Concentration Of Microtubules Research Articles

Molecular investigations into the unfoldase action of severing enzymes on microtubules.

Microtubule (MT)-associated proteins regulate the dynamic behavior of MTs during cellular processes. MT severing enzymes are the associated proteins which destabilize MTs by removing subunits from the lattice. One model for how severing enzymes remove tubulin dimers from the MT lattice is by unfolding its subunits through pulling on the carboxy-terminal tails of tubulin dimers. This model stems from the fact that severing enzymes are AAA+ unfoldases. To test this mechanism, we apply pulling forces on the carboxy-terminal regions of MT subunits using coarse grained molecular simulations. In our simulations, we used different MT lattices and concentrations of severing enzymes. We compare our simulation results with data from in vitro severing assays and find that the experimental data is best fit by a model of cooperative removal of protofilament fragments by severing enzymes, which depends on the severing enzyme concentration and placement on the MT lattice.

The formin Drosophila homologue of Diaphanous2 (Diaph2) controls microtubule dynamics in colorectal cancer cells independent of its FH2-domain

In this study, we analyzed the functional role of the formin Drosophila Homologue of Diaphanous2 (Diaph2) in colorectal cancer cells. We show that stable down-regulation of Diaph2 expression in HT29 cells decreased chromosome alignment and the velocity of chromosome movement during M-phase, thus reducing the proliferation rate and colony formation. In interphase cells, Diaph2 was diffusely distributed in the cytosol, while in metaphase cells the protein was located to spindle microtubules (MTs). Diaph2-depletion increased the concentration of stable spindle MTs, showing that the formin is required to control spindle MT-dynamics. Our cellular data indicate that Diaph2-controls spindle MT-dynamics independent of Cdc42 activity and our in vitro results reveal that bacterially produced full-length (FL) Diaph2 strongly altered MT-dynamics in absence of Cdc42, where its actin-nucleating activity is auto-inhibited. FL-Diaph2 mediates a 10-fold increase in MT-polymerization compared to the Diaph2-FH2-domain. Interestingly, a Diaph2-mutant lacking the FH2-domain (ΔFH2) increased MT-polymerization to a similar extent as the FH2-domain, indicating the existence of a second MT-binding domain. However, in contrast to FL-Diaph2 and the FH2-domain, ΔFH2 did not alter the density of taxol-stabilized MTs. Thus, the FH2-domain and the second Diaph2-binding domain appear to control MT-dynamics by different mechanisms. In summary, our data indicate that Diaph2 controls M-phase progression under basal conditions by regulating spindle MT-dynamics. In addition, a region outside of the canonical MT-regulating FH2-domain is involved in Diaph2-mediated control of MT-dynamics.

Response to Alternating Electric Fields of Tubulin Dimers and Microtubule Ensembles in Electrolytic Solutions

Microtubules (MTs), which are cylindrical protein filaments that play crucial roles in eukaryotic cell functions, have been implicated in electrical signalling as biological nanowires. We report on the small-signal AC (“alternating current”) conductance of electrolytic solutions containing MTs and tubulin dimers, using a microelectrode system. We find that MTs (212 nM tubulin) in a 20-fold diluted BRB80 electrolyte increase solution conductance by 23% at 100 kHz, and this effect is directly proportional to the concentration of MTs in solution. The frequency response of MT-containing electrolytes exhibits a concentration-independent peak in the conductance spectrum at 111 kHz (503 kHz FWHM that decreases linearly with MT concentration), which appears to be an intrinsic property of MT ensembles in aqueous environments. Conversely, tubulin dimers (42 nM) decrease solution conductance by 5% at 100 kHz under similar conditions. We attribute these effects primarily to changes in the mobility of ionic species due to counter-ion condensation effects, and changes in the solvent structure and solvation dynamics. These results provide insight into MTs’ ability to modulate the conductance of aqueous electrolytes, which in turn, has significant implications for biological information processing, especially in neurons, and for intracellular electrical communication in general.

Controlling the bias of rotational motion of ring-shaped microtubule assembly.

Biomolecular motor system microtubule (MT)-kinesin is considered a building block for developing artificial microdevices. Recently, an active self-organization method has been established to integrate MT filaments into ring-shaped assembly that can produce rotational motion both in the clockwise and in the counterclockwise directions. In this work, we have investigated the effect of parameters such as MT and kinesin concentration, length, and rigidity of MT and type of kinesin (structure of tail region) on the preferential rotation of the ring-shaped MT assembly produced in an active self-organization. We elucidated that these factors can significantly affect the bias of rotation of the ring-shaped MT assembly, which seems to be related to the fluctuation of leading tip of moving MT filaments. This new finding might be important for designing handedness regulated artificial biomachine using the ring-shaped MT assembly in future.

Nematic ordering pattern formation in the process of self-organization of microtubules in a gravitational field

By virtue of a functional scaling, the free energy for cytoskeletal microtubule (MT) solution system in the gravitational field has been proposed theoretically, and on this basis the influence of gravitational field on MT’s self-organization process is studied. A concentration gradient coupled with orientational order characteristic of nematic ordering pattern formation is the new feature emerging in the presence of gravity. Theoretical calculation results show that gravity facilitates the isotropic to nematic phase transition, which is reflected in a significantly broader transition region and the phase coexistence region increases with increasing g or MT concentration. We also discuss the numerical results obtained due to local MT concentration changing with the height of the vessel and some phase transition properties.

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded theCENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.

Interactions between Subunits in Heterodimeric Ncd Molecules

The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a single-headed molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.

The Homotetrameric Kinesin-5 KLP61F Preferentially Crosslinks Microtubules into Antiparallel Orientations

The segregation of genetic material during mitosis is coordinated by the mitotic spindle, whose action depends upon the polarity patterns of its microtubules (MTs). Homotetrameric mitotic kinesin-5 motors can crosslink and slide adjacent spindle MTs, but it is unknown whether they or other motors contribute to establishing these MT polarity patterns. Here, we explored whether the Drosophila embryo kinesin-5 KLP61F, which plausibly crosslinks both parallel and antiparallel MTs, displays a preference for parallel or antiparallel MT orientation. In motility assays, KLP61F was observed to crosslink and slide adjacent MTs, as predicted. Remarkably, KLP61F displayed a 3-fold higher preference for crosslinking MTs in the antiparallel orientation. This polarity preference was observed in the presence of ADP or ATP plus AMPPNP, but not AMPPNP alone, which induces instantaneous rigor binding. Also, a purified motorless tetramer containing the C-terminal tail domains displayed an antiparallel orientation preference, confirming that motor activity is not required. The results suggest that, during morphogenesis of the Drosophila embryo mitotic spindle, KLP61F's crosslinking and sliding activities could facilitate the gradual accumulation of KLP61F within antiparallel interpolar MTs at the equator, where the motor could generate force to drive poleward flux and pole-pole separation.

Self-Organization of Anastral Spindles by Synergy of Dynamic Instability, Autocatalytic Microtubule Production, and a Spatial Signaling Gradient

Assembly of the mitotic spindle is a classic example of macromolecular self-organization. During spindle assembly, microtubules (MTs) accumulate around chromatin. In centrosomal spindles, centrosomes at the spindle poles are the dominating source of MT production. However, many systems assemble anastral spindles, i.e., spindles without centrosomes at the poles. How anastral spindles produce and maintain a high concentration of MTs in the absence of centrosome-catalyzed MT production is unknown. With a combined biochemistry-computer simulation approach, we show that the concerted activity of three components can efficiently concentrate microtubules (MTs) at chromatin: (1) an external stimulus in form of a RanGTP gradient centered on chromatin, (2) a feed-back loop where MTs induce production of new MTs, and (3) continuous re-organization of MT structures by dynamic instability. The mechanism proposed here can generate and maintain a dissipative MT super-structure within a RanGTP gradient.

A Role for NuSAP in Linking Microtubules to Mitotic Chromosomes

The spindle apparatus is a microtubule (MT)-based machinery that attaches to and segregates the chromosomes during mitosis and meiosis. Self-organization of the spindle around chromatin involves the assembly of MTs, their attachment to the chromosomes, and their organization into a bipolar array. One regulator of spindle self-organization is RanGTP. RanGTP is generated at chromatin and activates a set of soluble, Ran-regulated spindle factors such as TPX2, NuMA, and NuSAP . How the spindle factors direct and attach MTs to the chromosomes are key open questions. Nucleolar and Spindle-Associated Protein (NuSAP) was recently identified as an essential MT-stabilizing and bundling protein that is enriched at the central part of the spindle . Here, we show by biochemical reconstitution that NuSAP efficiently adsorbs to isolated chromatin and DNA and that it can directly produce and retain high concentrations of MTs in the immediate vicinity of chromatin or DNA. Moreover, our data reveal that NuSAP-chromatin interaction is subject to Ran regulation and can be suppressed by Importin alpha (Impalpha) and Imp7. We propose that the presence of MT binding agents such as NuSAP, which can be directly immobilized on chromatin, are critical for targeting MT production to vertebrate chromosomes during spindle self-organization.

Verticillium dahliae toxin induced alterations of cytoskeletons and nucleoli in Arabidopsis thaliana suspension cells

In plant cells, cytoskeletons play important roles in response to biotic and abiotic stresses. However, little is known about the dynamics of cytoskeletons when cells are attacked by unphysical stress factors such as elicitors and toxins. We report here that the toxin of Verticillium dahliae (VD toxin) induced changes of microfilaments (MFs) and microtubules (MTs) in Arabidopsis thaliana suspension-cultured cells. When cells were treated with a low concentration of VD toxin, MFs were disrupted ordinally from the cortex to the perinuclear region, and then recovered spontaneously; but the MTs persisted. The MFs in the perinuclear region showed more resistance to VD toxin than the cortical ones. In contrast, when cells were treated with a high concentration of VD toxin, MFs and MTs were disrupted sooner and more severely and did not recover spontaneously. Treatments with high concentrations of VD toxin also induced changes of nucleoli. At the early stages of treatment, a nucleus had a single ring-shaped nucleolus. At the later stages, multiple smaller and more brightly fluorescing nucleoli emerged in a single nucleus. Disrupted MFs could be recovered by removing the VD toxin before the ring-shaped nucleoli appeared. All these results showed that MFs and MTs play important roles in the early defense responses against VD toxin in Arabidopsis suspension cells. The cytoskeletons may be used as sensors and effectors monitoring the defense reactions. The changes of nucleoli induced by VD toxin should be important characteristics of cell death.

Microtubule Binding and Clustering of Human Tau-4R and Tau-P301L Proteins Isolated from Yeast Deficient in Orthologues of Glycogen Synthase Kinase-3β or cdk5

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3beta and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Delta mds1 and Delta pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.

17] Expression, purification, and biochemical properties of rabkinesin-6 domains and their interactions with Rab6A

This chapter describes the purification of several Rabkinesin-6 domains from bacteria and eukaryotic systems, and their biochemical characterization (ATPase, MT-binding, hydrodynamic properties).The chapter describes an assay to visualize the interaction between Rab6A and Rabkinesin-6. For purification, Escherichia coli strain freshly transformed with pET-Nt or pTrc-Q665 plasmids are cultured. Bacterial pellets are obtained are sonicated, centrifuged, and chromatographed. Protein fractions are analyzed by SDS-PAGE and Western blot. This protocol leads to the purification of about 0.5–1 mg fusion proteins. α/β-Tubulin is purified from bovine brain with two cycles of polymerization in the presence of GTP, and microtubule (MT)-associated proteins (MAPs) are removed by phosphocellulose chromatography. Tubulin concentration is estimated by the Bradford assay and MT concentration is expressed per tubulin heterodimer. MT-induced ATPase activity of kinesins is often measured by using a NADH coupled enzymatic assay.

Embryo sac development in Arabidopsis thaliana II. The cytoskeleton during megagametogenesis

The microtubular and actin cytoskeletons have been investigated during megagametogenesis in Arabidopsis thaliana using immunofluorescence labelling of isolated coenocytic and mature embryo sacs. We found both actin and microtubules (MTs) to occur in abundance throughout megagametogenesis and in all constituent cells of the mature embryo sac. During many stages, the patterns of distribution of these cytoskeletal elements are congruent and may prove to be co-aligned. Many changes in the arrays of MTs and microfilaments take place and indicate varying roles of the cytoskeleton in the different stages and cell types of megagametogenesis. Two major populations of MTs recur throughout embryo sac formation: (1) Elaborate nuclear-based networks are found during the two-nucleate and four-nucleate developmental stages as well as in the egg cell. These arrays may function in positioning the nuclei. (2) Cytoplasmic MTs in longitudinal orientation in the two-nucleate embryo sac, synergids and part of the egg cell, or in a reticulate pattern in the four-nucleate embryo sac, egg and central cell probably participate in organization of the cytoplasm. Synergid MTs converge at the filiform apparatus. Preprophase bands of MTs are absent throughout megagametogenesis but phragmoplast arrays occur during cellularization of the embryo sac. Well developed arrays of cortical MTs are restricted to the antipodal cells. A large concentration of MTs in the part of the egg cell adjacent to the synergids is well placed for being involved with sperm cell movement within the degenerative synergid. On the basis of the morphology of the cytoskeleton, we concur with views that the shape of megagametophyte is largely determined by the surrounding tissues, including the integumentary tapetum.