伊藤 光二

Cytoplasmic streaming with extremely high velocity (∼70 μm s⁻¹) occurs in cells of the characean algae (<italic>Chara</italic>). Because cytoplasmic streaming is caused by myosin XI, it has been suggested that a myosin XI with a velocity of 70 μm s⁻¹, the fastest myosin measured so far, exists in <italic>Chara</italic> cells. However, the velocity of the previously cloned <italic>Chara corallina</italic> myosin XI (<italic>Cc</italic>XI) was about 20 μm s⁻¹, one-third of the cytoplasmic streaming velocity in <italic>Chara</italic>. Recently, the genome sequence of <italic>Chara braunii</italic> has been published, revealing that this alga has four myosin XI genes. We cloned these four myosin XI (<italic>Cb</italic>XI-1, 2, 3, and 4) and measured their velocities. While the velocities of <italic>Cb</italic>XI-3 and <italic>Cb</italic>XI-4 motor domains (MDs) were similar to that of <italic>Cc</italic>XI MD, the velocities of <italic>Cb</italic>XI-1 and <italic>Cb</italic>XI-2 MDs were 3.2 times and 2.8 times faster than that of <italic>Cc</italic>XI MD, respectively. The velocity of chimeric <italic>Cb</italic>XI-1, a functional, full-length <italic>Cb</italic>XI-1 construct, was 60 μm s⁻¹. These results suggest that <italic>Cb</italic>XI-1 and <italic>Cb</italic>XI-2 would be the main contributors to cytoplasmic streaming in <italic>Chara</italic> cells and show that these myosins are ultrafast myosins with a velocity 10 times faster than fast skeletal muscle myosins in animals. We also report an atomic structure (2.8-Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-electron microscopy structure of acto-myosin XI at low resolution (4.3-Å), it appears that the actin-binding region contributes to the fast movement of <italic>Chara</italic> myosin XI. Mutation experiments of actin-binding surface loops support this hypothesis.

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.

Background: Molecular properties of class VIII myosin are not characterized. Results:Arabidopsis class VIII myosin, ATM1, has low enzymatic activity and high affinity for actin and is primarily localized at the cell cortex. Conclusion: Our data suggest that ATM1 functions as a tension sensor/generator. Significance: This is the first report of enzymatic and motile properties of class VIII myosin. Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg2+-ATPase activity (V-max = 4 s(-1)), although their affinities for actin were high (K-actin = 4 m). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 m/s, respectively, from which the value for full-length ATM1 is calculated to be approximate to 0.2 m/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was approximate to 90%. ADP dissociation from the actinATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s(-1), respectively). Physiological concentrations of free Mg2+ modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis.

Cytoplasmic streaming is active transport widely occurring in plant cells ranging from algae to angiosperms. Although it has been revealed that cytoplasmic streaming is generated by organelle-associated myosin XI moving along actin bundles, the fundamental function in plants remains unclear. We generated high- and low-speed chimeric myosin XI by replacing the motor domains of Arabidopsis thaliana myosin XI-2 with those of Chara corallina myosin XI and Homo sapiens myosin Vb, respectively. Surprisingly, the plant sizes of the transgenic Arabidopsis expressing high- and low-speed chimeric myosin XI-2 were larger and smaller, respectively, than that of the wild-type plant. This size change correlated with acceleration and deceleration, respectively, of cytoplasmic streaming. Our results strongly suggest that cytoplasmic streaming is a key determinant of plant size. Furthermore, because cytoplasmic streaming is a common system for intracellular transport in plants, our system could have applications in artificial size control in plants.

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 V(max) and K(app) of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected K(app) and the velocity, although it did not affect V(max). 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 myosin&apos;s 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.

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 &gt; 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 &lt; 0.82 ms. This value is the shortest among known values for various myosins and yields the duty ratio of &lt; 0.3 with a V-max 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.

By using monopolar spindles artificially induced in sea urchin embryos, we examined whether or not the presence of two opposing poles was an indispensable condition for keeping chromosomes at a fixed distance from the pole at metaphase and for the anaphase chromosome movement. Chromosomes were stained with Hoechst dye 33342 and their behavior was followed in the monopolar and the control bipolar spindles. In the monopolar spindle, chromosomes were first arranged on a curved metaphase plate and then spread on a part of the imaginary surface of a sphere whose center was the monopole. The estimated chromosome-to-pole distance was similar to that of bipolar spindles at metaphase and remained fixed until chromosomes started to move toward the pole. The average duration of metaphase in the monopolar spindle was 6 times longer than that in the bipolar spindle. The poleward movement of chromosomes in the monopolar spindle was similar to the anaphase A (chromosome-to-pole movement) in the bipolar spindle with respect to the velocity, duration, distance, and synchronization of migration. These results show that even half of the normal spindle has capacities for the arrangement of chromosomes at metaphase and for the anaphase A chromosome movement. Based on these results, we were able to exclude some existing theories of metaphase, such as the one based on the balance of forces between the two poles.