Kohji Ito

Kinetic Mechanism of the Fastest Motor Protein, Chara Myosin

Chara corallina class XI myosin is by far the fastest molecular motor. To investigate the molecular mechanism of this fast movement, we performed a kinetic analysis of a recombinant motor domain of Chara myosin. We estimated the time spent in the strongly bound state with actin by measuring rate constants of ADP dissociation from actin·motor domain complex and ATP-induced dissociation of the motor domain from actin. The rate constant of ADP dissociation from acto-motor domain was >2800 s-1, and the rate constant of ATP-induced dissociation of the motor domain from actin at physiological ATP concentration was 2200 s-1. From these data, the time spent in the strongly bound state with actin was estimated to be <0.82 ms. This value is the shortest among known values for various myosins and yields the duty ratio of <0.3 with a Vmax value of the actin-activated ATPase activity of 390 s-1. The addition of the long neck domain of myosin Va to the Chara motor domain largely increased the velocity of the motility without increasing the ATP hydrolysis cycle rate, consistent with the swinging lever model. In addition, this study reveals some striking kinetic features of Chara myosin that are suited for the fast movement: a dramatic acceleration of ADP release by actin (1000-fold) and extremely fast ATP binding rate.

Kinetic Characterization of Tail Swing Steps in the ATPase Cycle of Dictyostelium Cytoplasmic Dynein

According to the power stroke model of dynein deduced from electron microscopic and fluorescence resonance energy transfer studies, the power stroke and the recovery stroke are expected to take place at the two isomerization steps of the ATPase cycle at the primary ATPase site. Here, we have conducted presteady-state kinetic analyses of these two isomerization steps with the single-headed motor domain of Dictyostelium cytoplasmic dynein by employing fluorescence resonance energy transfer to probe ATPase steps at the primary site and tail positions. Our results show that the recovery stroke at the first isomerization step proceeds quickly (∼180 s–1), whereas the power stroke at the second isomerization step is very slow (∼0.2 s–1) in the absence of microtubules, and that the presence of microtubules accelerates the second but not the first step. Moreover, a comparison of the microtubule-induced acceleration of the power stroke step and that of steady-state ATP hydrolysis implies the intriguing possibility that microtubules simultaneously accelerate the ATPase activity not only at the primary site but also at other site(s) in the motor domain.

Cooperativity between Two Heads of Dictyostelium Myosin II in in Vitro Motility and ATP Hydrolysis

To elucidate the significance of the two-headed structure of myosin II, we have engineered and characterized recombinant single-headed myosin II. A tail segment of a myosin II heavy chain fused with a His-tag was expressed in wild-type Dictyostelium cells. Single-headed myosin, which consists of a full length myosin heavy chain and a tagged tail, was isolated on the basis of the affinities for Nickel agarose and actin. Actin sliding velocity by the single-headed myosin was about half of the two-headed, whereas the minimum density of the heads to support continuous movement was twofold higher. Actin-activated MgATPase activity of the single-headed myosin in solution in the presence of 24 microM actin was less than half of the two headed. This decrease is primarily because of fourfold-elevated Kapp for actin and secondary to 40% lower Vmax. These results suggest that the two heads of a Dictyostelium myosin II molecule act cooperatively on an actin filament. We propose a mechanism by which two heads move actin efficiently based on the cooperativity.

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected Vmax and Kapp of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected Kapp and the velocity, although it did not affect Vmax. Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosins high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.

The molecular mechanism and physiological role of cytoplasmic streaming.

Cytoplasmic streaming occurs widely in plants ranging from algae to angiosperms. However, the molecular mechanism and physiological role of cytoplasmic streaming have long remained unelucidated. Recent molecular genetic approaches have identified specific myosin members (XI-2 and XI-K as major and XI-1, XI-B, and XI-I as minor motive forces) for the generation of cytoplasmic streaming among 13 myosin XIs in Arabidopsis thaliana. Simultaneous knockout of these myosin XI members led to a reduced velocity of cytoplasmic streaming and marked defects of plant development. Furthermore, the artificial modifications of myosin XI-2 velocity changed plant and cell sizes along with the velocity of cytoplasmic streaming. Therefore, we assume that cytoplasmic streaming is one of the key regulators in determining plant size.

Some motile properties of fast characean myosin.

We improved a motility assay system by using an affinity-purified antibody against the C-terminal globular domain of characean myosin. This improvement allowed us to study the sensitivity to ionic strength or the processivity of characean myosin. The sliding velocity of actin filaments on a characean myosin-coated surface was unaffected by ionic strength. This property is unlike that of skeletal or smooth muscle myosin and suggests that the binding manner of characean myosin to actin is different from that in other muscle myosins. The sliding velocity decreased when the MgADP concentration was raised. The extent of inhibition by MgADP on the motile activity of characean myosin was almost the same as in skeletal muscle or cardiac myosin. The number of sliding filaments on the characean myosin-coated surface decreased drastically with a decrease in the motor density. The motor density required to produce a successful movement of actin filament was about 200 molecules/microm(2). These results suggest that the characean myosin is not a processive motor protein.

Myosin XI-I is Mechanically and Enzymatically Unique Among Class-XI Myosins in Arabidopsis

Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator.

Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells

Flowering plants express multiple actin isoforms. Previous studies suggest that individual actin isoforms have specific functions; however, the subcellular localization of actin isoforms in plant cells remains obscure. Here, we transiently expressed and observed major Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, as fluorescent-fusion proteins. By optimizing the linker sequence between fluorescent protein and actin, we succeeded in observing filaments that contained these expressed actin isoforms fused with green fluorescent protein (GFP) in Arabidopsis protoplasts. Different colored fluorescent proteins fused with AtACT2 and AtACT7 and co-expressed in Nicotiana benthamiana mesophyll cells co-polymerized in a segregated manner along filaments. In epidermal cells, surprisingly, AtACT2 and AtACT7 tended to polymerize into different types of filaments. AtACT2 was incorporated into thinner filaments, whereas AtACT7 was incorporated into thick bundles. We conclude that different actin isoforms are capable of constructing unique filament arrays, depending on the cell type or tissue. Interestingly, staining patterns induced by two indirect actin filament probes, Lifeact and mTalin1, were different between filaments containing AtACT2 and those containing AtACT7. We suggest that filaments containing different actin isoforms bind specific actin-binding proteins in vivo, since the two probes comprise actin-binding domains from different actin-binding proteins.

Functional Diversity of Class XI Myosins in Arabidopsis thaliana

Abstract Plant myosin XI acts as a motive force for cytoplasmic streaming through interacting with actin filaments within the cell. Arabidopsis thaliana (At) has 13 genes belonging to the myosin XI family. Previous reverse genetic approaches suggest that At myosin XIs are partially redundant, but are functionally diverse for their specific tasks within the plant. However, the tissue-specific expression and enzymatic properties of myosin XIs have to date been poorly understood, primarily because of the difficulty in cloning and expressing large myosin XI genes and proteins. In this study, we cloned full-length cDNAs and promoter regions for all 13 At myosin XIs and identified tissue-specific expression (using promoter–reporter assays) and motile and enzymatic activities (using in vitro assays). In general, myosins belonging to the same class have similar velocities and ATPase activities. However, the velocities and ATPase activities of the 13 At myosin XIs are significantly different and are classified broadly into three groups based on velocity (high group, medium group and low group). Interestingly, the velocity groups appear roughly correlated with the tissue-specific expression patterns. Generally, ubiquitously expressed At myosin XIs belong to the medium-velocity group, pollen-specific At myosin XIs belong to the high-velocity group and only one At myosin XI (XI-I) is classified as belonging to the low-velocity group. In this study, we demonstrated the diversity of the 13 myosin XIs in Arabidopsis at the molecular and tissue levels. Our results indicate that myosin XIs in higher plants have distinct motile and enzymatic activities adapted for their specific roles.

Single-Molecule Analysis of Actomyosin in the Presence of Osmolyte

Actomyosin is a protein complex composed of myosin and actin, which is well known for being the minimal contractile unit of muscle. The chemical free energy of ATP is converted into mechanical work by the complex, and the single-molecule mechanical properties of myosin are well characterized in vitro. However, the aqueous solution environment in in vitro assay is far from that in cells, where biomolecules are crowded, which influences osmotic pressure, and processes such as folding, and association and diffusion of proteins. Here, to bridge the gap between in vitro and in-cell environment, we observed mechanical motion of actomyosin-V in the presence of the osmolyte sucrose, as a model system. Single-molecule observation of myosin-V motor domains (heads) on actin filament at varying sucrose concentration revealed modulated mechanical elementary processes suggesting increased affinity of heads with actin and more robust force generation possibly accompanied by a sliding motion of myosin head along actin.