Yoko Y. Toyoshima

Assays for actin sliding movement over myosin-coated surfaces.

Publisher Summary One important result from in vitro studies of the interaction of the major proteins of muscle, actin and myosin, has been the growing recognition that nearly any aspect of muscle mechanics can be studied in a model system consisting of purified proteins. This chapter is a compilation of techniques for purified in vitro motility assays for actin sliding movement over myosin. Several forms of myosin, including filaments, monomers, and soluble proteolytic fragments, have been found to work well in aetin sliding movement assays. The focus is limited to studies using skeletal muscle proteins, but only slight modification of these protocols may be necessary for proteins derived from smooth muscle and nonmuscle sources. The properties of the protein preparations used are critical to reproducibility of actin sliding movement assays. The methods presented in the chapter are trustworthy preparations but are not singularly successful. However, in particular it should be noted that myosin subfragment preparations that work well in solution experiments might not be optimal for use in movement assays.

Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia

Dynein, the force-generating enzyme that powers the movement of cilia and flagella, has been characterized biochemically, but no simple system has been available for examining its motile properties. Here we describe a quantitative in vitro motility assay in which dynein adsorbed onto a glass surface induces linear translocation of purified bovine microtubules. Using this assay, we show that both 22S and 14S dyneins from Tetrahymena cilia induce movement but have distinct motile properties. A unique property of 14S dynein, which has not been described for other motility proteins, is its ability to generate torque that causes microtubules to rotate during forward translocation. In the axoneme, 14S dynein-induced torque may induce rotation of central-pair microtubules and may play an important role in generating three-dimensional ciliary beating patterns.

Isolation and Characterization of pos Mutants Defective in Correct Positioning of Septum in Schizosaccharomyces pombe

Abstract We have isolated mutants of fission yeast defective in correct positioning of septum. In visual screening, we obtained 16 clones showing unequal septation at restrictive temperature, which were classified into three complementation groups. At restrictive temperature, all the mutants underwent nuclear division normally. In cytokinesis, however, a contractile ring was formed at the site independent of the mitotic spindle. These results suggest that positional information for cytokinesis are not accurately transmitted to the cell equator. Furthermore, all the mutants frequently displayed incorrect orientation and/or distortion of septum, which suggests that the septum positioning is closely related to correct orientation and organization of septum.

LIS1 and NDEL1 coordinate the plus-end-directed transport of cytoplasmic dynein

LIS1 was first identified as a gene mutated in human classical lissencephaly sequence. LIS1 is required for dynein activity, but the underlying mechanism is poorly understood. Here, we demonstrate that LIS1 suppresses the motility of cytoplasmic dynein on microtubules (MTs), whereas NDEL1 releases the blocking effect of LIS1 on cytoplasmic dynein. We demonstrate that LIS1, cytoplasmic dynein and MT fragments co‐migrate anterogradely. When LIS1 function was suppressed by a blocking antibody, anterograde movement of cytoplasmic dynein was severely impaired. Immunoprecipitation assay indicated that cytoplasmic dynein forms a complex with LIS1, tubulins and kinesin‐1. In contrast, immunoabsorption of LIS1 resulted in disappearance of co‐precipitated tubulins and kinesin. Thus, we propose a novel model of the regulation of cytoplasmic dynein by LIS1, in which LIS1 mediates anterograde transport of cytoplasmic dynein to the plus end of cytoskeletal MTs as a dynein‐LIS1 complex on transportable MTs, which is a possibility supported by our data.

NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment

ABSTRACT NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.

Kinesin–microtubule binding depends on both nucleotide state and loading direction

Kinesin is a motor protein that transports organelles along a microtubule toward its plus end by using the energy of ATP hydrolysis. To clarify the nucleotide-dependent binding mode, we measured the unbinding force for one-headed kinesin heterodimers in addition to conventional two-headed kinesin homodimers under several nucleotide states. We found that both a weak and a strong binding state exist in each head of kinesin corresponding to a small and a large unbinding force, respectively; that is, weak for the ADP state and strong for the nucleotide-free and adenosine 5′-[β,γ-imido]triphosphate states. Model analysis showed that (i) the two binding modes in each head could be explained by a difference in the binding energy and (ii) the directional instability of binding, i.e., dependence of unbinding force on loading direction, could be explained by a difference in the characteristic distance for the kinesin–microtubule interaction during plus- and minus-end-directed loading. Both these factors must play an important role in the molecular mechanism of kinesin motility.

Dynein and kinesin share an overlapping microtubule‐binding site

Dyneins and kinesins move in opposite directions on microtubules. The question of how the same‐track microtubules are able to support movement in two directions remains unanswered due to the absence of details on dynein–microtubule interactions. To address this issue, we studied dynein–microtubule interactions using the tip of the microtubule‐binding stalk, the dynein stalk head (DSH), which directly interacts with microtubules upon receiving conformational change from the ATPase domain. Biochemical and cryo‐electron microscopic studies revealed that DSH bound to tubulin dimers with a periodicity of 80 Å, corresponding to the step size of dyneins. The DSH molecule was observed as a globular corn grain‐like shape that bound the same region as kinesin. Biochemical crosslinking experiments and image analyses of the DSH–kinesin head–microtubule complex revealed competition between DSH and the kinesin head for microtubule binding. Our results demonstrate that dynein and kinesin share an overlapping microtubule‐binding site, and imply that binding at this site has an essential role for these motor proteins.

Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors

Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

Regulation of Mitochondrial Transport and Inter-Microtubule Spacing by Tau Phosphorylation at the Sites Hyperphosphorylated in Alzheimer's Disease

The microtubule-associated protein Tau is a major component of the neurofibrillary tangles that serve as a neuropathological hallmark of Alzheimers disease. Tau is a substrate for protein phosphorylation at multiple sites and occurs in tangles in a hyperphosphorylated state. However, the physiological functions of Tau phosphorylation or how it may contribute mechanistically to Alzheimers pathophysiology are not completely understood. Here, we examined the function of human Tau phosphorylation at three sites, Ser199, Ser202, and Thr205, which together comprise the AT8 sites that mark abnormal phosphorylation in Alzheimers disease. Overexpression of wild-type Tau or mutated forms in which these sites had been changed to either unphosphorylatable alanines or phosphomimetic aspartates inhibited mitochondrial movement in the neurite processes of PC12 cells as well as the axons of mouse brain cortical neurons. However, the greatest effects on mitochondrial translocation were induced by phosphomimetic mutations. These mutations also caused expansion of the space between microtubules in cultured cells when membrane tension was reduced by disrupting actin filaments. Thus, Tau phosphorylation at the AT8 sites may have meaningful effects on mitochondrial movement, likely by controlling microtubule spacing. Hyperphosphorylation of the AT8 sites may contribute to axonal degeneration by disrupting mitochondrial transport in Alzheimers disease.

A Novel Action of Terpendole E on the Motor Activity of Mitotic Kinesin Eg5

To reveal the mechanism of mitosis, the development of M phase-specific inhibitors is an important strategy. We have been screening microbial products to find specific M phase inhibitors that do not directly target tubulins, and rediscovered terpendole E (TerE) as a novel Eg5 inhibitor. TerE did not affect microtubule integrity in interphase, but induced formation of a monoastral spindle in M phase. TerE inhibited both motor and microtubule-stimulated ATPase activities of human Eg5, but did not affect conventional kinesin from either Drosophila or bovine brain. Although terpendoles have been reported as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT), the Eg5 inhibitory activity of TerE was independent of ACAT inhibition. Taken together, we demonstrate that TerE is a novel Eg5 inhibitor isolated from a fungal strain.