Keiko Hirose

The dynamin A ring complex: molecular organization and nucleotide-dependent conformational changes

Here we show that Dictyostelium discoideum dynamin A is a fast GTPase, binds to negatively charged lipids, and self‐assembles into rings and helices in a nucleotide‐dependent manner, similar to human dynamin‐1. Chemical modification of two cysteine residues, positioned in the middle domain and GTPase effector domain (GED), leads to altered assembly properties and the stabilization of a highly regular ring complex. Single particle analysis of this dynamin A* ring complex led to a three‐dimensional map, which shows that the nucleotide‐free complex consists of two layers with 11‐fold symmetry. Our results reveal the molecular organization of the complex and indicate the importance of the middle domain and GED for the assembly of dynamin family proteins. Nucleotide‐dependent changes observed with the unmodified and modified protein support a mechanochemical action of dynamin, in which tightening and stretching of a helix contribute to membrane fission.

A Novel System for Expressing Toxic Actin Mutants in Dictyostelium and Purification and Characterization of a Dominant Lethal Yeast Actin Mutant

We have developed a novel system for expressing recombinant actin in Dictyostelium. In this system, the C terminus of actin is fused to thymosin β via a glycine-based linker. The fusion protein is purified using a His tag attached to the thymosin β moiety and then cleaved by chymotrypsin immediately after the native final residue of actin to yield intact actin. Wild-type actin prepared in this way was functionally normal in terms of its polymerization kinetics and muscle myosin-mediated motility. We expected that this system would be particularly useful for expressing toxic actin mutants, because the actin moiety of the fusion protein is unlikely to interact with the actin cytoskeleton of the host cells. We therefore chose to express the E206A/R207A/E208A mutant, which appears to be dominant lethal in yeast, as a model case of a toxic actin mutant that is difficult to express. We found that the E206A/R207A/E208A mutant could be expressed and purified with a yield comparable to the wild-type molecule (3–4 mg/20 g cells), even though green fluorescent protein-fused actin carrying the E206A/R207A/E208A mutation was expressed at a much lower level than wild-type actin. Purified E206A/R207A/E208A actin did not polymerize, even in the presence of muscle actin; however, it accelerated polymerization of muscle actin and inhibited the nucleating and severing activities of gelsolin. Given that the location of the substituted residues is near the pointed end face of the mutant, we suggest that E206A/R207A/E208A actin behaves like a weak pointed end-capping protein that perturbs the actin cytoskeleton of the host cells.

Structural comparison of dimeric Eg5, Neurospora kinesin (Nkin) and Ncd head–Nkin neck chimera with conventional kinesin

Cryo‐electron microscopy and 3D image reconstruction of microtubules saturated with kinesin dimers has shown one head bound to tubulin, the other free. The free head of rat kinesin sits on the top right of the bound head (with the microtubule oriented plus‐end upwards) in the presence of 5′‐adenylylimido‐diphosphate (AMPPNP) and on the top left in nucleotide‐free solutions. To understand the relevance of this movement, we investigated other dimeric plus‐end‐directed motors: Neurospora kinesin (Nkin); Eg5, a slow non‐processive kinesin; and a chimera of Ncd heads attached to Nkin necks. In the AMPPNP (ATP‐like) state, all dimers have the free head to the top right. In the absence of nucleotide, the free head of an Nkin dimer appears to occupy alternative positions to either side of the bound head. Despite having the Nkin neck, the free head of the chimera was only seen to the top right of the bound head. Eg5 also has the free head mostly to the top right. We suggest that processive movement may require kinesins to move their heads in alternative ways.

Single‐headed mode of kinesin‐5

In most organisms, kinesin‐5 motors are essential for mitosis and meiosis, where they crosslink and slide apart the antiparallel microtubule half‐spindles. Recently, it was shown using single‐molecule optical trapping that a truncated, double‐headed human kinesin‐5 dimer can step processively along microtubules. However, processivity is limited (∼8 steps) with little coordination between the heads, raising the possibility that kinesin‐5 motors might also be able to move by a nonprocessive mechanism. To investigate this, we engineered single‐headed kinesin‐5 dimers. We show that a set of these single‐headed Eg5 dimers drive microtubule sliding at about 90% of wild‐type velocity, indicating that Eg5 can slide microtubules by a mechanism in which one head of each Eg5 head‐pair is effectively redundant. On the basis of this, we propose a muscle‐like model for Eg5‐driven microtubule sliding in spindles in which most force‐generating events are single‐headed interactions and alternate‐heads processivity is rare.

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.

Distinct Biochemical Properties of Arabidopsis thaliana Actin Isoforms.

Plants and animals express multiple actin isoforms in a manner that is dependent on tissues, organs and the stage of development. Previous genetic analyses suggested that individual actin isoforms have specific roles in cells, but there is little biochemical evidence to support this hypothesis. In this study, we purified four recombinant Arabidopsis actin isoforms, two major vegetative actin isoforms, ACT2 and ACT7, and two major reproductive isoforms, ACT1 and ACT11, and characterized them biochemically. Phalloidin bound normally to the filaments of the two reproductive actins as well as to the filaments of skeletal muscle actin. However, phalloidin bound only weakly to ACT7 filaments and hardly at all to ACT2 filaments, despite the conserved sequence of the phalloidin-binding site. Polymerization and phosphate release rates among these four actin isoforms were also significantly different. Moreover, interactions with profilin (PRF) were also different among the four Arabidopsis actin isoforms. PRF1 and PRF2 inhibited the polymerization of ACT1, ACT11 and ACT7, while ACT2 was only weakly affected. Plant actin isoforms have different biochemical properties. This result supports the idea that actin isoforms play specific roles to achieve multiple cell functions.

Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin, Leading to Dominant Toxicity in Vivo

Background: Mutation of Asp-11 is dominant negative in yeast and human actins. Results: Mutant actins exchange bound nucleotides rapidly, cannot bind cofilin, and cofilin-induced depolymerization of mutant and wild type copolymers is slow. Conclusion: Rapid nucleotide exchange with exogenous ATP inhibits cofilin-mediated depolymerization of copolymers, leading to dominant toxicity. Significance: Mechanism of a dominant negative actin mutation is elucidated. Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human α-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.

Gas7b (Growth Arrest Specific Protein 7b) Regulates Neuronal Cell Morphology by Enhancing Microtubule and Actin Filament Assembly

Background: Growth arrest specific protein 7b (Gas7b) protein levels are significantly reduced in the brain tissue of patients with Alzheimer disease. Results: Gas7b enhances microtubule bundling and microtubule cross-linking with the actin filament. Conclusion: Gas7b regulates neural cell morphology by altering the cytoskeletal organization. Significance: A decrease in Gas7b levels impairs neural cell function. Neurons undergo several morphological changes as a part of normal neuron maturation process. Alzheimer disease is associated with increased neuroproliferation and impaired neuronal maturation. In this study, we demonstrated that Gas7b (growth arrest specific protein 7b) expression in a neuronal cell line, Neuro 2A, induces cell maturation by facilitating formation of dendrite-like processes and/or filopodia projections and that Gas7b co-localizes with neurite microtubules. Molecular analysis was performed to evaluate whether Gas7b associates with actin filaments and microtubules, and the data revealed two novel roles of Gas7b in neurite outgrowth: we showed that Gas7b enhances bundling of several microtubule filaments and connects microtubules with actin filaments. These results suggest that Gas7b governs neural cell morphogenesis by enhancing the coordination between actin filaments and microtubules. We conclude that lower neuronal Gas7b levels may impact Alzheimer disease progression.

A Bidirectional Kinesin Motor in Live Drosophila Embryos

Spindle assembly and elongation involve poleward and away‐from‐the‐pole forces produced by microtubule dynamics and spindle‐associated motors. Here, we show that a bidirectional Drosophila Kinesin‐14 motor that moves either to the microtubule plus or minus end in vitro unexpectedly causes only minor spindle defects in vivo. However, spindles of mutant embryos are longer than wild type, consistent with increased plus‐end motor activity. Strikingly, suppressing spindle dynamics by depriving embryos of oxygen causes the bidirectional motor to show increased accumulation at distal or plus ends of astral microtubules relative to wild type, an effect not observed for a mutant motor defective in motility. Increased motor accumulation at microtubule plus ends may be due to increased slow plus‐end movement of the bidirectional motor under hypoxia, caused by perturbation of microtubule dynamics or inactivation of the only other known Drosophila minus‐end spindle motor, cytoplasmic dynein. Negative‐stain electron microscopy images are consistent with highly cooperative motor binding to microtubules, and gliding assays show dependence on motor density for motility. Mutant effects of the bidirectional motor on spindle function may be suppressed under normal conditions by motor: motor interactions and minus‐end movement induced by spindle dynamics. These forces may also bias wild‐type motor movement toward microtubule minus ends in live cells. Our findings link motor : motor interactions to function in vivo by showing that motor density, together with cellular dynamics, may influence motor function in live cells.

High-resolution structural analysis of the kinesin-microtubule complex by electron cryo-microscopy.

To understand the interaction of kinesin and microtubules, it is necessary to study the three-dimensional (3D) structures of the kinesin-microtubule complex at a high enough resolution to identify structural components such as alpha-helices and beta-sheets. Electron cryo-microscopy combined with computer image analysis is the most common method to study such complexes that cannot be crystallized. By selecting microtubules that have a helical symmetry, 3D structures of the complex can be calculated using the helical 3D reconstruction method. Details of the interaction are studied by docking the individual crystal structures of the kinesin motor domains and tubulin heterodimer into the 3D maps of the complex. To study the structural changes during ATP hydrolysis, structures of the complexes in the presence and absence of different nucleotides are compared.