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Calcium inhibition as an intracellular signal for actin-myosin interaction.

I. Introduction

The latter half of 1970s, when I started working with Physarum polycephalum, was an exciting era. The field of actomyosin expanded from muscle cells to non-muscle cells, including a variety of vertebrate and invertebrate cells. In muscle cells, actomyosin is a system responsible solely for contraction. However, in non-muscle cells, it is a major component of the cytoskeleton, and is responsible for various physiological functions. Our understanding of the regulatory role of [Ca.sup.2+] at the micromolar level was also developing owing to the discovery of Ca-binding proteins, such as troponin and calmodulin expanded our knowledge of the regulatory role of [Ca.sup.2+] beyond actomyosin regulation. (1)

I was educated as a protein biochemist of muscle tissues, and was interested in Physarum, a lower eukaryote that shows vigorous shuttle streaming in the cytoplasm, the force of which is generated by actomyosin. Physarum was a good model organism for protein chemistry, because it can be cultured in the lab in large quantities, (2) and because the procedures for purifying actin (3) and myosin (4) were similar to those for skeletal muscle. It was expected that [Ca.sup.2+] would regulate actin and myosin in Physarum in a similar way to muscle; namely, that [Ca.sup.2+] would activate the actin-myosin interaction. However, this review describes that the regulation is quite different in terms of [Ca.sup.2+] regulation.

At first, I purified actomyosin preparations, which were thought to be composed of actin, myosin, and regulatory proteins. The effect of [Ca.sup.2+] was examined by inducing ATP-dependent aggregation of actin and myosin under a spectrophotometer. When the aggregation as named by superprecipitation was induced in EGTA, i.e., in the absence of [Ca.sup.2+], it was more rapid than in the presence of [Ca.sup.2+]. The effect was the opposite of the known effect of [Ca.sup.2+], which activated the ATP-dependent interaction between actin and myosin. The effect was not reproducible when I first observed the inhibitory effect of [Ca.sup.2+]. However, Ca-inhibitory actomyosin was prepared consistently by repeated washing of the actomyosin preparation, by dissolution in a high-salt buffer followed by precipitation by dilution of the solution with cold water. The result was published in the Proceedings of the Japan Academy Series B in a communication by my supervisor, Professor Setsuro Ebashi. (5)

This review describes how the inhibitory effect of [Ca.sup.2+] on Physarum actomyosin has been discovered. As shown in Table 1, the cytoplasmic concentration of [Ca.sup.2+] ([[Ca.sup.2+]]i) is kept as low as possible by the extrusion of [Ca.sup.2+] through the cell membrane and by the sequestration of [Ca.sup.2+] in the cytoplasmic reticulum. When the cell is excited, [[Ca.sup.2+]]i increases owing to the entry of extracellular [Ca.sup.2+] and the release of sequestered [Ca.sup.2+]. The increase soon disappears, because the mechanisms for keeping [[Ca.sup.2+]]i low are triggered again. Cells use this transient increase in [[Ca.sup.2+]]i as a secondary messenger to regulate the actin--myosin interaction, (6) both in the animal and plant kingdoms. However, the modes of use are different; intracellular [Ca.sup.2+] is an inhibitor for plant cells and an activator for animal cells. One of the conclusions of this review is that the plant mode of use is observed in Physarum.

I wrote this review as a personal memoir of over 40 years' research on Physarum. It is based on our published reviews (2), (7)-(22) and on recent original publications. (23), (24) I would like to describe how [Ca.sup.2+] exerts a regulatory effect on actomyosin via the inhibitory mode of Ca regulation.

II. Purification of Physarum myosin

A method for preparing Ca-inhibitory myosin in its actin--myosin interaction was developed by Kohama and Kendrick-Jones. (25) It was based on the preparation of actomyosin, from which myosin is rapidly purified (Table 2).

The procedure for preparing actomyosin is a modification of the method published by Hatano and Tazawa. (26) Actomyosin is extracted by homogenizing Physarum plasmodial cells in a high-salt buffer (pH 7.8-8.0) containing EGTA, followed by the removal of cell debris and slime by centrifugation at 50,000 x g for 30min (Steps 1 and 2). Then, the crude actomyosin is recovered from the centrifugation supernatant as a precipitate produced by reducing the pH of the buffer to 6.5 and the ionic strength to about 50 mM. Native actomyosin is purified from the crude actomyosin preparation by repeating cycles of dissolution in high-salt buffer and precipitation in low-salt buffer (Step 3).

Myosin is purified by a modification of the method that Ebashi developed for removing actin from smooth muscle actomyosin preparation. (27) The native actomyosin is dissolved in 20 mM ATP containing DTT, mixed with concentrated Mg acetate solution to give a final concentration of 0.1 M, and centrifuged at 100,000 x g for 30min (Step 4). The supernatant is mixed with 2 volumes of cold water, and allowed to stand for 3-4h on ice; myosin is then recovered as a precipitate (Step 5). Because this myosin preparation was often contaminated with a trace amount of actin, as shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), we usually repeated Step 5.

Thus, myosin was purified rapidly (within 2 days) to a high level (>95% by SDS-PAGE) and in high yield (about 10mg/100g packed wet cells). The ratio of absorbance at 280 nm to that at 260 nm was about 1.5, indicating a low level of nucleotide or RNA contamination.

When Ca-inhibitory myosin was treated with Nethylmaleimide (NEM), the inhibitory effect of [Ca.sup.2+] on the myosin disappeared. (28) The role of highly reactive SH groups in myosin activity are known in skeletal muscle. (29) We proposed that the modification by NEM made Physarum myosin insensitive to [Ca.sup.2+], and that rapid preparative procedures that did not involve column chromatography and long centrifugation might contribute to obtaining intact native myosin.

III. Subunit composition

As shown in Table 3, (7), (8), (10)-(13), (31) myosin is resolved into three subunits by SDS-PAGE: a heavy chain (HC) with a molecular weight of 230 kDa, which is larger than the HC of skeletal muscle myosin; and two light chains (Lcs) with molecular weights of 16 and 18 kDa. Quantitative densitometry of SDS-PAGE shows that the three subunits are present in a 1:1:1 stoichiometry. (32) Myosin is eluted from a gel-filtration column under high salt conditions in the fraction where skeletal muscle myosin is eluted; thus, the myosin molecule is made up of six polypeptides, two of each subunit.

The 18kDa, phosphorylatable light chain (PLc) bound to skeletal muscle myosin that had 5,5-dithiobis(2-nitrobenzoic acid) (DTNB)-Lc removed by DTNB-treatment. (13) The 16kDa protein, a Cabinding light chain (CaLc), did not bind to the DTNB-treated myosin. However, CaLc was exchanged with the alkali Lc (Al1, Al2) of skeletal muscle by KSCN treatment followed by LiCl treatment. Thus, the CaLc of Physarum myosin is similar to the alkali Lc of skeletal muscle myosin, and the PLc of Physarum myosin is similar to the DTNB-Lc of skeletal muscle myosin.

IV. Head and tail structure

Figure 1D shows a myosin molecule visualized by electron microscopy after rotary shadow casting. The molecule consists of a long rod-shaped tail domain and two globular head domains. The HCs span the entire length of the molecule, whereas the Lcs are associated only with the head region (see Section X). The tail portion is involved in self-assembly; the head portion exhibits ATPase activity, and interacts with actin.

The properties related to the head and tail structures are similar to those of skeletal muscle myosin. Table 3 summarizes the differences in the head and tail structures between Physarum and skeletal muscle myosin. The main differences are as follows. (11), (33) (i) Physarum myosin has a longer tail (170 nm) than skeletal muscle myosin (150 nm), which is consistent with observations that it is more viscous (2.0dL/g) than skeletal muscle myosin (1.5dL/g). (ii) The Physarum myosin tail contains one or two sharp (<90[degrees]) bends, which are not usually observed in skeletal muscle myosin. (iii) Physarum myosin does not assemble in 150 mM KCl, a physiological salt concentration for skeletal muscle cells, but does assemble to form filaments in 30 mM KCl, a physiological salt concentration for Physarum plasmodial cells (see Section XI C). The extent of the Physarum myosin assembly is modified by its phosphorylated state. In contrast, skeletal muscle myosin assembles in 30 and 150 mM KCl irrespective of its phosphorylated state. (iv) Physarum myosin can be obtained as a recombinant protein. As discussed in Section X, these differences will be confirmed by using recombinant Physarum myosin.

V. Physarum myosin ATPase activities

A. Myosin ATPase activities in the absence of actin. Mg-ATPase activity in low-salt buffer containing EGTA ([Ca.sup.2+] <1nM) is 4-10 nmol [min.sup.-1] [mg.sup.-1] (Table 3), whereas Ca-ATPase activity is typically 70nmol [min.sup.-1] [mg.sup.-1]. When assayed at high salt concentrations (0.5M KCl), Mg-ATPase and Ca-ATPase activities increase to 30 and 800 nmol [min.sup.-1] [mg.sup.-1], respectively.

K-EDTA-ATPase activity is typically 100 nmol [min.sup.-1] [mg.sup.-1], which is three times that of the Mg-ATPase activity and 1/8 that of Ca-ATPase activity at high salt concentrations. K-EDTA-ATPase activities of myosins from vertebrate muscle and non-muscle cells show the highest myosin ATPase activities. However, Physarum myosin shows the opposite behavior; K-EDTA-ATPase activity is lower than that of Ca-ATPase, which may be a feature of myosin in lower eukaryotes. (19)

B. Myosin Mg-ATPase activity in the presence of actin. A typical experiment examining the effect of actin on myosin Mg-ATPase activity at low salt concentrations is shown in Fig. 1A. The maximum rates of reaction, [V.sub.max], in EGTA and 50 [micro]M [Ca.sup.2+] were 230.0 and 54.1 nmol [min.sup.-1] [mg.sup.-1] myosin, respectively. Thus, actin maximally activated Mg-ATPase activity 51.1-fold in the absence of [Ca.sup.2+] and 31.1-fold in the presence of 50[micro]M [Ca.sup.2+] (see, Section VII). The Michaelis constant, [K.sub.m], for actin was 0.6 [micro]M, irrespective of the presence or absence of [Ca.sup.2+], suggesting that [Ca.sup.2+] exerted its regulatory effect on myosin after myosin associated with actin. The relationship between [Ca.sup.2+] concentration and actin-activated Mg-ATPase activities (Fig. 1C) indicated that half-maximal inhibition occurred at 1-3[micro]M [Ca.sup.2+].

C. Inhibitory effect of [Ca.sup.2+]: myosin-linked Ca inhibition. Purified actin and myosin from skeletal muscle have no regulatory roles themselves. Mg-ATPase activity of skeletal muscle myosin activated by skeletal muscle actin is not inhibited by [Ca.sup.2+] (Fig. 1C). This Ca-insensitive Mg-ATPase activity of skeletal myosin was also observed when it was activated by Physarum actin. However, in Physarum myosin, the Mg-ATPase activity was inhibited by an increase in [Ca.sup.2+] concentration, irrespective of whether the actin was from Physarum or skeletal muscle. These hybrid experiments clearly demonstrated that the Ca inhibition of actin-activated Mg-ATPase activity of Physarum myosin was mediated by Physarum myosin. (28)

VI. Ca-binding properties of Physarum myosin

A. Inverse relationship of Ca-binding activity with the ATPase activity of Physarum myosin. Physarum myosin bound [sup.45][Ca.sup.2+] with a high affinity at micromolar dissociation constants (28) and with a binding capacity of 2 mol [Ca.sup.2+] per mole of myosin. Figure 1B shows that Ca-binding activity increases with [Ca.sup.2+] concentration. However, ATPase activity decreases with the increase of [Ca.sup.2+] concentration at micromolar levels. The inverse relationship between Ca-binding activity and ATPase activity indicates that Ca-binding activity inhibits ATPase activity.

B. Ca-binding light chain as a Ca-receptive subunit of Physarum myosin. Physarum myosin is composed of a pair of HCs and two pairs of Lcs of 18 and 16kDa (Fig. 2Aa). The domain structure of the HC showed that the binding sites for ATP, actin, and Lcs are all in the HCs, (12) which was confirmed by cloning and expressing HC cDNA (see Section X). The Ca binding site is localized in the 16-kDa CaLc. The primary structure of CaLc was determined by peptide analysis and cDNA cloning, (34) and its molecular weight was calculated to be 16,084 Da. The EF-hand structure, which is a consensus sequence for Ca-binding proteins, was identified at one position at the N terminal. The highest homology of CaLc was found in bovine brain calmodulin. (34)

The evidence that CaLc is a Ca-receptive subunit is as follows (Table 4). (i) The mobility of CaLc in SDS-PAGE of myosin was altered in the presence of [Ca.sup.2+]. (36) (ii) The CaLc band in SDS-PAGE of myosin bound to [sup.45][Ca.sup.2+], (35) and biochemical measurements using [sup.45][Ca.sup.2+] showed that CaLc bound 0.4mol [Ca.sup.2+] per mole. This figure is too low to be explained by Ca-binding (1.3 mol [Ca.sup.2+] per mole) of the parent myosin. It was proposed that the Ca-binding activity of CaLc increases when it is incorporated into the myosin molecule, which was later confirmed by expressing CaLc as a recombinant protein (see Section X).

VII. Phosphorylated states and Ca inhibition of Physarum myosin

A. Phosphorylation and dephosphorylation of Physarum myosin. 1. Phosphorylation sites of Physarum myosin. The total phosphate content after ashing Physarum myosin was 6.8-4.0 mol [P.sub.i] per mol myosin (500 kDa), indicating that the myosin is phosphorylated at multiple sites (Fig. 2B). The major phosphorylation sites were in the HC, because the myosin from plasmodial cells cultured in the presence of [H.sub.3][[sup.32]P][O.sub.4] incorporated the radioactive isotope only in the HC (Figs. 2Ba and b). However, a subspot on two-dimensional isoelectric focusing SDS-PAGE of myosin suggested the partial phosphorylation of PLc (Fig. 2Bc). PLc phosphorylation was also consistent with the observation that PLc was phosphorylated by an endogenous kinase contaminating the myosin preparation (Fig. 2Bb). Myosin from vertebrates can be purified in the dephosphorylated state, whereas myosins from lower eukaryotes, such as Physarum, (25) Dictyostelium, (37) and Acanthamoeba (38) were purified in the phosphorylated state.

2. Dephosphorylation and Ca inhibition. Physarum myosin was dephosphorylated with exogenous acid phosphatase, and its actin-activated ATPase activity was compared with that of untreated, phosphorylated myosin. (39), (40) The activity was high in the absence of [Ca.sup.2+] and decreased as [Ca.sup.2+] concentration increased. The activity of dephosphorylated myosin was high in the absence of [Ca.sup.2+] and remained low in the presence of [Ca.sup.2+] irrespective of changes in [Ca.sup.2+] concentration. We examined the Ca-binding activity of myosin following dephosphorylation. Dephosphorylated myosin binding to [Ca.sup.2+] was similar to that of phosphorylated myosin (Table 5). Therefore, dephosphorylation of Physarum myosin minimizes its ATPase activity, and thus the activity cannot be inhibited further by [Ca.sup.2+]. (39)

B. Role of actin in Ca inhibition. 1. Dephosphorylation decreases the affinity of Physarum myosin for actin. Figure 3A shows comparison of the ATPase activity of phosphorylated myosin with that of dephosphorylated myosin in the presence of various concentrations of actin. (40) The ATPase activity of phosphorylated myosin increased with the concentration of actin. A similar increase was observed with dephosphorylated myosin, although the increase required higher actin concentrations. Kinetic analysis (inset of Fig. 3A) showed that dephosphorylated myosin had a higher [K.sub.m] for actin. The [V.sub.max] values were the same for both types of myosin.

Figure 3B summarizes the effect of [Ca.sup.2+] by using the extent of Ca inhibition, 100 x [(ATPase activity in EGTA)--(ATPase activity in [Ca.sup.2+])]/ (ATPase activity in EGTA), as an index. (40) The extent of Ca-inhibition of phosphorylated myosin was high, irrespective of actin concentration. For dephosphorylated myosin, Ca-inhibition of actin-activated ATPase activity was low in the presence of low actin concentrations, and increased with the actin concentration, reaching a level comparable to that of the phosphorylated myosin. Thus, changes in the actin-activated ATPase activity associated with myosin phosphorylation can be explained by changes in the affinity of myosin for actin.

2. Ca binding mode rather than the phosphorylating mode is physiologically important. In the presence of actin at low concentrations, the ATPase activity of Physarum myosin was modified by [Ca.sup.2+] binding and by altering its phosphorylated state. However, in the presence at high actin concentrations, only [Ca.sup.2+] binding regulated the ATPase activity. Therefore, the crucial factor in determining which of the two modes is dominant in vivo is the concentration of actin in Physarum plasmodial cells. In skeletal muscles, the concentration of actin is comparable to that of myosin by weight. However, non-muscle tissues are expected to be exposed to high actin concentrations, because actin concentrations are much higher than myosin concentrations. (41) This is true in plasmodial cells, (42) and hence the mode of Ca binding should be physiological. Changes in the phosphorylated state of myosin may not play a major role in vivo. The conclusion was consistent with the data shown in Table 5 that the Ca-binding activity of myosin was not affected by the phosphorylation or dephosphorylation of myosin.

VIII. Inhibitory effect of [Ca.sup.2+] on Physarum myosin as compared with the activating effect of [Ca.sup.2+] on scallop myosin

The detection of the inhibitory effect has been described so far by measuring myosin ATPase activity in the presence of actin (Fig. 1A). (28) However, the inhibitory effect is now detected by a variety of methods (Table 6), as explained in the following paragraph.

The regulation by [Ca.sup.2+] binding to myosin was first established with myosin from the scallop adductor muscle (Fig. 4A). (31) However, [Ca.sup.2+] activates the activity of scallop myosin, whereas it inhibits that of Physarum myosin. (43) Both modes were observed by measuring the actin-activated ATPase activities of the two myosins. This section describes another method for detecting the ATPdependent sliding between actin and myosin, (44), (45) in which the movement of actin-filaments on myosin fixed to the surface of a coverslip was observed under a microscope. This method allows direct comparison of movement velocities in EGTA with those in [Ca.sup.2+], because solutions containing EGTA and solutions containing [Ca.sup.2+] can be applied sequentially to the same myosin-coated glass surface.

Physarum myosin was purified from plasmodia (Fig. 4A) by the method described in Table 2. Scallop myosin was purified from striated muscle of Patinopecten yessoensis (Fig. 4E). (46) Actin was purified from rabbit skeletal muscle and was polymerized to form filaments. Actin filaments labeled with rhodamine--phalloidin were allowed to move on Physarum or scallop myosin fixed to a nitro cellulose-coated glass surface. The ATP-dependent movement of the actin filaments was monitored under a fluorescent microscope equipped with a video camera.

The mean velocity of actin movement on Physarum myosin was 0.80 pm/s in the absence of [Ca.sup.2+] with EGTA (Fig. 4B). However, the velocity reduced to 0.31 pm/s in the presence of [Ca.sup.2+] (Fig. 4C), which agrees with the inhibitory effect of [Ca.sup.2+] measured by the actin-activated ATPase activity (Fig. 1A). On the scallop myosin-coated surface, the velocity was 1.28 [micro]m/s when [Ca.sup.2+] was present (Fig. 4C). In the presence of EGTA, almost no movement was detected (Fig. 4F). Thus, we confirmed the inhibitory effect of [Ca.sup.2+] by comparing the mechanical movement of actin filaments for Physarum and scallop myosin. (Figs. 4D and H). (46), (47)

IX. Actin-binding proteins involved in Ca inhibition of Physarum

Ca inhibition through myosin reached a maximum of about 60% (see Section VII, Fig. 3), suggesting that additional mechanisms may work for the Ca inhibition. This section describes Ca inhibition caused by modulating actin filaments in a [Ca.sup.2+]-dependent manner.

A. Fragmin. Fragmin, purified from Physarum plasmodium, bound to actin filaments at the barbed end to shorten them in the presence of [Ca.sup.2+]. This effect reduced the tension development in actomyosin threads in the presence of [Ca.sup.2+]. (48), (56) We speculated that this type of regulatory mechanism might also produce the inhibitory effect of [Ca.sup.2+] on the actin--myosin interaction of Physarum.

B. Cytoplasmic Ca-binding light chain in Physarum plasmodial cells. In Section VI B, we explained that CaLc is a [Ca.sup.2+]-binding subunit of Physarum myosin (Table 4). However, we noticed that Physarum plasmodial cells contained CaLc that was not incorporated into myosin molecules as a light chain, and proposed that CaLc enhanced the inhibitory effect of [Ca.sup.2+]. (35), (57) Later, we found that CaLc modulates the polymerization of actin in the absence of [Ca.sup.2+], namely CaLc polymerized actin, and this activity was abolished in the presence of [Ca.sup.2+]. (33) Because polymerized actin tends to activate myosin, we concluded that calcium light chain exerts an inhibitory effect on actin--myosin interactions through polymerized actin. Because CaLc can now be obtained as a recombinant protein (see Section X), the back-up mechanism for Ca inhibitory effects can be confirmed.

C. Caldesmon-like protein. Caldesmon (CaD)-like protein was purified from Physarum (Table 7). Similar to smooth muscle CaD, it is heat-stable and has an elongated shape. It reacted with the antibody against smooth muscle CaD, and was present in Physarum cells in association with stress fibers, which was composed of actin and myosin together with their binding proteins, if any. (42), (59) The CaD-like protein exerted a stimulatory effect on the interaction of Physarum myosin with actin. Because the stimulation was abolished by the calmodulin in the presence of [Ca.sup.2+], CaD-like protein enhanced the inhibitory effect on the interaction of Physarum myosin with actin. Further explanation was added in Supporting information section.

CaD was first obtained from smooth muscle, and exerted an inhibitory effect on the interaction of smooth muscle myosin; the inhibitory effect was released by calmodulin in the presence of [Ca.sup.2+]. (60) Later, Lin et al. found that the stimulatory effect was detected for smooth muscle CaD under specific conditions and that the stimulation was abolished by calmodulin in the presence of [Ca.sup.2+]. (61) Because this stimulation was similar to the effect of Physarum CaD-like protein, we should re-examine how Physarum CaD-like protein produced the stimulation and its abolition.

X. Studies with recombinant Physarum myosin and its mutants

A. Expression and purification of full-length of Physarum myosin, short-tailed heavy meromyosin, and Physarum myosin with mutant Ca-binding light chain. We cloned the full-length Physarum myosin HC (GenBank accession number AF335500), CaLc (GenBank accession number J03499; see Section VI B, Table 4), and PLc (GenBank accession number AB076705) from plasmodia, and then constructed baculovirus vectors with the wild types of HC, CaLc, and PLc. The Sf-9 cells cultured in Grace's insect culture medium were co-infected with three constructs of HC, CaLc, and PLc simultaneously to express Physarum myosin as a recombinant protein. Using his-tagged vectors, we purified Physarum myosin by Ni-NTA Superflow column chromatography. The purified Physarum myosin consisted of 230 kDa HC, 18kDa PLc, and 16kDa CaLc (SDS-PAGE shown in Fig. 5A). The electron micrograph of Physarum myosin showed the structure consisting of two globular heads and a tail. The SDS-PAGE and electron micrograph results were comparable with those in Figs. 2Aa and 1D, respectively, confirming that Physarum myosin was expressed. Physarum heavy meromyosin (HMM) was expressed with HC cDNA of Met 1-Lys 118, CaLc, and PLc, followed by similar purification procedures. The SDS-PAGE and electron micrograph results are shown in Figs. 5A and B, respectively. The identification of 135 kDa HC with two globular heads was consistent with the notion that we obtained Physarum HMM as a recombinant protein.

B. Examining the role of CaLc in Ca inhibition by mutating CaLc. We engineered mutant Physarum myosin with CaLc-3A that showed no Ca-binding activity (Figs. 6A and B). (62) We compared the effect of [Ca.sup.2+] on wild-type Physarum myosin and the mutant Physarum myosin by measuring the actin-activated ATPase activity (Fig. 7A) and detecting the motor activity (Fig. 7b). (22) The ATPase activity of wild-type Physarum myosin was reduced by increasing the [Ca.sup.2+] concentration (filled circles of Fig. 7A), whereas the ATPase activity of the mutant Physarum myosin was hardly affected by the [Ca.sup.2+] concentration (open circles of Fig. 7A). Similarly, we detected the inhibitory effect of [Ca.sup.2+] by measuring the movement velocity of actin filaments on the surface coated with wild-type Physarum myosin, whereas there was no inhibitory effect on the surface coated with mutant Physarum myosin (Fig. 7B).

C. Role of myosin tail length. Electron microscopy showed that recombinant full-length myosin has a 160nm tail (Fig. 5B1), and that HMM has a shorter tail (Fig. 5B3). The inhibitory effect of [Ca.sup.2+] on wild-type Physarum myosin was more pronounced than that on HMM (filled triangles, Fig. 7), although both myosin and HMM contained wild-type CaLc. Because this difference was detectable by both ATPase activity assay (Fig. 7A) and in vitro motility assay (Fig. 7B), we concluded that the length of the HC should change the effect of [Ca.sup.2+].

D. Reversibility of the effect of [Ca.sup.2+] on Physarum myosin. To test the reversibility of the [Ca.sup.2+] effect, we coated a glass coverslip surface with recombinant Physarum myosin, and mounted actin filaments in motility buffer containing EGTA or [Ca.sup.2+], and subjected to the observation of the movement of actin filaments in the following order; EGTA [??] C[a.sup.2+] [??] EGTA. The motility was inhibited when the motility buffer was changed from EGTA to [Ca.sup.2+]. Then, upon changing the buffer from [Ca.sup.2+] to EGTA, the movement of actin filaments recovered (Fig. 8A). The reversible effect of [Ca.sup.2+] was confirmed by reversing the order of the buffer (Fig. 8B). (24) The reversibility demonstrated both ways is consistent with the idea that [Ca.sup.2+] binding and release regulate myosin activity, which is shown in Fig. 7 of Ref. 25.

E. Role of CaLc in the inhibitory effect of [Ca.sup.2+]. To determine whether CaLc or PLc plays a more important role in exerting the inhibitory effect of [Ca.sup.2+] on myosin, we expressed and purified HMMs associated with scallop and Physarum Lcs (Fig. 9). We used cDNA fragment coding the HC of smooth muscle myosin, because [Ca.sup.2+] binding to and release from smooth muscle HMM were not involved in the regulation. (63)

As shown in Fig. 9, the actin-activated ATPase activity of HMM with smooth muscle Lcs (construct #1) remained at the basal level, irrespective of the presence of [Ca.sup.2+] or EGTA, confirming that smooth muscle Lcs are not regulated by calcium binding. (63) We confirmed that [Ca.sup.2+] exerted an activating effect on the recombinant HMM of scallop myosin Lcs, ScELc and ScRLc (construct #3). However, the effect of [Ca.sup.2+] was inhibitory on the recombinant HMM of Physarum myosin Lcs, PhCaLc, and PhPLc (construct #2). For heterogeneous Lcs, HMM with ScELc and PhPLc (construct #4), and HMM with PhCaLc and ScRLc (construct #5), we observed that the actin-activated ATPase activity was increased by [Ca.sup.2+] for scallop Lc, and that the activity was inhibited by [Ca.sup.2+] for Physarum Lc. Therefore, the key Lc for activation was ScELc and the key Lc for inhibition was PhCaLc. In conclusion, ScELc played an important role in the activity effect of [Ca.sup.2+] and PhCaLc for the inhibitory effect of [Ca.sup.2+], (23) and both are alkali Lc class (Table 3).

F. Crystal structure of the regulatory domain of Physarum myosin. CaLc has four Ca-binding sequences, which are EF-hand loops (Fig. 6A). We mutated cDNA of CaLc followed by expression in Escherichia coli cells, and then measured Ca binding to the recombinant CaLc (wild type) and mutant CaLc-3A (D15A/D17A/E26A). The Ca-binding activity of CaLc was lost in CaLc-3A (Fig. 6B). Further analysis indicated that the Ca binding site was localized in the first loop of CaLc. (62) The neck region of the myosin molecule (Fig. 5B), known as the regulatory domain (RD), acts as a lever arm during force generation, and studies with scallop myosin have shown that it is the site of [Ca.sup.2+] regulation. We expressed the RD of Physarum myosin, which is composed of CaLc, PLc, and a short HC fragment containing the binding site for the Lcs. RD was purified, crystallized, and its structure was determined. (64) The scallop ELC had an extra turn in the first loop compared with Physarum CaLc, as shown in Fig. 2 of Ref. 64. Thus, we confirmed the important role of the first EF-hand loop of Physarum CaLc based on structural analysis.

XI. Inhibitory mode for [Ca.sup.2+] regulation of contraction of Physarum plasmodium

A. Inverse relationship of [[Ca.sup.2+]]i with plasmodium contraction. The Physarum plasmodial cell is a giant single cell with multiple nuclei, and it consists of a sol-like inner protoplasm and an outer layer of gel protoplasm. The sol-like protoplasm shows streaming movements of the nuclei. Actomyosin system is responsible for the contraction--relaxation movement of the outer layer. (58), (65) It is possible to measure tension development by using the strand of plasmodial cell. Yoshimoto and Kamiya treated the strand with saponin to destroy its cell membrane, and they measured the tension development by changing [[Ca.sup.2+]]i from outside the cell. (50) This method detected stable isotonic tension when ATP was supplied. The tension was reduced with the increase in [Ca.sup.2+] concentration. We also measured [[Ca.sup.2+]]i of living plasmodium and related to the contraction and relaxation of the cell. The inverse relationship of [[Ca.sup.2+]]i with plasmodium contraction was in confirmation of the inversed relationship. (55)

B. Detection of the inhibitory mode with actomyosin preparations. Early studies on the Ca-control of the actin-myosin-ATP interaction in Physarum plasmodium were made with crude prep arations of actomyosin. The observation that the ATPase activity of this preparation was increased with the increase in [Ca.sup.2+] concentration led the authors to the conclusion that [Ca.sup.2+] activated the interaction in the similar way to the regulatory way of vertebrate skeletal muscle. (66)

We also observed that [Ca.sup.2+] elevated the MgATPase activity of crude actomyosin preparation of Table 2, Step 3 as shown by the filled circles in Fig. 10A. However, when the preparation was purified by repeated washing, the effect of [Ca.sup.2+] on the activity was eventually reversed. As shown by the open circles in Fig. 10A, the Mg-ATPase activity of purified actomyosin (Table 2, Step 4) was inhibited with the increase in [Ca.sup.2+] concentration. We interpreted that the reversed effect of [Ca.sup.2+] was brought about by removing ATP pyrophosphohydrolase(s), which were [Ca.sup.2+] activatable and abundant in plasmodium, (67) from the crude actomyosin preparation to the purified actomyosin preparation. (25) Because SDS-PAGE of the purified actomyosin was mainly consisted of actin and myosin (for SDS-PAGE, see Fig. 1A of Ref. 25), we reached to the conclusion that [Ca.sup.2+] controlled the actin-myosin-ATP interaction by inhibiting actin-activated ATPase activity of myosin as reviewed in Ref. 8.

C. Inhibitory mode detected in the ATPase activity of actin-activated Mg-ATPase activity of purified myosin under the physiological conditions of Physarum plasmodium. On a phylogenic basis, Physarum belongs to a species that is quite remote from vertebrate. Although a few reports are available for the intracellular ionic concentrations, (68), (69) we dare to subject living plasmodium to [sup.31]P-Nuclear magnetic resonance (NMR) study. (70) The spectrum of NMR (Insert of Fig. 10B) revealed that the intracellular pH, ATP concentration and free [Mg.sup.2+] concentration were pH 6.9, 0.2-0.5 mM, and about 1[micro]M, respectively.

Physarum myosin was purified from an actomyosin preparation as described in Section II. The ATPase activity of Physarum myosin activated by purified skeletal muscle actin was examined under physiological conditions. As shown in Fig. 10B, we measured the actin-activated Mg-ATPase activity of Physarum myosin under the physiological conditions for Physarum including the above data. The activity was reduced by [Ca.sup.2+] at micromolar concentrations, demonstrating that a myosin-linked inhibitory mode is present in living Physarum plasmodial cells. (71)

XII. [Ca.sup.2+]-dependent phosphorylation and dephosphorylation in the Physarum plasmodial cell

A. Model of myosin signal transduction. The phosphorylation of Physarum myosin at PLc and HC suggested the presence of kinases for myosin Lcs and HCs in the plasmodial cell (Section VII, Fig. 2). The removal of phosphate by an exogenous potato acid phosphatase caused the loss of the actin-activated ATPase activity of myosin (39) and the loss of phosphate content in the myosin. (40) However, the phosphatase treatment did not affect the [Ca.sup.2+]-binding activity of the myosin (Table 5), suggesting that the regulatory mechanism for myosin phosphorylation is independent from regulation by the Ca inhibitory mechanism. [[Ca.sup.2+]]i changes periodically with the periodic changes in the direction of cytoplasmic streaming because of the actin--myosin interaction. (55) The changes are so slow, on a time scale of minutes, suggesting that myosin phosphorylation does not play a physiological role in the plasmodial cell. As the first step in understanding the turnover of phosphate, we investigated whether the PLc of Physarum myosin was phosphorylated in vitro in low or high [[Ca.sup.2+]]i conditions. A crude fraction containing both myosin light chain kinase (MLCK) activity and [Ca.sup.2+]-binding protein was purified, and we identified the 38 kDa protein as the [Ca.sup.2+]-binding protein and the 55 kDa protein as MLCK. (72) The effect of [Ca.sup.2+] on MLCK phosphorylation of PLc was inhibitory, (72), (73) indicating that myosin is phosphorylated under low [[Ca.sup.2+]]i conditions in plasmodial cells. To estimate the dephosphorylation in vivo, the actomyosin preparation (Table 2, Step 3) is an excellent model to test the regulation related to actin and myosin. The model contains the kinase activities for actin and myosin subunits and the phosphatase activity for these proteins. We incubated the preparation in [[sup.32]P] [gamma]-ATP to allow the kinases to phosphorylate these proteins, and the phosphorylation was terminated by the kinase inhibitor staurosporine. We observed that the radioactivity incorporated was gradually decreased by the phosphatase. The decrease in radioactivity was terminated by the phosphatase inhibitor okadaic acid, as shown in Figs. 4-6 of Ref. 73. The experiments showed that the phosphatase activity was low in the absence of [Ca.sup.2+] and increased with the increase in [Ca.sup.2+] at micromolar concentrations. Furthermore, the calmodulin inhibitor trifluoperazine inhibited the phosphatase activities, indicating that calmodulin was involved in the phosphatase activity. Thus, myosin tends to be in an active, phosphorylated form in the absence of [Ca.sup.2+], whereas it is in an inactive, dephosphorylated form in the presence of [Ca.sup.2+].

Figures 11A and B summarize the signal transduction of myosin in a plasmodial cell. Myosin is active only in the phosphorylated form, and is affected by binding [Ca.sup.2+]. However, the phosphorylation of myosin does not affect the binding activity of myosin. These [Ca.sup.2+]-independent pathways are shown in A by the short arrows in (1). Myosin is phosphorylated at low [[Ca.sup.2+]]i in step (2) and is dephosphorylated at high [[Ca.sup.2+]]i in step (3). The Ca-binding protein responsible for the phosphorylation is the 38 kDa protein, (72) and Physarum calmodulin is responsible for the dephosphorylation. (34) In B, the sites of Ca-binding ([Ca.sup.2+]) and phosphorylation ([??]) are schematically expressed. They are located in myosin heads, where CaLc and PLc are both located (see Section X F). Phosphorylation of PLc is a prerequisite for myosin to be in an active form. When phosphorylated myosin binds [Ca.sup.2+] at CaLc, it is inactivated as described above.

B. Discrepancies in data on Physarum myosin. Inspired by the molecular cloning of MLCK from Dictyostelium amoeba, (74) we used the same approach in Physarum. Template cDNA prepared from Physarum plasmodium was subjected to the polymerase chain reaction (PCR) with degenerate PCR primers designed from MLCK and calmodulin kinases. (75) The resulting cDNA sequence predicted a 42,650 Da protein containing 376 amino acids. The cDNA was expressed in E. coli, demonstrating that the Physarum MLCK phosphorylated PLc in the presence of calmodulin and [Ca.sup.2+], and that [Ca.sup.2+] stimulated Physarum MLCK. Thus, we obtained an MLCK that exhibited [Ca.sup.2+]-inhibitory activity by the conventional purification procedure, and also obtained an MLCK that exhibited [Ca.sup.2+]-stimulatory activity by molecular cloning and molecular expression. It is unknown which MLCK plays a physiological role.

A similar question arises from the difference in myosin purification techniques. Physarum myosin can be obtained by a biochemical purification technique (Fig. 1D) or by cDNA cloning followed by expression in cultured cells (Fig. 5B1). Indeed, the specific activity of the actin-activated Mg-ATPase of purified myosin (Fig. 1A) were similar to that of recombinant myosin (Fig. 7A of Ref. 23). For example, the actin-activated Mg-ATPase activity of the biochemically purified myosin was about 150 nmol [mg.sup.-1][min.sup.-1] in EGTA (Figs. 3A and 10D), and that for recombinant myosin was 160 nmol [mg.sup.-1][min.sup.-1] in EGTA (Fig. 5A of Ref. 23). However, the two types of myosin showed different behavior. Purified myosin is at least partially in the phosphorylated form (Fig. 2B), and recombinant myosin is not phosphorylated. (75) Further, Ca inhibition was detectable in the both myosins by measuring actin-activated ATPase activity as well as by inducing in vitro motility, (47) although the phosphorylated state is quite different. The effect of phosphorylation on Dictyostelium myosin II differs between the PLc and HC; (76) stimulatory effect for the former and the inhibitory effect for the latter. Although no data are available for Physarum myosin, the phosphorylation approach may work (see Section VIII). The discrepancy between the myosin obtained by the conventional and recombinant methods remains unresolved.

XIII. Perspectives

The life cycle of Physarum consists of two phases with different modes of motility: uninucleate amoeba showing slow amoeboid movement; and multinucleated plasmodium showing rapid cytoplasmic streaming. Actomyosin was obtained from amoeba by methods similar to those used for plasmodium (Table 2), (77) and amoebal myosin was purified from the actomyosin preparation. (78) Plasmodial myosin in its two-headed and long-tailed shape was examined by electron microscopy, and its subunit composition of HC and CaLc was examined by SDS-PAGE. Peptide mapping showed that the HC and PLc differed between amoebal and plasmodial myosins whereas CaLc was identical. The actin-activated Mg-ATPase activities of amoebal myosin showed a similar [Ca.sup.2[+ or -]]-inhibitory effect to plasmodial myosin. Sonobe et al. (79) prepared a contractile model by permeabilizing the membrane of Amoeba proteus, and found that the ATP-dependent contraction inhibited by increasing [Ca.sup.2+]. Based on the results from A. proteus, it can be speculated that [Ca.sup.2+] inhibited the physiological motility of Physarum amoeba.

A similar myosin, the conventional myosin II isoform, was purified from Dictyostelium amoeba, and its actin-activated Mg-ATPase activity was modified by the phosphorylation mechanism. The effect of phosphorylating its Lc was stimulatory, whereas that of phosphorylating its HC was inhibitory. (76) It will be intriguing to see whether the Lc or HC subunit plays a physiological role in regulating amoeba motility.

Cytoplasmic streaming, which is usually observed in plant cells, is thought to be mediated by the actomyosin system. Streaming occurs in the resting state of the cells at low [Ca.sup.2+] concentrations, and stops when the intracellular [Ca.sup.2+] concentration is increased by excitation. [Ca.sup.2+] is used as a signal for transient cessation of streaming. (80) In contrast, the actomyosin system in animal cells is active only when [Ca.sup.2+] increases. As exemplified by activating muscle contractions by [Ca.sup.2+], (6) [Ca.sup.2+] is an activator. Thus, the actomyosin system of Physarum, a lower eukaryote, is akin to that of plant cells in terms of [Ca.sup.2+] regulation. Chara is a well-characterized plant because of the rapid movement of cellular organelles along its intracellular actin-filament cables. We cloned the cDNA of the motor protein responsible for the movement. (81) The structure differed from the conventional muscle myosin shown in Fig. 5B1, and was characterized as myosin XI. Myosin XI was purified biochemically and characterized from tobacco BY-2 cells. The effect of [Ca.sup.2+] on this myosin XI isoform was inhibitory, as shown by both ATPase activity and motor activity. (82) Among other unconventional myosins, (83) myosin I and myosin V were obtained from vertebrate cells by protein purification, and their motor activities were characterized. It will be intriguing to see the effect of [Ca.sup.2+] on the vesicle transportation of vertebrate cells using a similar method that was used in the study on BY-2 cells.

In this review, I proposes a question as if Ca inhibition of actomyosin activity never found in animal cell. The actomyosin system is also thought to be involved in the secretion of endocrine cells. As reported in Ref. 84, the secretion of renin from juxtaglomerular cells is inhibited by Ca. Therefore, the actomyosin systems in the cells are thought to be under inhibitory Ca control, although the conventional myosin of myosin II isoform is involved or not. I believe that more examples of such actomyosin systems will be found as the search for Ca inhibition proceeds.

Supporting Information

Is inhibitory effect of [Ca.sup.2+] as detected by ATPase and motility is sufficiently high enough to explain its physiological role? Ca-inhibition of the actin-activated ATPase activity of Physarum myosin was 960% at most (Fig. 3B). As shown in Figs. 4B and C, Ca-inhibition of the actin motility on a glass surface coated with Physarum myosin (Table 7) was 970%, indicating partial effect of [Ca.sup.2+]. The motility assay at the same experiment using scallop myosin, Ca-inhibition was 100% (Fig. 4FG).

To exert the inhibitory effect of [Ca.sup.2+] to Physarum myosin, the myosin must be activated in advance as described in Section VII. Phosphorylation of the myosin at PLc is responsible for the activation (Fig. 11B). Indeed, [Ca.sup.2+] exerts the inhibitory effect on the myosin activated in this way. Figure S1A relates schematically myosin activity to [Ca.sup.2+] concentration. The activity is high in the absence of [Ca.sup.2+] (--[Ca.sup.2+]), which is caused by myosin phosphorylation. Uppon addition of [Ca.sup.2+], myosin binds [Ca.sup.2+], and the activity is inhibited (+[Ca.sup.2+]).

The caldesmon-like protein of Physarum binds to actin and stimulates the actin-activated ATPase activity of Physarum myosin and the actin motility. Stimulation is abolished when calmodulin is mixed with the caldesmon-like protein in the present of [Ca.sup.2+]. Figure S1B schematically explains that the caldesmon-like protein enhances additionally the activity of myosin that is activated by phosphorylation in the absence of [Ca.sup.2+] (-[Ca.sup.2+]). Upon mixing calmodulin and [Ca.sup.2+], calmodulin abolishes the enhancement through actin to cause Ca-inhibition (+[Ca.sup.2+]). In addition to Ca-inhibition of myosin itself, we can detect larger Ca-inhibition. Such an actin-linked mode may back up the inhibitory effect of [Ca.sup.2+] on Physarum myosin, resulting in the augmentation of Ca-inhibition. As explained in Section IX of the text, a few actin-binding proteins including caldesmon-like protein are found in Physarum plasmodia. They may contribute to the augmentation of the effect.

Abbreviations: CaLc: calcium-binding light chain; CaM: calmodulin; DTNB: 5,5'-dithiobis (2-nitrobenzoic acid); DTT: dithiothreitol; EGTA: ethylene glycol-bis-([beta]-aminoethyl ether)N,N,N',N'-tetra-acetic acid; HC: heavy chain; HMM: heavy meromyosin; IEF: isoelectric focusing; Lc: light chain; MLCK: myosin Lc kinase; PLc: phosphorylatable Lc; RD: regulatory domain; Ph: Physarum; Sc: scallop; Sm: smooth muscle; S1: Subfragment 1.

doi: 10.2183/pjab.92.478

Acknowledgements

The publication costs of this article were defrayed in part by the grants from the Smoking Research Foundation and by Grants-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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(71) Kohama, K. and Kohama, T. (1984) Myosin confers inhibitory [Ca.sup.2+]-sensitivity on actin-myosin-ATP interaction of Physarum polycephalum under physiological conditions. Proc. Jpn. Acad., Ser. B (60, 435-439.

(72) Okagaki, T., Ishikawa, R. and Kohama, K. (1991) Purification of a novel Ca-binding protein that inhibits myosin light chain kinase activity in lower eukaryote Physarum polycephalum. Biochem. Biophys. Res. Commun. 176, 564-570.

(73) Okagaki, T., Ishikawa, R. and Kohama, K. (1991) Inhibitory [Ca.sup.2+]-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: Role of a [Ca.sup.2+]-dependent inhibitory factor. Eur. J. Cell Biol. 56, 113-122.

(74) Tan, J.L. and Spudich, J.A. (1991) Characterization and bacterial expression of the Dictyostelium myosin light chain kinase cDNA. Identification of an autoinhibitory domain. J. Biol. Chem. 266, (16044-16049.

(75) Nakamura, A., Hanyuda, Y., Okagaki, T., Takagi, T. and Kohama, K. (2005) A calmodulin-dependent protein kinase from lowereukaryote Physarum polycephalum. Biochem. Biophys. Res. Commun. (328, 838-844.

(76) Tan, J.L., Ravid, S. and Spudich, J.A. (1992) Control of nonmuscle myosins by phosphorylation. Annu. Rev. Biochem. 61, 721-759.

(77) Kohama, K. and Takano-Ohmuro, H. (1984) Stage specific myosins from amoeba and plasmodium of slime mold, Physarum polycephalum. Proc. Jpn. Acad., Ser. B 60, 431-434.

(78) Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, Y. and Kohama, T. (1986) Isolation and characterization of myosin from amoebae of Physarum polycephalum. J. Biol. Chem. 261, (8022-8027.

(79) Sonobe, S.S. and Kuroda, K. (1985) Cytoplasmic movement in a glycerinated model of Amoeba proteus. In Cell Motility: Mechanism and Regulation (eds. Ishikawa, H., Hatano, S. and Sato, H.). University of Tokyo Press, Tokyo, pp. 271-282.

(80) Shimmen, T. (1992) Inhibitory regulation of cyto plasmic streaming by [Ca.sup.2+] in plant cells. In Calcium Inhibition (ed. Kohama, K.). Japan Sci. Soc. Press, Tokyo and CRC Press, Boca Raton, pp. 69-90.

(81) Morimatsu, M., Nakamura, A., Sumiyoshi, H., Skaba, N., Taniguchi, H., Kohama, K. and Higashi-Fujime, S. (2000) The molecular structure of the fastest myosin from green algae, Chara. Biochem. Biopyhs. Res. Commun. 270, 147-152.

(82) Tominaga, M., Kojima, H., Yokota, E., Orii, H., Nakamori, R., Katayama, E., Anson, M., Shimmen, T. and Oiwa, K. (2003) Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity. EMBO J. 22, 1263-1272.

(83) Mooseker, M.S. and Cheney, R.E. (1995) Unconven tional myosins. Annu. Rev. Cell Dev. Biol. 11, (633-675.

(84) Matsumura, Y. and Morimoto, S. (1992) Calcium inhibition of renin secretion. In Calcium Inhibition (ed. Kohama, K.). Japan Sci. Soc. Press, Tokyo and CRC Press, Boca Raton, pp. 7-18.

(Received Aug. 10, 2016; accepted Oct. 26, 2016)

Kazuhiro KOHAMA [*1], [*2], ([dagger])

(Communicated by Masanori OTSUKA, M.J.A.)

[*1] Research Institute of Pharmaceutical Sciences, Musashino University, Nishitokyo, Tokyo, Japan.

[*2] Professor emeritus, Gunma University, Maebashi, Gunma, Japan.

([dagger]) Correspondence should be addressed: K. Kohama, 1-3-15 Shimokoide-machi, Maebashi, Gunma 371-0031, Japan (e-mail: kkohama@makikai.jp).

Profile

Kazuhiro Kohama was born in Suen-Wu, Manchuria (China) in 1944 and graduated from University of Tokyo, School of Medicine in 1971 to obtain MD degree.

He was educated in protein biochemistry under the guidance of Professor Seturo Ebashi as a graduate student at Department of Pharmacology, School of Medicine, University of Tokyo and received PhD degree. He was appointed to a research associate of the same department and started studies on the cytoplasmic streaming of Physarum polycephalum. In 1981-1983, he continued Physarum research under the supervision of Dr.John Kendrick-Jones as a postdoctoral bellow in MRC Laboratory of Molecular Biology, Cambridge, UK. In 1983, he was promoted to the associate professor of Pharmacology, University of Tokyo. In 1989, he was appointed to Professor and Chairman of Department of Pharmacology, Gunma University, School of Medicine. He further continued Physarum study in collaboration with Professor Andrew G.Szent-Gyorgyi at Brandeis University, USA and received the award of Inoue Prize for Science in 1994 for the discovery of calcium inhibition with Physarum. During the tenure in Gunma University, he extended his research field to the signal transduction in vertebrate smooth muscles partly in cooperation with Professor Gary L.Wright at Marshall University, School of Medicine, U.S.A. In 2010, he retired from Gunma University and promoted to Professor Emeritus, Gunma University. Since then, he joined to the reach group of Research Institute of Pharmaceutical Science, Musashino University in Tokyo as a visiting professor.

Table 1. Regulation of actin-myosin interaction by [Ca.sup.2+]

Cells                Resting           Excited
                 ([[Ca.sup.2+]]i   ([[Ca.sup.2+]]i
                   < [micro]M)       > [micro]M)

Plant Kingdom           +                 -
Animal Kingdom          -                 +

D, active interaction between actin and myosin; --, no active
interaction. [Ca.sup.2+] concentrations in the cells ([[Ca.sup.2+]]i)
of both kingdoms are kept as low as possible under the resting state,
and [[Ca.sup.2+]]i of both kingdoms increase transiently upon
excitation. In plant cells, (9) actin and myosin interact under
[[Ca.sup.2+]]i < [micro]M without requiring [Ca.sup.2+], but the
interaction of animal cell is activated by using [[Ca.sup.2+]]i that
is increased at >[micro]M. This review explains that Physarum
plasmodium should be classified to plant cells in terms of
[Ca.sup.2+] regulation of actinmyosin interaction.

Table 2. Purification of calcium inhibitory myosin

Step 1   Culture plasmodium of Physarum polycephalum in the
         dark on rolled oats.

Step 2   Extract in high salt buffer after homogenization of the
         plasmodium.

Step 3   * Precipitate actomyosin by diluting the extract,
         followed by dissoluting by the high salt buffer.

         * Repeat the dissolution-precipitation cycles, giving
         calcium inhibitory actomyosin.

Step 4   * Solubilize, the actomyosin preparation in the solution
         containing 20 mM ATP.

         * Add concentrated Mg acetate stock to give 0.1 M to
         assemble actin-filaments into bundles.

         * Remove bundles by the centrifugation.

Step 5   * Collect the supernatant of which myosin was almost
         actin free.

         * Dilute the supernate by adding 2 vol of cold water
         to precipitate myosin by forming myosin filaments.

         * The precipitate was used as Physarum myosin,
         showing calcium inhibition.

Note that column chromatographic procedures are not
employed in this preparation, which enables one to prepare
Physarum myosin rapidly, i.e., Physarum myosin is recovered
as a final purified form within 2 days. For detail, see Ref. 25.
We interpret reactive SH residues as explained in Section II in
the myosin heads is protected from the oxidation during the
short purification procedures.

Table 3. Comparison between Physarum myosin and skeletal muscle
myosin (7), (8), (10) (13), (30), (31)

              Physarum (a)                         Skeletal muscle (b)

Subunit       HC                  230 Kd (c)       HC
Composition   PLc                 18Kd (c)         DTNB-Lc
              CaLc                16 Kd (14 Kdc)   Al-Lc

Tail          Tail length         170 nm           150nm
Structure     Bends in the tail   >80%             <20%
              Rod ([M.sub.r])     130 Kd (e)       120 Kd (e)
              Viscosity (d)       2.0dl/g (d)      1.5dl/g (d)

Assembly      150 mM KCl          No               Yes
(see,         30 mM KCl           Yes (e)          Long
Section IV)   Bipolar filaments   Short            S (f)
              S[degrees]20,w      7.0 S (f)

Ca-binding                        2 mol/mol (g)    0 mol/mol (g)
capacity
(see,
Fig. 6)

                               Myosin ATPase activities

ATPase        Ca-ATPase >                          K-EDTA-ATPase >
activities    K-EDTA ATPase >                      Ca-ATPase >
(see,         Mg-ATPase                            Mg-ATPase
Section V)
                        Actin-activated Mg-ATPase activities

              Inhibited by                         Not affected by
              [micro]M levels                      [Ca.sup.2+]
              of [Ca.sup.2+]

                    Skeletal muscle (b)

Subunit                 200 Kd (c)
Composition              19Kd (c)
              [Al.sub.l] F 25 Kd (21 Kd (c))
                    [Al.sub.2] f 17 Kd

Tail
Structure

Assembly                    Yes
(see,                       6.0
Section IV)

Ca-binding
capacity
(see,
Fig. 6)

ATPase
activities
(see,
Section V)
                      Actin-activated
                   Mg-ATPase activities

(a) Physarum plasmodial cell (adopted from Ref. 10). (b) Fast white
muscle (adopted from Ref. 31). (c) Estimated by SDS-PAGE. (d) Compared
under the same conditions (0.5M KC1, 3.5mM Mg acetate, and 20mM
Tris-HCl pH 7.5). (e) Assembly is inhibited by the
dephosphorylation. (f) In 0.3 M KCl, 3.5 mM Mg acetate and 20 mM
Tris-HCl pH 7.5. (g) Expressed per mol of 2-headed myosin molecule.

Table 4. Summary of functional and structural properties of
calcium-binding light chain (CaLc) of Physarum myosin
(adopted from Ref. 13). See also recombinant CaLcs of wild
type and its mutant as shown in Fig. 6

* Binds [Ca.sup.2+] (see, Table 5)

* Interacts with actin calcium-dependently (33)

* Interacts with heavy chain of skeletal muscle myosin as
substitute for alkali light chain (13)

* Shows similarity with vertebrate calmodulin in the amino acid
sequence and in activating phosphodiesterase (34)

* Confers calcium inhibition on the actin-myosin interaction by
binding to actin (35)

Table 5. Calcium binding to Physarum myosin and its sub-units (40)

Myosin(phosphorylated)          1.27 [+ or -] 0.16mol/mol (n = 3)
Myosin(dephosphorylated)        1.21 [+ or -] 0.07mol/mol (n = 3)
Calcium-binding light chain     0.37 [+ or -] 0.092 mol/mol (n = 6)
Phosphorylatable light chain    ND

The binding of [Ca.sup.2+] to myosin and its subunits was measured
in 0.5M KCl, 1mM Mg[Cl.sub.2], 20mM Tris-HCl (pH 7.5), and
30[micro]M [Ca.sup.2+] containing [sup.45]Ca by equilibrium dialysis
method. ND, not detectable. Note that Ca-binding to Calcium-binding
light chain was enhanced when it is incorporated into myosin.

Table 6. Calcium inhibition of actin-myosin interaction of
Physarum plasmodium (modified from Ref. 20)

Actin-activated ATPase activity of myosin (28), (39)

Superprecipitation of actomyosin (28), (39) (see, Section I)

In vitro motility assay

  Nitella-basad motility assay (43)

  Myosin-coated surface assay (47)

Tension development of actomyosin threads (48)

Contraction of cell-free model (49)-(54) (see, Section XI)

Intact cell (55) (see, Section XI)

Note that mechanical aspects of actin-myosin interaction are
detected both by Nitella-based motility assay (44) and by
myosin-coated surface assay. (45) In the former assay, latex
beads about 2 pm in diameter are coated by Physarum myosin
and then are introduced to the bundles of Nitetta actinfilaments.
The ATP-depended movement of the beads are
observed in the presence of various concentrations of [Ca.sup.2+].

In the latter assay, the fluorescent actin-filaments prepared
from skeletal muscle are used. They are mounted on the glass
surface that is fixed by Physarum myosin. The ATP-dependent
movement of actin fluorescence is observed under
the fluorescent microscope.

Table 7. Effect of caldesmon-like protein and calmodulin on the
velocities of movement of actin filaments on coverslips coated
with Physarum myosin

Actin filament                             Velocity ([micro]m/
                                              sec, n = 30)

Control (0.1 mM EGTA)                      1.39 [+ or -] 0.49
  + Caldesmon-like protein (0.1 mM EGTA)   1.95 [+ or -] 0.66
Control (0.1 mM [Ca.sup.2+])               0.94 [+ or -] 0.42
  + Caldesmon-like protein (0.1 mM         1.26 [+ or -] 0.60
    [Ca.sup.2+])
  + Caldesmon-like protein + calmodulin    1.08 [+ or -] 0.50
    (0.1 mM [Ca.sup.2+])

Caldesmon-like protein was purified from Physarum plasmodia
as a heat-stable, actin-binding protein. Unlike smooth muscle
caldesmon, caldesmon-like protein stimulated the velocity of
movement. Because calmodulin in the presence of [Ca.sup.2+]
abolishes binding to actin, caldesmon-like protein works to
cause calcium inhibition. (58), (59)

Fig. 6. CaLc and its mutant. cDNA of CaLc was cloned and mutated,
followed by expression in E. coli. A. The amino acids substituted for
alanine or glutamic acid in the mutant CaLc-3A were marked with
asterisk (*). Consensus sequences of EF hand [Ca.sup.2+] binding
loops 1-4 were shown together with the six coordinating residues (x,
y, z, -y, -x, -z). O, oxygen-containing side group; #, hydrophobic
residue; X, any amino acid residue. B. Wild type CaLc (**) and mutant
CaLc-3A (***) were expressed in E. coli and were purified. The
[sup.45]Ca binding activities of CaLc and CaLc-3A were examined by
the flow dialysis method. Note that Ca-binding ability of CaLc-3A was
totally lost. (62)

A. Amino acid sequences of the EF-hand loops of CaLc

EF.1 (15-26)     D *   K   D *   N   D   G   K
EF.2 (41-52)     A     E   L     N   A   K   E
EF.3 (86-97)     D     K   E     G   N   G   T
EF.4 (111-122)   S     V   S     G   D   G   A
Consensus        D     X   O     X   O   G   X
                 x         y         z       y

A. Amino acid sequences of the EF-hand loops of CaLc

EF.1 (15-26)     V   S    I   E   E *
EF.2 (41-52)     F   D    L   A   T
EF.3 (86-97)     I   Q    E   A   E
EF.4 (111-122)   L   N    Y   E   S
Consensus        #   O    X   X   E
                     -x           -z
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Date:Dec 1, 2016
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