Kazuhiro Kohama

Regulation of Actin Binding and Actin Bundling Activities of Fascin by Caldesmon Coupled with Tropomyosin

Human fascin is an actin-bundling protein and is thought to play a role in the formation of microfilament bundles of microspikes and stress fibers in cultured cells. To explore the regulation of fascin-actin interaction, we have examined the effects of culture cell caldesmon and tropomyosin (TM) on actin binding activity of human fascin. Caldesmon alone or TM alone has little or no effect on the actin binding of fascin. However, caldesmon together with TM completely inhibits actin binding of human fascin. When calmodulin is added, the inhibition of fascin-actin interaction by caldesmon and TM becomes Ca2+ dependent because Ca2+/calmodulin blocks actin binding of caldesmon. Furthermore, as phosphorylation of caldesmon by cdc2 kinase inhibits actin binding of caldesmon, phosphorylation can also control actin binding of fascin in the presence of TM. As expected by the inhibition of fascin-actin binding, caldesmon coupled with TM also inhibits actin bundling activity of fascin. Whereas smooth muscle caldesmon alone or TM alone shows no effect, caldesmon together with TM completely inhibits actin bundling activity of fascin. This inhibition is again Ca2+ dependent when calmodulin is added to the system. These results suggest important roles for caldesmon and TM in the regulation of the function of human fascin.

Stable Transfectants of Smooth Muscle Cell Line Lacking the Expression of Myosin Light Chain Kinase and Their Characterization with Respect to the Actomyosin System

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U.u2009S.u2009A. 96, 6666–6671).

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

SummaryThe actin-activated ATPase activityPhysarum myosin was shown to be inhibited of μM levels of Ca2+. To determine if Ca2+ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 μm/sec; however, in perfusion solution containing Ca2+, the rate reduced to 0.0–0.7 μm/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca2+, was observed only in the perfusion solution containing Ca2+, indicating that myosin is responsible for the inhibitory effect of Ca2+ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca2+ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.

Direct activation by okadaic acid of the contractile elements in the smooth muscle of guinea-pig taenia coli

Summary1. Okadaic acid isolated from black sponge (Halichondria okadai), at the concentration of 10 μmol/l, caused contraction in saponin-treated skinned smooth muscle of guinea-pig taenia coli in the absence of Ca2+. In the presence of low concentration (0.3 μmol/l) of Ca2+ okadaic acid induced a greater contraction than in the absence of Ca2+ 2. Okadaic acid potentiated the contractions induced by Ca2+ and pCa2+-tension curve was shifted to the left as well as upward by 1 μmol/l okadaic acid. 3. Native actomyosin preparation (myosin B) containing calmodulinmyosin light chain kinase system and phosphatase was obtained from taenia coli. Okadaic acid (10 μmol/l) increased the actomyosin Mg2+-ATPase activity in the presence or absence of Ca2+. 4. Okadaic acid (1–100 μmol/l) had no effect on calmodulin activity as monitored by Ca2+-calmodulin activated cyclic nucleotide phosphodiesterase activity and the (Ca2+ + Mg2+)-ATPase activity of erythrocyte membranes. 5. These results suggest that okadaic acid directly activates contractile elements of smooth muscle.

CALCIUM REGULATION OF THE ACTIN-MYOSIN INTERACTION OF PHYSARUM POLYCEPHALUM

Plasmodia of Physarum polycephalum show vigorous cytoplasmic streaming, the motive force of which is supported by the actin-myosin interaction. Calcium is not required for the interaction but inhibits it. This calcium inhibition, a regulatory mode first discovered in Physarum, is the overwhelming mode of regulation of cytoplasmic streaming of plant cells and lower eukaryotes, and it is diametrically opposite to calcium activation of the interaction found in muscle and nonmuscle cells of the animal kingdom. Myosin, myosin II in myosin superfamily, is the most important protein for Ca2+ action. Its essential light chain, called calcium-binding light chain, is the sole protein that binds Ca2+. Although phosphorylation and dephosphorylation of myosin modify its properties, regulation of physiological significance is shown to be Ca-binding to myosin. The actin-binding protein of Physarum amplifies calcium inhibition when Ca2+ binds to calmodulin and other calcium-binding proteins. This review also includes characterization of this and other calcium-binding proteins of Physarum.

Ca-inhibitory myosins: Their structure and function

Actin-myosin-ATPase interaction of Physarum is under inhibitory Ca-control. Ca-binding to myosin inhibits the interaction, while Ca-release from myosin removes inhibition. However, this myosin-linked control system alone is not sufficient to account for in vivo contraction. A Ca-dependent inhibitory factor, whose active principle appears to be identical with or similar to Ca-Lc of myosin, augments myosin-linked control by interacting with actin. This novel regulatory system may not be specific for the actomyosin system in Physarum plasmodia, because some actomyosin-related processes, i.e., secretion and motility, are subject to inhibitory Ca-control. Accumulation of examples for the inhibitory Ca-control in higher and lower eukaryotes will clarify its phylogenic significance.

Myosin light chain kinase from skeletal muscle regulates an ATP-dependent interaction between actin and myosin by binding to actin

Myosin light chain kinase (MLCK) has been purified from various muscles as an enzyme to phosphorylate myosin light chains. While the regulatory role of smooth muscle MLCK is well understood, the role of skeletal muscle MLCK in the regulation of contraction has not been fully characterized. Such characterization of skeletal muscle MLCK is difficult because skeletal muscle myosin interacts with actin whether or not the myosin is phosphorylated. Taking the hint from our recent finding that smooth muscle MLCK inhibits the actin-myosin interaction by binding to actin (Kohama et al., Biochem Biophys Res Commun 184: 1204-1211, 1992), we investigated the regulatory role of the actin-binding activity of MLCK from chicken breast muscle in the actin-myosin interaction. The amount of MLCK that bound to actin increased with increases in the concentration of MLCK. However, MLCK hardly bound to myosin. The actin-binding activity of MLCK was affected when Ca2+ and calmodulin (Ca2+-CaM) were present. The effect of MLCK on the actin-myosin interaction was examined by an in vitro motility assay; the movement of actin-filaments on a myosin-coated glass surface was inhibited by increasing the concentration of MLCK. When CaM was present, the inhibition was overcome in a Ca2+-dependent manner at μM levels. The inhibition of the movement by MLCK and the recovery from the inhibition by Ca2+-CaM were not altered whether we use phosphorylated or unphosphorylated myosin for the assay, ruling out the involvement of the kinase activity of MLCK.

31P-nuclear magnetic resonance studies of intact plasmodia of Physarum polycephalum

31P‐nuclear magnetic resonance spectra were obtained from intact plasmodial cells of Physarum polycephalum, where cytoplasmic streaming is generated by actin‐myosin‐ATP interaction. Several peaks were resolved and identified. They included ATP, ADP, orthophosphate and polyphosphates. Peaks for phosphocreatine, phosphoarginine or AMP were not detected. The intracellular pH and concentrations of ATP and free Mg2+ were estimated to be pH 6.9, 0.2–0.5 mM, and about 1 mM, respectively.

Myosin switching during amoebo-plasmodial differentiation of slime mold, Physarum polycephalum.

We reported previously that myosins from amoebal and plasmodial stages in the life cycle of Physarum polycephalum differ in the primary structure of heavy chains and phosphorylatable 18,000 Mr light chains, while Ca-binding 14,000 Mr light chains are common to both myosins (Kohama & Takano-Ohmuro, Proc Jpn acad 60B (1984) 431; Kohama et al., J biol chem 260 (1986) 8022). We have carried out immunofluorescence microscopical studies upon differentiating cultures of amoebic colonies, which show apogamic amoebo-plasmodial differentiation as follows: Typical amoebae differentiate into mono-nucleate intermediate cells with swollen nuclei and then into two or multi-nucleate young plasmodia (Anderson et al., Protoplasma 89 (1976) 29. Antibodies against plasmodial myosin heavy chain (PMHC) and 18,000 Mr plasmodial myosin light chain (PMLC18) stained intermediate cells and young plasmodia, but not typical amoebae. On the other hand, antibody against amoebal myosin heavy chain (AMHC) stained typical amoebae and intermediate cells--but not young plasmodia. Thus staining was detected using antibodies against both PMHC and AMHC in intermediate cells. Intermediate cells were also stained by antibody against another plasmodium-specific cytoskeletal protein, viz., high molecular weight actin-binding protein (HMWP). We propose that synthesis of myosin subunits switches immediately from amoebal to plasmodial type in mono-nucleate cells with swollen nuclei. This myosin switching is associated with the initiation of HMWP synthesis.

Regulation of Muscle Contraction by Ca Ion

There is no doubt that the contractile processes of every kind of muscle are solely regulated by Ca ion. The fundamental evidence for this concept was furnished by the laboratory of Professor Fritz Lipmann in 1959.