Twitchin purified from molluscan catch muscles regulates interactions between actin and myosin filaments at rest in a phosphorylation-dependent manner

Abstract

Twitchin, also called mini-titin, is structurally related to the giant elastic protein connectin/titin, and has been found in not only striated but also smooth muscles of bivalves. Many bivalve smooth muscles such as byssus retractor muscles and the opaque part of adductor muscles are known as catch muscles that can maintain high passive tension with little expenditure of energy after they have actively contracted. Twitchin is phosphorylated when this high-tension state (catch state) ceases. Our recent studies revealed that the catch tension is due to interactions between thick and thin filaments in the presence of MgATP at low free Ca2+ concentrations, which can be visualized in vitro under a light microscope (Yamada et al., 2001 Proc Natl Acad Sci USA 98: 6635-6640). We also found that twitchin is essential for the interactions of the catch state in mussel (Mytilus galloprovincialis) catch muscles. In the presence of twitchin, actin filaments bound to purified myosin filaments when twitchin was dephosphorylated by Ser/Thr protein phosphatase 2B, while they did not when it was phosphorylated by cAMP-dependent protein kinase. In the current study we demonstrate the same essential components of the catch state for another bivalve that exhibits catch, i.e., Japanese oyster (Crassostrea gigas).

Striated Muscle Twitchin of Bivalves has “Catchability”, the Ability to Bind Thick Filaments Tightly to Thin Filaments, Representing the Catch State

“…As we have already reported, 22 the catch state can be reconstituted in our in vitro catch assay with myosin and twitchin purified from the oyster smooth adductor muscle (Figure 3(a)). When myosin filaments complexed with twitchin were mixed with fluorescence-labeled actin filaments in the presence of MgATP at a low free Ca 2+ concentration, they bound to the actin filaments (Figure 3(a), 1 Catch).…”

“…Myosin and twitchin were purified from the oyster's smooth and obliquely striated adductor muscles as described. 22 Myosin and twitchin were purified also from the scallop's transversely striated adductor muscle, with slight modifications. A 33% to 50% saturated (NH 4 ) 2 SO 4 precipitate and a 0% to 33% saturated (NH 4 ) 2 SO 4 precipitate of muscle extract were used for purification of the scallop myosin and twitchin, respectively.…”

“…In the absence of twitchin the myosin filaments did not bind to the actin filaments, even with catch treatment. 22 The same catch assay experiment was performed after replacing the oyster smooth muscle twitchin with oyster obliquely striated muscle twitchin (Figure 3(b)). Reversible binding of actin filaments in the same way was also observed with these hybrid filaments, indicating that oyster obliquely striated muscle twitchin has catchability.…”

“…In addition, we define catchability as the ability to facilitate the binding of catch muscle myosin filaments to actin filaments at low Ca 2+ concentrations, similar to that observed with the proteins of catch muscles under in vitro catch assay. We have already demonstrated the catch using myosin and twitchin purified from mussel and oyster catch muscles, 20,22 and we can now replace twitchin purified from catch muscle with that from another source. When tight binding between myosin filaments and actin filaments is observed in such an assay, we say that this twitchin has catchability.…”

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

“…The answer came in the demonstration that dephosphorylated, but not phosphorylated, twitchin bound the thin filament (Shelud'ko et al, 2004;Funabara et al, 2005;Tsutsui et al, 2005). Since twitchin is a thick filament protein, this would bind the two filaments and provide the necessary rigidity, and the phosphorylation dependence of the interaction explains how catch is controlled.…”