Kuniyoshi Kaseda

Long-range cooperative binding of kinesin to a microtubule in the presence of ATP

Interaction of kinesin-coated latex beads with a single microtubule (MT) was directly observed by fluorescence microscopy. In the presence of ATP, binding of a kinesin bead to the MT facilitated the subsequent binding of other kinesin beads to an adjacent region on the MT that extended for micrometers in length. This cooperative binding was not observed in the presence of ADP or 5′-adenylylimidodiphosphate (AMP-PNP), where binding along the MT was random. Cooperative binding also was induced by an engineered, heterodimeric kinesin, WT/E236A, that could hydrolyze ATP, yet remained fixed on the MT in the presence of ATP. Relative to the stationary WT/E236A kinesin on a MT, wild-type kinesin bound preferentially in close proximity, but was biased to the plus-end direction. These results suggest that kinesin binding and ATP hydrolysis may cause a long-range state transition in the MT, increasing its affinity for kinesin toward its plus end. Thus, our study highlights the active involvement of MTs in kinesin motility.

Coordination of kinesin's two heads studied with mutant heterodimers

A conventional kinesin molecule has two identical catalytic domains (heads) and is thought to use them alternately to move processively, with 8-nm steps. To clarify how each head contributes to the observed steps, we have constructed heterodimeric kinesins that consist of two distinct heads. The heterodimers in which one of the heads is mutated in a microtubule-binding loop moved processively, even when the parent mutant homodimers bound too weakly to retain microtubules in microtubule-gliding assays. The velocities of the heterodimers were only slightly higher than those of the mutant homodimers, although mixtures of these weak-binding mutant homodimers and the WT dimers moved microtubules at a velocity similar to the WT. Thus, the mutant head affects the motility of the WT head only when they are in the same molecule. The maximum force a single heterodimer produced in optical trapping nanometry was intermediate between the WT and mutant homodimers, indicating that both heads contribute to the maximum force at the same time. These results demonstrate close collaboration of kinesins two heads in producing force and motility.

Dual pathway spindle assembly increases both the speed and the fidelity of mitosis.

Summary Roughly half of all animal somatic cell spindles assemble by the classical prophase pathway, in which the centrosomes separate ahead of nuclear envelope breakdown (NEBD). The remainder assemble by the prometaphase pathway, in which the centrosomes separate following NEBD. Why cells use dual pathway spindle assembly is unclear. Here, by examining the timing of NEBD relative to the onset of Eg5-mEGFP loading to centrosomes, we show that a time window of 9.2 ± 2.9 min is available for Eg5-driven prophase centrosome separation ahead of NEBD, and that those cells that succeed in separating their centrosomes within this window subsequently show >3-fold fewer chromosome segregation errors and a somewhat faster mitosis. A longer time window would allow more cells to complete prophase centrosome separation and further reduce segregation errors, but at the expense of a slower mitosis. Our data reveal dual pathway mitosis in a new light, as a substantive strategy that increases both the speed and the fidelity of mitosis.

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.

A novel approach for purification of recombinant proteins using the dextran-binding domain.

Using the dextran‐binding domain (DBD) of a type of glucosyltransferase (GTF) from Streptococcus sobrinus, we have developed a novel method for purifying recombinant proteins. DBD‐tagged green and red fluorescent proteins as well as the parent GTF and DBD moiety were adsorbed well to commercially available cross‐linked dextran (such as Sephadex beads and Sephacryl beads), and eluted efficiently with water‐soluble dextran. The purity of the eluted proteins after this one‐step affinity purification was ∼90% or better. The results suggest that DBD can be used as a powerful carrier for purification of various recombinant proteins.

Single-Molecule Imaging of Interaction between Dextran and Glucosyltransferase from Streptococcus sobrinus

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constraints estimated for TMR-labeled GTF at pH 6.8 and 25 degrees C in the absence and presence of sucrose were 9.2 and 13.3 s(-1), respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (> or = 25 s(-1)) under comparable conditions.

Modulation of actomyosin motor function by 1-hexanol

This study examines the effects of 1-hexanol as a perturbing agent on actomyosin ATPase and its related functions in the concentration range between 0 and 20 mM. In this range the denaturation of myosin subfragment 1 (S1), as measured by the inactivation rate of its K-EDTA-ATPase, and depolymerization of F-actin were insignificant. Major findings showed that hexanol had the following effects which were fully reversible, (a) a marked activation of S1 MgATPase (≈10-fold at 20 mM) without greatly affecting the enhancement of tryptophan fluorescence by formation of S1·ADP·Pi intermediate and the rate of ADP release from S1·ADP; (b) an inhibition of the maximum actin-activated ATPase activity; (c) an increase in the affinity of S1 for actin in the presence of ATP and a decrease in the presence of ADP or the absence of nucleotide; (d) a reduction in the sliding velocity of actin filaments in in vitro motility assays with myosin, and (e) a decrease in isometric tension of single skinned muscle fibers. Thus, the effects of hexanol on actomyosin interaction are distinct for the weak and strong binding states, consistent with a change in the hydrophobic interaction in the interface between myosin and actin accompanying the transition from the weak to the strong binding state. Hexanol also accelerates the Pi release from S1·ADP·Pi, which is the transition step from the weak to the strong binding state. The fact that hexanol accelerates Pi release suggests that this alcohol perturbs the S1·ADP·Pi conformation. We speculate that this intermediate-specific structural perturbation is related to the inhibition of the maximum actin-activated ATPase, in vitro motility, and isometric tension.