Masaki Edamatsu

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.

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.

The human chromokinesin Kid is a plus end-directed microtubule-based motor

Kid is a kinesin‐like DNA‐binding protein known to be involved in chromosome movement during mitosis, although its actual motor function has not been demonstrated. Here, we describe the initial characterization of Kid as a microtubule‐based motor using optical trapping microscopy. A bacterially expressed fusion protein consisting of a truncated Kid fragment (amino acids 1–388 or 1–439) is indeed an active microtubule motor with an average speed of ∼160 nm/s, and the polarity of movement is plus end directed. We could not detect processive movement of either monomeric Kid or dimerizing chimeric Kid; however, low levels of processivity (a few steps) cannot be detected with our method. These results are consistent with Kid having a role in chromosome congression in vivo, where it would be responsible for the polar ejection forces acting on the chromosome arms.

Hybrid nanotransport system by biomolecular linear motors

We have demonstrated a novel micro/nanotransport system using biomolecular motors driven by adenosine triphosphate (ATP). For the driving mechanism, microtubule-kinesin system, which is one of the linear biomolecular motor systems was investigated. ATP dissolved in an aqueous condition is hydrolyzed to adenosine diphosphate (ADP) to energize the bionanoactuators in this mechanism. This means the system does not require an external electrical or mechanical energy source. Therefore, a purely chemical system which is similar to the in vivo transport will be realized. This paper reports some fundamental studies to integrate biomaterials and MEMS. The microtubules, or rail molecules, were patterned on a glass substrate with poly(dimethyl siloxane) (PDMS) using a regular soft lithography technique. Microbeads (320 nm in diameter) and a micromachined structure (2/spl times/3 /spl mu/m, 2 /spl mu/m in thickness) coated with kinesin molecules were transported along the microtubules at an average speed of 476/spl plusmn/56 and 308 nm/s, respectively. While ATP injection activated the transport system we have also managed to provide repetitive on/off control using hexokinase as an inhibitor. For the minimum response time in the repetitive control, the optimized concentration for ATP was 10/sup 2/ /spl mu/M and 10/sup 3/ U/L for hexokinase.

Fission yeast synaptobrevin is involved in cytokinesis and cell elongation

Synaptobrevin is a vesicle-associated membrane protein playing an essential role in regulated vesicle transport. In this study, we characterized Syb1, synaptobrevin of Schizosaccharomyces pombe. Syb1 was located on various sizes of vesicle-like structures in the cytoplasm and enriched in the medial region and cell ends. Transport of Syb1 to the medial region was mainly dependent on F-actin and Myo52/Myo4. Syb1 is essential for cell viability and most of the syb1-null cells showed a round or short cylindrical form. These results suggest that Syb1 is involved in membrane trafficking of cytokinesis and cell elongation.

Immunological detection of actin in the 14S ciliary dynein of Tetrahymena

The association of actin with Tetrahymena ciliary dyneins was examined using a polyclonal antibody against Tetrahymena actin. Western blotting shows that actin is present in the 14S dynein fraction, but not in the 22S dynein fraction, which comprises the outer arm. By anion‐exchange chromatography, 14S dynein can be further separated into three major fractions that contain four distinct heavy chains in total. When each fraction was tested by anti‐actin immunoblotting, all three fractions contained actin in nearly stoichiometric amounts with the heavy chain. Since Tetrahymena actin differs significantly from acting of other species, the association with inner‐arm dynein may be a conserved property of actin.

Diffusion and Directed Movement IN VITRO MOTILE PROPERTIES OF FISSION YEAST KINESIN-14 Pkl1

Fission yeast Pkl1 is a kinesin-14A family member that is known to be localized at the cellular spindle and is capable of hydrolyzing ATP. However, its motility has not been detected. Here, we show that Pkl1 is a slow, minus end-directed microtubule motor with a maximum velocity of 33 ± 9 nm/s. The Km,MT value of steady-state ATPase activity of Pkl1 was as low as 6.4 ± 1.1 nm, which is 20–30 times smaller than that of kinesin-1 and another kinesin-14A family member, Ncd, indicating a high affinity of Pkl1 for microtubules. However, the duty ratio of 0.05 indicates that Pkl1 spends only a small fraction of the ATPase cycle strongly associated with a microtubule. By using total internal reflection fluorescence microscopy, we demonstrated that single molecules of Pkl1 were not highly processive but only exhibited biased one-dimensional diffusion along microtubules, whereas several molecules of Pkl1, probably fewer than 10 molecules, cooperatively moved along microtubules and substantially reduced the diffusive component in the movement. Our results suggest that Pkl1 molecules work in groups to move and generate forces in a cooperative manner for their mitotic functions.

Bidirectional motility of the fission yeast kinesin-5, Cut7.

Kinesin-5 is a homotetrameric motor with its motor domain at the N-terminus. Kinesin-5 crosslinks microtubules and functions in separating spindle poles during mitosis. In this study, the motile properties of Cut7, fission yeast kinesin-5, were examined for the first time. In in vitro motility assays, full-length Cut7 moved toward minus-end of microtubules, but the N-terminal half of Cut7 moved toward the opposite direction. Furthermore, additional truncated constructs lacking the N-terminal or C-terminal regions, but still contained the motor domain, did not switch the motile direction. These indicated that Cut7 was a bidirectional motor, and microtubule binding regions at the N-terminus and C-terminus were not involved in its directionality.

The primary structure of Tetrahymena profilin

The cDNA of Tetrahymena profilin was cloned and sequenced. The deduced product has a molecular mass of 16,785 Da which is the largest among profilins known so far. Tetrahymena profilin shows higher homologies with lower eukaryotic profilins than with mammalian profilins. Although the homologies with mammalian and lower eukaryotic profilins are only 20-29% which is the lowest one among lower eukaryotic profilins, the N- and C-terminal regions of Tetrahymena profilin are considerably conserved as those in other profilins, suggesting that these regions are responsible for the essential properties common to profilins.