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Identification of an activity that inhibits tubulin polymerization in vitro . ( A ) Schematic representation of the purification strategy. ( B ) Effect of fractions on microtubule assembly after tubulin affinity chromatography. Mean numbers of microtubules, expressed as a percentage of a control experiment, are indicated. Note the inhibition of microtubule assembly in the 0.25 M KCl eluate from the tubulin column which is absent in the respective myoglobin column. Immunofluorescence micrographs of assembly reactions from a control experiment (buffer) and the respective 0.25 M KCl eluates are shown at the bottom. ( C ) Effect of the inhibitory activity in a microtubule sedimentation assay. A representative anti-tubulin dot-blot and the quantitation of pelleted material are shown. The mean value and standard deviation from three independent experiments are indicated. Affinity chromatography, microtubule assembly and microtubule sedimentation assays were performed as described in Materials and methods. Samples of the non-microtubule fraction after Sephadex G-10 chromatography corresponding to the material from 80 confluent 15 cm TC dishes were divided into two equal portions and loaded onto the myoglobin and tubulin affinity column, respectively. Then 1% (v/v) of each fraction was tested for inhibitory activity in microtubule assembly assays in the presence of 1.5 μ M tau. For the sedimentation assay, assembly was performed as described above, polymerized microtubules were separated by ultracentrifugation, immobilized on PVDF and immunodetected as described in Materials and methods. For quantitation, the amount of polymerized tubulin was normalized to a positive ( ϩ tau, –inhibitor, represented as 100%) and a negative (–tau, –inhibitor, represented as 0%) control.
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In eukaryotic cells, tubulin polymerization must be regulated precisely during cell division and differentiation. To identify new mechanisms involved in cellular microtubule formation, we isolated an activity that suppresses microtubule nucleation in vitro. The activity was due to a small acidic polypeptide of 4.7 kDa which we named MINUS (microtub...
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... interacts with tubulin but not with microtubules ( Figure 1A). As a sensitive assay, tau-dependent micro- tubule assembly assays were used to detect inhibitory activity (Fanara and Brandt, 1997). ...
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... purify further the inhibitory activity based on its interaction with dimeric tubulin, affinity chromatography on immobilized purified tubulin (PC-tubulin) was per- formed. Inhibitory activity, indicated by a strong decrease in microtubule number in our assay, was present in the 0.25 M KCl eluate from the tubulin column ( Figure 1B). No such activity was found in the corresponding eluate from a myoglobin column used as a control for unspecific binding. ...
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... such activity was found in the corresponding eluate from a myoglobin column used as a control for unspecific binding. The presence of the inhibitory activity was confirmed by a microtubule sedimentation assay used as an independent method ( Figure 1C); only ~5% of tubulin sedimented in polymeric form in the presence of the inhibitor compared with an assembly reaction in the presence of tau. In addition to inhibitory activity in the Table I. ...
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... brain was homogenized and cytosolic extract was prepared by differential centrifugation. The cytosol was subjected to the same purification steps as described in Figure 1A. After affinity chromatography, an activity that inhibited tau-dependent microtubule assembly was detected in the 0.1-0.25 M eluates ( Figure 6A). ...
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... portions of the protein eluates were dialyzed against BRB80 and loaded on the myoglobin and tubulin affinity column, respectively. Eluates were tested in standard microtubule assembly assays as described in the legend of Figure 1. Fractions eluting at 0.1 and 0.25 M KCl after tubulin affinity chromatography were combined and loaded onto the Superdex 200 HR and the eluted fractions tested in assembly assays as described in Figure 1. ...
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... were tested in standard microtubule assembly assays as described in the legend of Figure 1. Fractions eluting at 0.1 and 0.25 M KCl after tubulin affinity chromatography were combined and loaded onto the Superdex 200 HR and the eluted fractions tested in assembly assays as described in Figure 1. Active fractions were pooled, brought to 10 mM Tris-HCl and loaded on the MonoQ column. ...
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... fractions were pooled, brought to 10 mM Tris-HCl and loaded on the MonoQ column. Samples of the eluates were tested for activity as described in Figure 1. In (C), microtubules were assembled in the presence of tau (2 µM) and various amounts of the inhibitor after the MonoQ column as described in the legend of Figure 4. Active fractions were iodinated and separated by 2D-PAGE as described in Materials and methods. ...
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... In most non-neuronal cells, tubulin dimers and microtubule polymers require a rapid dynamic balance (Fanara et al. 2010). Whereas, in neurons, axonal and dendritic MT proved to be less dynamic in their rapid turnover due to the interaction with a specific subclass of microtubule-associated proteins that maintain the integrity of the microtubule-based axonal transport (Fanara et al. 1999(Fanara et al. , 2007. Stabilization of hyperdynamic MT is found to be related to neuroprotection in motor neuron degeneration (Fanara et al. 2007). ...
Extensive researches have deepened knowledge on the role of synaptic components in epileptogenesis, but limited attention has been devoted to the potential implication of the cytoskeleton. The study of the development of epilepsy and hyperexcitability states involves molecular, synaptic, and structural alterations of neuronal bioelectric activity. In this paper we aim to explore the neurobiological targets involved in microtubule functioning and cytoskeletal transport, i.e. how dynamic scaffolding of microtubules can influence neuronal morphology and excitability, in order to suggest a potential role for microtubule dynamics in the processes turning a normal neuronal network in a hyperexcited one. Pathophysiological alterations of microtubule dynamics inducing neurodegeneration, network remodeling and relative impairment on synaptic transmission were overviewed. Recent researches were reported on the phosphorylation state of microtubule-associated proteins such as tau in neurodegenerative diseases and epileptic states, but also on the effect of microtubule-active agents influencing cytoskeleton destabilization in epilepsy models. The manipulation of microtubule polymerization was found effective in the modulation of hyperexcitability. In addition, it was considered the importance of microtubules and related neurotrophic factors during neural development since they are essential for the formation of a properly functional neuronal network. Otherwise, this can lead to cognitive deficits, hyperexcitability phenomena and neurodevelopmental disorders. Lastly, we evaluated the role of microtubule dynamics on neuronal efficiency considering their importance in the transport of mitochondria, cellular elements fulfilling energy requirements for neuronal activity, and a putative influence on cannabinoid-mediated neuroprotection. This review provides novel perspectives for the implication of microtubule dynamics in the development of epileptic phenomena.
... In most non-neuronal cells, tubulin dimers and microtubule polymers exist in rapid dynamic equilibrium, as recently shown in vivo by isotopic labeling [6]. In neurons, however, this rapid turnover of axonal and dendritic microtubules is believed to be less dynamic due to their interactions with a specific subclass of microtubule-associated proteins [7]. This stability of microtubules is presumed to be necessary to maintain the integrity of the microtubule-based axonal transport [8]. ...
Approximately 30 % of epilepsy cases are refractory to current pharmacological treatments through unknown mechanisms. Much work has been done on the role of synaptic components in the pathogenesis of epilepsy, but relatively little attention has been given to the potential role of the microtubules. We investigated the level of microtubule dynamic in 30 human epileptic tissues and two different chronic epilepsy rat models. The administration of microtubule-modulating agent attenuated the progression of chronic epilepsy. By contrast, microtubule-depolymerizing agent aggravated the progression of chronic epilepsy. The electrophysiological index by whole-cell clamp was used to investigate the neuronal excitation and inhibitory synaptic transmission in brain slices after administration of microtubule-modulating agent and microtubule-depolymerizing agent. Interestingly, we found that microtubule-modulating agent significantly increased the frequency of action potential firing in interneurons, and significantly promoted the amplitudes and frequencies of miniature inhibitory postsynaptic currents. Microtubule-depolymerizing agent had an opposite effect. These findings suggest that modulating hyperdynamic microtubules may take an anti-epileptic effect via postsynaptic mechanisms in interneurons. It could represent a potential pharmacologic target in epilepsy treatment.
... The scraped cellular suspension was centrifuged at 4,000g at 20 °C for 20 min. The pellet was dissolved and homogenized in MSB-containing protease and phosphatase inhibitor cocktails as previously described (Fanara et al., 1999; Fanara et al., 2004; Fanara et al., 2007). ...
Synaptic plasticity plays a crucial role in learning, memory, and cognitive disorders. Cytoskeletal reorganization underlies neuronal synaptic plasticity, but little is known about the regulation of cytoskeletal dynamics in living animals. We used stable isotope labeling to measure the turnover of tubulin in defined microtubule (MT) populations in murine brain. Neuronal MTs generally exhibited low turnover rates in vivo. Basal turnover was highest in tau-associated MTs, intermediate in microtubule-associated protein 2 (MAP2)-associated MTs, and lowest in cold-stable MTs. Labeling of MTs in mature neurons in cell culture yielded similar turnover results. Intracerebroventricular glutamate injection stimulated, via N-methyl-D-aspartic acid receptors, label incorporation (turnover) in cold-stable, tau-associated, and MAP2-associated MTs, the last of which was shown to be dependent on cyclic adenosine-3', 5'-monophosphorothioate-protein kinase A. Contextual fear conditioning, a hippocampus-mediated form of memory formation, was accompanied by increased turnover of hippocampal MAP2-associated and cold-stable MTs. Treatment with the MT-depolymerizing drug nocodazole reversed the conditioning-induced increase in label incorporation in MAP2-associated MTs, reduced dendritic spine density, and impaired memory formation. The effects of nocodazole on MT turnover were prevented by the MT-stabilizing agent Taxol (Sigma-Aldrich, St. Louis, MO, USA) and by brain-derived nerve growth factor, both of which also restored dendritic spine density and memory formation in this model. In conclusion, these results suggest that changes in hippocampal MT turnover are required for, and are a biomarker of, the synaptic plasticity that is involved in memory formation.
... Stathmin inhibits microtubule polymerization in vitro by sequestering tubulin [2]. MINUS is a 4.7 kDa polypeptide which inhibits in vitro and in vivo microtubule nucleation and is inactivated by dephosphorylation [4]. Tubulin-folding cofactor D participates in tubulin heterodimer formation through a step involving the hydrolysis of GTP on β-tubulin [5]. ...
Microtubules play an essential role in eukaryotic cells, where they perform a wide variety of functions. In this paper, we describe the characterization of proteins associated to tubulin dimer in its native form, using affinity chromatography and mass spectrometry. We used an immunoaffinity column with coupled-monoclonal antibody directed against the alpha-tubulin C-terminus. Tubulin was first loaded onto the column, then interphase and mitotic cell lysates were chromatographed. Tubulin-binding proteins were eluted using a peptide mimicking the alpha-tubulin C-terminus. Elution fractions were analyzed by SDS-PAGE, and a total of 14 proteins were identified with high confidence by mass spectrometry. These proteins could be grouped in four classes: known tubulin-binding proteins, one microtubule-associated protein, heat shock proteins, and proteins that were not shown previously to bind tubulin dimer or microtubules.
... Crosslinked oligomers were recovered from disassembly products. For microtubule cross-linking, we followed previously published methods (23)(24)(25). Purified tubulin (100 M) was assembled for 20 min at 37°C in a buffer containing 80 mM Pipes (pH 6.7), 1 mM EGTA, 50% (v/v) glycerol, 5 mM MgCl 2 , and 1 mM GTP. EGS was then added at 3.4 mM final concentration for 15 min. To quench the EGS in excess, the mixture was diluted into 9 volumes of a buffer containing 80 mM Pipes (pH 6.7), 1 mM EGTA, 50% sucrose, 10 mM glutamate, and 1 mM MgCl 2 and incubated for 1 h at room temperature. ...
Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation
is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates
between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks
of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly. Here we have tested this
model directly by isolating nucleation-competent cross-linked tubulin oligomers. We show that such oligomers are composed
of 10–15 laterally associated tubulin dimers. In the presence of added free tubulin dimers, several oligomers combine to form
microtubule nuclei competent for elongation. We provide evidence that these nuclei have heterogeneous structures, indicating
unexpected flexibility in nucleation pathways. Our results suggest that microtubule nucleation in purified tubulin solution
is mechanistically similar to that templated by γ-tubulin ring complexes with the exception that in the absence of γ-tubulin
complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process.
... Micro-injecté dans des cellules, MINUS provoque une dépolymérisation transitoire des microtubules. MINUS est le premier effecteur du cytosquelette microtubulaire identifié agissant uniquement sur la nucléation (Fanara et al., 1999). ...
... Les fragments de microtubules ainsi obtenus sont stables et collectés par centrifugation puis repris en solution. Les observations en microscopie électronique montrent que les solutions ainsi préparées contiennent des microtubules mesurant environ 1 µm (Fanara et al., 1999;Symmons et al., 1996). Ces solutions de fragments de microtubules stimulent l'assemblage des microtubules dans des conditions où il n'y a pas d'assemblage spontané. ...
... Des études antérieures avaient décrit la production d'amorces de microtubules par pontage covalent de microtubules (Fanara et al., 1999;Koshland et al., 1988;Symmons et al., 1996). Ces auteurs ont utilisé des conditions de pontage par l'EGS qui stabilisaient des fragments de microtubules courts (environ 1 µm). ...
Microtubules play a crucial role in cellular organisation and function. Microtubule assembly display puzzling non-linear features, like dynamic instability. Here, we intended first to determine the limiting factors of microtubule assembly, and second to identify new effectors of this process. The kinetics of the reaction were studied in simple experimental conditions, allowing simultaneous and precise measurement of all the parameters of the reaction. Our results indicate that the microtubule nucleation occurs in two steps : rapid and irreversible formation of tubulin oligomers then aggregation of these oligomers into a microtubule. The elongation rate of the microtubule appears to be independent of the GTP-tubulin concentration : this elongation rate is probably limited by structural properties of the microtubules. Finally, microtubule disassembly would occur through catastrophes, and would be triggered by tubulin oligomers. The tubulin oligomers controlling disassembly could possibly be the same as those controlling microtubule nucleation. We then produced tubulin oligomers triggering microtubule nucleation by a moderate covalent crosslinking of microtubules. These oligomers are constituted by lateral interactions of 10 tubulin dimmers, four of which aggregate to form a microtubule. Microtubules assembled in our conditions in the presence of nucleating tubulin oligomers do not spontaneously disassemble : these conditions are suitable to look for catastrophe factors. Finally, we developed an affinity chromatographic procedure to purify tubulin-binding proteins in a native state. Thus, these tubulin partners are amenable to functional tests. Peptide mapping and mass spectrometry allowed us to identify the purified proteins. Some are known to control microtubule assembly, but others are possibly new effectors of microtubule dynamics.
... Ethylene glycol bis-succinimidyl succinate (EGS) cross-linked MTs were prepared (Fanara et al., 1999) in the presence of biotinylated tubulin (7.2 was further purified by Mono S chromatography and the resulting fractions were analyzed on a 10% SDS-PAGE gel and stained with Coomassie blue. (E) MT nucleating activity of the TuSC from the Mono S column as compared with corresponding fractions of a control Mono S purification of peptide alone (from one representative experiment ). ...
The γ-tubulin ring complex (γTuRC) is important for microtubule nucleation from the centrosome. In addition to γ-tubulin,
the Drosophila γTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both
the small γ-tubulin complex (γTuSC) and the γTuRC, while the remaining subunits are found only in the γTuRC. To study γTuRC
assembly and function, we first reconstituted γTuSC using the baculovirus expression system. Using the reconstituted γTuSC,
we showed for the first time that this subcomplex of the γTuRC has microtubule binding and capping activities. Next, we characterized
two new γTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known
γTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can
each interact with γTuSC. However, this interaction is insufficient for γTuRC assembly.
... Ethylene glycol bis-succinimidyl succinate (EGS) cross-linked MTs were prepared (Fanara et al., 1999) in the presence of biotinylated tubulin (7.2 for 15 min at 23ЊC followed by an additional 15 min of incubation with 5 l of streptavidin-coupled magnetic beads preblocked with 1 mg/ml BSA (Dynal). ␥TuSC in the supernatant and the beads were analyzed by Western blotting with antibodies against ␥-tubulin. ...
Paclitaxel is an anticancer drug that stabilizes microtubules by suppressing their dynamic instability. It binds to the lumen of the microtubules and enhances subunit interaction. In the present work, we show that the critical concentration of tubulins required to polymerize microtubules follows an inverse power law relation with the amount of paclitaxel added during polymerization. With an increase in paclitaxel concentration from 0.1 μM to 10 μM, the critical nucleation concentration decreases by 89%. We propose stabilization of microtubule nuclei in the presence of taxol for this observation. In addition, lowering in the critical nucleation concentration left fewer tubulins for growth, which resulted in shorter microtubules. Addition of 10 μM taxol resulted in reduced steady state length of microtubules by 37.5%. These observations are explained based on microtubule nuclei stabilization and subsequent depletion of tubulin pool. We also show mathematically, that taxol reduces the rate constant of shortening more prominently (by 100 folds), compared to the rate constant of elongation.
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."
