Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco.

Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.

Key residues on microtubule responsible for activation of kinesin ATPase.

Microtubule (MT) binding accelerates the rate of ATP hydrolysis in kinesin. To understand the underlying mechanism, using charged-to-alanine mutational analysis, we identified two independent sites in tubulin, which are critical for kinesin motility, namely, a cluster of negatively charged residues spanning the helix 11-12 (H11-12) loop and H12 of alpha-tubulin, and the negatively charged residues in H12 of beta-tubulin. Mutation in the alpha-tubulin-binding site results in a deceleration of ATP hydrolysis (k(cat)), whereas mutation in the beta-tubulin-binding site lowers the affinity for MTs (K(0.5)MT). The residue E415 in alpha-tubulin seems to be important for coupling MT binding and ATPase activation, because the mutation at this site results in a drastic reduction in the overall rate of ATP hydrolysis, largely due to a deceleration in the reaction of ADP release. Our results suggest that kinesin binding at a region containing alpha-E415 could transmit a signal to the kinesin nucleotide pocket, triggering its conformational change and leading to the release of ADP.

Strategies for selection and identification of rabbit single-chain Fv antibodies as ligand in affinity chromatography.

We developed affinity chromatographic resins that immobilized rabbit single-chain Fv antibodies (scFvs). By biopanning using antigen-coupled multilamellar vesicles (Ag-MLVs), 152 types of original scFv clones that specifically bind to human IgG were isolated and identified. Apparent dissociation rate constants, appkoff, of six different candidates were less than 10-3 s-1 and their dissociation constants, KDs, were ranged from 5.56 × 10-10 to 4.04 × 10-8 M. Consequently, the clones, R1-27, R2-18, and R3-26 were further investigated for use in affinity purification of human IgG. Both the clones, R1-27 and R3-26 maintained more than 40% of antigen-binding activities on the surface of affinity resins. Especially, R3-26 had a relatively high alkaline resistance. The direct separation of human IgG from 10% FBS-D-MEM by use of the column with R1-27 achieved 97.2% purity, while the column with R3-26 showed almost 100% recovery. The affinity resins at the densities between 4.32 and 15.19 mg-scFv/cm3 exhibited maximum binding amount of human IgG, while the highest ligand utilization was achieved by use of the resin at approximately 9 mg-scFv/cm3. The resin exhibited 7.69 mg/cm3 of equilibrium binding capacity (EBC) in affinity chromatography. It was expected that the EBC of affinity resins was strongly dependent on the specific surface area as well as the pore volume of the base resin. Therefore, the strategies to develop affinity ligands will be beneficial for development of on-demand affinity columns with higher affinity/selectivity, chemical resistance, while optimization of pore size and pore volume for scFv-coupled resins will further improve the EBC.

GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation.

Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers. With tubulin having the Y222F mutation in the ß subunit, the proportion of straight oligomers increases and nucleation accelerates. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.

Reversal of axonal growth defects in an extraocular fibrosis model by engineering the kinesin-microtubule interface.

Mutations in human ß3-tubulin (TUBB3) cause an ocular motility disorder termed congenital fibrosis of the extraocular muscles type 3 (CFEOM3). In CFEOM3, the oculomotor nervous system develops abnormally due to impaired axon guidance and maintenance; however, the underlying mechanism linking TUBB3 mutations to axonal growth defects remains unclear. Here, we investigate microtubule (MT)-based motility in vitro using MTs formed with recombinant TUBB3. We find that the disease-associated TUBB3 mutations R262H and R262A impair the motility and ATPase activity of the kinesin motor. Engineering a mutation in the L12 loop of kinesin surprisingly restores a normal level of motility and ATPase activity on MTs carrying the R262A mutation. Moreover, in a CFEOM3 mouse model expressing the same mutation, overexpressing the suppressor mutant kinesin restores axonal growth in vivo. Collectively, these findings establish the critical role of the TUBB3-R262 residue for mediating kinesin interaction, which in turn is required for normal axonal growth and brain development.

A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation.

Dynein is a motor protein that moves on microtubules (MTs) using the energy of adenosine triphosphate (ATP) hydrolysis. To understand its motility mechanism, it is crucial to know how the signal of MT binding is transmitted to the ATPase domain to enhance ATP hydrolysis. However, the molecular basis of signal transmission at the dynein-MT interface remains unclear. Scanning mutagenesis of tubulin identified two residues in α-tubulin, R403 and E416, that are critical for ATPase activation and directional movement of dynein. Electron cryomicroscopy and biochemical analyses revealed that these residues form salt bridges with the residues in the dynein MT-binding domain (MTBD) that work in concert to induce registry change in the stalk coiled coil and activate the ATPase. The R403-E3390 salt bridge functions as a switch for this mechanism because of its reversed charge relative to other residues at the interface. This study unveils the structural basis for coupling between MT binding and ATPase activation and implicates the MTBD in the control of directional movement.

Overexpression, purification, and functional analysis of recombinant human tubulin dimer.

Microtubules consisting of tubulin dimers play essential roles in various cellular functions. Investigating the structure-function relationship of tubulin dimers requires a method to prepare sufficient quantities of recombinant tubulin. To this end, we simultaneously expressed human α1- and ß3-tubulin using a baculovirus-insect cell expression system that enabled the purification of 5mg recombinant tubulin per litre of cell culture. The purified recombinant human tubulin could be polymerized into microtubules that glide on a kinesin-coated glass surface. The method provides a powerful tool for in vitro functional analyses of microtubules.

The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends.

During cell division the replicated chromosomes are segregated precisely towards the spindle poles. Although many cellular processes involving motility require ATP-fuelled force generation by motor proteins, most models of the chromosome movement invoke the release of energy stored at strained (owing to GTP hydrolysis) plus ends of microtubules. This energy is converted into chromosome movement through passive couplers, whereas the role of molecular motors is limited to the regulation of microtubule dynamics. Here we report, that the microtubule-depolymerizing activity of MCAK (mitotic centromere-associated kinesin), the founding member of the kinesin-13 family, is accompanied by the generation of significant tension-remarkably, at both microtubule ends. An MCAK-decorated bead strongly attaches to the microtubule side, but readily slides along it in either direction under weak external loads and tightly captures and disassembles both microtubule ends. We show that the depolymerization force increases with the number of interacting MCAK molecules and is ∼1 pN per motor. These results provide a simple model for the generation of driving force and the regulation of chromosome segregation by the activity of MCAK at both kinetochores and spindle poles through a 'side-sliding, end-catching' mechanism.

Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin.

The kinesin-binding site on the microtubule has not been identified because of the technical difficulties involved in the mutant analyses of tubulin. Exploiting the budding yeast expression system, we succeeded in replacing the negatively charged residues in the alpha-helix 12 of beta-tubulin with alanine and analyzed their effect on kinesin-microtubule interaction in vitro. The microtubule gliding assay showed that the affinity of the microtubules for kinesin was significantly reduced in E410A, D417A, and E421A, but not in E412A mutant. The unbinding force measurement revealed that in the former three mutants, the kinesin-microtubule interaction in the adenosine 5'-[beta,gamma-imido]triphosphate state (AMP-PNP state) became less stable when a load was imposed towards the microtubule minus end. In parallel with this decreased stability, the stall force of kinesin was reduced. Our results implicate residues E410, D417, and E421 as crucial for the kinesin-microtubule interaction in the strong binding state, thereby governing the size of kinesin stall force.

Effects of N-glycosylation and inositol on the ER stress response in yeast Saccharomyces cerevisiae.

IRE1 and HAC1 are essential for the unfolded protein response in the endoplasmic reticulum (ER). IRE1- and HAC1-disruptants require high concentrations of inositol for its normal growth. The ALG6, ALG8, and ALG10 genes encode the glucosyltransferases necessary for the completion of the synthesis of the lipid-linked oligosaccharide used for the asparagine-linked glycosylation of proteins in that order. Here we show that, given a combination of the hac1 defect with a disruption of ALG6, ALG8, and ALG10, no strains grow on inositol-free medium. However, the growth defect of the hac1-alg10 double disrupted was partially, but significantly, suppressed by the addition of inositol to the medium. These results indicate that inositol, according to the numbers of glucose residues in the oligosaccharide, plays an important role in the stress response and quality control of glycoproteins in the ER.