Smooth muscle-like Ca2+-regulation of actin-myosin interaction in adult jellyfish striated muscle

Abstract

Cnidaria is an animal phylum, whose members probably have the most ancestral musculature. We prepared and characterized, for the first time to our knowledge, native actomyosin from the striated myoepithelium of the adult moon jelly Aurelia sp. The actomyosin contained myosin, paramyosin-like protein, Ser/Thr-kinase, actin, and two isoforms of tropomyosin, but not troponin, which is known to activate contraction dependent on intracellular Ca²⁺ signaling in almost all striated muscles of bilaterians. Notably, the myosin comprised striated muscle-type heavy chain and smooth muscle-type regulatory light chains. In the presence of Ca²⁺, the Mg-ATPase activity of actomyosin was stimulated and Ser21 of the regulatory light chain was concomitantly phosphorylated by the addition of calmodulin and myosin light chain kinase prepared from chicken smooth muscle. Collectively, these results suggest that, similar to smooth muscle, the contraction of jellyfish striated muscle is regulated by Ca²⁺-dependent phosphorylation of the myosin light chain.

Figure 1

Electron micrograph of adult moon jelly striated muscle. (a) Longitudinal section of myofibrils at the plane of the muscular sheet. The A-band (A), I-band (I), M-line (M), Z-disc (Z), and adherence junctions (AJ) are indicated. White arrowheads indicate the point where the M-lines are attached to the inner cell surface. Black arrowheads indicate the vesicles. (b) Radial section of subumbrella. The striated myofibrils (Mf), mitochondria (Mi), and epithelial cells (E) are indicated.

Figure 2

SDS-PAGE of the native actomyosin fraction prepared from the striated muscle of adult Aurelia sp. The actomyosin fraction (8 μg) was applied to a 10% acrylamide gel. Eight major bands (1 to 8) of proteins were identified as indicated. The accession numbers of the nucleotide sequences of cDNA encoding each protein are shown.

Figure 3

Neighbor-joining analysis of myosin-II heavy chain (MHC-II) proteins. Bootstrap values in percentages are shown on the branch lines. Scale bar represents 0.1 substitutions per site. The accession numbers of amino acid sequences in the UniProtKB/TrEMBL or GenBank (for sponge MHC-II) database are shown. Tissues, organs, or body parts in which the expression of these proteins had been experimentally confirmed are represented by lower case letters.

Figure 4

Neighbor-joining analysis of myosin regulatory light chain (MRLC) proteins. (a) Neighbor-joining tree generated with amino acid sequences of MRLC proteins. The accession numbers of amino acid sequences in the UniProtKB/TrEMBL are shown. (b) Comparison of N-terminal sequences. The N-terminal extension (NTE) is indicated. Residues identical to those of the Aurelia MRLC are represented by dots. The serine residues known to be primarily phosphorylated by myosin light chain kinase (MLCK) in vertebrate smooth muscles are shaded in green. The sequences of the non-muscle/smooth, striated, and invertebrate MRLC are shaded in blue, red, and yellow, respectively. Ser35 and Ser45 of tarantula striated muscle MRLC, which are reported to be phosphorylated, are boxed. The basic four residues important for the substrate recognition by smooth muscle MLCK are indicated in red font.

Figure 5

Expression analysis by endpoint RT-PCR. (a) Samples used for analysis. 1, Polyps cultivated at 25 °C. 2, Polyps after incubation for 7 days at 15 °C. 3, Early strobila (after 23 days); arrowhead indicates transversal segmentation. 4, Late strobila (after 29 days); the rudiment of the sensory organ (arrow) has developed. 5, Liberated ephyra (after 34 days). 6, Juvenile medusa. 7, Adult jellyfish. (b) Qualitative RT-PCR performed on the samples. Whole-animal bodies of 5, 5, and 50 individuals of polyp (1 and 2), strobila (3 and 4), and ephyra (5) were used for analysis. The umbrella (6 A) and oral arms (6B) of a single juvenile medusa were separately analyzed, whereas only the umbrella of adult medusa (7) was analyzed. Prior to using the umbrellas as samples, the marginal part including smooth muscle tissues was removed. Expression of elongation factor 1α (EF1α) gene was measured as a positive control. The images have been cropped from different parts of the gel or different gels. The full-length gels, including DNA size markers, are presented in Supplementary Fig. S4 together with the photographic conditions. Abbreviations: CaM, calmodulin; MRLC, myosin regulatory light chain; MHC-II, myosin-II heavy chain; MELC, myosin essential light chain; Tm, tropomyosin.

Figure 6

Ca²⁺-dependent activation and phosphorylation of actomyosin. (a) Mg-ATPase activity of actomyosin in the absence and presence of Ca²⁺. Ca²⁺-dependent activation was enhanced by adding myosin light chain kinase (MLCK) and calmodulin (CaM). The values were presented as means and standard errors (n = 5). The values were also statistically compared by two-tailed t-test (n = 5), and the p-values are indicated. (b) Phos-Tag SDS-PAGE of actomyosin incubated for 10 min under the same conditions as the ATPase assay. Abbreviations: MRLC, myosin regulatory light chain; MHC-II, myosin-II heavy chain; PM, paramyosin-like protein; MELC, myosin essential light chain; Tm, tropomyosin. (c) Comparison of the mass spectra of tryptic digests of unphosphorylated (upper) and phosphorylated (lower) myosin regulatory light chain (MRLC). The mass increment of 79.94 Da corresponds to phosphorylation of a single hydroxyl group.