Mutant Microtubule Research Articles

Pectin Dependent Cell Adhesion Restored by a Mutant Microtubule Organizing Membrane Protein.

The cellulose- and pectin-rich plant cell wall defines cell structure, mediates defense against pathogens, and facilitates plant cell adhesion. An adhesion mutant screen of Arabidopsis hypocotyls identified a new allele of QUASIMODO2 (QUA2), a gene required for pectin accumulation and whose mutants have reduced pectin content and adhesion defects. A suppressor of qua2 was also isolated and describes a null allele of SABRE (SAB), which encodes a previously described plasma membrane protein required for longitudinal cellular expansion that organizes the tubulin cytoskeleton. sab mutants have increased pectin content, increased levels of expression of pectin methylesterases and extensins, and reduced cell surface area relative to qua2 and Wild Type, contributing to a restoration of cell adhesion.

Microtubule Severing by Katanin p60 AAA+ ATPase Requires the C-terminal Acidic Tails of Both α- and β-Tubulins and Basic Amino Acid Residues in the AAA+ Ring Pore

The microtubule (MT) network is highly dynamic and undergoes dramatic reorganizations during the cell cycle. Dimers of α- and β-tubulins rapidly polymerize to and depolymerize from the end of MT fibrils in an intrinsic GTP-dependent manner. MT severing by ATP-driven enzymes such as katanin and spastin contributes significantly to microtubule dynamics, and it has been shown that katanin p60, a AAA+ family protein, has ATPase and MT-severing activities. The mechanism of MT severing by katanin p60 is poorly understood, and the residues in katanin p60 and tubulins important for severing activity were therefore explored in this study. MT-severing activity, but not ATPase activity, was inhibited by mutations of the conserved aromatic residue and the flanking basic residues in the pore region of the katanin p60 hexameric ring. When the acidic residue-rich C-terminal unstructured segment of either α- or β-tubulin was removed, polymerized MTs were resistant to katanin p60 treatment. Interactions between katanin p60 and the mutant MTs, on the other hand, were unaffected. Taken together, these findings led us to propose that the interactions between the positively charged residues of katanin p60 and the acidic tails of both tubulins are essential for efficient severing of MTs.

Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

The spindle matrix protein, Chromator, is a novel tubulin binding protein that can interact with both microtubules and free tubulin.

The chromodomain protein, Chromator, is localized to chromosomes during interphase; however, during cell division together with other nuclear proteins Chromator redistributes to form a macro molecular spindle matrix complex that embeds the microtubule spindle apparatus. It has been demonstrated that the CTD of Chromator is sufficient for localization to the spindle matrix and that expression of this domain alone could partially rescue Chro mutant microtubule spindle defects. Furthermore, the presence of frayed and unstable microtubule spindles during mitosis after Chromator RNAi depletion in S2 cells indicated that Chromator may interact with microtubules. In this study using a variety of biochemical assays we have tested this hypothesis and show that Chromator not only has binding activity to microtubules with a Kd of 0.23 µM but also to free tubulin. Furthermore, we have mapped the interaction with microtubules to a relatively small stretch of 139 amino acids in the carboxy-terminal region of Chromator. This sequence is likely to contain a novel microtubule binding interface since database searches did not find any sequence matches with known microtubule binding motifs.

Dissecting the cellular functions of plant microtubules using mutant tubulins

α- and β-tubulins, the building blocks of the microtubule (MT) polymer, are encoded by multiple genes that are largely functionally redundant in plants. Null tubulin mutants are thus phenotypically indistinguishable from the wild type, but miss-sense or deletion mutations of critical amino acid residues that are important for the assembly, stability, or dynamics of the polymer disrupt the proper organization and function of the resultant MT arrays. Mutant tubulins co-assemble with wild-type tubulins into mutant MTs with compromised functions, and thus mechanistically act as dominant-negative MT poisons. Cortical MT arrays in interphase plant cells are most sensitive to tubulin mutations, and are transformed into helical structures or random orientation, which produce twisted or radially swollen cells. Mutant plants resistant to MT-targeted herbicides may possess tubulin mutations at the binding sites of the herbicides. Tubulin mutants are valuable tools for investigating how individual MTs are organized into particular patterns in cortical arrays, and for defining the functional contribution of MTs to various MT-dependent or -assisted cellular processes in plant cells.

Human Disease-Related Mutation at R262 of β3-Tubulin Critical for Kinesin Motility and ATP Hydrolysis

Kinesin-microtubule interaction plays essential roles in brain development and function. In human neuronal cells, a point mutation, R262C/H, in β3-tubulin causes a disrupton in the interaction of microtubules with kinesin, leading to developmental disorders collectively termed TUBB3 syndrome (Tischfield, 2010). To clarify the molecular mechanism underlying the disease, in vitro functional analysis of mutant microtubules is essential. Aiming to examine the effect of tubulin mutation on kinesin motility and ATPase activity, here we developed a method to express and purify recombinant human tubulin using a baculovirus-insect-cell expression system. The method yields >1 mg of human α1β3-tubulin with a purity of >95% from 1 L culture of High Five cell. In a single-molecule motility assay, while kinesin moved along wild type (WT) human microtubules at a velocity comparable to that on porcine brain microtubules, it scarcely bound to β3-R262A microtubules. In parallel with this observation, in β-R262A mutants, the maximum ATPase rate (kcat) was reduced four-fold and the Michaelis constant (kM,MT) was 40 times larger than that of WT. These results clearly demonstrate the involvement of β-R262 in kinesin-microtubule interaction. Compared with other critical residues previously identified using yeast mutant microtubules (Uchimura 2010), β-R262 is unusual because it is the only positively charged residue critical for kinesin-microtubule interaction. Whether the β-R262 residue directly binds to a putatively negatively charged residue in kinesin or modulates kinesin-microtubule interaction through repulsion will be discussed at the poster session. Our new method provides a powerful tool to perform molecular level analyses on the function of tubulin/microtubules associated with human diseases.

Characterization of the Microtubules Modified with Photochromic Molecules

Previously we have introduced photochromic molecules into the functional region of kinesin and succeeded to regulate the ATPase activity of kinesin reversibly by light irradiation. However, the regulation efficiency of the ATPase activity was not significant. It is well known that the contraction of skeletal muscle is regulated by calcium binding to the rail protein actin filament. The regulating system achieves high efficient switching. Therefore, it is expected that introducing artificial switching system into the rail protein microtubules enables high efficient regulation of kinesin motor activity. In this study, we tried to introduce photochromic molecules into the microtubules to regulate kinesin movement. First we modified the microtubules composed of wild type tubulin and measured gliding speed for kinesin using invitro motility assay under UV or VIS light irradiation, respectively. Gliding speeds of the microtubules modified with 4-phenylazophenyl maleimide (PAM) or 3,3-dimethyl-6’-nitro −1-[2- [3-(3- pyrroline −2,5-dion) −1-yl] propanoyloxy] ethylindoline −2-spiro-2’ (2H)- chromene (MASP) were almost identical to intact microtubules and also were not influenced by UV or VIS light irradiation. This result suggested that that these photochromic molecules incorporated into intrinsic reactive cysteine residues on the surface of microtubules did not affect kinesin motor activity. Subsequently, we prepared the 4 types of α-tubulin mutants (V406C, V410C, M414C, G417C), which have additional cysteine residues in the kinesin binding region. Microtubule concentration dependent kinesin ATPase activity for the mutant microtubules, V406C, V410C and M414 showed almost similar values to that of WT microtubules. On the other hand, ATPase activity for G417C mutant microtubule drastically decreased. The mutant microtubules were modified with photochromic molecules and the effect upon UV-VIS light irradiation for gliding speed and kinesin ATPase activity were studied.

KIF1A Repeats Cycle of ‘FREE Diffusion’ and ‘SPECIFIC Binding’ during Weak Binding State

The nature of intermolecular interaction between motor and cytoskeletal filament during the weak binding state is not fully understood. In the case of kinesin, while structural analyses revealed that kinesin binds to a specific binding site on tubulin, motility data suggested that kinesin undergoes diffusion, searching for its next binding site. To understand how specific binding and diffusion are compatible in a single ADP state, we analyzed the motion of the single-headed kinesin KIF1A on various mutant microtubules (MTs) in the presence of ADP, using the single molecule motility assay. We prepared two series of mutant MTs. The first is a series with increased/decreased negative charges at the C-terminal tails (CTTs) of tubulin, reported to be indispensable for the weak binding of KIF1A to the MT (Okada et al., 2000). The second is a series of charged-to-alanine mutants in the H11-12 loop and H12 of tubulin (α-E415, -E416, -E418, -E421 and β-E410, -D417), found to be critical for kinesin motility and ATPase (Uchimura et al., 2010). The analyses of KIF1A movement showed that a reduction of negative charges in CTTs leads to a reduction in both the duration of interaction and the diffusion length of KIF1A, yet the diffusion constant was not greatly changed. In contrast, in most of the charged-to-alanine tubulin mutants, the diffusion constant of KIF1A increased and the duration shortened, but the diffusion length was unaffected. These results indicate that KIF1A-MT interaction in the ADP state can be modeled as an equilibrium between two substates: a dynamic ‘diffusion state’ and a static ‘binding state’. While CTTs stabilize the former, the critical residues in the H11-12 loop and H12 of tubulin stabilize the latter. This model is applicable to dimeric kinesin.

Photo-Regulation of Kinesin ATPase Using Microtubules Modified with Photochromic Molecule

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Previously we have introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin and succeeded to regulate the ATPase activity of kinesin reversibly by light irradiation. However, the regulation efficiency of the ATPase activity was not significant. It is well known that the contraction of skeletal muscle myosin is regulated by calcium binding to the rail protein actin. The regulating system achieves high efficient switching. Therefore, it is expected that introducing artificial switching system into the microtubules enables high efficient regulation of kinesin motor activity. In this study, we have done the experiments on incorporation of photochromic molecule into wild type microtubules and gliding assay of them under UV and VIS light irradiation. The gliding speed of the wild type microtubules modified with PAM or 3,3-dimethyl-6’-nitro −1-[2- [3-(3- pyrroline −2,5-dion) −1-yl] propanoyloxy]ethylindoline −2-spiro-2’(2H)- chromene (MASP) did not change. The results suggested that modification of the intrinsic reactive cysteine residues on the surface of microtubules does not affect motor activity of kinesin. Therefore, currently we are trying to prepare mutant microtubules that have an additional single reactive cysteine residue at the region near the kinesin binding site.

Cytoplasmic Dynein Travel Cut Short by a Neurodegenerative Mutation in its Tail

Cytoplasmic dynein is a minus-end directed microtubule motor responsible for cellular functions including fast retrograde axonal transport. The Loa mouse strain carries a mutation within the tail domain of the dynein heavy chain gene, and develops progressive motor neuron degeneration similar to amyotrophic lateral sclerosis, associated with a decreased rate of retrograde axonal transport. To understand the molecular basis for transport impairment and its role in motor neuron degeneration, we have conducted a detailed biochemical and biophysical study of purified mutant and wild-type mouse dynein. The mutant dynein was identical in subunit composition to the wild type protein but showed mild, but reproducible dissociation during sucrose gradient centrifugation (Ori-McKenney et al., 2009, MBC abstr., in press). ATPase activity for the mutant dynein exhibited a higher Km for microtubules, and the mutant's microtubule binding was reduced in the presence of ATP. Using optical trap and quantum dot assays, we found that Loa mutation drastically reduced the single motor processivity of dynein without affecting its velocity or force production. Surprisingly, small increase in buffer ionic strength further exaggerated the difference between the mutant and wild-type travel. Our analysis of motor motion under load showed no difference in the stepping behavior between mutant and wild-type dynein. We propose that the Loa mutation introduces defects in the interhead communication leading to decreased processivity, possibly via weakened heavy chain-intermediate chain interaction. Our study provides the first indication that a tail mutation can affect dynein function. Furthermore, our results provide the first link between altered processivity and disease. Supported by RO1GM070676, AHA0825278F, GM47434, and the CUMC MNC.Authors Jing Xu and Kassandra M. Ori-McKenney contributed equally to this work. Steven P. Gross and Richard B. Vallee are co-senior authors.

Three-State Model for One-Dimensional Brownian Motion of Charged Nanoparticles Along Microtubules

A variety of motor proteins, such as dynein, KIF1A and MCAK, are known to exhibit one-dimensional (1D) Brownian motion along microtubules. The electrostatic interaction between the proteins and microtubules appears to be crucial for 1D Brownian motion, although the underlying mechanism has not been fully clarified. We examined the interactions of positively-charged nanoparticles composed of polyacrylamide gels with microtubules. These hydrophilic nanoparticles bound to the microtubules and displayed 1D Brownian motion in a charge-dependent manner, which indicates that nonspecific electrostatic interaction is sufficient for 1D Brownian motion. While the diffusion coefficient decreased exponentially with an increasing particle charge (with the exponent being 0.10 kBTper charge), the duration of the interaction increased exponentially (with an exponent of 0.22 kBT per charge). These results can be consistently explained if one assumes that a particle repeats a cycle of ‘binding’ and ‘diffusion’ along a microtubule until it finally ‘dissociates’ from the microtubule. This entire process can be described by a three-state model analogous to the Michaelis-Menten scheme, in which two parameters - the equilibrium constant between ‘binding’ and ‘diffusion’, and the rate of ‘dissociation’ from the microtubule - are derived as a function of the particle charge density.Further, to understand the molecular basis for this ‘binding’ and ‘diffusion’ mechanism, we engineered microtubules with variable charges at the tubulin C-terminal tail (CTT) and measured 1D Brownian motion of the particles along these mutant microtubules. The measurements revealed an unexpected result: the negative charges in the CTT did not significantly affect the diffusion coefficient of the particles, but the equilibrium between ‘binding’ and ‘diffusion’ shifted more towards ‘diffusion’ with increasing charges of the CTT. These results indicate that the negatively-charged CTT provides a field that facilitates the ‘diffusion’ of charged particles.

Hypersensitivity to cytoskeletal antagonists demonstrates microtubule–microfilament cross‐talk in the control of root elongation in Arabidopsis thaliana

Elongation of diffusely expanding plant cells is thought to be mainly under the control of cortical microtubules. Drug treatments that disrupt actin microfilaments, however, can reduce elongation and induce radial swelling. To understand how microfilaments assist growth anisotropy, we explored their functional interactions with microtubules by measuring how microtubule disruption affects the sensitivity of cells to microfilament-targeted drugs. We assessed the sensitivity to actin-targeted drugs by measuring the lengths and diameters of expanding roots and by analysing microtubule and microfilament patterns in the temperature-sensitive Arabidopsis thaliana mutant microtubule organization 1 (mor1-1), along with other mutants that constitutively alter microtubule arrays. At the restrictive temperature of mor1-1, root expansion was hypersensitive to the microfilament-disrupting drugs latrunculin B and cytochalasin D, while immunofluorescence microscopy showed that low doses of latrunculin B exacerbated microtubule disruption. Root expansion studies also showed that the botero and spiral1 mutants were hypersensitive to latrunculin B. Hypersensitivity to actin-targeted drugs is a direct consequence of altered microtubule polymer status, demonstrating that cross-talk between microfilaments and microtubules is critical for regulating anisotropic cell expansion.