Development of an expression system in Tetrahymena inner arm dyneins and motile properties of the single-headed subspecies (Dyh8p and Dyh12p).

Diverse inner arm dyneins cooperate with outer arm dyneins to produce ciliary beating. This study demonstrates an expression system for inner arm dyneins in Tetrahymena. The motor domain of inner arm dynein (Dyh8p or Dyh12p) was fused with the tail of outer arm dynein (Dyh3p) and expressed in viable DYH3-knockout (vKO-DYH3) cells. The chimeric dyneins were observed in the oral apparatus and cilia on the cell bodies, and did not change the swimming speed of vKO-DYH3 cells. In a gliding assay, the motor domains of Dyh8p and Dyh12p moved toward the minus ends of microtubules at 0.8 and 0.3 µm/s, respectively. The gliding velocities of Dyh8p and Dyh12p were decreased in 5 mM ATP but not increased in 0.1 or 0.5 mM ADP. This expression system will be useful for molecular studies on diverse inner arm dyneins.

Functional characterization of lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p).

Mutational analyses of axonemal dyneins are useful for elucidating the molecular mechanism of ciliary motility. This study demonstrates a mutation system for characterizing lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p). The viable DYH3-knockout (vKO-DYH3) cells isolated in this study enabled the examination of lethal mutations in P-loops 1 and 2. The P1 mutant dynein localized in the oral apparatus and the proximal region of the cilia, and the P2 mutant dynein localized only in the oral apparatus. Both results are different from the localization of wild-type Dyh3p. In addition, a co-precipitation assay showed that the P1 and P2 mutant dyneins did not dissociate from microtubules in ATP plus vanadate or in no-ATP conditions, in contrast to wild-type Dyh3p. This mutation system is useful for further molecular studies of axonemal dyneins and ciliary motility.

Establishment of a mutation system in Tetrahymena outer arm dynein and P-loop functions of the alpha heavy chain (Dyh3p).

Axonemal dyneins are large AAA+ type motor proteins that exhibit unique motor properties during ciliary beating. This study established a mutation system for Tetrahymena outer arm dynein and characterized four nucleotide-binding loops (P-loops; P1-P4) in the alpha heavy chain (Dyh3p). Macronuclear transformation of the mutant DYH3 genes in DYH3-knockout (KO-DYH3) cells enabled P-loop mutations that abolish the ability of nucleotide binding to be stably maintained in the polyploid genome. This mutation system revealed that the P3 and P4 mutant dyneins rescued lethality in macronuclear KO-DYH3 cells and exhibited normal ciliary localization. Intriguingly, however, an in vitro motility assay showed that the P3 mutation abolished the motor activity of Dyh3p, whereas the P4 mutation did not affect the gliding velocity or gliding index of Dyh3p. In contrast, no P1 or P2 mutant cells were isolated from the KO-DYH3 cells, which suggests that nucleotide binding at the P1 and P2 sites is required for the intracellular function of Dyh3p. This mutation system will be useful for further molecular studies of diverse axonemal dyneins and ciliary motility.

Motor domain-based motility system and motile properties of alpha heavy chain in Tetrahymena outer arm dynein.

Axonemal dynein plays an essential role in ciliary motility, and impaired ciliary motility causes human diseases such as primary ciliary dyskinesia (PCD). The motor domain of axonemal dynein powers ciliary motility and its function is regulated by several accessary proteins bound to the tail region. Therefore, to understand the essential properties of dynein motility, examining the motile properties of the motor domain without the tail is necessary. In this study, the functional motor domain of the alpha heavy chain in Tetrahymena outer arm dynein was purified, and its motile properties were examined using an in vitro motility system. The purified protein caused microtubules to glide at a velocity of 5.0µm/s with their minus-end trailing, and motility was inhibited in an ATP concentration-dependent manner, which is in contrast with kinesin1. This method could be applicable to other axonemal dyneins and will enable further molecular studies on diverse axonemal dyneins and ciliary motility.

Identification of biotin carboxyl carrier protein in Tetrahymena and its application in in vitro motility systems of outer arm dynein.

Axonemal dynein plays a central role in ciliary beating. Recently, a functional expression system of axonemal dynein was established in the ciliated protozoan Tetrahymena. This study identifies biotin carboxyl carrier protein (BCCP) in Tetrahymena and demonstrates its application in in vitro motility systems of outer arm dynein.

The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena.

Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement.

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.

Nucleotide-dependent behavior of single molecules of cytoplasmic dynein on microtubules in vitro.

We visualized the nucleotide-dependent behavior of single molecules of mammalian native cytoplasmic dynein using fragments of dynactin p150 with or without its N-terminal microtubule binding domain. The results indicate that the binding affinity of dynein for microtubules is high in AMP-PNP, middle in ADP or no nucleotide, and low in ADP.Pi conditions. It is also demonstrated that the microtubule binding domain of dynactin p150 maintains the association of dynein with microtubules without altering the motile property of dynein in the weak binding state. In addition, we observed bidirectional movement of dynein in the presence of ATP as well as in ADP/Vi condition, suggesting that the bidirectional movement is driven by diffusion rather than active transport.

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.

Direction and speed of microtubule movements driven by kinesin motors arranged on catchin thick filaments.

Conventional kinesin (Kinesin-1) is a microtubule-based molecular motor that supports intracellular vesicle/organelle transport in various eukaryotic cells. To arrange kinesin motors similarly to myosin motors on thick filaments in muscles, the motor domain of rat conventional kinesin (amino acid residues 1-430) fused to the C-terminal 829 amino acid residues of catchin (KHC430Cat) was bacterially expressed and attached to catchin filaments that can attach to and arrange myosin molecules in a bipolar manner on their surface. Unlike the case of myosin where actin filaments move toward the center much faster than in the opposite direction along the catchin filaments, microtubules moved at the same speed in both directions. In addition, many microtubules moved across the filaments at the same speed with various angles between the axes of the microtubule and catchin filament. Kinesin/catchin chimera proteins with a shorter kinesin neck domain were also prepared. Those without the whole hinge 1 domain and the C-terminal part of the neck helix moved microtubules toward the center of the catchin filaments significantly, but only slightly, faster than in the opposite direction, although the movements in both directions were slower than those of the KHC430Cat construct. The results suggest that kinesin has substantial mechanical flexibility within the motor domain, possibly within the neck linker, enabling its interaction with microtubules having any orientation.

The dynein stalk head, the microtubule binding-domain of dynein: NMR assignment and ligand binding.

Dynein is a motor ATPase, and the C-terminal two-thirds of its heavy chain form a ring structure. One of protrudings from this ring structure is a stalk whose tip, the dynein stalk head (DSH), is thought to be the microtubule-binding domain. As a first step toward elucidating the functional mechanisms of DSH, we aimed at the NMR structural analysis of an isolated DSH from mouse cytoplasmic dynein. The DSH expressed in bacteria and purified was coprecipitated with microtubules, suggesting its proper folding. Chemical shifts of the DSH were obtained from NMR measurements, and backbone assignment identified 94% of the main-chain N-H signals. Secondary structural prediction programs showed that about 60% of the residues formed alpha-helices. A region with cationic residues K58 and R61 (and possibly R66 as well), and another with R86, K88, K90, and K91, were found to form alpha-helices. Both of these regions may be important in the formation of the DSH-binding site to a microtubule that has a low pI with a number of acidic residues. Two synthetic peptides containing the sequence of the alpha-helix 12 of beta-tubulin, considered to be important in binding to DSH, were investigated. Of these two peptides, the one with higher helix-formation propensity appeared to bind to DSH, since it precipitated with DSH in a nearly stoichiometric manner. This suggested that the alpha-helicity of this region would be important in its binding to DSH.

Microtubule-binding properties of dynactin p150 expedient for dynein motility.

Dynactin is a hetero-oligomeric protein complex that has an important role in dynein-based intracellular transport. The expressed N-terminal fragments of dynactin p150 bound to microtubules in the ratio of one to one tubulin dimer, independent from the binding of dynein stalk head. Single molecule observation revealed that these fragments moved around on microtubules by Brownian motion. When the dynein-dynactin complex moves on microtubules, p150 can support dynein to maintain contact with microtubules and does not interfere with the motility of dynein, and thus, the dynein-dynactin complex can efficiently achieve long-distance carriage of the cargo.

Removal of tightly bound ADP induces distinct structural changes of the two tryptophan-containing regions of the ncd motor domain.

ncd is a molecular motor belonging to the kinesin superfamily. In solution, it is a homo-dimer of a 700 amino acid polypeptide. The C-terminus of each polypeptide forms a globular domain of about 40 kDa, the motor domain with ATPase activity. The ATPase site of the motor domain of kinesin family members, including ncd, binds ADP tightly, the release of which is facilitated by microtubules during the mechanochemical ATPase cycle. Previously, we studied the spectroscopic characteristics of the ncd motor domain, focusing on interactions of the transition-moment-dipoles between ADP and aromatic amino acid side chains using circular dichroism (CD) spectroscopy. In the present study, we generated several ncd motor domain mutants. In each, a tryptophanyl or specific tyrosyl residue was mutated. We found that Trp370 and Tyr442, the latter of which stacks directly with the adenine moiety of bound ADP, caused the bound ADP to exhibit peculiar CD signals. In addition, fluorescence measurements revealed that Trp370, but not Trp473, was responsible for the emission intensity change depending on the presence or absence of bound ADP. This fluorescence result implies that the structural change induced at the ADP-binding site (on the release of the ADP) is transmitted to the region that includes Trp370, which is relatively close to the ADP-binding site but not in direct contact with the ADP-binding region. In contrast, Trp473 in the region that is in contact with the alpha-helical coiled coil stalk did not experience the structural changes caused on removal of ADP. The distinct behavior of these two tryptophanyl residues suggests that the ncd motor domain has a bifacial architecture made up of a relatively deformable side including the nucleotide binding site and a more rigid one.

Multiple ATP-hydrolyzing sites that potentially function in cytoplasmic dynein.

Cytoplasmic dynein is a minus-end-directed microtubule motor involved in numerous essential processes within eukaryotic cells, such as nuclear segregation and trafficking of intracellular particles. The motor domain of the dynein heavy chain comprises six tandemly linked AAA (ATPase associated with diverse cellular activities) modules (AAA1-AAA6). The first four modules include nucleotide-binding sites (Walker A or P-loop motifs), and each of the four sites appears to bind ATP. However, the role and the function of each binding site are unknown. Especially, the question of which P-loops are ATP-hydrolyzing sites has not been answered, because it is difficult to measure the ATPase activity of each P-loop. Here, we purified several truncated Saccharomyces cerevisiae cytoplasmic dynein fragments and their mutants expressed in Escherichia coli and then measured their ATPase activities. Our results suggest that there are multiple ATP-binding sites that have abilities to hydrolyze ATP in cytoplasmic dynein. Furthermore, a single AAA module is insufficient for ATP hydrolysis, and the adjacent module facing the ATP-binding site is necessary for ATP-hydrolyzing activity.

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 A, 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 second microtubule-binding site of monomeric kid enhances the microtubule affinity.

Chromokinesin Kid (kinesin-like DNA-binding protein) localizes on spindles and chromosomes and has important roles in generating polar ejection force on microtubules in the metaphase. To understand these functions of Kid at the molecular level, we investigated molecular properties of Kid, its oligomeric state, interaction with microtubules, and physiological activity in vitro. Kid expressed in mammalian cells, as well as Kid expressed in Escherichia coli, was found to be monomeric. However, Kid cross-linked microtubules in an ATP-sensitive manner, suggesting that Kid has a second microtubule-binding site in addition to its motor domain. This was ascertained by binding of Kid fragments lacking the motor domain to microtubules. The interaction of the second microtubule-binding site was weak in a nucleotide-insensitive manner. KmMT of the ATPase activity of Kid was lower than that of the fragments lacking the second microtubule-binding site. Moreover, the velocity of Kid movement in vitro was not affected by the second microtubule-binding site, which is consistent with the weak binding of this site to microtubules. The second microtubule-binding site would be important to enhance the affinity to microtubules for the monomeric motor, Kid. Because the amino acid sequence of this region is highly conserved among species, it seems to have essential roles for the functions of Kid in vivo.

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.

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 approximately 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.

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'-[beta,gamma-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.