Maki Yoshio

An in vitro assay reveals essential protein components for the "catch" state of invertebrate smooth muscle.

“Catch,” a state where some invertebrate muscles sustain high tension over long periods of time with little energy expenditure (low ATP hydrolysis rate) is similar to the “latch” state of vertebrate smooth muscles. Its induction and release involve Ca2+-dependent phosphatase and cAMP-dependent protein kinase, respectively. Molecular mechanisms for catch remain obscure. Here, we describe a quantitative microscopic in vitro assay reconstituting the catch state with proteins isolated from catch muscles. Thick filaments attached to glass coverslips and pretreated with ≈10−4 M free Ca2+ and soluble muscle proteins bound fluorescently labeled native thin filaments tightly in catch at ≈10−8 M free Ca2+ in the presence of MgATP. At ≈10−4 M free Ca2+, the thin filaments moved at ≈4 μm/s. Addition of cAMP and cAMP-dependent protein kinase at ≈10−8 M free Ca2+ caused their release. Rabbit skeletal muscle F-actin filaments completely reproduced the results obtained with native thin filaments. Binding forces >500 pN/μm between thick and F-actin filaments were measured by glass microneedles, and were sufficient to explain catch tension in vivo. Synthetic filaments of purified myosin and twitchin bound F-actin in catch, showing that other components of native thick filaments such as paramyosin and catchin are not essential. The binding between synthetic thick filaments and F-actin filaments depended on phosphorylation of twitchin but not of myosin. Cosedimentation experiments showed that twitchin did not bind directly to F-actin in catch. These results show that catch is a direct actomyosin interaction regulated by twitchin phosphorylation.

Bi-directional movement of actin filaments along long bipolar tracks of oriented rabbit skeletal muscle myosin molecules

In actomyosin in vitro motility assays, orientation of myosin molecules affects their interaction with actin. We obtained long tracks of myosin molecules with uniform orientation. Bipolar filaments about 50 μm long were made from myosin rod prepared from molluscan smooth muscles, to which rabbit skeletal‐muscle myosin bound, creating long synthetic thick‐filaments. Movement of F‐actin toward their center was much faster (4.7±0.6 μm s−1) than in the opposite direction (1.9±0.2 μm s−1), indicating that myosin molecules were arranged in the same orientation along each half of the bipolar filament. These complex thick‐filaments permit measurement of actin movement over 20 μm of oriented skeletal myosin tracks facilitating mechanistic studies of actomyosin motility.

Creating biomolecular motors based on dynein and actin-binding proteins

Biomolecular motors such as myosin, kinesin and dynein are protein machines that can drive directional movement along cytoskeletal tracks and have the potential to be used as molecule-sized actuators. Although control of the velocity and directionality of biomolecular motors has been achieved, the design and construction of novel biomolecular motors remains a challenge. Here we show that naturally occurring protein building blocks from different cytoskeletal systems can be combined to create a new series of biomolecular motors. We show that the hybrid motors-combinations of a motor core derived from the microtubule-based dynein motor and non-motor actin-binding proteins-robustly drive the sliding movement of an actin filament. Furthermore, the direction of actin movement can be reversed by simply changing the geometric arrangement of these building blocks. Our synthetic strategy provides an approach to fabricating biomolecular machines that work along artificial tracks at nanoscale dimensions.

Myosin Mg-ATPase of molluscan muscles is slightly activated by F-actin under catch state in vitro

Molluscan muscle twitchin, a titin/connectin-related giant protein, regulates interactions between actin and myosin filaments at low Ca2+ concentrations. When it is dephosphorylated, actin filaments tightly bind to myosin filaments, resulting in the catch state known as the state of high passive tension with very low energy consumption. Yet when twitchin is phosphorylated actin filaments detach from the myosin filaments, resulting in relaxation of the catch. Here, steady-state Mg-ATPase activities of purified myosin were measured under various conditions: without twitchin, with dephosphorylated twitchin, or with phosphorylated twitchin; with or without phalloidin-stabilized F-actin; and at various Ca2+ concentrations. At low Ca2+ concentration, Mg-ATPase was activated by F-actin only in the presence of dephosphorylated twitchin (catch state). The activation was about two orders lower than that fully activated by Ca2+ and F-actin. In the absence of F-actin, twitchin and its phosphorylation state did not affect Mg-ATPase activities in any of the conditions we tested. Based on these results, we propose a molecular mechanism for the catch, where twitchin alone does not interact with the myosin catalytic motor domain but its complex with F-actin does, forming the bridge between actin and myosin filaments and the myosin slowly hydrolyzes Mg-ATP in the catch state.

‘Catch Mechanism’ found in Molluscan Animals other than Bivalves

Some muscles of bivalve molluscs (phylum Mollusca, class Bivalvia) such as the smooth part of the adductor and the byssus retractor are known as ‘catch muscles’ that can maintain high passive tension for long periods with little energy expenditure after their active contraction. The state of this high passive tension is called ‘catch’ and it has been hypothesized that some special ‘catch mechanism’ exists for the maintenance of the tension. We have developed an in vitro catch assay in which the catch state is reconstituted by using proteins purified from bivalve catch muscles. With this assay, we have revealed that the catch tension is due to binding of actin filaments to myosin filaments which can be directly visualized under a light microscope. We have also found that twitchin, a giant protein associated with myosin filaments structurally related to connectin/titin, and to less extent, vertebrate cardiac myosin binding protein C, is an essential key component for the catch mechanism. Twitchin was originally found in a nematode Caenorhabditis elegans, and was later found widely in many invertebrates. This fact raises a question whether animals having twitchin other than bivalves also have the catch mechanism. To answer this, we prepared synthetic thick filaments containing myosin and twitchin from organs of molluscan animal species other than bivalves. We used foot of a spiral shellfish Monodonta labio (class Gastropoda) and a chiton Liolophura japonica (class Polyplacophora), and inner wall of suckers of an octopus Octopus vulgaris (class Cephalopoda). We performed the in vitro catch assay and found that the catch mechanism, at least at the molecular level, exists also in these molluscan animals. The results suggest that the catch mechanism is distributed more widely in Animal Kingdom than we know.