原口 武士

Abstract Myosins are a crucial motor protein associated with the actin cytoskeleton in eukaryotic cells. Structurally, myosins form heteromeric complexes, with smaller light chains such as calmodulin (CaM) bound to isoleucine–glutamine (IQ) domains in the neck region. These interactions facilitate mechano-enzymatic activity. Recently, we identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors with proteins containing multiple IQ domains, that function as the myosin VIII light chains. This study demonstrates that CaM, CML13, and CML14 specifically bind to the neck region of all 13 Arabidopsis myosin XI isoforms, with some preference among the CaM/CML-IQ domains. Additionally, we observed distinct residue preferences within the IQ domains for CML13, CML14, and CaM.In vitroexperiments revealed that recombinant CaM, CML13, and CML14 exhibit calcium-independent binding to the IQ domains of myosin XIs. Furthermore, when co-expressed with MAP65-1–myosin fusion proteins containing the IQ domains of myosin XIs, CaM, CML13, and CML14 co-localize to microtubules.In vitroactin motility assays demonstrated that recombinant CML13, CML14, and CaM function as myosin XI light chains. Acml13T-DNA mutant exhibited a shortened primary root phenotype that was complemented by the wild-type CML13 and was similar to that observed in a triple myosin XI mutant (xi3KO). Overall, our data indicate that Arabidopsis CML13 and CML14 are novel myosin XI light chains that likely participate in a breadth of myosin XI functions. Highlight Myosin XI proteins play a crucial role in the plant cytoskeleton, but their associated light chains have remained unidentified. Here, we show that calmodulin-like proteins, CML13 and CML14, serve as light chains for myosin XI, similar to their role for myosin VIII proteins

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.

Summary 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.