamic interplay of plasma membrane domains and cortical microtubules in secondary cell wall patterning

Directory of Open Access Journals (Sweden)

Yoshihisa eOda

2013-12-01

Full Text Available Patterning of the cellulosic cell wall underlies the shape and function of plant cells. The cortical microtubule array plays a central role in the regulation of cell wall patterns. However, the regulatory mechanisms by which secondary cell wall patterns are established through cortical microtubules remain to be fully determined. Our recent study in xylem vessel cells revealed that a mutual inhibitory interaction between cortical microtubules and distinct plasma membrane domains leads to distinctive patterning in secondary cell walls. Our research revealed that the recycling of active and inactive ROP proteins by a specific GAP and GEF pair establishes distinct de novo plasma membrane domains. Active ROP recruits a plant-specific microtubule-associated protein, MIDD1, which mediates the mutual interaction between cortical microtubules and plasma membrane domains. In this mini review, we summarize recent research regarding secondary wall patterning, with a focus on the emerging interplay between plasma membrane domains and cortical microtubules through MIDD1 and ROP.

The engine of microtubule dynamics comes into focus.

Science.gov (United States)

Mitchison, T J

2014-05-22

In this issue, Alushin et al. report high-resolution structures of three states of the microtubule lattice: GTP-bound, which is stable to depolymerization; unstable GDP-bound; and stable Taxol and GDP-bound. By comparing these structures at near-atomic resolution, they are able to propose a detailed model for how GTP hydrolysis destabilizes the microtubule and thus powers dynamic instability and chromosome movement. Destabilization of cytoskeleton filaments by nucleotide hydrolysis is an important general principle in cell dynamics, and this work represents a major step forward on a problem with a long history. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

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.

Stabilization, not polymerization, of microtubules inhibits the nuclear translocation of STATs in adipocytes

International Nuclear Information System (INIS)

Gleason, Evanna L.; Hogan, Jessica C.; Stephens, Jacqueline M.

2004-01-01

Signal transducers and activators of transcriptions (STATs) are a family of latent transcription factors which are activated by a variety of growth factors and cytokines in many cell types. However, the mechanism by which these transcription factors translocate to the nucleus is poorly understood. The goal of this study was to determine the requirement of microfilaments and microtubules for cytokine induced STAT activation in cultured adipocytes. We used seven different actin-specific and microtubule-specific agents that are well-established effectors of these cytoskeletal networks. Our results clearly demonstrate that inhibition of microfilaments or the prevention of microtubule polymerization has no effect on the ability of STATs to be tyrosine phosphorylated or to translocate to the nucleus. However, we observed that paclitaxel, a microtubule stabilizer, resulted in a significant decrease in the nuclear translocation of STATs without affecting the cytosolic tyrosine phosphorylation of these transcription factors. In summary, our results demonstrate that the dynamic instability, but not the polymerization, of microtubules contributes to nuclear translocation of STAT proteins in adipocytes

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.

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 this complex polymerization-depolymerization cycle. The model reproduces well the qualitatively different time series that result from different experimental conditions, and illuminates the role and importance of individual processes in the cycle. Simple experiments are suggested that can further test...... and define the model and the polymer's reaction cycle....

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.

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

Directory of Open Access Journals (Sweden)

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.

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

Gravity resistance, another graviresponse in plants - role of microtubule-membrane-cell wall continuum

Science.gov (United States)

Hoson, T.; Saito, Y.; Usui, S.; Soga, K.; Wakabayashi, K.

Resistance to the gravitational force has been a serious problem for plants to survive on land, after they first went ashore more than 400 million years ago. Thus, gravity resistance is the principal graviresponse in plants comparable to gravitropism. Nevertheless, only limited information has been obtained for this second gravity response. We have examined the mechanism of gravity resistance using hypergravity conditions produced by centrifugation. The results led a hypothesis on the mechanism of plant resistance to the gravitational force that the plant constructs a tough body by increasing the cell wall rigidity, which are brought about by modification of the cell wall metabolism and cell wall environment, especially pH. The hypothesis was further supported by space experiments during the Space Shuttle STS-95 mission. On the other hand, we have shown that gravity signal may be perceived by mechanoreceptors (mechanosensitive ion channels) on the plasma membrane and amyloplast sedimentation in statocytes is not involved in gravity resistance. Moreover, hypergravity treatment increased the expression levels of genes encoding alpha-tubulin, a component of microtubules and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols. The expression of HMGR and alpha- and beta-tubulin genes increased within several hours after hypergravity treatment, depending on the magnitude of gravity. The determination of levels of gene products as well as the analysis with knockout mutants of these genes by T-DNA insertions in Arabidopsis supports the involvement of both membrane sterols and microtubules in gravity resistance. These results suggest that structural or physiological continuum of microtubule-cell membrane-cell wall is responsible for plant resistance to the gravitational force.

Moonlighting microtubule-associated proteins: regulatory functions by day and pathological functions at night.

Science.gov (United States)

Oláh, J; Tőkési, N; Lehotzky, A; Orosz, F; Ovádi, J

2013-11-01

The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeletal network. Cytoskeleton comprises fibrous protein networks of microtubules, actin, and intermediate filaments. These filamentous polymer structures are highly dynamic and undergo constant and rapid reorganization during cellular processes. The microtubular system plays a crucial role in the brain, as it is involved in an enormous number of cellular events including cell differentiation and pathological inclusion formation. These multifarious functions of microtubules can be achieved by their decoration with proteins/enzymes that exert specific effects on the dynamics and organization of the cytoskeleton and mediate distinct functions due to their moonlighting features. This mini-review focuses on two aspects of the microtubule cytoskeleton. On the one hand, we describe the heteroassociation of tubulin/microtubules with metabolic enzymes, which in addition to their catalytic activities stabilize microtubule structures via their cross-linking functions. On the other hand, we focus on the recently identified moonlighting tubulin polymerization promoting protein, TPPP/p25. TPPP/p25 is a microtubule-associated protein and it displays distinct physiological or pathological (aberrant) functions; thus it is a prototype of Neomorphic Moonlighting Proteins. The expression of TPPP/p25 is finely controlled in the human brain; this protein is indispensable for the development of projections of oligodendrocytes that are responsible for the ensheathment of axons. The nonphysiological, higher or lower TPPP/p25 level leads to distinct CNS diseases. Mechanisms contributing to the control of microtubule stability and dynamics by metabolic enzymes and TPPP/p25 will be discussed. Copyright © 2013 Wiley Periodicals, Inc.

TONNEAU2/FASS Regulates the Geometry of Microtubule Nucleation and Cortical Array Organization in Interphase Arabidopsis Cells[C][W

Science.gov (United States)

Kirik, Angela; Ehrhardt, David W.; Kirik, Viktor

2012-01-01

Organization of microtubules into ordered arrays involves spatial and temporal regulation of microtubule nucleation. Here, we show that acentrosomal microtubule nucleation in plant cells involves a previously unknown regulatory step that determines the geometry of microtubule nucleation. Dynamic imaging of interphase cortical microtubules revealed that the ratio of branching to in-bundle microtubule nucleation on cortical microtubules is regulated by the Arabidopsis thaliana B′′ subunit of protein phosphatase 2A, which is encoded by the TONNEAU2/FASS (TON2) gene. The probability of nucleation from γ-tubulin complexes localized at the cell cortex was not affected by a loss of TON2 function, suggesting a specific role of TON2 in regulating the nucleation geometry. Both loss of TON2 function and ectopic targeting of TON2 to the plasma membrane resulted in defects in cell shape, suggesting the importance of TON2-mediated regulation of the microtubule cytoskeleton in cell morphogenesis. Loss of TON2 function also resulted in an inability for cortical arrays to reorient in response to light stimulus, suggesting an essential role for TON2 and microtubule branching nucleation in reorganization of microtubule arrays. Our data establish TON2 as a regulator of interphase microtubule nucleation and provide experimental evidence for a novel regulatory step in the process of microtubule-dependent nucleation. PMID:22395485

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

2017-01-01

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. PMID:28906251

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

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

Science.gov (United States)

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

2011-10-01

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.

GDP-to-GTP exchange on the microtubule end can contribute to the frequency of catastrophe.

Science.gov (United States)

Piedra, Felipe-Andrés; Kim, Tae; Garza, Emily S; Geyer, Elisabeth A; Burns, Alexander; Ye, Xuecheng; Rice, Luke M

2016-11-07

Microtubules are dynamic polymers of αβ-tubulin that have essential roles in chromosome segregation and organization of the cytoplasm. Catastrophe-the switch from growing to shrinking-occurs when a microtubule loses its stabilizing GTP cap. Recent evidence indicates that the nucleotide on the microtubule end controls how tightly an incoming subunit will be bound (trans-acting GTP), but most current models do not incorporate this information. We implemented trans-acting GTP into a computational model for microtubule dynamics. In simulations, growing microtubules often exposed terminal GDP-bound subunits without undergoing catastrophe. Transient GDP exposure on the growing plus end slowed elongation by reducing the number of favorable binding sites on the microtubule end. Slower elongation led to erosion of the GTP cap and an increase in the frequency of catastrophe. Allowing GDP-to-GTP exchange on terminal subunits in simulations mitigated these effects. Using mutant αβ-tubulin or modified GTP, we showed experimentally that a more readily exchangeable nucleotide led to less frequent catastrophe. Current models for microtubule dynamics do not account for GDP-to-GTP exchange on the growing microtubule end, so our findings provide a new way of thinking about the molecular events that initiate catastrophe. © 2016 Piedra 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).

Numerical simulation and analysis of fuzzy PID and PSD control methodologies as dynamic energy efficiency measures

International Nuclear Information System (INIS)

Ardehali, M.M.; Saboori, M.; Teshnelab, M.

2004-01-01

Energy efficiency enhancement is achieved by utilizing control algorithms that reduce overshoots and undershoots as well as unnecessary fluctuations in the amount of energy input to energy consuming systems during transient operation periods. It is hypothesized that application of control methodologies with characteristics that change with time and according to the system dynamics, identified as dynamic energy efficiency measures (DEEM), achieves the desired enhancement. The objective of this study is to simulate and analyze the effects of fuzzy logic based tuning of proportional integral derivative (F-PID) and proportional sum derivative (F-PSD) controllers for a heating and cooling energy system while accounting for the dynamics of the major system components. The procedure to achieve the objective includes utilization of fuzzy logic rules to determine the PID and PSD controllers gain coefficients so that the control laws for regulating the heat exchangers heating or cooling energy inputs are determined in each time step of the operation period. The performances of the F-PID and F-PSD controllers are measured by means of two cost functions that are based on quadratic forms of the energy input and deviation from a set point temperature. It is found that application of the F-PID control algorithm, as a DEEM, results in lower costs for energy input and deviation from a set point temperature by 24% and 17% as compared to a PID and 13% and 8% as compared to a PSD, respectively. It is also shown that the F-PSD performance is better than that of the F-PID controller

A computational framework for cortical microtubule dynamics in realistically shaped plant cells.

Directory of Open Access Journals (Sweden)

Bandan Chakrabortty

2018-02-01

Full Text Available 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.

A computational framework for cortical microtubule dynamics in realistically shaped plant cells

KAUST Repository

Chakrabortty, Bandan; Blilou, Ikram; Scheres, Ben; Mulder, Bela M.

2018-01-01

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.

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.

GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

Directory of Open Access Journals (Sweden)

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.

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.

PSD Permit for the Marblehead Lime Company

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Classification of Ethanol Fuel Plants under PSD

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

NARCIS (Netherlands)

Atherton, Joseph; Jiang, Kai; Stangier, Marcel M.; Luo, Yanzhang; Hua, Shasha; Houben, Klaartje; Van Hooff, Jolien J.E.; Joseph, Agnel Praveen; Scarabelli, Guido; Grant, Barry J.; Roberts, Anthony J.; Topf, Maya; Steinmetz, Michel O.; Baldus, Marc; Moores, Carolyn A.; Akhmanova, Anna

2017-01-01

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among

The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force

Science.gov (United States)

Trushko, Anastasiya; Schäffer, Erik; Howard, Jonathon

2013-01-01

The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip. PMID:23964126

Effects of ultraviolet radiation on microtubule organisation and morphogenesis in plants

Energy Technology Data Exchange (ETDEWEB)

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. 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. 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{sub 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 appeared due to stimulation of cell division in the leaf meristems. 111 refs, 5 figs, 2 tabs.

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

Science.gov (United States)

Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

2016-01-01

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

Prickle isoforms control the direction of tissue polarity by microtubule independent and dependent mechanisms

Directory of Open Access Journals (Sweden)

Katherine A. Sharp

2016-03-01

Full Text Available Planar cell polarity signaling directs the polarization of cells within the plane of many epithelia. While these tissues exhibit asymmetric localization of a set of core module proteins, in Drosophila, more than one mechanism links the direction of core module polarization to the tissue axes. One signaling system establishes a polarity bias in the parallel, apical microtubules upon which vesicles containing core proteins traffic. Swapping expression of the differentially expressed Prickle isoforms, Prickle and Spiny-legs, reverses the direction of core module polarization. Studies in the proximal wing and the anterior abdomen indicated that this results from their differential control of microtubule polarity. Prickle and Spiny-legs also control the direction of polarization in the distal wing (D-wing and the posterior abdomen (P-abd. We report here that this occurs without affecting microtubule polarity in these tissues. The direction of polarity in the D-wing is therefore likely determined by a novel mechanism independent of microtubule polarity. In the P-abd, Prickle and Spiny-legs interpret at least two directional cues through a microtubule-polarity-independent mechanism.

Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells

OpenAIRE

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

2015-01-01

Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies sugges...

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.

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.

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.

Multi-channel PSD Estimators for Speech Dereverberation

DEFF Research Database (Denmark)

Kuklasinski, Adam; Doclo, Simon; Gerkmann, Timo

2015-01-01

densities (PSDs). We first derive closed-form expressions for the mean square error (MSE) of both PSD estimators and then show that one estimator – previously used for speech dereverberation by the authors – always yields a better MSE. Only in the case of a two microphone array or for special spatial...... distributions of the interference both estimators yield the same MSE. The theoretically derived MSE values are in good agreement with numerical simulation results and with instrumental speech quality measures in a realistic speech dereverberation task for binaural hearing aids....

Internal Exposure of a Seoul Subway Passenger due to Radon Inhalation: Before and After PSD Installation

Energy Technology Data Exchange (ETDEWEB)

Shin, Ji-Yong; Kim, Eun-Hee [Seoul National University, Seoul (Korea, Republic of)

2015-05-15

Radon is the major source of public exposure to natural radiation and is also known to cause lung cancer. Platform screen doors (PSD) were installed primarily for passenger's safety purposes. Radon concentration and aerosol distribution have been changed since PSD installation. In this study, we have assessed the annual effective dose of regular subway passengers, before and after PSD installation, by employing current available data on air concentration of radon in Seoul subways with aerosol size distributions taken into account. ICRP recommends that the reference value for internal dose from radon be between 1.0 and 20.0 mSv. Korean Ministry of Environment enacted the indoor radon regulation, which requires the indoor radon level should not exceed 148 Bq/m{sup 3}. Radon concentrations in Seoul subways and annual dose estimates meet the requirements.

Internal Exposure of a Seoul Subway Passenger due to Radon Inhalation: Before and After PSD Installation

International Nuclear Information System (INIS)

Shin, Ji-Yong; Kim, Eun-Hee

2015-01-01

Radon is the major source of public exposure to natural radiation and is also known to cause lung cancer. Platform screen doors (PSD) were installed primarily for passenger's safety purposes. Radon concentration and aerosol distribution have been changed since PSD installation. In this study, we have assessed the annual effective dose of regular subway passengers, before and after PSD installation, by employing current available data on air concentration of radon in Seoul subways with aerosol size distributions taken into account. ICRP recommends that the reference value for internal dose from radon be between 1.0 and 20.0 mSv. Korean Ministry of Environment enacted the indoor radon regulation, which requires the indoor radon level should not exceed 148 Bq/m 3 . Radon concentrations in Seoul subways and annual dose estimates meet the requirements

Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

International Nuclear Information System (INIS)

Arachchige Don, Aruni S.; Dallapiazza, Robert F.; Bennin, David A.; Brake, Tiffany; Cowan, Colleen E.; Horne, Mary C.

2006-01-01

Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G 1 /S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the

Tank Farm WM-182 and WM-183 Heel Slurry Samples PSD Results

International Nuclear Information System (INIS)

Batcheller, T.A.; Huestis, G.M.

2000-01-01

Particle size distribution (PSD) analysis of INTEC Tank Farm WM-182 and WM-183 heel slurry samples were performed using a modified Horiba LA-300 PSD analyzer at the RAL facility. There were two types of testing performed: typical PSD analysis, and setting rate testing. Although the heel slurry samples were obtained from two separate vessels, the particle size distribution results were quite similar. The slurry solids were from approximately a minimum particle size of 0.5 mm to a maximum of 230 mm with about 90% of the material between 2-to-133 mm, and the cumulative 50% value at approximately 20 mm. This testing also revealed that high frequency sonication with an ultrasonic element may break-up larger particles in the WM-182 and WM-183 tank from heel slurries. This finding represents useful information regarding ultimate tank heel waste processing. Settling rate testing results were also fairly consistent with material from both vessels in that it appears that most of the mass of solids settle to an agglomerated, yet easily redispersed layer at the bottom. A dispersed and suspended material remained in the ''clear'' layer above the settled layer after about one-half an hour of settling time. This material had a statistical mode of approximately 5 mm and a maximum particle size of 30 mm

CEP295 interacts with microtubules and is required for centriole elongation.

Science.gov (United States)

Chang, Ching-Wen; Hsu, Wen-Bin; Tsai, Jhih-Jie; Tang, Chieh-Ju C; Tang, Tang K

2016-07-01

Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules. © 2016. Published by The Company of Biologists Ltd.

Challenges and opportunities in the high-resolution cryo-EM visualization of microtubules and their binding partners.

Science.gov (United States)

Nogales, Eva; Kellogg, Elizabeth H

2017-10-01

As non-crystallizable polymers, microtubules have been the target of cryo-electron microscopy (cryo-EM) studies since the technique was first established. Over the years, image processing strategies have been developed that take care of the unique, pseudo-helical symmetry of the microtubule. With recent progress in data quality and data processing, cryo-EM reconstructions are now reaching resolutions that allow the generation of atomic models of microtubules and the factors that bind them. These include cellular partners that contribute to microtubule cellular functions, or small ligands that interfere with those functions in the treatment of cancer. The stage is set to generate a family portrait for all identified microtubule interacting proteins and to use cryo-EM as a drug development tool in the targeting of tubulin. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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.

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

OpenAIRE

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

2005-01-01

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ...

Stabilization of microtubules by inorganic phosphate and its structural analogues, the fluoride complexes of aluminum and beryllium

International Nuclear Information System (INIS)

Carlier, M.F.; Didry, D.; Melki, R.; Chabre, M.; Pantaloni, D.

1988-01-01

In order to elucidate how the elementary reactions of GTP cleavage and subsequent inorganic phosphate (P/sub i/) release, which accompany microtubule assembly, regulate microtubule dynamics, the effect of P/sub i/ and of its structural analogues AlF 4 - and BeF 3 - on the stability of GDP-microtubules has been investigated. Inorganic phosphate binds to microtubules with a low affinity (K/sub D/ = 25 mM) and slows down the rate of GDP-subunit dissociation by about 2 orders of magnitude. AlF 4 - and BeF 3 - exhibit phosphate-like effects with 1000-fold higher affinity. Evidence has been obtained for direct binding of BeF 3 - to microtubules with a stoichiometry of 1 mol of BeF 3 - per mole of GDP-subunit and an equilibrium dissociation constant of 12-15 μM. AlF 4 - and P/sub i/ compete for this site. Phosphate analogues abolish oscillatory polymerization kinetics and slow down microtubule turnover at steady state. In view of these results, the authors propose that P/sub i/ and its structural analogues bind to the site of the γ-phosphate of GTP in the E site and reconstitute a GDP-P/sub i/-microtubule, from which tubulin subunits dissociate very slowly. They therefore understand that, following GTP cleavage on microtubules, P/sub i/ release in the medium is accompanied by a structural change resulting in a large destabilization of the polymer. A cap of slowly dissociating GDP-P/sub i/-subunits prevents depolymerization of the microtubule GDP-core at steady state. The similarity with the actin system is studied

Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis.

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Belteton, Samuel A; Sawchuk, Megan G; Donohoe, Bryon S; Scarpella, Enrico; Szymanski, Daniel B

2018-01-01

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 ( Arabidopsis thaliana ) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived 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. © 2018 American Society of Plant Biologists. All Rights Reserved.

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

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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.

Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing.

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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.

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 ...

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

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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.

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

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Eun-Mi Hur

2014-12-01

Full Text Available 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.

Lateral motion and bending of microtubules studied with a new single-filament tracking routine in living cells.

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Pallavicini, Carla; Levi, Valeria; Wetzler, Diana E; Angiolini, Juan F; Benseñor, Lorena; Despósito, Marcelo A; Bruno, Luciana

2014-06-17

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells

MVL-PLA2, a snake venom phospholipase A2, inhibits angiogenesis through an increase in microtubule dynamics and disorganization of focal adhesions.

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Amine Bazaa

Full Text Available Integrins are essential protagonists of the complex multi-step process of angiogenesis that has now become a major target for the development of anticancer therapies. We recently reported and characterized that MVL-PLA2, a novel phospholipase A2 from Macrovipera lebetina venom, exhibited anti-integrin activity. In this study, we show that MVL-PLA2 also displays potent anti-angiogenic properties. This phospholipase A2 inhibited adhesion and migration of human microvascular-endothelial cells (HMEC-1 in a dose-dependent manner without being cytotoxic. Using Matrigel and chick chorioallantoic membrane assays, we demonstrated that MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both in vitro and in vivo. We have also found that the actin cytoskeleton and the distribution of alphav beta3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. In order to further investigate the mechanism of action of this protein on endothelial cells, we analyzed the dynamic instability behavior of microtubules in living endothelial cells. Interestingly, we showed that MVL-PLA2 significantly increased microtubule dynamicity in HMEC-1 cells by 40%. We propose that the enhancement of microtubule dynamics may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration.

Calmodulin immunolocalization to cortical microtubules is calcium independent

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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.

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.

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.

An agent-based model contrasts opposite effects of dynamic and stable microtubules on cleavage furrow positioning.

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Odell, Garrett M; Foe, Victoria E

2008-11-03

From experiments by Foe and von Dassow (Foe, V.E., and G. von Dassow. 2008. J. Cell Biol. 183:457-470) and others, we infer a molecular mechanism for positioning the cleavage furrow during cytokinesis. Computer simulations reveal how this mechanism depends on quantitative motor-behavior details and explore how robustly this mechanism succeeds across a range of cell sizes. The mechanism involves the MKLP1 (kinesin-6) component of centralspindlin binding to and walking along microtubules to stimulate cortical contractility where the centralspindlin complex concentrates. The majority of astral microtubules are dynamically unstable. They bind most MKLP1 and suppress cortical Rho/myosin II activation because the tips of unstable microtubules usually depolymerize before MKLP1s reach the cortex. A subset of astral microtubules stabilizes during anaphase, becoming effective rails along which MKLP1 can actually reach the cortex. Because stabilized microtubules aim statistically at the equatorial spindle midplane, that is where centralspindlin accumulates to stimulate furrow formation.

Pyk2 modulates hippocampal excitatory synapses and contributes to cognitive deficits in a Huntington’s disease model

KAUST Repository

Giralt, Albert; Brito, Veronica; Chevy, Quentin; Simonnet, Clé mence; Otsu, Yo; Cifuentes-Dí az, Carmen; Pins, Benoit de; Coura, Renata; Alberch, Jordi; Giné s, Sí lvia; Poncer, Jean-Christophe; Girault, Jean-Antoine

2017-01-01

The structure and function of spines and excitatory synapses are under the dynamic control of multiple signalling networks. Although tyrosine phosphorylation is involved, its regulation and importance are not well understood. Here we study the role of Pyk2, a non-receptor calcium-dependent protein-tyrosine kinase highly expressed in the hippocampus. Hippocampal-related learning and CA1 long-term potentiation are severely impaired in Pyk2-deficient mice and are associated with alterations in NMDA receptors, PSD-95 and dendritic spines. In cultured hippocampal neurons, Pyk2 has autophosphorylation-dependent and -independent roles in determining PSD-95 enrichment and spines density. Pyk2 levels are decreased in the hippocampus of individuals with Huntington and in the R6/1 mouse model of the disease. Normalizing Pyk2 levels in the hippocampus of R6/1 mice rescues memory deficits, spines pathology and PSD-95 localization. Our results reveal a role for Pyk2 in spine structure and synaptic function, and suggest that its deficit contributes to Huntington’s disease cognitive impairments.

Pyk2 modulates hippocampal excitatory synapses and contributes to cognitive deficits in a Huntington’s disease model

KAUST Repository

Giralt, Albert

2017-05-30

The structure and function of spines and excitatory synapses are under the dynamic control of multiple signalling networks. Although tyrosine phosphorylation is involved, its regulation and importance are not well understood. Here we study the role of Pyk2, a non-receptor calcium-dependent protein-tyrosine kinase highly expressed in the hippocampus. Hippocampal-related learning and CA1 long-term potentiation are severely impaired in Pyk2-deficient mice and are associated with alterations in NMDA receptors, PSD-95 and dendritic spines. In cultured hippocampal neurons, Pyk2 has autophosphorylation-dependent and -independent roles in determining PSD-95 enrichment and spines density. Pyk2 levels are decreased in the hippocampus of individuals with Huntington and in the R6/1 mouse model of the disease. Normalizing Pyk2 levels in the hippocampus of R6/1 mice rescues memory deficits, spines pathology and PSD-95 localization. Our results reveal a role for Pyk2 in spine structure and synaptic function, and suggest that its deficit contributes to Huntington’s disease cognitive impairments.

Coupling of kinesin ATP turnover to translocation and microtubule regulation: one engine, many machines.

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Friel, Claire T; Howard, Jonathon

2012-12-01

The cycle of ATP turnover is integral to the action of motor proteins. Here we discuss how variation in this cycle leads to variation of function observed amongst members of the kinesin superfamily of microtubule associated motor proteins. Variation in the ATP turnover cycle among superfamily members can tune the characteristic kinesin motor to one of the range of microtubule-based functions performed by kinesins. The speed at which ATP is hydrolysed affects the speed of translocation. The ratio of rate constants of ATP turnover in relation to association and dissociation from the microtubule influence the processivity of translocation. Variation in the rate-limiting step of the cycle can reverse the way in which the motor domain interacts with the microtubule producing non-motile kinesins. Because the ATP turnover cycle is not fully understood for the majority of kinesins, much work remains to show how the kinesin engine functions in such a wide variety of molecular machines.

GIT1 enhances neurite outgrowth by stimulating microtubule assembly

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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.

Caffeine and modafinil given during 48 h sleep deprivation modulate object recognition memory and synaptic proteins in the hippocampus of the rat.

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Wadhwa, M; Sahu, S; Kumari, P; Kauser, H; Ray, K; Panjwani, U

2015-11-01

We aimed to evaluate the effect of caffeine/modafinil on sleep deprivation (SD) induced alterations in recognition memory and synaptic proteins. The data revealed a beneficial effect of caffeine/modafinil against deficit in the familiar object retrieval performance and object exploration ratio after 48 h SD. Caffeine treatment prevented the SD induced down-regulation of synaptophysin and synapsin I proteins with no change in PSD-95 protein in hippocampus. However, modafinil administration improved the down-regulation of synaptophysin, synapsin I and PSD-95 proteins in hippocampus. Hence, caffeine/modafinil can serve as counter measures in amelioration of SD induced consequences at behavioural and protein levels. Copyright © 2015 Elsevier B.V. All rights reserved.

Influence of carbon nanotubes on the buckling of microtubule bundles in viscoelastic cytoplasm using nonlocal strain gradient theory

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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

Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study

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Zhu, Jie; Burakov, Anton; Rodionov, Vladimir

2010-01-01

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces—dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles—is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility. PMID:20980619

Three-dimensional fine structure of the organization of microtubules in neurite varicosities by ultra-high voltage electron microscope tomography.

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Nishida, Tomoki; Yoshimura, Ryoichi; Endo, Yasuhisa

2017-09-01

Neurite varicosities are highly specialized compartments that are involved in neurotransmitter/ neuromodulator release and provide a physiological platform for neural functions. However, it remains unclear how microtubule organization contributes to the form of varicosity. Here, we examine the three-dimensional structure of microtubules in varicosities of a differentiated PC12 neural cell line using ultra-high voltage electron microscope tomography. Three-dimensional imaging showed that a part of the varicosities contained an accumulation of organelles that were separated from parallel microtubule arrays. Further detailed analysis using serial sections and whole-mount tomography revealed microtubules running in a spindle shape of swelling in some other types of varicosities. These electron tomographic results showed that the structural diversity and heterogeneity of microtubule organization supported the form of varicosities, suggesting that a different distribution pattern of microtubules in varicosities is crucial to the regulation of varicosities development.

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.

The Altered Hepatic Tubulin Code in Alcoholic Liver Disease.

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Groebner, Jennifer L; Tuma, Pamela L

2015-09-18

The molecular mechanisms that lead to the progression of alcoholic liver disease have been actively examined for decades. Because the hepatic microtubule cytoskeleton supports innumerable cellular processes, it has been the focus of many such mechanistic studies. It has long been appreciated that α-tubulin is a major target for modification by highly reactive ethanol metabolites and reactive oxygen species. It is also now apparent that alcohol exposure induces post-translational modifications that are part of the natural repertoire, mainly acetylation. In this review, the modifications of the "tubulin code" are described as well as those adducts by ethanol metabolites. The potential cellular consequences of microtubule modification are described with a focus on alcohol-induced defects in protein trafficking and enhanced steatosis. Possible mechanisms that can explain hepatic dysfunction are described and how this relates to the onset of liver injury is discussed. Finally, we propose that agents that alter the cellular acetylation state may represent a novel therapeutic strategy for treating liver disease.

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.

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.

SDF1 Reduces Interneuron Leading Process Branching through Dual Regulation of Actin and Microtubules

Science.gov (United States)

Lysko, Daniel E.; Putt, Mary

2014-01-01

Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process. PMID:24695713

SDF1 reduces interneuron leading process branching through dual regulation of actin and microtubules.

Science.gov (United States)

Lysko, Daniel E; Putt, Mary; Golden, Jeffrey A

2014-04-02

Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process.

Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

Directory of Open Access Journals (Sweden)

Jing Wang

2014-10-01

Full Text Available AIM:To investigate the morphological altering effect of transforming growth factor-β2 (TGF-β2 on untransfected human corneal endothelial cells (HCECs in vitro.METHODS: After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology, cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy, immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2 (9 μg/L altered HCE cell morphology after treatment for 36h, increased the mean optical density (P<0.01 and the length of F-actin, reduced the mean optical density (P<0.01 of the collagen type IV in extracellular matrix (ECM and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72h. CONCLUTION:TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

A comparison of different discrimination parameters for the DFT-based PSD method in fast scintillators

International Nuclear Information System (INIS)

Liu, G.; Yang, J.; Luo, X.L.; Lin, C.B.; Peng, J.X.; Yang, Y.

2013-01-01

Although the discrete Fourier transform (DFT) based pulse shape discrimination (PSD) method, realized by transforming the digitized scintillation pulses into frequency coefficients by using DFT, has been proven to effectively discriminate neutrons and γ rays, its discrimination performance depends strongly on the selection of the discrimination parameter obtained by the combination of these frequency coefficients. In order to thoroughly understand and apply the DFT-based PSD in organic scintillation detectors, a comparison of three different discrimination parameters, i.e. the amplitude of zero-frequency component, the amplitude difference between the amplitude of zero-frequency component and the amplitude of base-frequency component, and the ratio of the amplitude of base-frequency component to the amplitude of zero-frequency component, is described in this paper. An experimental setup consisting of an Americium–Beryllium (Am–Be) source, a BC501A liquid scintillator detector, and a 5Gsample/s 8-bit oscilloscope was built to assess the performance of the DFT-based PSD with each of these discrimination parameters in terms of the figure-of-merit (based on the separation of the event distributions). The third technique, which uses the ratio of the amplitude of base-frequency component to the amplitude of zero-frequency component as the discrimination parameter, is observed to provide the best discrimination performance in this research. - Highlights: • The spectrum difference between neutron pulse and γ-ray pulse was investigated. • The DFT-based PSD with different parameter definitions was assessed. • The way of using the ratio of magnitude spectrum provides the best performance. • The performance differences were explained from noise suppression features

Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups.

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Stefania Piersanti

Full Text Available Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD canine adenovirus type 2 vectors (CAV-2 are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.

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

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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.

Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis1[OPEN

Science.gov (United States)

Sawchuk, Megan G.; Scarpella, Enrico

2018-01-01

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 (Arabidopsis thaliana) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived 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. PMID:29192026

Microtubule dynamics of the centrosome-like polar organizers from the basal land plant Marchantia polymorpha.

Science.gov (United States)

Buschmann, Henrik; Holtmannspötter, Michael; Borchers, Agnes; O'Donoghue, Martin-Timothy; Zachgo, Sabine

2016-02-01

The liverwort Marchantia employs both modern and ancestral devices during cell division: it forms preprophase bands and in addition it shows centrosome-like polar organizers. We investigated whether polar organizers and preprophase bands cooperate to set up the division plane. To this end, two novel green fluorescent protein-based microtubule markers for dividing cells of Marchantia were developed. Cells of the apical notch formed polar organizers first and subsequently assembled preprophase bands. Polar organizers were formed de novo from multiple mobile microtubule foci localizing to the nuclear envelope. The foci then became concentrated by bipolar aggregation. We determined the comet production rate of polar organizers and show that microtubule plus ends of astral microtubules polymerize faster than those found on cortical microtubules. Importantly, it was observed that conditions increasing polar organizer numbers interfere with preprophase band formation. The data show that polar organizers have much in common with centrosomes, but that they also have specialized features. The results suggest that polar organizers contribute to preprophase band formation and in this way are involved in controlling the division plane. Our analyses of the basal land plant Marchantia shed new light on the evolution of plant cell division. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Cationic membranes complexed with oppositely charged microtubules: hierarchical self-assembly leading to bio-nanotubes

International Nuclear Information System (INIS)

Raviv, Uri; Needleman, Daniel J; Safinya, Cyrus R

2006-01-01

The self-assembly of microtubules and charged membranes has been studied, using x-ray diffraction and electron microscopy. Polyelectrolyte lipid complexes usually form structures templated by the lipid phase, when the polyelectrolyte curvature is much larger than the membrane spontaneous curvature. When the polyelectrolyte curvature approaches the membrane spontaneous curvature, as in microtubules, two types of new structures emerge. Depending on the conditions, vesicles either adsorb onto the microtubule, forming a 'beads on a rod' structure, or coat the microtubule, which now forms the template. Tubulin oligomers then coat the external lipid layer, forming a lipid protein nanotube. The tubulin oligomer coverage at the external layer is determined by the membrane charge density. The energy barrier between the beads on a rod and the lipid-protein nanotube states depends on the membrane bending rigidity and membrane charge density. By controlling the lipid/tubulin stoichiometry we can switch between lipid-protein nanotubes with open ends to lipid-protein nanotubes with closed end with lipid cups. This forms the basis for controlled drug encapsulation and release

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.

Indicine N-oxide binds to tubulin at a distinct site and inhibits the assembly of microtubules: a mechanism for its cytotoxic activity.

Science.gov (United States)

Appadurai, Prakash; Rathinasamy, Krishnan

2014-02-10

Indicine N-oxide, a pyrrolizidine alkaloid present in the plant Heliotropium indicum had shown promising cytotoxic activity in various tumor models. The compound exhibited severe toxicity to hepatocytes and bone marrow cells. The present work was aimed to evaluate the molecular mechanism of the toxicity of indicine N-oxide. We found that indicine N-oxide inhibited the proliferation of various cancer cell lines in a concentration dependent manner with IC50 ranging from 46 to 100 μM. At the half maximal inhibitory concentration it blocked the cell cycle progression at mitosis without significantly altering the organization of the spindle and interphase microtubules. The toxicities of the compound at higher concentrations are attributed to its severe depolymerizing effect on both the interphase and spindle microtubules. Binding studies using purified goat brain tubulin indicated that indicine N-oxide binds to tubulin at a distinct site not shared by colchicine or taxol. It decreased the polymer mass of both purified tubulin and MAP-rich tubulin. It was found to induce cleavage of DNA using pUC18 plasmid. The interactions of indicine N-oxide on DNA were also confirmed by computational analysis; which predicted its binding site at the minor groove of DNA. These studies bring to light that the toxicities of indicine N-oxide were due to its DNA damaging effects and depolymerization of microtubules. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

Microtubules can regulate GPCR (G protein-coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the β-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance...... 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...

ZiBuPiYin recipe protects db/db mice from diabetes-associated cognitive decline through improving multiple pathological changes.

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Jing Chen

Full Text Available Multiple organ systems, including the brain, which undergoes changes that may increase the risk of cognitive decline, are adversely affected by diabetes mellitus (DM. Here, we demonstrate that type 2 diabetes mellitus (T2DM db/db mice exhibited hippocampus-dependent memory impairment, which might associate with a reduction in dendritic spine density in the pyramidal neurons of brain, Aβ1-42 deposition in the prefrontal cortex (PFC and hippocampus, and a decreased expression of neurostructural proteins including microtubule-associated protein (MAP2, a marker of dendrites, and postsynaptic density 95 (PSD95, a marker of excitatory synapses. To investigate the effects of the ZiBuPiYin recipe (ZBPYR, a traditional Chinese medicine recipe, on diabetes-related cognitive decline (DACD, db/db mice received daily administration of ZBPYR over an experimental period of 6 weeks. We then confirmed that ZBPYR rescued learning and memory performance impairments, reversed dendritic spine loss, reduced Aβ1-42 deposition and restored the expression levels of MAP2 and PSD95. The present study also revealed that ZBPYR strengthened brain leptin and insulin signaling and inhibited GSK3β overactivity, which may be the potential mechanism or underlying targets of ZBPYR. These findings conclude that ZBPYR prevents DACD, most likely by improving dendritic spine density and attenuating brain leptin and insulin signaling pathway injury. Our findings provide further evidence for the effects of ZBPYR on DACD.

Role of proBDNF and BDNF in dendritic spine plasticity and depressive-like behaviors induced by an animal model of depression.

Science.gov (United States)

Qiao, Hui; An, Shu-Cheng; Xu, Chang; Ma, Xin-Ming

2017-05-15

Major depressive disorder (MDD) is one of the most common psychiatric disorder, but the underlying mechanisms are largely unknown. Increasing evidence shows that brain-derived neurotrophic factor (BDNF) plays an important role in the structural plasticity induced by depression. Considering the opposite effects of BDNF and its precursor proBDNF on neural plasticity, we hypothesized that the balance of BDNF and proBDNF plays a critical role in chronic unpredicted mild stress (CUMS)-induced depressive-like behaviors and structural plasticity in the rodent hippocampus. The aims of this study were to compare the functions of BDNF and proBDNF in the CUMS-induced depressive-like behaviors, and determine the effects of BDNF and proBDNF on expressions of kalirin-7, postsynaptic density protein 95 (PSD95) and NMDA receptor subunit NR2B in the hippocampus of stressed and naïve control rats, respectively. Our results showed that CUMS induced depressive-like behaviors, caused a decrease in the ratio of BDNF/proBDNF in the hippocampus and resulted in a reduction in spine density in hippocampal CA1 pyramidal neurons; these alterations were accompanied by a decrease in the levels of kalirin-7, PSD95 and NR2B in the hippocampus. Injection of exogenous BDNF into the CA1 area of stressed rats reversed CUMS-induced depressive-like behaviors and prevented CUMS-induced spine loss and decrease in kalirin-7, NR2B and PSD95 levels. In contrast, injection of exogenous proBDNF into the CA1 region of naïve rats caused depressive-like behavior and an accompanying decrease in both spine density and the levels of kalirin-7, NR2B and PSD95. Taken together, our results suggest that the ratio of BDNF to proBDNF in the hippocampus plays a key role in CUMS-induced depressive-like behaviors and alterations of dendritic spines in hippocampal CA1 pyramidal neurons. Kalirin-7 may play an important role during this process. Copyright © 2017 Elsevier B.V. All rights reserved.

Microtubule self-organisation by reaction-diffusion processes causes collective transport and organisation of cellular particles

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Demongeot Jacques

2004-06-01

Full Text Available Abstract Background The transport of intra-cellular particles by microtubules is a major biological function. Under appropriate in vitro conditions, microtubule preparations behave as a 'complex' system and show 'emergent' phenomena. In particular, they form dissipative structures that self-organise over macroscopic distances by a combination of reaction and diffusion. Results Here, we show that self-organisation also gives rise to a collective transport of colloidal particles along a specific direction. Particles, such as polystyrene beads, chromosomes, nuclei, and vesicles are carried at speeds of several microns per minute. The process also results in the macroscopic self-organisation of these particles. After self-organisation is completed, they show the same pattern of organisation as the microtubules. Numerical simulations of a population of growing and shrinking microtubules, incorporating experimentally realistic reaction dynamics, predict self-organisation. They forecast that during self-organisation, macroscopic parallel arrays of oriented microtubules form which cross the reaction space in successive waves. Such travelling waves are capable of transporting colloidal particles. The fact that in the simulations, the aligned arrays move along the same direction and at the same speed as the particles move, suggest that this process forms the underlying mechanism for the observed transport properties. Conclusions This process constitutes a novel physical chemical mechanism by which chemical energy is converted into collective transport of colloidal particles along a given direction. Self-organisation of this type provides a new mechanism by which intra cellular particles such as chromosomes and vesicles can be displaced and simultaneously organised by microtubules. It is plausible that processes of this type occur in vivo.

Microtubule self-organisation by reaction-diffusion processes causes collective transport and organisation of cellular particles

Science.gov (United States)

Glade, Nicolas; Demongeot, Jacques; Tabony, James

2004-01-01

Background The transport of intra-cellular particles by microtubules is a major biological function. Under appropriate in vitro conditions, microtubule preparations behave as a 'complex' system and show 'emergent' phenomena. In particular, they form dissipative structures that self-organise over macroscopic distances by a combination of reaction and diffusion. Results Here, we show that self-organisation also gives rise to a collective transport of colloidal particles along a specific direction. Particles, such as polystyrene beads, chromosomes, nuclei, and vesicles are carried at speeds of several microns per minute. The process also results in the macroscopic self-organisation of these particles. After self-organisation is completed, they show the same pattern of organisation as the microtubules. Numerical simulations of a population of growing and shrinking microtubules, incorporating experimentally realistic reaction dynamics, predict self-organisation. They forecast that during self-organisation, macroscopic parallel arrays of oriented microtubules form which cross the reaction space in successive waves. Such travelling waves are capable of transporting colloidal particles. The fact that in the simulations, the aligned arrays move along the same direction and at the same speed as the particles move, suggest that this process forms the underlying mechanism for the observed transport properties. Conclusions This process constitutes a novel physical chemical mechanism by which chemical energy is converted into collective transport of colloidal particles along a given direction. Self-organisation of this type provides a new mechanism by which intra cellular particles such as chromosomes and vesicles can be displaced and simultaneously organised by microtubules. It is plausible that processes of this type occur in vivo. PMID:15176973

Microtubule organization in three-dimensional confined geometries: Evaluating the role of elasticity through a combined in vitro and modeling approach

NARCIS (Netherlands)

Cosentino Lagomarsino, M.; Tanase, C.; Vos, J.W.; Emons, A.M.C.; Mulder, B.; Dogterom, M.

2007-01-01

Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a

Does usnic acid affect microtubules in human cancer cells? O ácido úsnico pode afetar microtúbulos em células cancerosas humanas?

Directory of Open Access Journals (Sweden)

MA. O'Neill

2010-08-01

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.O ácido úsnico, um metabólito de liquens, é conhecido por sua atividade antimitótica e antiproliferativa em células humanas normais e malignas. Muitos quimioterápicos exercem suas atividades bloqueando a progressão do ciclo celular e induzindo morte celular por apoptose. Os microtúbulos, estruturas protéicas envolvidas na segregação dos cromossomos durante a mitose, servem como alvo quimioterapêutico devido ao seu importante papel tanto na divisão celular quanto nos mecanismos de morte celular por apoptose. O objetivo deste trabalho foi investigar se o ácido úsnico afeta a formação e/ou estabilização dos microtúbulos, a partir da visualização de microtúbulos e determinação de índices mitóticos após o tratamento. Células de câncer de mama MCF7 e de c

Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules

International Nuclear Information System (INIS)

Mithieux, G.; Rousset, B.

1989-01-01

We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes, a process which is inhibited by ATP. Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells

Capu and Spire Assemble a Cytoplasmic Actin~Mesh that Maintains Microtubule Organization in the Drosophila Oocyte

DEFF Research Database (Denmark)

Dahlgaard, K.; Raposo, A.A.S.F.; Niccoli, T.

2007-01-01

Mutants in the actin nucleators Cappuccino and Spire disrupt the polarized microtubule network in the Drosophila oocyte that defines the anterior-posterior axis, suggesting that microtubule organization depends on actin. Here, we show that Cappuccino and Spire organize an isotropic mesh of actin...

National Asphalt Pavement Association Questions and Answers on PSD

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Classification of the Bardstown Fuel Alcohol Company under PSD

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Interaction of the Tobacco mosaic virus movement protein with microtubules during the cell cycle in tobacco BY-2 cells.

Science.gov (United States)

Boutant, Emmanuel; Fitterer, Chantal; Ritzenthaler, Christophe; Heinlein, Manfred

2009-10-01

Cell-to-cell movement of Tobacco mosaic virus (TMV) involves the interaction of virus-encoded 30-kDa movement protein (MP) with microtubules. In cells behind the infection front that accumulate high levels of MP, this activity is reflected by the formation of stabilized MP/microtubule complexes. The ability of MP to bind along and stabilize microtubules is conserved upon expression in mammalian cells. In mammalian cells, the protein also leads to inhibition of mitosis and cell division through a microtubule-independent process correlated with the loss of centrosomal gamma-tubulin and of centrosomal microtubule-nucleation activity. Since MP has the capacity to interact with plant factors involved in microtubule nucleation and dynamics, we used inducible expression in BY-2 cells to test whether MP expression inhibits mitosis and cell division also in plants. We demonstrate that MP:GFP associates with all plant microtubule arrays and, unlike in mammalian cells, does not interfere with mitosis. Thus, MP function and the interaction of MP with factors of the cytoskeleton do not entail an inhibition of mitosis in plants. We also report that the protein targets primary plasmodesmata in BY-2 cells immediately upon or during cytokinesis and that the accumulation of MP in plasmodesmata occurs in the presence of inhibitors of the cytoskeleton and the secretory pathway.

Centromere Protein (CENP)-W Interacts with Heterogeneous Nuclear Ribonucleoprotein (hnRNP) U and May Contribute to Kinetochore-Microtubule Attachment in Mitotic Cells

Science.gov (United States)

Chun, Younghwa; Kim, Raehyung; Lee, Soojin

2016-01-01

Background Recent studies have shown that heterogeneous nuclear ribonucleoprotein U (hnRNP U), a component of the hnRNP complex, contributes to stabilize the kinetochore-microtubule interaction during mitosis. CENP-W was identified as an inner centromere component that plays crucial roles in the formation of a functional kinetochore complex. Results We report that hnRNP U interacts with CENP-W, and the interaction between hnRNP U and CENP-W mutually increased each other’s protein stability by inhibiting the proteasome-mediated degradation. Further, their co-localization was observed chiefly in the nuclear matrix region and at the microtubule-kinetochore interface during interphase and mitosis, respectively. Both microtubule-stabilizing and microtubule-destabilizing agents significantly decreased the protein stability of CENP-W. Furthermore, loss of microtubules and defects in microtubule organization were observed in CENP-W-depleted cells. Conclusion Our data imply that CENP-W plays an important role in the attachment and interaction between microtubules and kinetochore during mitosis. PMID:26881882

Phenylbutyrate ameliorates cognitive deficit and reduces tau pathology in an Alzheimer's disease mouse model.

Science.gov (United States)

Ricobaraza, Ana; Cuadrado-Tejedor, Mar; Pérez-Mediavilla, Alberto; Frechilla, Diana; Del Río, Joaquin; García-Osta, Ana

2009-06-01

Chromatin modification through histone acetylation is a molecular pathway involved in the regulation of transcription underlying memory storage. Sodium 4-phenylbutyrate (4-PBA) is a well-known histone deacetylase inhibitor, which increases gene transcription of a number of genes, and also exerts neuroprotective effects. In this study, we report that administration of 4-PBA reversed spatial learning and memory deficits in an established mouse model of Alzheimer's disease (AD) without altering beta-amyloid burden. We also observed that the phosphorylated form of tau was decreased in the AD mouse brain after 4-PBA treatment, an effect probably due to an increase in the inactive form of the glycogen synthase kinase 3beta (GSK3beta). Interestingly, we found a dramatic decrease in brain histone acetylation in the transgenic mice that may reflect an indirect transcriptional repression underlying memory impairment. The administration of 4-PBA restored brain histone acetylation levels and, as a most likely consequence, activated the transcription of synaptic plasticity markers such as the GluR1 subunit of the AMPA receptor, PSD95, and microtubule-associated protein-2. The results suggest that 4-PBA, a drug already approved for clinical use, may provide a novel approach for the treatment of AD.

and its allicin on microtubule and cancer cell lines

African Journals Online (AJOL)

STORAGESEVER

2009-10-05

Oct 5, 2009 ... microtubule protein polymer that treated by A. hirtifolium. (A), and allicin (B) in .... with a chromogenic thiol: reaction of 4-mercaptopyridine with ... transformed tumor growth in vivo by diallyl disulfide is associated with inhibition ...

Novel mitochondrial extensions provide evidence for a link between microtubule-directed movement and mitochondrial fission

International Nuclear Information System (INIS)

Bowes, Timothy; Gupta, Radhey S.

2008-01-01

Mitochondrial dynamics play an important role in a large number of cellular processes. Previously, we reported that treatment of mammalian cells with the cysteine-alkylators, N-ethylmaleimide and ethacrynic acid, induced rapid mitochondrial fusion forming a large reticulum approximately 30 min after treatment. Here, we further investigated this phenomenon using a number of techniques including live-cell confocal microscopy. In live cells, drug-induced fusion coincided with a cessation of fast mitochondrial movement which was dependent on microtubules. During this loss of movement, thin mitochondrial tubules extending from mitochondria were also observed, which we refer to as 'mitochondrial extensions'. The formation of these mitochondrial extensions, which were not observed in untreated cells, depended on microtubules and was abolished by pretreatment with nocodazole. In this study, we provide evidence that these extensions result from of a block in mitochondrial fission combined with continued application of motile force by microtubule-dependent motor complexes. Our observations strongly suggest the existence of a link between microtubule-based mitochondrial trafficking and mitochondrial fission

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

Explaining the Microtubule Energy Balance: Contributions Due to Dipole Moments, Charges, van der Waals and Solvation Energy

Directory of Open Access Journals (Sweden)

Ahmed Taha Ayoub

2017-09-01

Full Text Available Microtubules are the main components of mitotic spindles, and are the pillars of the cellular cytoskeleton. They perform most of their cellular functions by virtue of their unique dynamic instability processes which alternate between polymerization and depolymerization phases. This in turn is driven by a precise balance between attraction and repulsion forces between the constituents of microtubules (MTs—tubulin dimers. Therefore, it is critically important to know what contributions result in a balance of the interaction energy among tubulin dimers that make up microtubules and what interactions may tip this balance toward or away from a stable polymerized state of tubulin. In this paper, we calculate the dipole–dipole interaction energy between tubulin dimers in a microtubule as part of the various contributions to the energy balance. We also compare the remaining contributions to the interaction energies between tubulin dimers and establish a balance between stabilizing and destabilizing components, including the van der Waals, electrostatic, and solvent-accessible surface area energies. The energy balance shows that the GTP-capped tip of the seam at the plus end of microtubules is stabilized only by − 9 kcal/mol, which can be completely reversed by the hydrolysis of a single GTP molecule, which releases + 14 kcal/mol and destabilizes the seam by an excess of + 5 kcal/mol. This triggers the breakdown of microtubules and initiates a disassembly phase which is aptly called a catastrophe.

A Novel MAPT Mutation, G55R, in a Frontotemporal Dementia Patient Leads to Altered Tau Function

Science.gov (United States)

Guzman, Elmer; Barczak, Anna; Chodakowska-Żebrowska, Małgorzata; Barcikowska, Maria; Feinstein, Stuart

2013-01-01

Over two dozen mutations in the gene encoding the microtubule associated protein tau cause a variety of neurodegenerative dementias known as tauopathies, including frontotemporal dementia (FTD), PSP, CBD and Pick's disease. The vast majority of these mutations map to the C-terminal region of tau possessing microtubule assembly and microtubule dynamics regulatory activities as well as the ability to promote pathological tau aggregation. Here, we describe a novel and non-conservative tau mutation (G55R) mapping to an alternatively spliced exon encoding part of the N-terminal region of the protein in a patient with the behavioral variant of FTD. Although less well understood than the C-terminal region of tau, the N-terminal region can influence both MT mediated effects as well as tau aggregation. The mutation changes an uncharged glycine to a basic arginine in the midst of a highly conserved and very acidic region. In vitro, 4-repeat G55R tau nucleates microtubule assembly more effectively than wild-type 4-repeat tau; surprisingly, this effect is tau isoform specific and is not observed in a 3-repeat G55R tau versus 3-repeat wild-type tau comparison. In contrast, the G55R mutation has no effect upon the abilities of tau to regulate MT growing and shortening dynamics or to aggregate. Additionally, the mutation has no effect upon kinesin translocation in a microtubule gliding assay. Together, (i) we have identified a novel tau mutation mapping to a mutation deficient region of the protein in a bvFTD patient, and (ii) the G55R mutation affects the ability of tau to nucleate microtubule assembly in vitro in a 4-repeat tau isoform specific manner. This altered capability could markedly affect in vivo microtubule function and neuronal cell biology. We consider G55R to be a candidate mutation for bvFTD since additional criteria required to establish causality are not yet available for assessment. PMID:24086739

The Altered Hepatic Tubulin Code in Alcoholic Liver Disease

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Jennifer L. Groebner

2015-09-01

Full Text Available The molecular mechanisms that lead to the progression of alcoholic liver disease have been actively examined for decades. Because the hepatic microtubule cytoskeleton supports innumerable cellular processes, it has been the focus of many such mechanistic studies. It has long been appreciated that α-tubulin is a major target for modification by highly reactive ethanol metabolites and reactive oxygen species. It is also now apparent that alcohol exposure induces post-translational modifications that are part of the natural repertoire, mainly acetylation. In this review, the modifications of the “tubulin code” are described as well as those adducts by ethanol metabolites. The potential cellular consequences of microtubule modification are described with a focus on alcohol-induced defects in protein trafficking and enhanced steatosis. Possible mechanisms that can explain hepatic dysfunction are described and how this relates to the onset of liver injury is discussed. Finally, we propose that agents that alter the cellular acetylation state may represent a novel therapeutic strategy for treating liver disease.

The nucleoporin MEL-28 promotes RanGTP-dependent γ-tubulin recruitment and microtubule nucleation in mitotic spindle formation.

Science.gov (United States)

Yokoyama, Hideki; Koch, Birgit; Walczak, Rudolf; Ciray-Duygu, Fulya; González-Sánchez, Juan Carlos; Devos, Damien P; Mattaj, Iain W; Gruss, Oliver J

2014-01-01

The GTP-bound form of the Ran GTPase (RanGTP), produced around chromosomes, drives nuclear envelope and nuclear pore complex (NPC) re-assembly after mitosis. The nucleoporin MEL-28/ELYS binds chromatin in a RanGTP-regulated manner and acts to seed NPC assembly. Here we show that, upon mitotic NPC disassembly, MEL-28 dissociates from chromatin and re-localizes to spindle microtubules and kinetochores. MEL-28 directly binds microtubules in a RanGTP-regulated way via its C-terminal chromatin-binding domain. Using Xenopus egg extracts, we demonstrate that MEL-28 is essential for RanGTP-dependent microtubule nucleation and spindle assembly, independent of its function in NPC assembly. Specifically, MEL-28 interacts with the γ-tubulin ring complex and recruits it to microtubule nucleation sites. Our data identify MEL-28 as a RanGTP target that functions throughout the cell cycle. Its cell cycle-dependent binding to chromatin or microtubules discriminates MEL-28 functions in interphase and mitosis, and ensures that spindle assembly occurs only after NPC breakdown.

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

An antitubulin agent BCFMT inhibits proliferation of cancer cells and induces cell death by inhibiting microtubule dynamics.

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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

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)

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

Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii.

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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.

The proteasome of the differently-diverged eukaryote Giardia lamblia and its role in remodeling of the microtubule-based cytoskeleton.

Science.gov (United States)

Ray, Atrayee; Sarkar, Srimonti

2017-08-01

Giardia lamblia is the causative agent of the diarrheal disease giardiasis, against which only a limited number of drugs are currently available. Increasing reports of resistance to these drugs makes it necessary to identify new cellular targets for designing the next generation of anti-giardial drugs. Towards this goal, therapeutic agents that target the parasitic cellular machinery involved in the functioning of the unique microtubule-based cytoskeleton of the Giardia trophozoites are likely to be effective as microtubule function is not only important for the survival of trophozoites within the host, but also their extensive remodeling is necessary during the transition from trophozoites to cysts. Thus, drugs that affect microtubule remodeling have the potential to not only kill the disease-causing trophozoites, but also inhibit transmission of cysts in the community. Recent studies in other model organisms have indicated that the proteasome plays an integral role in the formation and remodeling of the microtubule-based cytoskeleton. This review draws attention to the various processes by which the giardial proteasome may impact the functioning of its microtubule cytoskeleton and highlights the possible differences of the parasitic proteasome and some of other cellular machinery involved in microtubule remodeling, compared to that of the higher eukaryotic host.

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.

Neuronal microtubule organization: from minus end to plus end

NARCIS (Netherlands)

Yau, K.W.

2016-01-01

Neurons are highly polarized cells consisting of a dendritic part and axonal part. Dendrites receive signals from other cells while axons transmit signals to other cells. In this thesis, mostly hippocampal neurons from rat embryos are used to study fundamental aspects of the microtubule organization

C-terminal region of MAP7 domain containing protein 3 (MAP7D3 promotes microtubule polymerization by binding at the C-terminal tail of tubulin.

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Saroj Yadav

Full Text Available MAP7 domain containing protein 3 (MAP7D3, a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7 family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD with the C-terminal tail (CT of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0 ± 0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics.

Application of quasi-steady state methods to molecular motor transport on microtubules in fungal hyphae.

Science.gov (United States)

Dauvergne, Duncan; Edelstein-Keshet, Leah

2015-08-21

We consider bidirectional transport of cargo by molecular motors dynein and kinesin that walk along microtubules, and/or diffuse in the cell. The motors compete to transport cargo in opposite directions with respect to microtubule polarity (towards the plus or minus end of the microtubule). In recent work, Gou et al. (2014) used a hierarchical set of models, each consisting of continuum transport equations to track the evolution of motors and their cargo (early endosomes) in the specific case of the fungus Ustilago maydis. We complement their work using a framework of quasi-steady state analysis developed by Newby and Bressloff (2010) and Bressloff and Newby (2013) to reduce the models to an approximating steady state Fokker-Plank equation. This analysis allows us to find analytic approximations to the steady state solutions in many cases where the full models are not easily solved. Consequently, we can make predictions about parameter dependence of the resulting spatial distributions. We also characterize the overall rates of bulk transport and diffusion, and how these are related to state transition parameters, motor speeds, microtubule polarity distribution, and specific assumptions made. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy

NARCIS (Netherlands)

Flex, Elisabetta; Niceta, Marcello; Cecchetti, Serena; Thiffault, Isabelle; Au, Margaret G.; Capuano, Alessandro; Piermarini, Emanuela; Ivanova, Anna A.; Francis, Joshua W.; Chillemi, Giovanni; Chandramouli, Balasubramanian; Carpentieri, Giovanna; Haaxma, Charlotte A.; Ciolfi, Andrea; Pizzi, Simone; Douglas, Ganka V.; Levine, Kara; Sferra, Antonella; Dentici, Maria Lisa; Pfundt, Rolph R.; Le Pichon, Jean-Baptiste; Farrow, Emily; Baas, Frank; Piemonte, Fiorella; Dallapiccola, Bruno; Graham, John M.; Saunders, Carol J.; Bertini, Enrico; Kahn, Richard A.; Koolen, David A.; Tartaglia, Marco

2016-01-01

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause

Ocean thermal energy conversion power system development. Final design report: PSD-I, Phase II

Energy Technology Data Exchange (ETDEWEB)

None

1980-06-30

The PSD-I program provides a heat exchanger sytem consisting of an evaporator, condenser and various ancillaries with ammonia used as a working fluid in a closed simulated Rankine cycle. It is to be installed on the Chepachet Research Vessel for test and evaluation of a number of OTEC concepts in a true ocean environment. It is one of several test articles to be tested. Primary design concerns include control of biofouling, corrosion and erosion of aluminum tubes, selection of materials, and the development of a basis for scale-up to large heat exchangers so as to ultimately demonstrate economic feasibility on a commercial scale. The PSD-I test article is devised to verify thermodynamic, environmental, and mechanical performance of basic design concepts. The detailed design, development, fabrication, checklist, delivery, installation support, and operation support for the Test Article Heat Exchangers are described. (WHK)

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.

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.

Particle-size distribution (PSD) of pulverized hair: A quantitative approach of milling efficiency and its correlation with drug extraction efficiency.

Science.gov (United States)

Chagas, Aline Garcia da Rosa; Spinelli, Eliani; Fiaux, Sorele Batista; Barreto, Adriana da Silva; Rodrigues, Silvana Vianna

2017-08-01

Different types of hair were submitted to different milling procedures and their resulting powders were analyzed by scanning electron microscopy (SEM) and laser diffraction (LD). SEM results were qualitative whereas LD results were quantitative and accurately characterized the hair powders through their particle size distribution (PSD). Different types of hair were submitted to an optimized milling conditions and their PSD was quite similar. A good correlation was obtained between PSD results and ketamine concentration in a hair sample analyzed by LC-MS/MS. Hair samples were frozen in liquid nitrogen for 5min and pulverized at 25Hz for 10min, resulting in 61% of particles sample extracted after pulverization comparing with the same sample cut in 1mm fragments. When milling time was extended to 25min, >90% of particles were sample retesting and quality control procedures. Copyright © 2017 Elsevier B.V. All rights reserved.

Invited review: Frontotemporal dementia caused by microtubule-associated protein tau gene (MAPT) mutations: a chameleon for neuropathology and neuroimaging.

Science.gov (United States)

Ghetti, B; Oblak, A L; Boeve, B F; Johnson, K A; Dickerson, B C; Goedert, M

2015-02-01

Hereditary frontotemporal dementia associated with mutations in the microtubule-associated protein tau gene (MAPT) is a protean disorder. Three neuropathologic subtypes can be recognized, based on the presence of inclusions made of tau isoforms with three and four repeats, predominantly three repeats and mostly four repeats. This is relevant for establishing a correlation between structural magnetic resonance imaging and positron emission tomography using tracers specific for aggregated tau. Longitudinal studies will be essential to determine the evolution of anatomical alterations from the asymptomatic stage to the various phases of disease following the onset of symptoms. © 2014 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.

USDA Forest Service, Rocky Mountain Region PSD Permit Completeness Determination

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Applicability of PSD to Pennsylvania Power and Light Auxiliary Boiler

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Request for Guidance on PSD Applicability Determinations for Boiler Emissions

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Two Types of Genetic Interaction Implicate the Whirligig Gene of Drosophila Melanogaster in Microtubule Organization in the Flagellar Axoneme

Science.gov (United States)

Green, L. L.; Wolf, N.; McDonald, K. L.; Fuller, M. T.

1990-01-01

The mutant nc4 allele of whirligig (3-54.4) of Drosophila melanogaster fails to complement mutations in an α-tubulin locus, α1t, mutations in a β-tubulin locus, B2t, or a mutation in the haywire locus. However, wrl fails to map to any of the known α- or β-tubulin genes. The extragenic failure to complement could indicate that the wrl product participates in structural interactions with microtubule proteins. The whirligig locus appears to be haploinsufficient for male fertility. Both a deficiency of wrl and possible loss of function alleles obtained by reverting the failure to complement between wrl(nc4) and B2t(n) are dominant male sterile in a genetic background wild type for tubulin. The dominant male sterility of the revertant alleles is suppressed if the flies are also heterozygous for B2t(n), for a deficiency of α1t, or for the hay(nc2) allele. These results suggest that it is not the absolute level of wrl gene product but its level relative to tubulin or microtubule function that is important for normal spermatogenesis. The phenotype of homozygous wrl mutants suggests that the whirligig product plays a role in postmeiotic spermatid differentiation, possibly in organizing the microtubules of the sperm flagellar axoneme. Flies homozygous for either wrl(nc4) or revertant alleles are viable and female fertile but male sterile. Premeiotic and meiotic stages of spermatogenesis appear normal. However, in post-meiotic stages, flagellar axonemes show loss of the accessory microtubule on the B-subfiber of outer doublet microtubules, outer triplet instead of outer doublet microtubules, and missing central pair microtubules. PMID:2127579

MAGUKs: multifaceted synaptic organizers.

Science.gov (United States)

Won, Sehoon; Levy, Jon M; Nicoll, Roger A; Roche, Katherine W

2017-04-01

The PSD-95 family of proteins, known as MAGUKs, have long been recognized to be central building blocks of the PSD. They are categorized as scaffolding proteins, which link surface-expressed receptors to the intracellular signaling molecules. Although the four members of the PSD-95 family (PSD-95, PSD-93, SAP102, and SAP97) have many shared roles in regulating synaptic function, recent studies have begun to delineate specific binding partners and roles in plasticity. In the current review, we will highlight the conserved and unique roles of these proteins. Published by Elsevier Ltd.

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

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.

Involvement of microtubules in lipoprotein degradation and utilization for steroidogenesis in cultured rat luteal cells

International Nuclear Information System (INIS)

Rajan, V.P.; Menon, K.M.

1985-01-01

Cells isolated from superovulated rat ovaries metabolize low density lipoprotein (LDL) and high density lipoprotein (HDL) of human or rat origin and use the lipoprotein-derived cholesterol as a precursor for progesterone production. Under in vitro conditions, both lipoproteins are internalized and degraded in the lysosomes, although degradation of HDL is of lower magnitude than that of LDL. In this report we have examined the role of cellular microtubules in the internalization and degradation of human LDL and HDL in cultured rat luteal cells. The microtubule depolymerizing agents colchicine, podophyllotoxin, vinblastine, and nocodazole as well as taxol, deuterium oxide, and dimethyl sulfoxide, which are known to rapidly polymerize cellular tubulin into microtubules, were used to block the function of microtubules. When these antimicrotubule agents were included in the incubations, degradation of the apolipoproteins of [ 125 I]iodo-LDL and [ 125 I]iodo-HDL by the luteal cells was inhibited by 50-85% compared to untreated control values. Maximum inhibitory effects were observed when the cells were preincubated with the inhibitor for at least 4 h at 37 C before treatment with the labeled lipoprotein. Lipoprotein-stimulated progesterone production by luteal cells was also inhibited by 50% or more in the presence of antimicrotubule agents. However, basal and hCG-stimulated progesterone production were unaffected by these inhibitors. The binding of [ 125 I]iodo-LDL and [ 125 I]iodo-HDL to luteal cell plasma membrane receptors was not affected by the microtubule inhibitors. Although binding was unaffected and degradation was impaired in the presence of the inhibitors, there was no detectable accumulation of undegraded lipoprotein within the cells during the 24 h of study

Phosphatase PP2A and microtubule-mediated pulling forces disassemble centrosomes during mitotic exit

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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.

Image-based compound profiling reveals a dual inhibitor of tyrosine kinase and microtubule polymerization.

Science.gov (United States)

Tanabe, Kenji

2016-04-27

Small-molecule compounds are widely used as biological research tools and therapeutic drugs. Therefore, uncovering novel targets of these compounds should provide insights that are valuable in both basic and clinical studies. I developed a method for image-based compound profiling by quantitating the effects of compounds on signal transduction and vesicle trafficking of epidermal growth factor receptor (EGFR). Using six signal transduction molecules and two markers of vesicle trafficking, 570 image features were obtained and subjected to multivariate analysis. Fourteen compounds that affected EGFR or its pathways were classified into four clusters, based on their phenotypic features. Surprisingly, one EGFR inhibitor (CAS 879127-07-8) was classified into the same cluster as nocodazole, a microtubule depolymerizer. In fact, this compound directly depolymerized microtubules. These results indicate that CAS 879127-07-8 could be used as a chemical probe to investigate both the EGFR pathway and microtubule dynamics. The image-based multivariate analysis developed herein has potential as a powerful tool for discovering unexpected drug properties.

Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation

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Duangmano Suwit

2012-10-01

Full Text Available Abstract Background Cucurbitacin B, an oxygenated tetracyclic triterpenoid compound extracted from the Thai medicinal plant Trichosanthes cucumerina L., has been reported to have several biological activities including anti-inflammatory, antimicrobial and anticancer. Cucurbitacin B is great of interest because of its biological activity. This agent inhibits growth of various types of human cancer cells lines. Methods In this study, we explored the novel molecular response of cucurbitacin B in human breast cancer cells, MCF-7 and MDA-MB-231. The growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay. The effects of cucurbitacin B on microtubules morphological structure and tubulin polymerization were analyzed using immunofluorescence technique and tubulin polymerization assay kit, respectively. Proteomic analysis was used to identify the target-specific proteins that involved in cucurbitacin B treatment. Some of the differentially expressed genes and protein products were validated by real-time RT-PCR and western blot analysis. Cell cycle distributions and apoptosis were investigated using flow cytometry. Results Cucurbitacin B exhibited strong antiproliferative effects against breast cancer cells in a dose-dependent manner. We show that cucurbitacin B prominently alters the cytoskeletal network of breast cancer cells, inducing rapid morphologic changes and improper polymerization of the microtubule network. Moreover, the results of 2D-PAGE, real-time RT-PCR, and western blot analysis revealed that the expression of nucleophosmin/B23 and c-Myc decreased markedly after cucurbitacin B treatment. Immunofluorescence microscopy showed that cucurbitacin B induced translocation of nucleophosmin/B23 from the nucleolus to nucleoplasm. Treatment with cucurbitacin B resulted in cell cycle arrest at G2/M phase and the enhancement of apoptosis. Conclusions Our findings suggest that cucurbitacin B may inhibit the

Precise mapping of the CD95 pre-ligand assembly domain.

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Valérie Edmond

Full Text Available Pre-association of CD95 at the plasma membrane is mandatory for efficient death receptor signaling. This homotrimerization occurs through self-association of an extracellular domain called the pre-ligand assembly domain (PLAD. Using novel molecular and cellular tools, we confirmed that CD95-PLAD is necessary to promote CD95 multimerization and plays a pivotal role in the transmission of apoptotic signals. However, while a human CD95 mutant deleted of the previously described PLAD domain (amino acids 1 to 66 fails to interact with its wild-type counterpart and trigger autonomous cell death, deletion of amino acids 1 to 42 does not prevent homo- or hetero (human/mouse-oligomerization of CD95, and thus does not alter transmission of the apoptotic signal. Overall, these findings indicate that the region between amino acids 43 to 66 corresponds to the minimal motif involved in CD95 homotypic interaction and is necessary to convey an efficient apoptotic signal. Interfering with this PLAD may represent a new therapeutic strategy for altering CD95-induced apoptotic and non-apoptotic signals.

Wolbachia utilizes host microtubules and Dynein for anterior localization in the Drosophila oocyte.

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Patrick M Ferree

2005-10-01

Full Text Available To investigate the role of the host cytoskeleton in the maternal transmission of the endoparasitic bacteria Wolbachia, we have characterized their distribution in the female germ line of Drosophila melanogaster. In the germarium, Wolbachia are distributed to all germ cells of the cyst, establishing an early infection in the cell destined to become the oocyte. During mid-oogenesis, Wolbachia exhibit a distinct concentration between the anterior cortex and the nucleus in the oocyte, where many bacteria appear to contact the nuclear envelope. Following programmed rearrangement of the microtubule network, Wolbachia dissociate from this anterior position and become dispersed throughout the oocyte. This localization pattern is distinct from mitochondria and all known axis determinants. Manipulation of microtubules and cytoplasmic Dynein and Dynactin, but not Kinesin-1, disrupts anterior bacterial localization in the oocyte. In live egg chambers, Wolbachia exhibit movement in nurse cells but not in the oocyte, suggesting that the bacteria are anchored by host factors. In addition, we identify mid-oogenesis as a period in the life cycle of Wolbachia in which bacterial replication occurs. Total bacterial counts show that Wolbachia increase at a significantly higher rate in the oocyte than in the average nurse cell, and that normal Wolbachia levels in the oocyte depend on microtubules. These findings demonstrate that Wolbachia utilize the host microtubule network and associated proteins for their subcellular localization in the Drosophila oocyte. These interactions may also play a role in bacterial motility and replication, ultimately leading to the bacteria's efficient maternal transmission.

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

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

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

Microtubule dynamics. II. Kinetics of self-assembly

DEFF Research Database (Denmark)

Flyvbjerg, H.; Jobs, E.

1997-01-01

Inverse scattering theory describes the conditions necessary and sufficient to determine an unknown potential from known scattering data. No similar theory exists for when and how one may deduce the kinetics of an unknown chemical reaction from quantitative information about its final state and i...... to analyze the self-assembly of microtubules from tubulin are general, and many other reactions and processes may be studied as inverse problems with these methods when enough experimental data are available....

The Microtubule Plus-End Tracking Protein CLASP2 Is Required for Hematopoiesis and Hematopoietic Stem Cell Maintenance

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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.

Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrix secretion and deposition by affecting microtubule dynamics.

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Hadil F Al-Jallad

2011-01-01

Full Text Available Transglutaminase activity, arising potentially from transglutaminase 2 (TG2 and Factor XIIIA (FXIIIA, has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.

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

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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.

In vivo FRET imaging revealed a regulatory role of RanGTP in kinetochore-microtubule attachments via Aurora B kinase.

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Yoke-Peng Lee

Full Text Available Under the fluctuating circumstances provided by the innate dynamics of microtubules and opposing tensions resulted from microtubule-associated motors, it is vital to ensure stable kinetochore-microtubule attachments for accurate segregation. However, a comprehensive understanding of how this regulation is mechanistically achieved remains elusive. Using our newly designed live cell FRET time-lapse imaging, we found that post-metaphase RanGTP is crucial in the maintenance of stable kinetochore-microtubule attachments by regulating Aurora B kinase via the NES-bearing Mst1. More importantly, our study demonstrates that by ensuring stable alignment of metaphase chromosomes prior to segregation, RanGTP is indispensible in governing the genomic integrity and the fidelity of cell cycle progression. Our findings suggest an additional role of RanGTP beyond its known function in mitotic spindle assembly during the prometaphase-metaphase transition.

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.

Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs

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Francehuli Dagger

2013-02-01

Full Text Available The trypanosomatid cytoskeleton is responsible for the parasite's shape and it is modulated throughout the different stages of the parasite's life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD. We studied the effects of anti-microtubule (Mt drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite's hypotonic stress-induced shape change, as well as its recovery.

Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine

International Nuclear Information System (INIS)

Rathinasamy, Krishnan; Jindal, Bhavya; Asthana, Jayant; Singh, Parminder; Balaji, Petety V; Panda, Dulal

2010-01-01

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 αβ intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic

Insight into microtubule destabilization mechanism of 3,4,5-trimethoxyphenyl indanone derivatives using molecular dynamics simulation and conformational modes analysis

Science.gov (United States)

Tripathi, Shubhandra; Srivastava, Gaurava; Singh, Aastha; Prakasham, A. P.; Negi, Arvind S.; Sharma, Ashok

2018-03-01

Colchicine site inhibitors are microtubule destabilizers having promising role in cancer therapeutics. In the current study, four such indanone derivatives (t1, t9, t14 and t17) with 3,4,5-trimethoxyphenyl fragment (ring A) and showing significant microtubule destabilization property have been explored. The interaction mechanism and conformational modes triggered by binding of these indanone derivatives and combretastatin at colchicine binding site (CBS) of αβ-tubulin dimer were studied using molecular dynamics (MD) simulation, principle component analysis and free energy landscape analysis. In the MD results, t1 showed binding similar to colchicine interacting in the deep hydrophobic core at the CBS. While t9, t14 and t17 showed binding conformation similar to combretastatin, with ring A superficially binding at the CBS. Results demonstrated that ring A played a vital role in binding via hydrophobic interactions and got anchored between the S8 and S9 sheets, H8 helix and T7 loop at the CBS. Conformational modes study revealed that twisting and bending conformational motions (as found in the apo system) were nearly absent in the ligand bound systems. Absence of twisting motion might causes loss of lateral contacts in microtubule, thus promoting microtubule destabilization. This study provides detailed account of microtubule destabilization mechanism by indanone ligands and combretastatin, and would be helpful for designing microtubule destabilizers with higher activity.

A Novel Plasma Membrane-Anchored Protein Regulates Xylem Cell-Wall Deposition through Microtubule-Dependent Lateral Inhibition of Rho GTPase Domains.

Science.gov (United States)

Sugiyama, Yuki; Wakazaki, Mayumi; Toyooka, Kiminori; Fukuda, Hiroo; Oda, Yoshihisa

2017-08-21

Spatial control of cell-wall deposition is essential for determining plant cell shape [1]. Rho-type GTPases, together with the cortical cytoskeleton, play central roles in regulating cell-wall patterning [2]. In metaxylem vessel cells, which are the major components of xylem tissues, active ROP11 Rho GTPases form oval plasma membrane domains that locally disrupt cortical microtubules, thereby directing the formation of oval pits in secondary cell walls [3-5]. However, the regulatory mechanism that determines the planar shape of active Rho of Plants (ROP) domains is still unknown. Here we show that IQD13 associates with cortical microtubules and the plasma membrane to laterally restrict the localization of ROP GTPase domains, thereby directing the formation of oval secondary cell-wall pits. Loss and overexpression of IQD13 led to the formation of abnormally round and narrow secondary cell-wall pits, respectively. Ectopically expressed IQD13 increased the presence of parallel cortical microtubules by promoting microtubule rescue. A reconstructive approach revealed that IQD13 confines the area of active ROP domains within the lattice of the cortical microtubules, causing narrow ROP domains to form. This activity required the interaction of IQD13 with the plasma membrane. These findings suggest that IQD13 positively regulates microtubule dynamics as well as their linkage to the plasma membrane, which synergistically confines the area of active ROP domains, leading to the formation of oval secondary cell-wall pits. This finding sheds light on the role of microtubule-plasma membrane linkage as a lateral fence that determines the planar shape of Rho GTPase domains. Copyright © 2017 Elsevier Ltd. All rights reserved.

The polarity protein Par6 is coupled to the microtubule network during molluscan early embryogenesis

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Homma, Taihei [Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Shimizu, Miho [Kuroda Chiromorphology Team, ERATO-SORST, JST, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Kuroda, Reiko, E-mail: ckuroda@mail.ecc.u-tokyo.ac.jp [Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Kuroda Chiromorphology Team, ERATO-SORST, JST, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902 (Japan)

2011-01-07

Research highlights: {yields} The cDNAs encoding Par6 and aPKC homologues were cloned from the snail Lymnaea stagnalis. {yields} L. stagnalis Par6 directly interacts with tubulin and microtubules and localizes to the microtubule cytoskeleton during the early embryogenesis. {yields} Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of body handedness. -- Abstract: Cell polarity, which directs the orientation of asymmetric cell division and segregation of fate determinants, is a fundamental feature of development and differentiation. Regulators of polarity have been extensively studied, and the critical importance of the Par (partitioning-defective) complex as the polarity machinery is now recognized in a wide range of eukaryotic systems. The Par polarity module is evolutionarily conserved, but its mechanism and cooperating factors vary among different systems. Here we describe the cloning and characterization of a pond snail Lymnaea stagnalis homologue of partitioning-defective 6 (Lspar6). The protein product LsPar6 shows high affinity for microtubules and localizes to the mitotic apparatus during embryonic cell division. In vitro assays revealed direct binding of LsPar6 to tubulin and microtubules, which is the first evidence of the direct interaction between the two proteins. The interaction is mediated by two distinct regions of LsPar6 both located in the N-terminal half. Atypical PKC, a functional partner of Par6, was also found to localize to the mitotic spindle. These results suggest that the L. stagnalis Par complex employs the microtubule network in cell polarity processes during the early embryogenesis. Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of handedness.

The polarity protein Par6 is coupled to the microtubule network during molluscan early embryogenesis

International Nuclear Information System (INIS)

Homma, Taihei; Shimizu, Miho; Kuroda, Reiko

2011-01-01

Research highlights: → The cDNAs encoding Par6 and aPKC homologues were cloned from the snail Lymnaea stagnalis. → L. stagnalis Par6 directly interacts with tubulin and microtubules and localizes to the microtubule cytoskeleton during the early embryogenesis. → Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of body handedness. -- Abstract: Cell polarity, which directs the orientation of asymmetric cell division and segregation of fate determinants, is a fundamental feature of development and differentiation. Regulators of polarity have been extensively studied, and the critical importance of the Par (partitioning-defective) complex as the polarity machinery is now recognized in a wide range of eukaryotic systems. The Par polarity module is evolutionarily conserved, but its mechanism and cooperating factors vary among different systems. Here we describe the cloning and characterization of a pond snail Lymnaea stagnalis homologue of partitioning-defective 6 (Lspar6). The protein product LsPar6 shows high affinity for microtubules and localizes to the mitotic apparatus during embryonic cell division. In vitro assays revealed direct binding of LsPar6 to tubulin and microtubules, which is the first evidence of the direct interaction between the two proteins. The interaction is mediated by two distinct regions of LsPar6 both located in the N-terminal half. Atypical PKC, a functional partner of Par6, was also found to localize to the mitotic spindle. These results suggest that the L. stagnalis Par complex employs the microtubule network in cell polarity processes during the early embryogenesis. Identical sequence and localization of LsPar6 for the dextral and the sinistral snails exclude the possibility of the gene being the primary determinant of handedness.

Microtubules in cell migration, morphogenesis and metabolism: Making the connections

NARCIS (Netherlands)

Noordstra, I.

2017-01-01

Cell polarity refers to a fundamental property of eukaryotic cells, in which cellular components and structures are organized in an asymmetric fashion. In order to control their polarity, cells make use of microtubules, hollow polymers that extend throughout the cytoplasm. Due to the asymmetry of

Chlamydomonas outer arm dynein alters conformation in response to Ca2+.

Science.gov (United States)

Sakato, Miho; Sakakibara, Hitoshi; King, Stephen M

2007-09-01

We have previously shown that Ca(2+) directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the beta and gamma heavy chains (HCs). The gamma HC-associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca(2+) with K(Ca) = 3 x 10(-5) M in vitro, suggesting it may act as a Ca(2+) sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and gamma HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the gamma heavy chain. In vitro experiments indicate that LC4 undergoes a Ca(2+)-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca(2+). The sedimentation profile of the gamma HC subunit changed subtly upon Ca(2+) addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated gamma subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca(2+) addition. We propose that Ca(2+)-dependent conformational change of LC4 has a direct effect on the stem domain of the gamma HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm.

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

Energy Technology Data Exchange (ETDEWEB)

Wei, Ren-Jie [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Lin, Su-Shuan [Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Wu, Wen-Ren [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Chen, Lih-Ren [Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan (China); Division of Physiology, Livestock Research Institute, Council of Agriculture, Taiwan (China); Li, Chien-Feng [Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan (China); Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan (China); National Institute of Cancer Research, National Health Research Institute, Tainan, Taiwan (China); Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Chen, Han-De; Chou, Chien-Ting; Chen, Ya-Chun [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Liang, Shih-Shin [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Chien, Shang-Tao [Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Shiue, Yow-Ling, E-mail: ylshiue@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan (China)

2016-11-15

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G{sub 2}/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G{sub 2}/M cell cycle regulators, ABT-751 induced G{sub 2}/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. - Highlights: • An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells. �

75 FR 27643 - Prevention of Significant Deterioration (PSD) and Nonattainment New Source Review (NSR): Aggregation

Science.gov (United States)

2010-05-18

... once the delay is no longer necessary. ADDRESSES: Docket: The final rule, the petition for...): Aggregation AGENCY: Environmental Protection Agency (EPA). ACTION: Delay of effective date. SUMMARY: EPA is delaying the effective date of the final rule titled ``Prevention of Significant Deterioration (PSD) and...

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.

Influence of catch up growth on spatial learning and memory in a mouse model of intrauterine growth restriction.

Directory of Open Access Journals (Sweden)

Cristina Duran Fernandez-Feijoo

Full Text Available Intrauterine growth restriction (IUGR and rapid postnatal weight gain or catch up growth (CUG increase the susceptibility to metabolic syndrome during adult life. Longitudinal studies have also revealed a high incidence of learning difficulties in children with IUGR. The aim of the present study was to investigate the effect of nutrition and CUG on learning memory in an IUGR animal model. We hypothesized that synaptic protein expression and transcription, an essential mechanism for memory consolidation, might be affected by intrauterine undernutrition.IUGR was induced by 50% maternal caloric undernutrition throughout late gestation. During the suckling period, dams were either fed ad libitum or food restricted. The pups were divided into: Normal prenatal diet-Normal postnatal diet (NN, Restricted prenatal diet- Normal postnatal diet + catch up growth (RN+, Normal prenatal diet-Restricted postnatal diet (NR and Restricted prenatal diet-Restricted postnatal diet (RR. At 4 weeks of age, memory was assessed via a water maze test. To evaluate synaptic function, 2 specific synaptic proteins (postsynaptic density-95 [PSD95], synaptophysin as well as insulin receptors (IR were tested by Western Blot and quantitative polymerase chain reaction (qPCR. Brain-derived neurotrophic factor and serum insulin levels were also studied.The RN+ group presented a learning curve similar to the NN animals. The RR animals without CUG showed learning disabilities. PSD95 was lower in the RR group than in the NN and RN+ mice. In contrast, synaptophysin was similar in all groups. IR showed an inverse expression pattern to that of the PSD95. In conclusion, perinatal nutrition plays an important role in learning. CUG after a period of prenatal malnutrition seems to improve learning skills. The functional alterations observed might be related to lower PSD95 activity and a possible dysfunction in the hormone regulation of synaptic plasticity.

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

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 cellula...

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

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.

Glucose regulated proteins 78 and 75 bind to the receptor for hyaluronan mediated motility in interphase microtubules

International Nuclear Information System (INIS)

Kuwabara, Hiroko; Yoneda, Masahiko; Hayasaki, Hana; Nakamura, Toshiya; Mori, Hiroshi

2006-01-01

The receptor for hyaluronan mediated motility (RHAMM), which is a hyaluronan-binding protein, is a centrosomal and microtubal protein. Here, we have identified two RHAMM-binding proteins, glucose regulated protein (GRP) 78 and GRP75, using co-immunoprecipitation analysis. These two proteins directly bound to glutathione-S-transferase-RHAMM fusion proteins. By double immunostaining, GRP78 and GRP75 colocalized with RHAMM in interphase microtubules, but were separated in mitotic spindles. Prevention of microtubule polymerization by TN-16 and vincristine sulfate induced RHAMM overexpression without a significant change in GRP78/75. Taken together, GRP78/75 and RHAMM complexes may stabilize microtubules in the interphase, associated with a downregulation of RHAMM. These results reveal a new biochemical activity of RHAMM

Non-equilibrium assembly of microtubules: from molecules to autonomous chemical robots.

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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.

PSD Determination: Sun Oil Company and the Definition of Major Modification

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Interim Policy Determination Related to NSR/PSD Significance Level for ODS

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Selective synaptic targeting of the excitatory and inhibitory presynaptic organizers FGF22 and FGF7.

Science.gov (United States)

Terauchi, Akiko; Timmons, Kendall M; Kikuma, Koto; Pechmann, Yvonne; Kneussel, Matthias; Umemori, Hisashi

2015-01-15

Specific formation of excitatory and inhibitory synapses is crucial for proper functioning of the brain. Fibroblast growth factor 22 (FGF22) and FGF7 are postsynaptic-cell-derived presynaptic organizers necessary for excitatory and inhibitory presynaptic differentiation, respectively, in the hippocampus. For the establishment of specific synaptic networks, these FGFs must localize to appropriate synaptic locations - FGF22 to excitatory and FGF7 to inhibitory postsynaptic sites. Here, we show that distinct motor and adaptor proteins contribute to intracellular microtubule transport of FGF22 and FGF7. Excitatory synaptic targeting of FGF22 requires the motor proteins KIF3A and KIF17 and the adaptor protein SAP102 (also known as DLG3). By contrast, inhibitory synaptic targeting of FGF7 requires the motor KIF5 and the adaptor gephyrin. Time-lapse imaging shows that FGF22 moves with SAP102, whereas FGF7 moves with gephyrin. These results reveal the basis of selective targeting of the excitatory and inhibitory presynaptic organizers that supports their different synaptogenic functions. Finally, we found that knockdown of SAP102 or PSD95 (also known as DLG4), which impairs the differentiation of excitatory synapses, alters FGF7 localization, suggesting that signals from excitatory synapses might regulate inhibitory synapse formation by controlling the distribution of the inhibitory presynaptic organizer. © 2015. Published by The Company of Biologists Ltd.

A preliminary examination of the validity and reliability of a new brief rating scale for symptom domains of psychosis: Brief Evaluation of Psychosis Symptom Domains (BE-PSD).

Science.gov (United States)

Takeuchi, Hiroyoshi; Fervaha, Gagan; Lee, Jimmy; Agid, Ofer; Remington, Gary

2016-09-01

Brief assessments have the potential to be widely adopted as outcome measures in research but also routine clinical practice. Existing brief rating scales that assess symptoms of schizophrenia or psychosis have a number of limitations including inability to capture five symptom domains of psychosis and a lack of clearly defined operational anchor points for scoring. We developed a new brief rating scale for five symptom domains of psychosis with clearly defined operational anchor points - the Brief Evaluation of Psychosis Symptom Domains (BE-PSD). To examine the psychometric properties of the BE-PSD, fifty patients with schizophrenia or schizoaffective disorder were included in this preliminary cross-sectional study. To test the convergent and discriminant validity of the BE-PSD, correlational analyses were employed using the consensus Positive and Negative Syndrome Scale (PANSS) five-factor model. To examine the inter-rater reliability of the BE-PSD, single measures intraclass correlation coefficients (ICCs) were calculated for 11 patients. The BE-PSD domain scores demonstrated high convergent validity with the corresponding PANSS factor score (rs = 0.81-0.93) as well as good discriminant validity, as evidenced by lower correlations with the other PANSS factors (rs = 0.23-0.62). The BE-PSD also demonstrated excellent inter-rater reliability for each of the domain scores and the total scores (ICC(2,1) = 0.79-0.96). The present preliminary study found the BE-PSD measure to be valid and reliable; however, further studies are needed to establish the psychometric properties of the BE-PSD because of the limitations such as the small sample size and lacking data on test-retest reliability or sensitivity to change. Copyright © 2016 Elsevier Ltd. All rights reserved.

Role of membrane sterols and cortical microtubules in gravity resistance in plants

Science.gov (United States)

Hoson, T.; Koizumi, T.; Matsumoto, S.; Kumasaki, S.; Soga, K.; Wakabayashi, K.; Sakaki, T.

Resistance to the gravitational force is a principal graviresponse in plants comparable to gravitropism Nevertheless only limited information has been obtained for this graviresponse We have examined mechanisms of signal perception transformation and transduction of the perceived signal and response to the transduced signal in gravity resistance using hypergravity conditions produced by centrifugation In Arabidopsis hypocotyls hypergravity treatment greatly increased the expression level of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase HMGR which catalyzes a reaction producing mevalonic acid a key precursor of terpenoids such as membrane sterols Geranyl diphosphate synthase gene was also up-regulated by hypergravity whereas the expression of other genes involved in membrane lipid metabolism was not influenced Hypergravity caused an increase in sterol content in azuki bean epicotyls but not in phospholipid glycolipid or fatty acid content Also hypergravity did not influence fatty acid composition in any lipid class Thus the effect of hypergravity on membrane lipid metabolism was specific for sterol synthesis On the other hand alpha- and beta-tubulin genes were up-regulated by hypergravity treatment in Arabidopsis hypocotyls Hypergravity also induced reorientation of cortical microtubules in azuki epicotyls the percentage of epidermal cells with transverse microtubles was decreased whereas that with longitudinal microtubules was increased Inhibitors of HMGR action and microtubule-disrupting agents completely prevented the gravity resistance

The centrosomal linker and microtubules provide dual levels of spatial coordination of centrosomes.

Directory of Open Access Journals (Sweden)

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.

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

Duplication in the microtubule-actin cross-linking factor 1 gene causes a novel neuromuscular condition

DEFF Research Database (Denmark)

Jørgensen, Louise H; Mosbech, Mai-Britt; Færgeman, Nils J

2014-01-01

Spectrins and plakins are important communicators linking cytoskeletal components to each other and to cellular junctions. Microtubule-actin cross-linking factor 1 (MACF1) belongs to the spectraplakin family and is involved in control of microtubule dynamics. Complete knock out of MACF1 in mice...... muscles and diminished motor skills, with heterogeneous presentation among the affected family members. To corroborate these findings we used RNA interference to knock down the VAB-10 locus containing the MACF1 homologue in C. elegans, and we could show that this also causes movement disturbances...

Early weaning PCB 95 exposure alters the neonatal endocrine system: thyroid adipokine dysfunction.

Science.gov (United States)

Ahmed, R G

2013-12-01

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that can severely disrupt the endocrine system. In the present study, early-weaned male rats were administered a single dose of 2,3,6-2',5'-pentachlorinated biphenyl (PCB 95; 32 mg/kg per day, by i.p. injection) for two consecutive days (postnatal days (PNDs) 15 and 16) and killed 24 and 48 h after the administration of the last dose. Compared with the control group, administration of PCB 95 induced a reduction (Pcolloidal contents at PND 18. The dyshormonogenesis and thyroid dysgenesis may be attributed to the elevation of DNA fragmentation at PNDs 17 and 18. Furthermore, this hypothyroid state revealed higher (Pinsulin at both PNDs compared with the control group. Interestingly, the body weight of the neonates in the PCB 95 group exhibited severe decreases throughout the experimental period in relation to that of the control group. These results imply that PCB 95 may act as a disruptor of the developmental hypothalamic-pituitary-thyroid axis. Hypothyroidism caused by PCB 95 may impair the adipokine axis, fat metabolism, and in general postnatal development. Thus, further studies need to be carried out to understand this concept.

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.

Effects of colchicine treatment on the microtubule cytoskeleton and total protein during microsporogenesis in ginkgo biloba

International Nuclear Information System (INIS)

Cao, Q.J.; Mei, F.F.

2015-01-01

The purpose of this study was to examine the effects of colchicine treatment on the microtubule cytoskeleton and the expression of proteins during microsporogenesis in G. biloba, as observed by immunofluorescence and 2-DE analysis in microsporangia treated with colchicine. The results showed the microtubule structures were affected by the colchicine in Ginkgo biloba, but the treatment effect of the colchicine had certain limitation in G. biloba. The percentage of microsporocytes whose microtubule structures were affected by the colchicine treatment was less than that observed in other plant species, not higher than 10 %. It was also found that the expression level of several endogenous proteins were changed in G. biloba when the microsporangia were treated with colchicine. Although we only tested colchicines was only tested in the present study, G. biloba appeared to possess factors that restricted the effect of such chemical agents. Our observations led us to speculate that the endogenous proteins are possibly responsible for the reduced effects of colchicine treatment in G. biloba. (author)

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

Science.gov (United States)

Schatten, H.; Ripple, M.; Balczon, R.; Weindruch, R.; Chakrabarti, A.; Taylor, M.; Hueser, C. N.

2000-01-01

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well. Copyright 2000 Wiley-Liss, Inc.

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...... microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine...... the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in ¿-tubulins...

Birefringence of single and bundled microtubules.

Science.gov (United States)

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

1998-01-01

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

The research of PSD location method in micro laser welding fields

Science.gov (United States)

Zhang, Qiue; Zhang, Rong; Dong, Hua

2010-11-01

In the field of micro laser welding, besides the special requirement in the parameter of lasers, the locating in welding points accurately is very important. The article adopt position sensitive detector (PSD) as hard core, combine optic system, electric circuits and PC and software processing, confirm the location of welding points. The signal detection circuits adopt the special integrate circuit H-2476 to process weak signal. It is an integrated circuit for high-speed, high-sensitivity optical range finding, which has stronger noiseproof feature, combine digital filter arithmetic, carry out repair the any non-ideal factors, increasing the measure precision. The amplifier adopt programmable amplifier LTC6915. The system adapt two dimension stepping motor drive the workbench, computer and corresponding software processing, make sure the location of spot weld. According to different workpieces to design the clamps. The system on-line detect PSD 's output signal in the moving processing. At the workbench moves in the X direction, the filaments offset is detected dynamic. Analyze the X axes moving sampling signal direction could be estimate the Y axes moving direction, and regulate the Y axes moving values. The workbench driver adopt A3979, it is a stepping motor driver with insert transducer and operate easily. It adapts the requirement of location in micro laser welding fields, real-time control to adjust by computer. It can be content up 20 μm's laser micro welding requirement on the whole. Using laser powder cladding technology achieve inter-penetration welding of high quality and reliability.

Contingency Plan for FGD Systems During Downtime as a Function of PSD

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

PSD Applicability of SO2 Emissions from Incineration of Total Reduced Sulfur

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This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

PSD Applicability for Ashland Chemical's Maleic Anhydride Plant in Neal, West Virginia

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Tire Derived Fuel Classified as Municipal Solid Waste for a PSD Exemption

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Thermodynamics of TMPC/PSd/Fullerene Nanocomposites: SANS Study

KAUST Repository

Chua, Yang-Choo

2010-11-23

Wereport a small angle neutron scattering study of the thermodynamics of a polymer mixture in the presence of nanoparticles, both in equilibrium and during phase separation. Neutron cloud point measurements and random phase approximation (RPA) analysis demonstrate that 1-2 mass % of C60 fullerenes destabilizes a highly interacting mixture of poly(tetramethyl bisphenol A polycarbonate) and deuterated polystyrene (TMPC/PSd). We unequivocally corroborate these findings with time-resolved temperature jump experiments that, in identical conditions, result in phase separation for the nanocomposite and stability for the neat polymer mixture. At lower C 60 loadings (viz. 0.2-0.5 mass %), stabilization of the mixture is observed. The nonmonotonic variation of the spinodal temperature with fullerene addition suggests a competitive interplay of asymmetric component interactions and nanoparticle dispersion. The stability line shift depends critically on particle dispersion and vanishes upon nanoparticle agglomeration. © 2010 American Chemical Society.

Learning and memory disabilities in IUGR babies: Functional and molecular analysis in a rat model.

Science.gov (United States)

Camprubí Camprubí, Marta; Balada Caballé, Rafel; Ortega Cano, Juan A; Ortega de la Torre, Maria de Los Angeles; Duran Fernández-Feijoo, Cristina; Girabent-Farrés, Montserrat; Figueras-Aloy, Josep; Krauel, Xavier; Alcántara, Soledad

2017-03-01

1Intrauterine growth restriction (IUGR) is the failure of the fetus to achieve its inherent growth potential, and it has frequently been associated with neurodevelopmental problems in childhood. Neurological disorders are mostly associated with IUGR babies with an abnormally high cephalization index (CI) and a brain sparing effect. However, a similar correlation has never been demonstrated in an animal model. The aim of this study was to determine the correlations between CI, functional deficits in learning and memory and alterations in synaptic proteins in a rat model of IUGR. 2Utero-placental insufficiency was induced by meso-ovarian vessel cauterization (CMO) in pregnant rats at embryonic day 17 (E17). Learning performance in an aquatic learning test was evaluated 25 days after birth and during 10 days. Some synaptic proteins were analyzed (PSD95, Synaptophysin) by Western blot and immunohistochemistry. 3Placental insufficiency in CMO pups was associated with spatial memory deficits, which are correlated with a CI above the normal range. CMO pups presented altered levels of synaptic proteins PSD95 and synaptophysin in the hippocampus. 4The results of this study suggest that learning disabilities may be associated with altered development of excitatory neurotransmission and synaptic plasticity. Although interspecific differences in fetal response to placental insufficiency should be taken into account, the translation of these data to humans suggest that both IUGR babies and babies with a normal birth weight but with intrauterine Doppler alterations and abnormal CI should be closely followed to detect neurodevelopmental alterations during the postnatal period.

LDLR expression and localization are altered in mouse and human cell culture models of Alzheimer's disease.

Directory of Open Access Journals (Sweden)

Jose F Abisambra

Full Text Available BACKGROUND: Alzheimer's disease (AD is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the epsilon-4 allele of apolipoprotein E (apoE, the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR has the highest affinity for apoE and plays an important role in brain cholesterol metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Abeta-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of gamma- and alpha-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network. CONCLUSIONS/SIGNIFICANCE: These data suggest that increased APP expression and Abeta exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.

Aggregation of SND1 in Stress Granules is Associated with the Microtubule Cytoskeleton During Heat Shock Stimulus.

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Shao, Jie; Gao, Fei; Zhang, Bingbing; Zhao, Meng; Zhou, Yunli; He, Jinyan; Ren, Li; Yao, Zhi; Yang, Jie; Su, Chao; Gao, Xingjie

2017-12-01

Stress granules (SGs) are dynamic dense structures in the cytoplasm that form in response to a variety of environmental stress stimuli. Staphylococcal nuclease and Tudor domain containing 1 (SND1) is a type of RNA-binding protein and has been identified as a transcriptional co-activator. Our previous studies have shown that SND1 is a component of the stress granule, which forms under stress conditions. Here, we observed that SND1 granules were often surrounded by ɑ-tubulin-microtubules in 45°C-treated HeLa cells at 15 min or colocalized with microtubules at 30 or 45 min. Furthermore, Nocodazole-mediated microtubule depolymerization could significantly affect the efficient recruitment of SND1 proteins to the SGs during heat shock stress. In addition, the 45°C heat shock mediated the enhancement of eIF2α phosphorylation, which was not affected by treatment with Nocodazole, an agent that disrupts the cytoskeleton. The intact microtubule cytoskeletal tracks are important for the efficient assembly of SND1 granules under heat shock stress and may facilitate SND1 shuttling between cytoplasmic RNA foci. Anat Rec, 300:2192-2199, 2017. © 2017 The Authors The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists. Copyright © 2017 The Authors The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD

Science.gov (United States)

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

2001-01-01

Arabidopsis thaliana plants were transformed with GFP-MBD (J. Marc et al., Plant Cell 10: 1927-1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP-specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35S GFP-MBD plants grown on kanamycin-containing media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the two processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more defined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubule-orienting factor(s) may be sensitive to growth acceleration, rather than growth per se.

Spatiotemporal Regulation of Nuclear Transport Machinery and Microtubule Organization

Science.gov (United States)

Okada, Naoyuki; Sato, Masamitsu

2015-01-01

Spindle microtubules capture and segregate chromosomes and, therefore, their assembly is an essential event in mitosis. To carry out their mission, many key players for microtubule formation need to be strictly orchestrated. Particularly, proteins that assemble the spindle need to be translocated at appropriate sites during mitosis. A small GTPase (hydrolase enzyme of guanosine triphosphate), Ran, controls this translocation. Ran plays many roles in many cellular events: nucleocytoplasmic shuttling through the nuclear envelope, assembly of the mitotic spindle, and reorganization of the nuclear envelope at the mitotic exit. Although these events are seemingly distinct, recent studies demonstrate that the mechanisms underlying these phenomena are substantially the same as explained by molecular interplay of the master regulator Ran, the transport factor importin, and its cargo proteins. Our review focuses on how the transport machinery regulates mitotic progression of cells. We summarize translocation mechanisms governed by Ran and its regulatory proteins, and particularly focus on Ran-GTP targets in fission yeast that promote spindle formation. We also discuss the coordination of the spatial and temporal regulation of proteins from the viewpoint of transport machinery. We propose that the transport machinery is an essential key that couples the spatial and temporal events in cells. PMID:26308057

CYP Suppression in Human Hepatocytes by Monomethyl Auristatin E, the Payload in Brentuximab Vedotin (Adcetris®), is Associated with Microtubule Disruption.

Science.gov (United States)

Wolenski, Francis S; Xia, Cindy Q; Ma, Bingli; Han, Tae H; Shyu, Wen C; Balani, Suresh K

2018-06-01

Monomethyl auristatin E (MMAE), the toxin linked to CD30-specific monoclonal antibody of Adcetris ® (brentuximab vedotin), is a potent anti-microtubule agent. Brentuximab vedotin has been approved for the treatment of relapsed or refractory Hodgkin lymphoma and anaplastic large cell lymphoma. Cytochrome P450 (CYP) induction assessment of MMAE was conducted in human hepatocytes to assess DDI potentials and its translation to clinic. MMAE was incubated at 1-1000 nM with cultured primary human hepatocytes for 72 h, and CYP1A2, CYP2B6, and CYP3A4 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction and CYP-specific probe substrate by liquid chromatography coupled with mass spectrometry, along with microtubule disruption by immunofluorescence staining using anti-β-tubulin antibody and imaging. MMAE up to 10 nM had no significant effect on CYP1A2, CYP2B6, and CYP3A4 mRNA expression and activity, whereas at higher concentrations of 100- and 1000-nM MMAE, the CYP mRNA expression and activity were diminished substantially. Further investigation showed that the degree of CYP suppression was paralleled by that of microtubule disruption by MMAE, as measured by increase in the number of β-tubulin-positive aggregates. At the clinical dose, the concentration of MMAE was 7 nM which did not show any significant CYP suppression or microtubule disruption in hepatocytes. MMAE was not a CYP inducer in human hepatocytes. However, it caused a concentration-dependent CYP mRNA suppression and activity. The CYP suppression was associated with microtubule disruption, supporting the reports that intact microtubule architecture is required for CYP regulations. The absence of CYP suppression and microtubule disruption in vitro at the clinical plasma concentrations of MMAE (< 10 nM) explains the lack of pharmacokinetic drug interaction between brentuximab vedotin and midazolam, a sensitive CYP3A substrate, reported in patients.

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

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.

Cytoplasmic Overexpression of CD95L in Esophageal Adenocarcinoma Cells Overcomes Resistance to CD95-Mediated Apoptosis

Directory of Open Access Journals (Sweden)

Gregory A. Watson

2011-03-01

Full Text Available Introduction: The CD95/CD95L pathway plays a critical role in tissue homeostasis and immune system regulation; however, the function of this pathway in malignancy remains poorly understood. We hypothesized that CD95L expression in esophageal adenocarcinoma confers advantages to the neoplasm other than immune privilege. Methods: CD95L expression was characterized in immortalized squamous esophagus (HET-1A and Barrett esophagus (BAR-T cells; adenocarcinoma cell lines FLO-1, SEG-1, and BIC-1, and MDA468 (- control; and KFL cells (+ control. Analyses included reverse transcription-polymerase chain reaction, immunoblots of whole cell and secretory vesicle lysates, FACScan analysis, laser scanning confocal microscopy of native proteins and fluorescent constructs, and assessment of apoptosis and ERK1/2 pathways. Results: Cleaved, soluble CD95L is expressed at both the RNA and protein levels in these cell lines derived from esophageal adenocarcinoma and other human tissues. CD95L was neither trafficked to the cell membrane nor secreted into the media or within vesicles, rather the protein seems to be sequestered in the cytoplasm. CD95 and CD95L colocalize by immunofluorescence, but an interaction was not proven by immunoprecipitation. Overexpression of CD95L in the adenocarcinoma cell lines induced robust apoptosis and, under conditions of pan-caspase inhibition, resulted in activation of ERK signaling. Conclusions: CD95L localization in EA cells is inconsistent with the conference of immune privilege and is more consistent with a function that promotes tumor growth through alternative CD95 signaling. Reduced cell surface expression of CD95 affects cell sensitivity to extracellular apoptotic signals more significantly than alterations in downstream modulators of apoptosis.

Torsion of the central pair microtubules in eukaryotic flagella due to bending-driven lateral buckling

International Nuclear Information System (INIS)

Li, C.; Ru, C.Q.; Mioduchowski, A.

2006-01-01

Inspired by recent interest in torsion of the central pair microtubules in eukaryotic flagella, a novel thin-walled elastic beam model is suggested to study critical condition under which uniform bending of a flagellum will cause lateral/torsional buckling of the central pair. The model is directed to the central pair itself and the role of all surrounding cross-linkings inside the flagellum is modeled as an equivalent surrounding elastic medium. The model predicts that bending-driven torsion of the central pair does occur when the radius of curvature of the bent flagellum reduces to a moderate critical value typically of tens of microns. In particular, this critical value is almost independent of the flagellum length, and more sensitive to the parameters defining the surrounding elastic medium than the shear modulus of microtubules. The predicted wavelengths of the torsional buckling mode are insensitive to the flagellum length and comparable to some known related experimental data. These results indicate that torsion of the central pair microtubules in flagella is inevitable as a result of bending-driven lateral buckling. This offers an entirely new insight into the ongoing research on the mechanism of the central pair torsion

Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells

Science.gov (United States)

Zhang, Hui-ming; Talbot, Mark J.; McCurdy, David W.; Patrick, John W.; Offler, Christina E.

2015-01-01

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca2+ levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane–microtubule inter-relationship is discussed. PMID:26136268

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

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 ...

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

Quantitative analysis and efficiency study of PSD methods for a LaBr{sub 3}:Ce detector

Energy Technology Data Exchange (ETDEWEB)

Zeng, Ming; Cang, Jirong [Key Laboratory of Particle & Radiation Imaging(Tsinghua University), Ministry of Education (China); Department of Engineering Physics, Tsinghua University, Beijing 100084 (China); Zeng, Zhi, E-mail: zengzhi@tsinghua.edu.cn [Key Laboratory of Particle & Radiation Imaging(Tsinghua University), Ministry of Education (China); Department of Engineering Physics, Tsinghua University, Beijing 100084 (China); Yue, Xiaoguang; Cheng, Jianping; Liu, Yinong; Ma, Hao; Li, Junli [Key Laboratory of Particle & Radiation Imaging(Tsinghua University), Ministry of Education (China); Department of Engineering Physics, Tsinghua University, Beijing 100084 (China)

2016-03-21

The LaBr{sub 3}:Ce scintillator has been widely studied for nuclear spectroscopy because of its optimal energy resolution (<3%@ 662 keV) and time resolution (~300 ps). Despite these promising properties, the intrinsic radiation background of LaBr{sub 3}:Ce is a critical issue, and pulse shape discrimination (PSD) has been shown to be an efficient potential method to suppress the alpha background from the {sup 227}Ac. In this paper, the charge comparison method (CCM) for alpha and gamma discrimination in LaBr{sub 3}:Ce is quantitatively analysed and compared with two other typical PSD methods using digital pulse processing. The algorithm parameters and discrimination efficiency are calculated for each method. Moreover, for the CCM, the correlation between the CCM feature value distribution and the total charge (energy) is studied, and a fitting equation for the correlation is inferred and experimentally verified. Using the equations, an energy-dependent threshold can be chosen to optimize the discrimination efficiency. Additionally, the experimental results show a potential application in low-activity high-energy γ measurement by suppressing the alpha background.

Nitrogen Concentrations and Isotopic Compositions of Seafloor-Altered Terrestrial Basaltic Glass: Implications for Astrobiology

Science.gov (United States)

Banerjee, N.R.; Izawa, M.R.M.; Kobayashi, K.; Lazzeri, K.; Ranieri, L.A.; Nakamura, E.

2018-01-01

Abstract Observed enrichments of N (and the δ15N of this N) in volcanic glasses altered on Earth's modern and ancient seafloor are relevant in considerations of modern global N subduction fluxes and ancient life on Earth, and similarly altered glasses on Mars and other extraterrestrial bodies could serve as valuable tracers of biogeochemical processes. Palagonitized glasses and whole-rock samples of volcanic rocks on the modern seafloor (ODP Site 1256D) contain 3–18 ppm N with δ15Nair values of up to +4.5‰. Variably altered glasses from Mesozoic ophiolites (Troodos, Cyprus; Stonyford volcanics, USA) contain 2–53 ppm N with δ15N of −6.3 to +7‰. All of the more altered glasses have N concentrations higher than those of fresh volcanic glass (for MORB, smectite, illite) in both the palagonitized cracks and the microtubules. These phyllosilicates (particularly illite), and possibly also zeolites, are the likely hosts for N in these glasses. Key Words: Nitrogen—Nitrogen isotope—Palagonite—Volcanic glass—Mars. Astrobiology 18, 330–342. PMID:29106312

Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

Science.gov (United States)

Zhang, Hui-ming; Talbot, Mark J; McCurdy, David W; Patrick, John W; Offler, Christina E

2015-09-01

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

-NH-dansyl isocolchicine exhibits a significantly improved tubulin-binding affinity and microtubule inhibition in comparison to isocolchicine by binding tubulin through its A and B rings.

Science.gov (United States)

Das, Lalita; Datta, Ajit B; Gupta, Suvroma; Poddar, Asim; Sengupta, Suparna; Janik, Mark E; Bhattacharyya, Bhabatarak

2005-03-08

Structure-activity relationship studies have established that the A and C rings of colchicine comprise the minimum structural feature necessary for high affinity drug-tubulin binding. Thus, colchicine acts as a bifunctional ligand by making two points of attachment to the protein. Furthermore, analogues belonging to the iso series of colchicine are virtually inactive in binding to tubulin and inhibiting microtubule assembly. In the present study, we found that the substitution of a hydrophobic dansyl group on the B-ring side chain (C7 position) of isocolchicine reverses the structural alterations at the C ring and the newly synthesized -NH-dansyl isocolchicine restores the lost biological activity of the compound. It inhibits microtubule assembly efficiently with an IC(50) value of 10 microM and competes with [(3)H]colchicine for binding to tubulin. Moreover, although -NH-dansyl colchicine binding to tubulin involves two steps, the -NH-dansyl isocolchicine-tubulin interaction has been found to occur via a one-step process. Also, the affinity constant of the -NH-dansyl isocolchicine-tubulin interaction is roughly only 3 times lower than that of the -NH-dansyl colchicine-tubulin interaction. These results suggest that the enhanced microtubule inhibitory ability of -NH-dansyl isocolchicine is therefore related to the affinity of the drug-tubulin interaction and not to any conformational changes upon binding tubulin. We also observed that the competition of -NH-dansyl isocolchicine with [(3)H]colchicine for binding to tubulin was dependent on the tubulin concentration. In conclusion, this paper for the first time indicates that a biologically active bifuntional colchicine analogue can be designed where the drug binds tubulin through its A and B rings, while the C ring remains inactive.

Application of PSD Review to a Portland Cement Manufacturing Operation, Texas Industries, Inc.

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Automatic or Blanket Exemptions for Excess Emissions During Startup, and Shutdowns Under PSD

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

PSD Applicability Determination for Multiple Owner/Operator Point Sources Within a Single Facility

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Electron tomography of the microtubule cytoskeleton in multinucleated hyphae of Ashbya gossypii.

Science.gov (United States)

Gibeaux, Romain; Lang, Claudia; Politi, Antonio Z; Jaspersen, Sue L; Philippsen, Peter; Antony, Claude

2012-12-01

We report the mechanistic basis guiding the migration pattern of multiple nuclei in hyphae of Ashbya gossypii. Using electron tomography, we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the spindle microtubules of four mitotic nuclei. Each spindle pole body (SPB) nucleates three cMTs and most cMTs above a certain length grow according to their plus-end structure. Long cMTs closely align for several microns along the cortex, presumably marking regions where dynein generates pulling forces on nuclei. Close proximity between cMTs emanating from adjacent nuclei was not observed. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, in most cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forth movements. Only occasionally do all six cMTs orient in one direction, a precondition for long-range nuclear bypassing. Following mitosis, daughter nuclei carry a single SPB with three cMTs. The increased probability that all three cMTs orient in one direction explains the high rate of nuclear bypassing observed in these nuclei. The A. gossypii mitotic spindle was found to be structurally similar to that of Saccharomyces cerevisiae in terms of nuclear microtubule (nMT) number, length distribution and three-dimensional organization even though the two organisms differ significantly in chromosome number. Our results suggest that two nMTs attach to each kinetochore in A. gossypii and not only one nMT like in S. cerevisiae.

Physical Limits on the Precision of Mitotic Spindle Positioning by Microtubule Pushing forces: Mechanics of mitotic spindle positioning.

Science.gov (United States)

Howard, Jonathon; Garzon-Coral, Carlos

2017-11-01

Tissues are shaped and patterned by mechanical and chemical processes. A key mechanical process is the positioning of the mitotic spindle, which determines the size and location of the daughter cells within the tissue. Recent force and position-fluctuation measurements indicate that pushing forces, mediated by the polymerization of astral microtubules against- the cell cortex, maintain the mitotic spindle at the cell center in Caenorhabditis elegans embryos. The magnitude of the centering forces suggests that the physical limit on the accuracy and precision of this centering mechanism is determined by the number of pushing microtubules rather than by thermally driven fluctuations. In cells that divide asymmetrically, anti-centering, pulling forces generated by cortically located dyneins, in conjunction with microtubule depolymerization, oppose the pushing forces to drive spindle displacements away from the center. Thus, a balance of centering pushing forces and anti-centering pulling forces localize the mitotic spindles within dividing C. elegans cells. © 2017 The Authors. BioEssays published by Wiley Periodicals, Inc.

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.

Microtubule depolymerization normalizes in vivo myocardial contractile function in dogs with pressure-overload left ventricular hypertrophy

Science.gov (United States)

Koide, M.; Hamawaki, M.; Narishige, T.; Sato, H.; Nemoto, S.; DeFreyte, G.; Zile, M. R.; Cooper G, I. V.; Carabello, B. A.

2000-01-01

BACKGROUND: Because initially compensatory myocardial hypertrophy in response to pressure overloading may eventually decompensate to myocardial failure, mechanisms responsible for this transition have long been sought. One such mechanism established in vitro is densification of the cellular microtubule network, which imposes a viscous load that inhibits cardiocyte contraction. METHODS AND RESULTS: In the present study, we extended this in vitro finding to the in vivo level and tested the hypothesis that this cytoskeletal abnormality is important in the in vivo contractile dysfunction that occurs in experimental aortic stenosis in the adult dog. In 8 dogs in which gradual stenosis of the ascending aorta had caused severe left ventricular (LV) pressure overloading (gradient, 152+/-16 mm Hg) with contractile dysfunction, LV function was measured at baseline and 1 hour after the intravenous administration of colchicine. Cardiocytes obtained by biopsy before and after in vivo colchicine administration were examined in tandem. Microtubule depolymerization restored LV contractile function both in vivo and in vitro. CONCLUSIONS: These and additional corroborative data show that increased cardiocyte microtubule network density is an important mechanism for the ventricular contractile dysfunction that develops in large mammals with adult-onset pressure-overload-induced cardiac hypertrophy.

Sensitivity of docetaxel-resistant MCF-7 breast cancer cells to microtubule-destabilizing agents including vinca alkaloids and colchicine-site binding agents.

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Richard C Wang

Full Text Available One of the main reasons for disease recurrence in the curative breast cancer treatment setting is the development of drug resistance. Microtubule targeted agents (MTAs are among the most commonly used drugs for the treatment of breaset cancer and therefore overcoming taxane resistance is of primary clinical importance. Our group has previously demonstrated that the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitivity to docetaxel due to the distinct expression profiles of β-tubulin isotypes in addition to the high expression of p-glycoprotein (ABCB1. In the present investigation we examined whether taxane-resistant breast cancer cells are more sensitive to microtubule destabilizing agents including vinca alkaloids and colchicine-site binding agents (CSBAs than the non-resistant cells.Two isogenic MCF-7 breast cancer cell lines were selected for resistance to docetaxel (MCF-7TXT and the wild type parental cell line (MCF-7CC to examine if taxane-resistant breast cancer cells are sensitive to microtubule-destabilizing agents including vinca alkaloids and CSBAs. Cytotoxicity assays, immunoblotting, indirect immunofluorescence and live imaging were used to study drug resistance, apoptosis, mitotic arrest, microtubule formation, and microtubule dynamics.MCF-7TXT cells were demonstrated to be cross resistant to vinca alkaloids, but were more sensitive to treatment with colchicine compared to parental non-resistant MCF-7CC cells. Cytotoxicity assays indicated that the IC50 of MCF-7TXT cell to vinorelbine and vinblastine was more than 6 and 3 times higher, respectively, than that of MCF-7CC cells. By contrast, the IC50 of MCF-7TXT cell for colchincine was 4 times lower than that of MCF-7CC cells. Indirect immunofluorescence showed that all MTAs induced the disorganization of microtubules and the chromatin morphology and interestingly each with a unique pattern. In terms of microtubule and chromain morphology, MCF-7TXT cells were

Daple Coordinates Planar Polarized Microtubule Dynamics in Ependymal Cells and Contributes to Hydrocephalus

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Maki Takagishi

2017-07-01

Full Text Available Motile cilia in ependymal cells, which line the cerebral ventricles, exhibit a coordinated beating motion that drives directional cerebrospinal fluid (CSF flow and guides neuroblast migration. At the apical cortex of these multi-ciliated cells, asymmetric localization of planar cell polarity (PCP proteins is required for the planar polarization of microtubule dynamics, which coordinates cilia orientation. Daple is a disheveled-associating protein that controls the non-canonical Wnt signaling pathway and cell motility. Here, we show that Daple-deficient mice present hydrocephalus and their ependymal cilia lack coordinated orientation. Daple regulates microtubule dynamics at the anterior side of ependymal cells, which in turn orients the cilial basal bodies required for the directional cerebrospinal fluid flow. These results demonstrate an important role for Daple in planar polarity in motile cilia and provide a framework for understanding the mechanisms and functions of planar polarization in the ependymal cells.

S. pombe CLASP needs dynein, not EB1 or CLIP170, to induce microtubule instability and slows polymerization rates at cell tips in a dynein-dependent manner

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Grallert, Agnes; Beuter, Christoph; Craven, Rachel A.; Bagley, Steve; Wilks, Deepti; Fleig, Ursula; Hagan, Iain M.

2006-01-01

The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and a screen for molecules that antagonized EB1 function. The conditional peg1.1 mutant enabled us to identify key features of Peg1 function. First, Peg1 was required to form a spindle and astral microtubules, yet destabilized interphase microtubules. Second, Peg1 was required to slow the polymerization rate of interphase microtubules that establish end-on contact with the cortex at cell tips. Third, Peg1 antagonized the action of S. pombe CLIP170 (Tip1) and EB1 (Mal3). Fourth, although Peg1 resembled higher eukaryotic CLASPs by physically associating with both Mal3 and Tip1, neither Tip1 nor Mal3 was required for Peg1 to destabilize interphase microtubules or for it to associate with microtubules. Conversely, neither Mal3 nor Tip1 required Peg1 to associate with microtubules or cell tips. Consistently, while mal3.Δ and tip1.Δ disrupted linear growth, corrupting peg1 + did not. Fifth, peg1.1 phenotypes resembled those arising from deletion of the single heavy or both light chains of fission yeast dynein. Furthermore, all interphase phenotypes arising from peg1 + manipulation relied on dynein function. Thus, the impact of S. pombe CLASP on interphase microtubule behavior is more closely aligned to dynein than EB1 or CLIP170. PMID:16951255

The Ndc80 internal loop is required for recruitment of the Ska complex to establish end-on microtubule attachment to kinetochores

DEFF Research Database (Denmark)

Zhang, Gang; Kelstrup, Christian D; Hu, Xiao-Wen

2012-01-01

The Ndc80 complex establishes end-on attachment of kinetochores to microtubules essential for chromosome segregation. The Ndc80 subunit is characterized by an N-terminal region, that binds directly to microtubules, and a long coiled-coil region that interacts with Nuf2. A loop region in Ndc80 tha...... chromosome segregation through the recruitment of specific proteins to the kinetochore....

Instrumentation for PSD based neutron diffractometers at Dhruva reactor

International Nuclear Information System (INIS)

Pande, S.S.; Borkar, S.P.; Prafulla, S.; Srivastava, V.D.; Behare, A.; Mukhopadhyay, P.K.; Ghodgaonkar, M.D.; Kataria, S.K.

2004-01-01

Linear position sensitive detectors (PSDs) are widely used to configure neutron diffractometers and other instruments. Necessary front-end electronics and a data acquisition system is developed to cater to such instruments built around the Dhruva research reactor in BARC. These include three diffractometers with multiple PSDs and four with single PSD. The front-end electronics consists of high voltage units, preamplifiers, shaping amplifiers, ratio ADCs (RDC). The data acquisition system consists of an interface card and software. Commercially available hardware like temperature controller or stepper motor controller connected over GPIB or RS232 are also integrated in the data acquisition system. The data acquisition is automated so that it can continue unattended for control parameter like temperature, thus enabling optimum utilization of available beam time. The instrumentation is scalable and can be easily configured for various instrumental requirements. The front-end electronics and the data acquisition system are described here. (author)

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.

Social defeat stress induces depression-like behavior and alters spine morphology in the hippocampus of adolescent male C57BL/6 mice

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Sergio D. Iñiguez

2016-12-01

Hippocampi were then dissected and Western blots were conducted to quantify protein levels for various markers important for synaptic plasticity including protein kinase M zeta (PKMζ, protein kinase C zeta (PKCζ, the dopamine-1 (D1 receptor, tyrosine hydroxylase (TH, and the dopamine transporter (DAT. Furthermore, we examined the presence of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-receptor subunit GluA2 as well as colocalization with the post-synaptic density 95 (PSD95 protein, within different spine subtypes (filopodia, stubby, long-thin, mushroom using an immunohistochemistry and Golgi-Cox staining technique. The results revealed that social defeat induced a depression-like behavioral profile, as inferred from decreased social interaction levels, increased immobility on the tail suspension test, and decreases in body weight. Whole hippocampal immunoblots revealed decreases in GluA2, with a concomitant increase in DAT and TH levels in the stressed group. Spine morphology analyses further showed that defeated mice displayed a significant decrease in stubby spines, and an increase in long-thin spines within the CA1 stratum radiatum. Further evaluation of GluA2/PSD95 containing-spines demonstrated a decrease of these markers within long-thin and mushroom spine types. Together, these results indicate that juvenile social stress induces GluA2- and dopamine-associated dysregulation in the hippocampus – a neurobiological mechanism potentially underlying the development of mood-related syndromes as a consequence of adolescent bullying.

Vitamin K3 disrupts the microtubule networks by binding to tubulin: a novel mechanism of its antiproliferative activity.

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Acharya, Bipul R; Choudhury, Diptiman; Das, Amlan; Chakrabarti, Gopal

2009-07-28

Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.

Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants.

Science.gov (United States)

Sotiriou, P; Giannoutsou, E; Panteris, E; Apostolakos, P; Galatis, B

2016-03-01

This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: : 1067-1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril

Experimental protocols for and studies of the effects of surface passivation and water isotopes on the gliding speed of microtubules propelled by kinesin-1

Science.gov (United States)

Maloney, Roger Andrew

This dissertation explores how the kinesin-1 and microtubule system is affected by surface passivation and water isotopes. Surface passivation was found to affect the gliding speed that microtubules exhibit in the gliding motility assay and the lengths of microtubules supported by the passivation. It was also found that gliding speeds of microtubules are very sensitive to temperature changes. Studies changing the water isotope were a first attempt to investigate if changing the solvent changed the osmotic pressure of the solution kinesin and microtubules were in. No osmotic pressure changes were observed, however, the experiments using different isotopes of water did illuminate the possibility that kinesin may be sensitive to viscosity changes in the solvent. This experiment also suggests further experiments that can be specifically designed to probe osmotic pressure changes. This thesis was also the first thesis ever, to the best of the author's knowledge, to be done in a completely open format. All information and notebook entries that are related to it, as well as the thesis itself, can be found on the website OpenWetWare. The thesis can also be found there including all the different versions that went into its editing. The philosophy and process of making data open and accessible to every one is also discussed.

Positioning of AMPA Receptor-Containing Endosomes Regulates Synapse Architecture

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Marta Esteves da Silva

2015-11-01

Full Text Available Lateral diffusion in the membrane and endosomal trafficking both contribute to the addition and removal of AMPA receptors (AMPARs at postsynaptic sites. However, the spatial coordination between these mechanisms has remained unclear, because little is known about the dynamics of AMPAR-containing endosomes. In addition, how the positioning of AMPAR-containing endosomes affects synapse organization and functioning has never been directly explored. Here, we used live-cell imaging in hippocampal neuron cultures to show that intracellular AMPARs are transported in Rab11-positive recycling endosomes, which frequently enter dendritic spines and depend on the microtubule and actin cytoskeleton. By using chemically induced dimerization systems to recruit kinesin (KIF1C or myosin (MyosinV/VI motors to Rab11-positive recycling endosomes, we controlled their trafficking and found that induced removal of recycling endosomes from spines decreases surface AMPAR expression and PSD-95 clusters at synapses. Our data suggest a mechanistic link between endosome positioning and postsynaptic structure and composition.

Integrins Regulate Apical Constriction via Microtubule Stabilization in the Drosophila Eye Disc Epithelium

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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.

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.

A novel spiroindoline targets cell cycle and migration via modulation of microtubule cytoskeleton.

Science.gov (United States)

Kumar, Naveen; Hati, Santanu; Munshi, Parthapratim; Sen, Subhabrata; Sehrawat, Seema; Singh, Shailja

2017-05-01

Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.

Microtubule and Cell Contact Dependency of ER-bound PTP1B Localization in Growth Cones

Science.gov (United States)

Fuentes, Federico

2009-01-01

PTP1B is an ER-bound protein tyrosine phosphatase implied in the regulation of cell adhesion. Here we investigated mechanisms involved in the positioning and dynamics of PTP1B in axonal growth cones and evaluated the role of this enzyme in axons. In growth cones, PTP1B consistently localizes in the central domain, and occasionally at the peripheral region and filopodia. Live imaging of GFP-PTP1B reveals dynamic excursions of fingerlike processes within the peripheral region and filopodia. PTP1B and GFP-PTP1B colocalize with ER markers and coalign with microtubules at the peripheral region and redistribute to the base of the growth cone after treatment with nocodazole, a condition that is reversible. Growth cone contact with cellular targets is accompanied by invasion of PTP1B and stable microtubules in the peripheral region aligned with the contact axis. Functional impairment of PTP1B causes retardation of axon elongation, as well as reduction of growth cone filopodia lifetime and Src activity. Our results highlight the role of microtubules and cell contacts in the positioning of ER-bound PTP1B to the peripheral region of growth cones, which may be required for the positive role of PTP1B in axon elongation, filopodia stabilization, and Src activity. PMID:19158394

Interaction of microtubules with active principles of Xanthium strumarium.

Science.gov (United States)

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

2001-01-01

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

Instrumentation for PSD-based neutron diffractometers at Dhruva reactor

Science.gov (United States)

Pande, S. S.; Borkar, S. P.; Prafulla, S.; Srivastava, V. D.; Behare, A.; Mukhopadhyay, P. K.; Ghodgaonkar, M. D.; Kataria, S. K.

2004-08-01

Linear position sensitive detectors (PSDs) are widely used to configure neutron diffractometers and other instruments. Necessary front-end electronics and a data acquisition system [1] is developed to cater to such instruments built around the Dhruva research reactor in BARC. These include three diffractometers with multiple PSDs and four with single PSD. The front-end electronics consists of high voltage units, preamplifiers [2], shaping amplifiers, ratio ADCs (RDC) [3]. The data acquisition system consists of an interface card and software. Commercially available hardware like temperature controller or stepper motor controller connected over GPIB or RS232 are also integrated in the data acquisition system. The data acquisition is automated so that it can continue unattended for control parameter like temperature, thus enabling optimum utilization of available beam time. The instrumentation is scalable and can be easily configured for various instrumental requirements. The front-end electronics and the data acquisition system are described here.

PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

International Nuclear Information System (INIS)

Seo, Kang-Sik; Hwang, Byung-Doo; Kim, Jong-Seok; Park, Ji-Hoon; Song, Kyoung-Sub; Yun, Eun-Jin; Park, Jong-Il; Kweon, Gi Ryang; Yoon, Wan-Hee; Lim, Kyu

2014-01-01

Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells. Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively. We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G 1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner. These results suggest that the synergy between PMA and apicularen A is involved by

Epothilones as lead structures for the synthesis-based discovery of new chemotypes for microtubule stabilization.

Science.gov (United States)

Feyen, Fabian; Cachoux, Frédéric; Gertsch, Jürg; Wartmann, Markus; Altmann, Karl-Heinz

2008-01-01

Epothilones are macrocyclic bacterial natural products with potent microtubule-stabilizing and antiproliferative activity. They have served as successful lead structures for the development of several clinical candidates for anticancer therapy. However, the structural diversity of this group of clinical compounds is rather limited, as their structures show little divergence from the original natural product leads. Our own research has explored the question of whether epothilones can serve as a basis for the development of new structural scaffolds, or chemotypes, for microtubule stabilization that might serve as a basis for the discovery of new generations of anticancer drugs. We have elaborated a series of epothilone-derived macrolactones whose overall structural features significantly deviate from those of the natural epothilone scaffold and thus define new structural families of microtubule-stabilizing agents. Key elements of our hypermodification strategy are the change of the natural epoxide geometry from cis to trans, the incorporation of a conformationally constrained side chain, the removal of the C3-hydroxyl group, and the replacement of C12 with nitrogen. So far, this approach has yielded analogs 30 and 40 that are the most advanced, the most rigorously modified, structures, both of which are potent antiproliferative agents with low nanomolar activity against several human cancer cell lines in vitro. The synthesis was achieved through a macrolactone-based strategy or a high-yielding RCM reaction. The 12-aza-epothilone ("azathilone" 40) may be considered a "non-natural" natural product that still retains most of the overall structural characteristics of a true natural product but is structurally unique, because it lies outside of the general scope of Nature's biosynthetic machinery for polyketide synthesis. Like natural epothilones, both 30 and 40 promote tubulin polymerization in vitro and at the cellular level induce cell cycle arrest in mitosis. These

PSD and NSPS Applicability Determination for Guardian Industries' Flat Glass Plant in Corsicana, Texas

Science.gov (United States)

This document may be of assistance in applying the New Source Review (NSR) air permitting regulations including the Prevention of Significant Deterioration (PSD) requirements. This document is part of the NSR Policy and Guidance Database. Some documents in the database are a scanned or retyped version of a paper photocopy of the original. Although we have taken considerable effort to quality assure the documents, some may contain typographical errors. Contact the office that issued the document if you need a copy of the original.

Design and Performance Analysis of Laser Displacement Sensor Based on Position Sensitive Detector (PSD)

International Nuclear Information System (INIS)

Song, H X; Wang, X D; Ma, L Q; Cai, M Z; Cao, T Z

2006-01-01

By using PSD as sensitive element, and laser diode as emitting element, laser displacement sensor based on triangulation method has been widely used. From the point of view of design, sensor and its performance were studied. Two different sensor configurations were described. Determination of the dimension, sensing resolution and comparison of the two different configurations were presented. The factors affecting the performance of the laser displacement sensor were discussed and two methods, which can eliminate the affection of dark current and environment light, are proposed

Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex.

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Nakamura, Masayoshi; Yagi, Noriyoshi; Kato, Takehide; Fujita, Satoshi; Kawashima, Noriyuki; Ehrhardt, David W; Hashimoto, Takashi

2012-07-01

Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat

Directory of Open Access Journals (Sweden)

Muller Sylviane

2008-07-01

Full Text Available Abstract Background During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. Results To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20°C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37°C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. Conclusion Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

Cell elasticity with altered cytoskeletal architectures across multiple cell types.

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Grady, Martha E; Composto, Russell J; Eckmann, David M

2016-08-01

The cytoskeleton is primarily responsible for providing structural support, localization and transport of organelles, and intracellular trafficking. The structural support is supplied by actin filaments, microtubules, and intermediate filaments, which contribute to overall cell elasticity to varying degrees. We evaluate cell elasticity in five different cell types with drug-induced cytoskeletal derangements to probe how actin filaments and microtubules contribute to cell elasticity and whether it is conserved across cell type. Specifically, we measure elastic stiffness in primary chondrocytes, fibroblasts, endothelial cells (HUVEC), hepatocellular carcinoma cells (HUH-7), and fibrosarcoma cells (HT 1080) subjected to two cytoskeletal destabilizers: cytochalasin D and nocodazole, which disrupt actin and microtubule polymerization, respectively. Elastic stiffness is measured by atomic force microscopy (AFM) and the disruption of the cytoskeleton is confirmed using fluorescence microscopy. The two cancer cell lines showed significantly reduced elastic moduli values (~0.5kPa) when compared to the three healthy cell lines (~2kPa). Non-cancer cells whose actin filaments were disrupted using cytochalasin D showed a decrease of 60-80% in moduli values compared to untreated cells of the same origin, whereas the nocodazole-treated cells showed no change in elasticity. Overall, we demonstrate actin filaments contribute more to elastic stiffness than microtubules but this result is cell type dependent. Cancer cells behaved differently, exhibiting increased stiffness as well as stiffness variability when subjected to nocodazole. We show that disruption of microtubule dynamics affects cancer cell elasticity, suggesting therapeutic drugs targeting microtubules be monitored for significant elastic changes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hepatocyte cotransport of taurocholate and bilirubin glucuronides: Role of microtubules

International Nuclear Information System (INIS)

Crawford, J.M.; Gollan, J.L.

1988-01-01

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

CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions

DEFF Research Database (Denmark)

Shahbazi, Marta N; Megias, Diego; Epifano, Carolina

2013-01-01

Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. In this paper, we report that the ...

Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

OpenAIRE

Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

2009-01-01

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one o...

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

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of AMPA receptor signaling

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Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-01-01

Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. PMID:27687706

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor signaling.

Science.gov (United States)

Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-09-01

Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Perinatal exposure to lead (Pb) induces ultrastructural and molecular alterations in synapses of rat offspring.

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Gąssowska, Magdalena; Baranowska-Bosiacka, Irena; Moczydłowska, Joanna; Frontczak-Baniewicz, Małgorzata; Gewartowska, Magdalena; Strużyńska, Lidia; Gutowska, Izabela; Chlubek, Dariusz; Adamczyk, Agata

2016-12-12

noticed in the cerebellum, while the expression of postsynaptic PSD-95 was significantly decreased in forebrain cortex and cerebellum, and raised in hippocampus. Additionally, we observed the lower level of BDNF in all brain structures in comparison to control animals. In conclusion, perinatal exposure to low doses of Pb caused pathological changes in nerve endings associated with the alterations in the level of key synaptic proteins. All these changes can lead to synaptic dysfunction, expressed by the impairment of the secretory mechanism and thereby to the abnormalities in neurotransmission as well as to the neuronal dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Assay of Calcium Transients and Synapses in Rat Hippocampal Neurons by Kinetic Image Cytometry and High-Content Analysis: An In Vitro Model System for Postchemotherapy Cognitive Impairment.

Science.gov (United States)

McDonough, Patrick M; Prigozhina, Natalie L; Basa, Ranor C B; Price, Jeffrey H

2017-07-01

Postchemotherapy cognitive impairment (PCCI) is commonly exhibited by cancer patients treated with a variety of chemotherapeutic agents, including the endocrine disruptor tamoxifen (TAM). The etiology of PCCI is poorly understood. Our goal was to develop high-throughput assay methods to test the effects of chemicals on neuronal function applicable to PCCI. Rat hippocampal neurons (RHNs) were plated in 96- or 384-well dishes and exposed to test compounds (forskolin [FSK], 17β-estradiol [ES]), TAM or fulvestrant [FUL], aka ICI 182,780) for 6-14 days. Kinetic Image Cytometry™ (KIC™) methods were developed to quantify spontaneously occurring intracellular calcium transients representing the activity of the neurons, and high-content analysis (HCA) methods were developed to quantify the expression, colocalization, and puncta formed by synaptic proteins (postsynaptic density protein-95 [PSD-95] and presynaptic protein Synapsin-1 [Syn-1]). As quantified by KIC, FSK increased the occurrence and synchronization of the calcium transients indicating stimulatory effects on RHN activity, whereas TAM had inhibitory effects. As quantified by HCA, FSK also increased PSD-95 puncta and PSD-95:Syn-1 colocalization, whereas ES increased the puncta of both PSD-95 and Syn-1 with little effect on colocalization. The estrogen receptor antagonist FUL also increased PSD-95 puncta. In contrast, TAM reduced Syn-1 and PSD-95:Syn-1 colocalization, consistent with its inhibitory effects on the calcium transients. Thus TAM reduced activity and synapse formation by the RHNs, which may relate to the ability of this agent to cause PCCI. The results illustrate that KIC and HCA can be used to quantify neurotoxic and neuroprotective effects of chemicals in RHNs to investigate mechanisms and potential therapeutics for PCCI.

The imidazopyridine derivative JK184 reveals dual roles for microtubules in Hedgehog signaling.

Science.gov (United States)

Cupido, Tommaso; Rack, Paul G; Firestone, Ari J; Hyman, Joel M; Han, Kyuho; Sinha, Surajit; Ocasio, Cory A; Chen, James K

2009-01-01

Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).

Microtubule affinity-regulating kinases are potential druggable targets for Alzheimer's disease.

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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.

TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

Science.gov (United States)

Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

2013-01-01

Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

Development of the laser alignment system with PSD used for shaft calibration

Science.gov (United States)

Jiao, Guohua; Li, Yulin; Hu, Baowen

2006-02-01

Shaft calibration is an important technique during installation and maintenance of a rotating machine. It requires unique and high-precision measurement instruments with calculation capability, and relies on experience on heavy, high-speed, or high-temperature machines. A high-precision laser alignment system has been designed using PSD (Position Sensing Detector) to change traditional manual way of shaft calibration and to make the measurement easier and more accurate. The system is comprised of two small measuring units (Laser transmitter and detector) and a hand operated control unit or a PC. Such a laser alignment system has been used in some actual shaft alignment with offset resolution 1.5μm and angular resolution 0.1°.

The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.

Science.gov (United States)

Kolobova, Elena; Roland, Joseph T; Lapierre, Lynne A; Williams, Janice A; Mason, Twila A; Goldenring, James R

2017-12-15

Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.

High Dynamics and Precision Optical Measurement Using a Position Sensitive Detector (PSD in Reflection-Mode: Application to 2D Object Tracking over a Smart Surface

Directory of Open Access Journals (Sweden)

Ioan Alexandru Ivan

2012-12-01

Full Text Available When related to a single and good contrast object or a laser spot, position sensing, or sensitive, detectors (PSDs have a series of advantages over the classical camera sensors, including a good positioning accuracy for a fast response time and very simple signal conditioning circuits. To test the performance of this kind of sensor for microrobotics, we have made a comparative analysis between a precise but slow video camera and a custom-made fast PSD system applied to the tracking of a diffuse-reflectivity object transported by a pneumatic microconveyor called Smart-Surface. Until now, the fast system dynamics prevented the full control of the smart surface by visual servoing, unless using a very expensive high frame rate camera. We have built and tested a custom and low cost PSD-based embedded circuit, optically connected with a camera to a single objective by means of a beam splitter. A stroboscopic light source enhanced the resolution. The obtained results showed a good linearity and a fast (over 500 frames per second response time which will enable future closed-loop control by using PSD.

Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein

DEFF Research Database (Denmark)

Sørensen, Rikke Kruse; Krantz, James; Barker, Natalie

2017-01-01

. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and also revealed MARK2 can co-IP SOGA1......, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and also with tubulin, which identifies SOGA1 as a new microtubule-associated protein....... These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology....

Comparative study of large samples (2'' × 2'') plastic scintillators and EJ309 liquid with pulse shape discrimination (PSD) capabilities

International Nuclear Information System (INIS)

Iwanowska-Hanke, J; Moszynski, M; Swiderski, L; Sibczynski, P; Szczesniak, T; Krakowski, T; Schotanus, P

2014-01-01

In the paper we reported on the scintillation properties and pulse shape discrimination (PSD) performance of new plastic scintillators. The samples with dimension of 2 inches × 2 inches were tested: EJ299-34, EJ299-34G, EJ299-33 and EJ299-33G. They are the first commercially available plastics with neutron/gamma discrimination properties. The paper covers the measurements of emission spectra, photoelectron yield, analysis of the light pulse shapes originating from events related to gamma-rays and fast neutrons as well as neutron/gamma discrimination. The tested plastics are characterized by a photoelectron yield on a level of approximately 1600-2500 phe/MeV, depending on the sample. The highest value, measured for EJ299-34, is similar to the number of photoelectrons measured for EJ309 (2600 phe/MeV). The figure of merit (FOM) calculated for narrow energy cuts — indicating the PSD performance — showed that the PSD capabilities of the plastics are significantly lower than of EJ309. These scintillators are still under development in order to optimize the composition and manufacturing procedures. At this time the results obtained with the new plastics suggest their possible use as an alternative for liquid scintillators, especially if we consider their inflammability and non-toxicity

Microtubule Abnormalities Underlying Gulf War Illness in Neurons from Human-Induced Pluripotent Cells