Microtubules In Solution Research Articles

Modified heptapeptide from tau binds both tubulin and microtubules.

BackgroundMicrotubules are the major cytoskeletal component in eukaryotes which are essential for a large spectrum of cellular activities. Monitoring the behavior of microtubules is helpful for a better understanding of the regulatory mechanism governing microtubule architecture and microtubule‐based activities. Here, we characterized the binding capability of a modified heptapeptide from tau to both tubulin and microtubules and sought to develop it as a fluorescent peptide for monitoring microtubules.MethodsTo deliver the fluorescent peptide into the cells, a cell‐penetrating peptide was conjugated to the modified heptapeptide from tau and synthesized. The affinity of the modified heptapeptide was determined by microscale thermophoresis. The microtubule labeling ability was determined by adding the peptide into the polymerized microtubule solutions or cultured HeLa cells.;ResultsAffinity determination revealed that the tau‐derived peptide specifically bound to tubulin. In addition, the peptide was able to label polymerized microtubules in solution, although no obvious microtubule filaments were observed clearly in living cells, probably due to the inadequate affinity.ConclusionsThese results suggest that using a peptide‐based strategy for imaging microtubules might be plausible and attempts to improve its affinity is warranted in the future.

Second harmonic generation polarization microscopy as a tool for protein structure analysis.

Cryo-electron microscopy and X-ray crystallography have been the major tools of protein structure analysis for decades and will certainly continue to be essential in the future. Moreover, nuclear magnetic resonance or Förster resonance energy transfer can measure structural dynamics. Here, we propose to add optical second-harmonic generation (SHG), which is a nonlinear optical scattering process sensitive to molecular structures in illuminated materials, to the tool-kit of structural analysis methodologies. SHG can be expected to probe the structural changes of proteins in the physiological condition, and thus link protein structure and biological function. We demonstrate that a conformational change as well as its dynamics in protein macromolecular assemblies can be detected by means of SHG polarization measurement. To prove the capability of SHG polarization measurement with regard to protein structure analysis, we developed an SHG polarization microscope to analyze microtubules in solution. The difference in conformation between microtubules with different binding molecules was successfully observed as polarization dependence of SHG intensity. We also succeeded in capturing the temporal variation of structure in a photo-switchable protein crystal in both activation and inactivation processes. These results illustrate the potential of this method for protein structure analysis in physiological solutions at room temperature without any labeling.

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.

Molecular Recognition of Epothilones by Microtubules and Tubulin Dimers Revealed by Biochemical and NMR Approaches

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.

Microtubule Structural Modeling and Analysis Using Small Angle X-Ray Scattering

Microtubules (MTs) play many central roles in cellular function. Understanding their interactions in solutions leads to greater understanding of their function. Observing MTs in solution is a trivial matter using small angle x-ray scattering (SAXS), however the data analysis can be quite complex. Here, we present the analysis of MTs using different geometric models to describe them and their advantages and disadvantages using X+, a software solution developed for general SAXS data analysis. When high resolution data are available, the simpler geometric models break down in the higher resolution regions. This allows for a finer definition of the geometric parameters of the protein's supra-molecular structure, i.e. the wall thickness. In addition, certain thermal fluctuations can be observed and quantified. The effects of isotropic fluctuations of the tubulin monomers, modeled as spheres, can readily be observed. Ref. Székely et al. Langmuir (2010) 26 (16), 13110-13129; Ben-Nun et al. Journal of Applied Crystallography (2010) in press.

Decoration of microtubules in solution by the kinesin-14, Ncd.

The kinesin-14, Ncd, is a cellular motor involved in microtubule spindle assembly and contraction during mitosis and meiosis. Like other members of the kinesin superfamily, Ncd consists of two motor heads connected by a linker and a long cargo-carrying stalk. The motor heads hydrolyze ATP to ADP to provide the power stroke that moves them and the cargo along the microtubule. Whereas conventional kinesins move processively along the sense of the microtubule right-handed helix, Ncd moves in the opposite direction, apparently using a different motive mechanism. According to the current model, the microtubule-binding state of Ncd is bound by one head and then released during the motive cycle. This is distinguished from the binding states of conventional kinesins, in which the motor heads are always bound in the motive cycle with alternating one-head and two-head binding. The objective was to determine the extent of binding, the binding states of Ncd in the presence of an ATP analogue, AMPPNP, and whether the binding is cooperative. Small-angle neutron scattering (SANS) of microtubules decorated with a deuterated Ncd construct, Ncd281, in solution containing 42% D(2)O was used. These conditions render the microtubule `invisible' to SANS, while amplifying the SANS from the Ncd constructs. In the presence of AMPPNP, 75% of Ncd281 was not bound. The remainder was bound cooperatively by one of its motor heads to the microtubule.

Effects of C-Termini distribution on ion transport along microtubules

Recent experiments demonstrated that microtubules in solution serve as directional pathways for ionic conduction. Biochemical assays have revealed the existence of numerous isoforms of the building block of microtubules, the protein called tubulin. Each isoform differs structurally, especially in the composition of their C-terminal tails. We developed a computational model for ionic conduction along a microtubular structure. The model allows for ionic hopping and trapping by the oppositely charged C-termini tails. Our simulations include various scenarios of isoform distribution within a given microtubule. We show that a critical number of ions is required for the onset of conduction and that it obeys scaling laws with respect to the length and isoform composition. We also found the formation of complex conduction pathways that depend on the distribution of tubulin isoforms.

Microtubule Alignment and Manipulation Using AC Electrokinetics

The kinesin-microtubule system plays an important role in intracellular transport and is a model system for integrating biomotor-driven transport into microengineered devices. AC electrokinetics provides a novel tool for manipulating and organizing microtubules in solution, enabling new experimental geometries for investigating and controlling the interactions of microtubules and microtubule motors in vitro. By fabricating microelectrodes on glass substrates and generating AC electric fields across solutions of microtubules in low-ionic-strength buffers, bundles of microtubules are collected and aligned and the electrical properties of microtubules in solution are measured. The AC electric fields result in electro-osmotic flow, electrothermal flow, and dielectrophoresis of microtubules, which can be controlled by varying the solution conductivity, AC frequency, and electrode geometry. By mapping the solution conductivity and AC frequency over which positive dielectrophoresis occurs, the apparent conductivity of taxol-stabilized bovine-brain microtubules in PIPES buffer is measured to be 250 mS m(-1). By maximizing dielectrophoretic forces and minimizing electro-osmotic and electrothermal flow, microtubules are assembled into opposed asters. These experiments demonstrate that AC electrokinetics provides a powerful new tool for kinesin-driven transport applications and for investigating the role of microtubule motors in development and maintenance of the mitotic spindle.

Collision induced spatial organization of microtubules

The dynamic behavior of microtubules in solution can be strongly modified by interactions with walls or other structures. We examine here a microtubule growth model where the increase in size of the plus-end is perturbed by collisions with other microtubules. We show that such a simple mechanism of constrained growth can induce ordered structures and patterns from an initially isotropic and homogeneous suspension. We find that microtubules self-organize locally in randomly oriented domains that grow and compete with each other. A weak orientation bias, similar to the one induced by gravity or cellular boundaries is enough to influence the domain growth direction, eventually leading to a macroscopic sample orientation.

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.

Location and properties of the taxol binding center in microtubules: a picosecond laser study with fluorescent taxoids.

The interaction of two bioactive, fluorescent analogues of the anticancer drug Taxol, Flutax1 [7-O-[N-(fluorescein-4'-carbonyl)-L-alanyl]taxol] and Flutax2 [7-O-[N-(2,7-difluorofluorescein-4'-carbonyl)-L-alanyl]taxol], with microtubules in solution has been studied with picosecond laser methods. As shown here, although a mixture of the fluorescein mono- and dianion species of Flutax1 is present in solution, the bound taxoid contains only the dianion form of the dye. This indicates strong electrostatic interactions at the microtubule lattice with the appending dye, most likely with charged residues of the M-loop of the beta-tubulin subunit. Moreover, analysis of the dynamic depolarization of microtubule-bound Flutax at low binding site occupancy was consistent with a protein active center with significant conformational flexibility. On the other hand, for microtubules fully saturated with the taxoid, a new, additional depolarizing process was observed, with relaxation times of 14 ns (Flutax1) and 8 ns (Flutax2), which is due to Förster resonance energy homotransfer (FREHT) between neighboring dye molecules. Application of a detailed analysis of FREHT-induced depolarization in a circular array of dye molecules presented here yielded a separation between nearest-neighbor Flutax moieties of 40 +/- 5 A, for microtubules made up of between 12 and 14 protofilaments, a value that is only compatible with the Taxol binding site being located at the inner wall of the microtubule. The internal position of the drug molecular target as measured here is also consistent with other spectroscopic observations and confirms existing predictions based on microtubule structures modeled from high-resolution, electron density maps of alphabeta-tubulin.

Translocation of microtubules caused by the αβ, β and γ outer arm dynein subparticles of Chlamydomonas

Three kinds of subparticles of Chlamydomonas outer-arm dynein containing the alphabeta, beta and gamma heavy chains were isolated and assayed for their activities to translocate microtubules in vitro. All of them had activities to form bundles of microtubules in solution in an ATP-dependent manner and, when adsorbed on an appropriate glass surface, translocated microtubules. The alphabeta subparticle readily translocated microtubules on a silicone-coated glass surface with a velocity of 4.6 micron/second at 1 mM ATP. The beta subparticle translocated microtubules after it had been preincubated with tubulin dimer and when the Brownian movement of microtubules was suppressed by addition of methylcellulose. The velocity was on average 0.7 micron/second. The gamma subparticle translocated microtubules after being preincubated with tubulin dimer and adsorbed onto a silicone-coated glass surface. The velocity was about 3.8 micron/second. The tubulin dimer appeared to facilitate in vitro motility by blocking the ATP-insensitive binding of dynein subparticles to microtubule. The alphabeta, beta and gamma subparticles were thus found to have different properties as motor proteins. In addition, these subparticles showed different dependencies upon the potassium acetate concentration. Hence the outer-arm dynein of Chlamydomonas is a complex of motor proteins with different properties.

Structural and functional domains of the Drosophila ncd microtubule motor protein.

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein that is required for proper chromosome distribution in Drosophila. Despite its sequence similarity to kinesin heavy chain, ncd translocates with the opposite polarity as kinesin, toward microtubule minus ends. We have expressed different regions of the protein in bacteria and analyzed the proteins for function. Results indicate that ncd consists of three domains: a basic, proline-rich N-terminal "tail," a central alpha-helical coiled-coil stalk, and a C-terminal motor domain. The ncd N terminus proteins bundle microtubules in motility assays and show ATP-independent binding to microtubules in solution. Truncated proteins, lacking the tail but containing the predicted motor domain and differing lengths of the stalk, did not support microtubule gliding in in vitro assays but showed microtubule-stimulated MgATPase activity in solution. Addition of a nonspecific N terminus to two of the truncated proteins restored directional gliding and rotation of microtubules in motility assays, demonstrating that these properties map to the predicted mechanochemical domain of ncd. Physical properties of the C terminus proteins indicate that the stalk region is important for dimerization and that the ncd protein probably exists and functions as a dimer.

Low resolution structure of microtubules in solution: Synchrotron X-ray scattering and electron microscopy of taxol-induced microtubules assembled from purified tubulin in comparison with glycerol and MAP-induced microtubules

The structure of microtubules has been characterized to 3 nm resolution employing time-resolved X-ray scattering. This has revealed detailed structural features of microtubules not observed before in solution. The polymerization of highly purified tubulin, induced by the antitumour drug taxol, has been employed as a microtubule model system. This assembly reaction requires Mg 2+, is optimal at a 1:1 taxol to tubulin heterodimer molar ratio, proceeds with GTP or GDP and is intrinsically reversible. The X-ray scattering profiles are consistent with identical non-globular α and β-tubulin monomers ordered within the known helical surface lattice of microtubules. Purified tubulin-taxol microtubules have a smaller mean diameter (approx. 22 nm) than those induced by microtubule associated proteins or glycerol (approx. 24 nm), but nearly identical wall substructure to the resolution of the measurements. This is because the majority of the former consist of only 12 protofilaments instead of the typical 13 protofilaments, as confirmed by electron microscopy of thin-sectioned, negatively stained and ice-embedded taxol microtubules. It may be concluded that taxol induces a slight reduction of the lateral contact curvature between tubulin monomers. The main fringe pattern observed in cryo-electron micrographs is consistent with a simple 12 protofilament 3-start skewed lattice model. Cylindrical closure of this lattice can be achieved by tilting the lattice 0.8 ° with respect to the microtubule axis. The closure implies a discontinuity in the type of lateral contacts between the tubulin monomers (regardless of whether these are of the -α-β- or the -α-α-/-β-β- type), which indicates that lateral contacts and the subunit specificity of taxol binding are, to a large degree, equivalent.

Kinetics of the spontaneous organization of microtubules in solution

Optically anisotropic zones occur spontaneously in solutions of microtubules. These tactoids, in which microtubules are arranged in parallel arrays, can be visualized by their birefringence. With microtubules assembled in the presence of associated proteins (MAPs), birefringence appears immediately after nucleation of polymerization, even at relatively low protein concentrations. It is not dependent on whether the assembly is initiated by temperature jump or by isothermal addition of GTP. With pure tubulin, assembled in buffers containing 25% glycerol or 4% dimethylsulfoxide and/or taxol, birefringence appears within a few hours, but it can be speeded up by gentle agitation. With tubulin assembled in the presence of MAPs, spontaneous orientation occurs simultaneously with polymerization. This may be due to the existence of more pronounced repulsive forces between microtubules when they are covered with MAPs. A simple calculation of the covolume, suggests that tactoid formation is expected for microtubules of lengths of 5 to 10 microns at protein concentrations in the range 1 to 3 mg/ml (as observed), and that repulsive forces will promote tactoid formation at even lower protein concentrations.

Microtubule solutions display nematic liquid crystalline structure.

We report a study of the spontaneous formation of ordered arrays of microtubules in solution. Form birefringence and anisotropic light-scattering appear rapidly and spontaneously when tubulin, initially present in homogeneous solution, self-assembles into microtubules. This phenomenon is reversible and occurs at protein concentrations of a few milligrams per ml, in the presence or absence of microtubule-associated proteins. Light and electron microscopic examination reveals that extensive regions of these birefringent solutions consist of nearly parallel microtubules. Measurement of the order parameter, S, yields a value of 0.81 +/- 0.05, indicating a high degree of alignment. Comparison of these observations to qualitative predictions developed from the theory of Onsager ((1949) Ann. N.Y. Acad. Sci. 51, 627-659) leads to the conclusion that microtubules form a nematic liquid crystalline phase in vitro under ordinary conditions. Simultaneous spectrophotometric observation of turbidity (a measure of microtubule assembly) and birefringence shows that the parallel ordering lags only slightly behind assembly, thus demonstrating that much microtubule growth must occur by addition of tubulin to the ends of microtubules that are already aligned. These observations of anisotropy are important to the understanding of microtubule dynamics in vitro.

Direct observation of microtubules with the scanning tunneling microscope.

To observe surface topography of microtubules, we have applied scanning tunneling microscopy (STM), which can image metal and semiconductive surfaces with atomic resolution. Isolated microtubules fixed in 0.1% glutaraldehyde in reassembly buffer containing 0.8 M glycerol were imaged in air on a graphite substrate. The presence of microtubules in solution was verified by electron microscopy. At atmospheric pressure and room temperature, STM probing of both freeze-dried and hydrated microtubules reveals structures approximately 25 nm in width, consisting of longitudinal filaments about 4 nm in width. These structures match electron microscopy images of microtubules and their component protofilaments. Microtubules imaged by STM frequently appear buckled and semiflattened. Top-view shaded scans show what appear to be individual tubulin subunits within protofilaments. We believe these results represent the first direct STM observation of protein assemblies in which components can be identified. Although the microtubule image resolution described here is no better than that presently obtainable by other techniques (e.g., electron microscopy with freeze-drying, shadowing, and/or negative staining), it is significant that suitably prepared biomolecules may be sufficiently conductive and stable for STM imaging, which is ultimately capable of atomic resolution. Further development of STM technology, computer-enhanced image processing, and elucidation of optimal STM sample preparation indicate that STM and related applications will offer unique opportunities for the study of biomolecular surfaces.

Theoretical treatment of microtubules disappearing in solution.

The origin of the two-phase (cap, no cap) macroscopic kinetic model of the end of a microtubule is reviewed. The model is then applied to a new theoretical problem, namely, the Mitchison-Kirschner [Mitchison, T. & Kirschner, M. W. (1984) Nature (London) 312, 237-242] experiment in which aggregated microtubules in solution spontaneously decrease in number (shorten to disappearance) while the surviving microtubules increase in length. The model fits the experiments without difficulty.

Phase-change kinetics for a microtubule with two free ends.

The two-phase macroscopic kinetic model of the end of a microtubule is extended to microtubules in solution, with two free ends. The theoretical treatment of this system is complicated by the possibility of microtubules shortening all the way to disappearance. Another possibility, if a microtubule is shortening from one end only (and has a GTP cap on the other end), is that completed shortening will leave a residual cap from which growth can then take place at both ends. Two approximations are introduced.

Promotion of microtubule assembly by neurofilament-associated microtubule-associated proteins.

Intact neurofilaments (NF) purified from mammalian brain and spinal cord promote the assembly of microtubules in solutions of pure phosphocellulose (PC)-purified tubulin. This assembly is temperature-dependent and is inhibited by mitotic spindle inhibitors. The ability of NF to induce microtubule formation is 20% of that of purified microtubule-associated proteins (MAPs), whereas MAPs comprise less than 5% of the protein in the NF preparations. The inducing activity of NF is rapidly lost on boiling. When intact NF are incubated with PC-tubulin and then centrifuged, tubulin is sedimented together with the filaments. This association is inhibited by colchicine and podophyllotoxin and is cold-sensitive. NF purified to homogeneity under denaturing conditions and then reassembled completely lack the ability to promote the assembly of PC-tubulin or to bind tubulin on a centrifugation assay. No MAPs are present in these preparations, though these filaments have the ability to bind exogenous MAPs. While these experiments do not rule out an intrinsic microtubule-assembly-promoting activity, they suggest that this activity is due to nontriplet proteins in the preparation, most likely filament-associated MAPs.