Tubulin Sheets Research Articles

Revealing and Attenuating the Electrostatic Properties of Tubulin and Its Polymers.

Tubulin is an electrostatically negative protein that forms cylindrical polymers termed microtubules, which are crucial for a variety of intracellular roles. Exploiting the electrostatic behavior of tubulin and microtubules within functional microfluidic and optoelectronic devices is limited due to the lack of understanding of tubulin behavior as a function of solvent composition. This work displays the tunability of tubulin surface charge using dimethyl sulfoxide (DMSO) for the first time. Increasing the DMSO volume fractions leads to the lowering of tubulin's negative surface charge, eventually causing it to become positive in solutions >80% DMSO. As determined by electrophoretic mobility measurements, this change in surface charge is directionally reversible, i.e., permitting control between -1.5 and + 0.2cm2 (Vs)-1 . When usually negative microtubules are exposed to these conditions, the positively charged tubulin forms tubulin sheets and aggregates, as revealed by an electrophoretic transport assay. Fluorescence-based experiments also indicate that tubulin sheets and aggregates colocalize with negatively charged g-C3 N4 sheets while microtubules do not, further verifying the presence of a positive surface charge. This study illustrates that tubulin and its polymers, in addition to being mechanically robust, are also electrically tunable.

Actin filaments modulate electrical activity of brain microtubule protein two-dimensional sheets.

The cytoskeleton of eukaryotic cells contains networks of actin filaments and microtubules (MTs) that are jointly implicated in various cell functions, including cell division, morphogenesis, and migration. In neurons, this synergistic activity drives both the formation of axons during development and synaptic activity in mature neurons. Both actin filaments and MTs also are highly charged polyelectrolytes that generate and conduct electrical signals. However, no information is presently available on a potential electrical crosstalk between these two cytoskeletal networks. Herein we tested the effect of actin polymerization on the electrical oscillations generated by two-dimensional sheets of bovine brain microtubule protein (2D-MT). The voltage-clamped 2D-MT sheets displayed spontaneous electrical oscillations representing a synchronous 224% change in conductance, and a fundamental frequency of 38 Hz. At 60 mV, a 4.15 nC of integrated charge transferred per second increased by 72.3% (7.15 nC) after addition of monomeric (G)-actin. This phenomenon had a 2-min lag time, and was prevented by the presence of the G-actin-binding protein DNAse I. Addition of prepolymerized F-actin, however, had a rapid onset (<10 s) and a higher effect on the tubulin sheets (~100% increase, 8.25 nC). The data are consistent with an interaction between the actin cytoskeleton and tubulin structures, in what seems to be an electrostatic effect. Because actin filaments and MTs interact with each other in neurons, it is possible for this phenomenon to be present, and of relevance in the processing of intracellular signaling, including the gating and activation of actin cytoskeleton-regulated excitable ion channels in neurons.

Structural Changes in Tubulin Sheets Caused by Immobilization on Solid Supports.

In the presence of zinc, the protein tubulin assembles into two-dimensional sheets that are a useful model system for the study of both tubulin and microtubule structure. Tubulin sheets present an ideal protein structure for study with atomic force microscopy because they contain a two-dimensional crystalline protein lattice and retain many of the structural features of tubulin and microtubules. However, high-resolution imaging requires nonperturbative immobilization onto an appropriate imaging substrate. In this report, several substrates commonly used for scanning probe microscopy are evaluated for their ability to effectively immobilize tubulin sheets: mica, gold, highly ordered pyrolytic graphite, and carbon-coated electron microscopy grids. We hypothesize that the different intermolecular interactions presented by these substrates will affect the morphology of adsorbed tubulin sheets as well as the amount of other contaminating adsorbates. Tubulin sheets were successfully imaged on all of these substrates and structural characterization is reported. The most consistent results were obtained on carbon-coated electron microscopy grids, which preserved fine structural features of the sheets and had the least amount of contamination from the adsorption of unpolymerized tubulin. Images of tubulin sheets obtained with atomic force microscopy also compare favorably with published electron micrographs of sheets produced using similar procedures. This work demonstrates the importance of assessing substrate effects when studying two-dimensional protein crystals and identifies suitable substrates for immobilizing tubulin sheets.

EB1 interacts with outwardly curved and straight regions of the microtubule lattice.

EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTPcap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region.

A Single Protofilament Is Sufficient to Support Unidirectional Walking of Dynein and Kinesin

Cytoplasmic dynein and kinesin are two-headed microtubule motor proteins that move in opposite directions on microtubules. It is known that kinesin steps by a ‘hand-over-hand’ mechanism, but it is unclear by which mechanism dynein steps. Because dynein has a completely different structure from that of kinesin and its head is massive, it is suspected that dynein uses multiple protofilaments of microtubules for walking. One way to test this is to ask whether dynein can step along a single protofilament. Here, we examined dynein and kinesin motility on zinc-induced tubulin sheets (zinc-sheets) which have only one protofilament available as a track for motor proteins. Single molecules of both dynein and kinesin moved at similar velocities on zinc-sheets compared to microtubules, clearly demonstrating that dynein and kinesin can walk on a single protofilament and multiple rows of parallel protofilaments are not essential for their motility. Considering the size and the motile properties of dynein, we suggest that dynein may step by an inchworm mechanism rather than a hand-over-hand mechanism.

A coarse grain model of microtubules

This paper proposes a 3-dimensional coarse grain model of microtubules and treats the tubulin monomer as a sphere of multiple patches, with parameters chosen to yield experimental values of bending and stretching stiffness. The model has demonstrated the ability to produce the bistability of tubulin sheets, elastic deformation near the tip, and cracking and peeling of protofilaments. This model is expected to take into account the structural and mechanical aspects underlying the physical mechanism of polymerization/depolymerization and dynamic instability of microtubules.

Direct Optical Microscopic Observation of the Microtubule Polymerization Intermediate Sheet Structure in the Presence of Gas7

The process of microtubule elongation is thought to consist of two stages—formation of a tubulin sheet structure and its closure into a tube. However, real-time observation of this process has been difficult. Here, by utilizing phospho-tau binding protein Gas7 (growth-arrest-specific protein 7), we visualized the polymer transformation process by dark-field microscopy. Upon elongation, thin and flexible structures, often similar to a curved hook, appeared at the end of microtubules. Electron microscopic observations supported the idea that these flexible structures are tubulin sheets. They maintained their length until they gradually became thick and rigid beginning in the central portion, resulting in straight microtubules. In the absence of Gas7, the sheet-like structure was rarely observed; moreover, when observed, it was fragile and engaged in typical dynamic instability. With Gas7, no catastrophe was observed. These results suggest that Gas7 enhances microtubule polymerization by stabilizing sheet intermediates and is a useful tool for analyzing microtubule transformation.

Deposition of Palladium Nanoparticles on Self-Assembled, Zinc-Induced Tubulin Macrotubes and Sheets

Co-factors control the GTP-induced assembly of tubulin protein into a variety of superstructures with defined geometry at the nanometre scale: microtubules, macrotubes, sheets, or spirals/rings. We report the Zn2+ ion-induced assembly of tubulin protein into sheet-like or tubular structures. Free functional groups of amino acids on the surface of the protein biopolymer provide nucleation sites for further deposition of small metal nanoparticles. This study describes the synthesis of metal particle--protein hybrids by a two-step chemical process that directs metal nanoparticle nucleation at specific surface sites by applying these tubulin assemblies as biotemplates. The hybrids are characterized by transmission electron microscopy (TEM) and scanning force microscopy (SFM). The present study demonstrates the potential and general applicability of tubulin assemblies as tools for the nanofabrication of nanoparticle arrays exhibiting various geometries.

The C-termini of tubulin and the specific geometry of tubulin substrates influence the depolymerization activity of MCAK

MCAK is a Kinesin-13 that depolymerizes microtubules (MTs) and regulates MT dynamics. We used subtilisin-treated MTs (MTs lacking the C-termini of α- and β-tubulin) and alternative tubulin substrates to study which structural and geometrical features of the MT are critical for MCAK activity. We found that removal of the C-termini significantly decreased the efficiency of MCAK-induced depolymerization, which was not due to a reduction of end-specific binding. We also found that depolymerization of SMTs led to an increase in the stabilization of curved oligomeric tubulin products. Using alternative tubulin substrates with different geometries, we found that MCAK depolymerized parallel and anti-parallel tubulin sheets. However, MCAK did not depolymerize tubulin rings regardless of the presence or absence of the tubulin C-termini. We propose that localization of MCAK to the ends of MTs is independent of tubulin C-termini, that MCAK stabilizes a curved conformation at the end of the MT, and that efficient release of this complex is dependent on the presence of the C-termini of tubulin.αβ

EB1 regulates microtubule dynamics and tubulin sheet closure in vitro

End binding 1 (EB1) is a plus-end-tracking protein (+TIP) that localizes to microtubule plus ends where it modulates their dynamics and interactions with intracellular organelles. Although the regulating activity of EB1 on microtubule dynamics has been studied in cells and purified systems, the molecular mechanisms involved in its specific activity are still unclear. Here, we describe how EB1 regulates the dynamics and structure of microtubules assembled from pure tubulin. We found that EB1 stimulates spontaneous nucleation and growth of microtubules, and promotes both catastrophes (transitions from growth to shrinkage) and rescues (reverse events). Electron cryomicroscopy showed that EB1 induces the initial formation of tubulin sheets, which rapidly close into the common 13-protofilament-microtubule architecture. Our results suggest that EB1 favours the lateral association of free tubulin at microtubule-sheet edges, thereby stimulating nucleation, sheet growth and closure. The reduction of sheet length at microtubule growing-ends together with the elimination of stressed microtubule lattices may account for catastrophes. Conversely, occasional binding of EB1 to the microtubule lattice may induce rescues.

The Schizosaccharomyces pombe EB1 Homolog Mal3p Binds and Stabilizes the Microtubule Lattice Seam

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

HURP Wraps Microtubule Ends with an Additional Tubulin Sheet That Has a Novel Conformation of Tubulin

HURP is a newly discovered microtubule-associated protein (MAP) required for correct spindle formation both in vitro and in vivo. HURP protein is highly charged with few predicted secondary and tertiary folding domains. Here we explore the effect of HURP on pure tubulin, and describe its ability to induce a new conformation of tubulin sheets that wrap around the ends of intact microtubules, thereby forming two concentric tubes. The inner tube is a normal microtubule, while the outer one is a sheet composed of tubulin protofilaments that wind around the inner tube with a 42.5 degrees inclination. We used cryo-electron microscopy and unidirectional surface shadowing to elucidate the structure and conformation of HURP-induced tubulin sheets and their interaction with the inner microtubule. These studies clarified that HURP-induced sheets are composed of anti-parallel protofilaments exhibiting P2 symmetry. HURP is a unique MAP that not only stabilizes and bundles microtubules, but also polymerizes free tubulin into a new configuration.

Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of β-tubulin (Asp26Glu) and less stable microtubules

Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 micromol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of paclitaxel makes contact with Asp26 of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.

Structural rearrangements in tubulin following microtubule formation.

Microtubules are essential cytoskeletal structures that mediate several dynamic processes in a cell. To shed light on the structural processes relating to microtubule formation and dynamic instability, we investigated microtubules composed of 15 protofilaments using cryo-electron microscopy, helical image reconstruction and computational modelling. Analysis of the configuration of the alpha beta-tubulin heterodimer shows distinct structural differences in both subunits, and illustrates that the tubulin subunits have different roles in the microtubule lattice. Our modelling data suggest that after GTP hydrolysis microtubules, adopt a conformational state somewhere between a straight protofilament conformation--as found in zinc-induced tubulin sheets--and an outward curved conformation--as found in tubulin-stathmin complexes. The tendency towards a curved conformation seems to be mediated mostly by beta-tubulin, whereas alpha-tubulin resembles a state more related to the straight structure. Our data suggest a possible explanation of dynamic instability of microtubules, and for nucleotide-sensitive microtubule-binding properties of microtubule-associated proteins and molecular motors.

Modeling the interaction of paclitaxel with beta-tubulin.

The natural product cytotoxic agent, paclitaxel, partly induces cell death through its ability to disrupt mitosis by binding to the microtubule protein beta-tubulin. Structural characterization of the paclitaxel-beta-tubulin complex, a first stage in the design of new antimitotics, has been complicated by contradictory observations obtained from different experimental techniques [electron crystallography, fluorescence resonance energy transfer (FRET), and photo-affinity labeling (PAL)] used to examine the complex. In this study we have used a range of molecular modeling techniques including restrained conformational searching and computer-assisted docking to propose that these contradictions may be resolved by the hypothesis that the nature of the interaction of paclitaxel with beta-tubulin depends on the physical form of the tubulin examined. In particular, our analysis identified a binding mode that is consistent with available data for the interaction of paclitaxel with beta-tubulin in polymerized tubulin or microtubules. This orientation is characterized by an alternate conformation (inverted orientation of side chains) and inverted orientation of the taxane core of paclitaxel within its tubulin binding site compared with the electron crystallographic structure. The proposed structure, however, is only marginally consistent with electron crystallographic data for the interaction of paclitaxel with beta-tubulin in Zn-induced tubulin sheets. Similarly, the electron crystallographic structure shows poor correlation with FRET, solid-state NMR, and some observed SAR relationships for paclitaxel interacting with polymerized tubulin or microtubules. These observations suggest to us that the interaction of paclitaxel with Zn-induced tubulin sheets may not reflect paclitaxel's interaction with tubulin in microtubules and hence may not be an appropriate guide for rational drug design programs.

823 The CDK2 inhibitor CYC202 (R-roscovitine) inhibits DNA synthesis and cell proliferation in rat liver after partial hepatectomy

Kinesin and ncd (non-claret disjunctional) are microtubule associated motor proteins which share several structural features: both motors are dimers; each monomer is composed of a stalk region, a cargo binding domain and a motor domain; the motor domains have ∼41% sequence identity. Despite these similarities the two motors have strikingly different movement properties: kinesin is a plus-end directed molecular motor, while ncd is minus-end directed. Here we compare the structure and the microtubule-binding properties of these oppositely directed molecular motors. We determined the three-dimensional structure of tubulin sheets decorated with the motor domains of either kinesin or ncd to a resolution of <20 Å by negative stain electron microscopy and tilt series reconstruction. Comparisons with a control structure of tubulin alone revealed that in both cases the motor domain binds to the outer crest of a single protofilament making contacts with both α and β tubulin. Despite their opposite directionality, the geometry of attachment of the motor domain to the protofilament in the presence of AMP-PNP is very similar for both motors. These data rule out models for directionality which have the motors binding in an opposite orientation to the microtubules. Binding of the ncd as well as the kinesin motor domain appears to induce conformational changes in tubulin. This observation suggests an active role of tubulin in motor movement and/or in the determination of directionality.

Relating Molecular Flexibility to Function: A Case Study of Tubulin

Microtubules (MT), along with a variety of associated motor proteins, are involved in a range of cellular functions including vesicle movement, chromosome segregation, and cell motility. MTs are assemblies of heterodimeric proteins, αβ-tubulins, the structure of which has been determined by electron crystallography of zinc-induced, pacilitaxel-stabilized tubulin sheets. These data provide a basis for examining relationships between structural features and protein function. Here, we study the fluctuation dynamics of the tubulin dimer with the aim of elucidating its functional motions relevant to substrate binding, polymerization/depolymerization and MT assembly. A coarse-grained model, harmonically constrained according to the crystal structure, is used to explore the global dynamics of the dimer. Our results identify six regions of collective motion, comprised of structurally close but discontinuous sequence fragments, observed only in the dimeric form, dimerization being a prerequisite for domain identification. Boundaries between regions of collective motions appear to act as linkages, found primarily within secondary-structure elements that lack sequence conservation, but are located at minima in the fluctuation curve, at positions of hydrophobic residues. Residue fluctuations within these domains identify the most mobile regions as loops involved in recognition of the adjacent regions. The least mobile regions are associated with nucleotide binding sites where lethal mutations occur. The functional coupling of motions between and within regions identifies three global motions: torsional and wobbling movements, en bloc, between the α- and β-tubulin monomers, and stretching longitudinally. Further analysis finds the antitumor drug pacilitaxel (TaxotereR) to reduce flexibility in the M loop of the β-tubulin monomer; an effect that may contribute to tightening lateral interactions between protofilaments assembled into MTs. Our analysis provides insights into relationships between intramolecular tubulin movements of MT organization and function.

Refined structure of αβ-tubulin at 3.5 Å resolution

We present a refined model of the αβ-tubulin dimer to 3.5 Å resolution. An improved experimental density for the zinc-induced tubulin sheets was obtained by adding 114 electron diffraction patterns at 40-60 ° tilt and increasing the completeness of structure factor amplitudes to 84.7 %. The refined structure was obtained using maximum-likelihood including phase information from experimental images, and simulated annealing Cartesian refinement to an R-factor of 23.2 and free R-factor of 29.7. The current model includes residues α:2-34, α:61-439, β:2-437, one molecule of GTP, one of GDP, and one of taxol, as well as one magnesium ion at the non-exchangeable nucleotide site, and one putative zinc ion near the M-loop in the α-tubulin subunit. The acidic C-terminal tails could not be traced accurately, neither could the N-terminal loop including residues 35-60 in the α-subunit. There are no major changes in the overall fold of tubulin with respect to the previous structure, testifying to the quality of the initial experimental phases. The overall geometry of the model is, however, greatly improved, and the position of side-chains, especially those of exposed polar/charged groups, is much better defined. Three short protein sequence frame shifts were detected with respect to the non-refined structure. In light of the new model we discuss details of the tubulin structure such as nucleotide and taxol binding sites, lateral contacts in zinc-sheets, and the significance of the location of highly conserved residues.

Individual expression of recombinant alpha- and beta-tubulin from Haemonchus contortus: polymerization and drug effects.

Three tubulin isotypes from the parasitic nematode Haemonchus contortus were individually expressed in Escherichia coli, purified, and induced to polymerize into microtubules in the absence of microtubule-associated proteins. The effect of different conditions on the rate of polymerization of pure tubulin was assessed. This is the first time that recombinant alpha-tubulin has been shown to be capable of polymerization into microtubule-like structures when incubated with recombinant beta-tubulin. In addition, the present study has shown that: (1) microtubule-associated proteins are not required for tubulin polymerization; and (2) pure beta-tubulin isotype, beta12-16, alone was capable of forming microtubule-like structures in the absence of alpha-tubulin. Polymerization of the recombinant invertebrate tubulin, as measured by a spectrophotometric assay, was found to be enhanced by a concentration of tubulin >0.25 mg/mL; temperature > or =20 degrees C; 2 mM GTP; glycerol; EGTA; and Mg(2+). Polymerization was inhibited by GTP (>2 mM) and albendazole. Calcium ions and a pH range of 6 to 8.5 had no measurable effect on polymerization. Individual isotypes of tubulin polymerized to approximately the same extent as an alpha-/beta-tubulin mixture. Samples of tubulin assembled under the above conditions for 60 min were also examined under a transmission electron microscope. Although the spectrophotometric assay indicated polymerization, it did not predict the structure of the polymer. In many cases tubulin sheets, folded sheets, and rings were observed in addition to, or instead of, microtubule-like structures.

Surface topography of microtubule walls decorated with monomeric and dimeric kinesin constructs.

The surface topography of opened-up microtubule walls (sheets) decorated with monomeric and dimeric kinesin motor domains was investigated by freeze-drying and unidirectional metal shadowing. Electron microscopy of surface-shadowed specimens produces images with a high signal/noise ratio, which enable a direct observation of surface features below 2 nm detail. Here we investigate the inner and outer surface of microtubules and tubulin sheets with and without decoration by kinesin motor domains. Tubulin sheets are flattened walls of microtubules, keeping lateral protofilament contacts intact. Surface shadowing reveals the following features: (i) when the microtubule outside is exposed the surface relief is dominated by the bound motor domains. Monomeric motor constructs generate a strong 8 nm periodicity, corresponding to the binding of one motor domain per alpha-beta-tubulin heterodimer. This surface periodicity largely disappears when dimeric kinesin motor domains are used for decoration, even though it is still visible in negatively stained or frozen hydrated specimens. This could be explained by disorder in the binding of the second (loosely tethered) kinesin head, and/or disorder in the coiled-coil tail. (ii) Both surfaces of undecorated sheets or microtubules, as well as the inner surface of decorated sheets, reveal a strong 4 nm repeat (due to the periodicity of tubulin monomers) and a weak 8 nm repeat (due to slight differences between alpha- and beta-tubulin). The differences between alpha- and beta-tubulin on the inner surface are stronger than expected from cryo-electron microscopy of unstained microtubules, indicating the existence of tubulin subdomain-specific surface properties that reflect the surface corrugation and hence metal deposition during evaporation. The 16 nm periodicity visible in some negatively stained specimens (caused by the pairing of cooperatively bound kinesin dimers) is not detected by surface shadowing.