Kenji Kikushima

Slow ADP-dependent acceleration of microtubule translocation produced by an axonemal dynein

Dynein has four nucleotide binding sites, of which the functional significance is unknown except for the single catalytic site. To obtain clues to the function of non‐catalytic nucleotide binding, we examined the effect of ADP on the in vitro motility of Chlamydomonas inner‐arm dynein species ‘a’. Upon continuous perfusion with ATP and ADP, microtubules glided on a dynein‐coated glass surface with a velocity that gradually increased over a few minutes. The velocity increased faster at higher ADP concentrations. These results suggest that this dynein is activated by nucleotide binding to regulatory site(s) through an extremely slow process.

Clockwise translocation of microtubules by flagellar inner-arm dyneins in vitro.

Cilia and flagella are equipped with multiple species of dyneins that have diverse motor properties. To assess the properties of various axonemal dyneins of Chlamydomonas, in vitro microtubule translocation by isolated dyneins was examined with and without flow of the medium. With one inner-arm dynein species, dynein c, most microtubules became aligned parallel to the flow and translocated downstream after the onset of flow. When the flow was stopped, the gliding direction was gradually randomized. In contrast, with inner-arm dyneins d and g, microtubules tended to translocate at a shallow right angle to the flow. When the flow was stopped, each microtubule turned to the right, making a curved track. The clockwise translocation was not accompanied by lateral displacement, indicating that these dyneins generate torque that bends the microtubule. The torque generated by these dyneins in the axoneme may modulate the relative orientation between adjacent doublet microtubules and lead to more efficient functioning of total dyneins.

The allosteric transition of GroEL induced by metal fluoride-ADP complexes.

To understand the mechanism of a functionally important ATP-induced allosteric transition of GroEL, we have studied the effect of a series of metal fluoride-ADP complexes and vanadate-ADP on GroEL by kinetic fluorescence measurement of pyrene-labeled GroEL and by small-angle X-ray scattering measurement of wild-type GroEL. The metal fluorides and vanadate, complexed with ADP, are known to mimic the gamma-phosphate group of ATP, but they differ in geometry and size; it is expected that these compounds will be useful for investigating the strikingly high specificity of GroEL for ATP that enables the induction of the allosteric transition. The kinetic fluorescence measurement revealed that aluminium, beryllium, and gallium ions, when complexed with the fluoride ion and ADP, induced a biphasic fluorescence change of pyrenyl GroEL, while scandium and vanadate ions did not induce any kinetically observed change in fluorescence. The burst phase and the first phase of the fluorescence kinetics were reversible, while the second phase and subsequent changes were irreversible. The dependence of the burst-phase and the first-phase fluorescence changes on the ADP concentration indicated that the burst phase represents non-cooperative nucleotide binding to GroEL, and that the first phase represents the allosteric transition of GroEL. Both the amplitude and the rate constant of the first phase of the fluorescence kinetics were well understood in terms of a kinetic allosteric model, which is a combination of transition state theory and the Monod-Wyman-Changeux allosteric model. From the kinetic allosteric model analysis, the relative free energy of the transition state in the metal fluoride-ADP-induced allosteric transition of GroEL was found to be larger than the corresponding free energy of the ATP-induced allosteric transition by more than 5.5kcal/mol. However, the X-ray scattering measurements indicated that the allosteric state induced by these metal fluoride-ADP complexes is structurally equivalent to the allosteric state induced by ATP. These results suggested that both the size and coordination geometry of gamma-phosphate (and its analogs) are related to the allosteric transition of GroEL. It was therefore concluded that the tetrahedral geometry of gamma-phosphate (or its analogs) and the inter-atomic distance ( approximately 1.6A) between phosphorus (vanadium, or metal atom) and oxygen (or fluorine) are both important for inducing the allosteric transition of GroEL, leading to the high selectivity of GroEL for ATP about ligand adenine nucleotides, which function as the preferred allosteric ligand.

Central pair apparatus enhances outer-arm dynein activities through regulation of inner-arm dyneins.

The beating of eukaryotic cilia and flagella is controlled by multiple species of inner-arm and outer-arm dyneins. To clarify the regulation on axonemal beating by nucleotide conditions and central-pair microtubules, microtubule sliding in disintegrating Chlamydomonas axonemes of various mutants and in vitro microtubule gliding by isolated axonemal dyneins were examined. In the in vitro motility assays with outer-arm dyneins (alphabeta and gamma), microtubule translocation velocity decreased at high concentrations of ATP, while this inhibition was canceled by the simultaneous presence of ADP or ribose-modified analogues, mantATP/ADP. In contrast, motility of inner-arm dyneins was rather insensitive to these nucleotides. The velocity of sliding disintegration in axonemes lacking the central pair was less than that in wild-type axonemes at high ATP concentrations, but was overcome by the presence of ADP or mantATP/ADP. While these nucleotides did not activate the sliding velocity in other mutant axonemes, they increased the extent of sliding, except for axonemes lacking outer-arm dynein. Experiments with axonemes lacking inner-arm dynein f using casein kinase 1 inhibitor suggest that the regulation of outer-arm dynein by the central pair is effected through the activation of inner-arm dynein f, and possibly by other interactions. These results indicate that the central pair activates outer-arm dyneins on specific outer-doublet, resulting in amplification of the axonemal bending force.

A non-invasive imaging for the in vivo tracking of high-speed vesicle transport in mouse neutrophils

Neutrophils play an essential role in the innate immune response. To understand neutrophil activity, the development of a new technique to observe neutrophils in situ is required. Here, we report the development of a non-invasive technique for the in vivo imaging of neutrophils labeled with quantum dots, up to 100 μm below the skin surface of mice. Upon inflammation neutrophils began to extravasate from blood vessels and locomoted in interstitial space. Most intriguingly, the quantum dots were endocytosed into vesicles in the neutrophils, allowing us to track the vesicles at 12.5 msec/frame with 15–24 nm accuracy. The vesicles containing quantum dots moved as “diffuse-and-go” manner and were transported at higher speed than the in vitro velocity of a molecular motor such as kinesin or dynein. This is the first report in which non-invasive techniques have been used to visualize the internal dynamics of neutrophils.

Ratchetlike properties of in vitro microtubule translocation by a Chlamydomonas inner-arm dynein species c in the presence of flow.

To investigate the force generation properties of Chlamydomonas axonemal inner-arm dyneins in response to external force, we analyzed microtubule gliding on dynein-coated surfaces under shear flow. When inner-arm dynein c was used, microtubule translocation in the downstream direction accelerated with increasing flow speed in a manner that depended on the dynein density and ATP concentration. In contrast, the microtubule translocation velocity in the upstream direction was unaffected by the flow speed. The number of microtubules on the glass surface was almost constant with and without flow, suggesting that gliding acceleration was not simply caused by weakened dynein-microtubule binding. With other inner-arm dynein species, the microtubule gliding velocity was unaffected by the flow regardless of the flow direction or nucleotide concentration. The flow-generated force acting on a single dynein was estimated to be as small as approximately 0.03 pN/dynein. These results indicate that dynein c possesses a ratchetlike property that allows acceleration only in one direction by a very small external force. This property should be important for slow- and fast-moving dyneins to function simultaneously within the axoneme.

A novel approach for purification and selective capture of membrane vesicles of the periodontopathic bacterium, Porphyromonas gingivalis: membrane vesicles bind to magnetic beads coated with epoxy groups in a noncovalent, species-specific manner.

Membrane vesicles (MVs) of Porphyromonas gingivalis are regarded as an offensive weapon of the bacterium, leading to tissue deterioration in periodontal disease. Therefore, isolation of highly purified MVs is indispensable to better understand the pathophysiological role of MVs in the progression of periodontitis. MVs are generally isolated by a conventional method based on ultracentrifugation of the bacterial culture supernatant. However, the resulting MVs are often contaminated with co-precipitating bacterial appendages sheared from the live bacteria. Here, we report an intriguing property of P. gingivalis MVs–their ability to bind superparamagnetic beads coated with epoxy groups (SB-Epoxy). Analysis of fractions collected during the purification revealed that all MVs of five tested P. gingivalis stains bound to SB-Epoxy. In contrast, free fimbriae in the crude MV preparation did not bind to the SB-Epoxy. The SB-Epoxy-bound MVs were easily dissociated from the SB-Epoxy using a mild denaturation buffer. These results suggest that the surface chemistry conferred by epoxy on the beads is responsible for the binding, which is mediated by noncovalent bonds. Both the structural integrity and purity of the isolated MVs were confirmed by electron microscopy. The isolated MVs also caused cell detachment from culture dishes at a physiologically relevant concentration. Assays of competitive binding between the SB-Epoxy and mixtures of MVs from five bacterial species demonstrated that only P. gingivalis MVs could be selectively eliminated from the mixtures. We suggest that this novel approach enables efficient purification and selective elimination of P. gingivalis MVs.