原口 武士

Abstract Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins, CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein-interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with RFP-myosin fusion proteins containing IQ- and tail-domains of myosin VIIIs. In vitro actin-motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.

Myosin IC, a single-headed member of the myosin I family, specifically interacts with anionic phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) in the cell membrane via the pleckstrin homology domain located in the myosin IC tail. Myosin IC is widely expressed and physically links the cell membrane to the actin cytoskeleton; it plays various roles in membrane-associated physiological processes, including establishing cellular chirality, lipid transportation, and mechanosensing. In this study, we evaluated the motility of full-length myosin IC of Drosophila melanogaster via the three-dimensional tracking of quantum dots bound to actin filaments that glided over a membrane-bound myosin IC-coated surface. The results revealed that myosin IC drove a left-handed rotational motion in the gliding actin filament around its longitudinal axis, indicating that myosin IC generated a torque perpendicular to the gliding direction of the actin filament. The quantification of the rotational motion of actin filaments on fluid membranes containing different PI(4,5)P2 concentrations revealed that the rotational pitch was longer at lower PI(4,5)P2 concentrations. These results suggest that the torque generated by membrane-bound myosin IC molecules can be modulated based on the phospholipid composition of the cell membrane.

<jats:title>Abstract</jats:title><jats:p><jats:italic>Arabidopsis thaliana</jats:italic> has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in <jats:italic>Arabidopsis</jats:italic> cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD). When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining total internal reflection fluorescence microscopy (TIRFM) and the fluorescence imaging with one-nanometer accuracy (FIONA) method, revealed that the MYA2 processively moved on actin with three different step sizes: − 28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes; hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2–3 pN). These results indicated that MYA2 has different transport properties from that of the myosin V responsible for vesicle transport in animal cells. Such properties may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.</jats:p>

Significance It has been suggested for more than 50 y that the fastest myosin in the biological world with a velocity of 70 μm s ⁻¹ exists in the alga Chara , because cytoplasmic streaming with a velocity of 70 μm s ⁻¹ occurs in Chara cells. However, a myosin with that velocity has not yet been identified. In this work, we succeeded in cloning a myosin XI with a velocity of 60 μm s ⁻¹ , which was measured using a chimeric myosin. We also successfully crystallized myosin XI. Structural comparison of various myosins and mutation experiments of actin-binding regions suggests that the central regions that define the fast movement of Chara myosin XI are the actin-binding sites.

Previous studies have revealed duplications and diversification of myosin XI genes between angiosperms and bryophytes; however, the functional differentiation and conservation of myosin XI between them remain unclear. Here, we identified a single myosin XI gene from the liverwort Marchantia polymorpha (Mp). The molecular properties of Mp myosin XI are similar to those of Arabidopsis myosin XIs responsible for cytoplasmic streaming, suggesting that the motor function of myosin XI is able to generate cytoplasmic streaming. In cultured Arabidopsis cells, transiently expressed green fluorescent protein (GFP)-fused Mp myosin XI was observed as some intracellular structures moving along the F-actin. These intracellular structures were co-localized with motile endoplasmic reticulum (ER) strands, suggesting that Mp myosin XI binds to the ER and generates intracellular transport in Arabidopsis cells. The tail domain of Mp myosin XI was co-localized with that of Arabidopsis myosin XI-2 and XI-K, suggesting that all these myosin XIs bind to common cargoes. Furthermore, expression of GFP-fused Mp myosin XI rescued the defects of growth, cytoplasmic streaming and actin organization in Arabidopsis multiple myosin XI knockout mutants. The heterologous expression experiments demonstrated the cellular and physiological competence of Mp myosin XI in Arabidopsis. However, the average velocity of organelle transport in Marchantia rhizoids was 0.04 ± 0.01 μm s^-1 , which is approximately one-hundredth of that in Arabidopsis cells. Taken together, our results suggest that the molecular properties of myosin XI are conserved, but myosin XI-driven intracellular transport in vivo would be differentiated from bryophytes to angiosperms.

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.

There are two classes of myosin, XI and VIII, in higher plants. Myosin XI moves actin filaments at high speed and its enzyme activity is also very high. In contrast, myosin VIII moves actin filaments very slowly with very low enzyme activity. Because most of these enzymatic and motile activities were measured using animal skeletal muscle α-actin, but not plant actin, they would not accurately reflect the actual activities in plant cells. We thus measured enzymatic and motile activities of the motor domains of two Arabidopsis myosin XI isoforms (MYA2, XI-B), and one Arabidopsis myosin VIII isoform (ATM1), by using three Arabidopsis actin isoforms (ACT1, ACT2, and ACT7). The measured activities were different from those measured by using muscle actin. Moreover, Arabidopsis myosins showed different enzymatic and motile activities when using different Arabidopsis actin isoforms. Our results suggest that plant actin should be used for measuring enzymatic and motile activities of plant myosins and that different actin isoforms in plant cells might function as different tracks along which affinities and velocities of each myosin isoform are modulated.

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.

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 Mg(2+)-ATPase activity (Vmax = 4 s(-1)), although their affinities for actin were high (Kactin = 4 μM). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 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 Mg(2+) 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.

All class II myosins have the conserved amino acid sequence Pro-Leu-Leu at their head-tail junctions. We systematically altered this sequence in smooth muscle heavy meromyosin (HMM) by site-directed mutagenesis and examined the effects of these mutations on actin-myosin interactions. Deletion of the proline and second leucine did not cause any noticeable change in either actin-activated ATPase activity or actin-sliding velocity. In contrast, deletion of the two leucine residues and substitution of the first leucine with alanine resulted in a 14-fold and 5-fold decrease, respectively, in actin-activated ATPase activity. However, both these mutations did not appreciably affect actin-sliding velocity, which was consistent with a result that there was no considerable change in the ADP release rate from acto-HMM in the deletion mutant. In contrast to double-headed HMM, a single-headed subfragment-1 (S1) with a Leu-Leu deletion mutation exhibited actin activated ATPase activity similar to that by wild type S1. Our results suggest that the first leucine of the conserved Leu-Leu sequence at the head-tail junction profoundly affects the cooperativity between the two heads involved in the actin activated ATPase activity of myosin II.