The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.     

The functional analysis of Type I postplasmic/PEM mRNAs in embryos of the ascidian Halocynthia roretzi

Maternal factors, such as a muscle determinant macho-1 mRNA that is localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, are crucial for embryonic axis formation and cell fate specification. Maternal mRNAs that show an identical posterior localization pattern to that of macho-1 in eggs and embryos are called Type I postplasmic/PEM mRNAs. We investigated the functions of five of the nine Type I mRNAs so far known in Halocynthia roretzi: Hr-Wnt-5, Hr-GLUT, Hr-PEM3, Hr-PEN1, and Hr-PEN2. Suppression of their functions with specific antisense morpholino oligonucleotides (MOs) had effects on the formation of various tissues: Hr-Wnt-5 on notochord, muscle, and mesenchyme, although zygotic function of Hr-Wnt-5 is responsible for notochord formation; Hr-GLUT on notochord, mesenchyme, and endoderm; and Hr-PEN2 on muscle, mesenchyme, and endoderm. On the other hand, Hr-PEM3 and Hr-PEN1 MOs seemed to have no effect. We conclude that the functions of at least some localized maternal Type I postplasmic/PEM mRNAs are necessary for early embryonic patterning in ascidians.     

Sulfated O-linked glycans of the vitelline coat as ligands in gamete interaction in the ascidian, Halocynthia roretzi

In the ascidian Halocynthia roretzi, sperm-egg binding is probably mediated through the interaction between alpha-L-fucosidase present on the sperm surface and anionic saccharide chains of the egg vitelline coat. To characterize biologically active glycans, total glycans were chemically released from the glycopeptide fraction of the vitelline coat. The fraction of uncharged glycans and two fractions of negatively charged glycans were separated by diethylaminoethyl-anion exchange chromatography. In a competitive inhibition assay of fertilization, both anionic fractions showed inhibitory activity, with more anionic glycans being most potent, while uncharged glycans were biologically inactive. Chemical desulfation combined with a competitive inhibition assay of fertilization and ion analysis determined that sulfate groups were responsible for anionic character and crucial for biological activity. Monosaccharide analysis of anionic fractions showed a high content of N-acetylgalactosamine, galactose, xylose and the presence of arabinose, mannose, N-acetylglucosamine, glucose and rhamnose. Glycans were O-linked and galactose and xylose residues were detected at reducing termini. Linkage analysis suggested that 1,4-linked xylose, 1,3-linked galactose and N-acetylgalactosamine residues, substituted to different degrees by sulfate groups on the C-3 and C-4 carbons, respectively, constituted the core structures of anionic glycans.     

Hypertrophic smooth muscle

Summary The ultrastructure of filaments is studied in the hypertrophic musculature of the small intestine of the guinea pig oral to an experimental stenosis. No structural change is observed in the thin and the thick myofilaments. However, there is a remarkable and consistent increase in the number of intermediate (10 nm) filaments; they are the predominant type of filament in many hypertrophic muscle cells. Experiments, in which the force developed in vitro by strips of control and hypertrophic musculature upon stimulation with carbachol, indicate that the force per unit sectional area is slightly less in the hypertrophic muscle than in the control tissue.The author thanks Miss Eva Franke for excellent technical assistance. This work was supported by grants from the Medical Research Council and the Central Research Funds of the University of London     

Voltage-dependent calcium current in dissociated smooth muscle cells of the buccal mass of Aplysia

Summary Isolated smooth muscle cells of the buccal mass of Aplysia contracted in response to depolarization elicited by a patch electrode in whole-cell configuration. With cesium-containing pipet solution and tetraethylammonium and 4-aminopyridine in the external solution depolarization elicited inward current. The voltage-dependent inward current was blocked completely by lanthanum (10 mmol·1^-1), inhibited 80–90% by nifedipine (1 mol·l^-1), and was dependent upon extracellular calcium. These results showed that the voltage-dependent inward current was due to activation of voltage-dependent calcium channels (VDCaCH). Minimal depolarization to begin activating VDCaCH was-60 to-30 mV. Inward current peaked within 8 ms and then decreased rapidly to a lower level of relatively non-inactivating current. The initial peak current could be mostly inactivated by a depolarization to-20 mV for 500 ms. Nifedipine reduced both the peak current and the relatively non-inactivating current. Nifedipine inhibited high potassium-elicited contractions of both intact and dissociated muscle. These results suggested that VDCaCH mediates calcium influx which triggers contraction in molluscan smooth muscle fibers.Abbreviations ACh acetylcholine - ATP adenosine triphosphate - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - GTP guanosine triphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MCG metacerebral giant cell - RNI relatively non-inactivating - SCP small cardioactive peptide - TEA-4AP-IO external solution containing Instant Ocean, tetraethylammonium chloride, and 4-aminopyridine (described in Methods) - TEA tetraethylammonium chloride - VDCaCH voltagedependent calcium channel - 4-AP 4-aminopyridine - 5-HT 5-hydroxytryptamine or serotonin     

Quantitative X-ray microanalysis of the morula cell of the blood of the ascidian Halocynthia papillosa (Stolidobranchiata)

Summary The elemental composition of the morula cell of Halocynthia papillosa blood was studied by X-ray microanalysis with respect to the possible iron accumulation in this cell type. We found various amounts of Na, Mg, P, S, Cl, K, Ca, Fe and Br in the cytoplasm, nucleus and vacuoles. With the exception of a few cells, Ca, Fe and Br were not detected. Thus, the morula cells of the studied species are not iron-rich cells.     

Functional states and fine structure of the contractile apparatus of the penis retractor muscle (PRM) of Helix pomatia L.

Summary The ultrastructure of the isolated glycerinated penis retractor muscle (PRM) of Helix pomatia was investigated. The diameter distributions of thick myofilaments from fibre cross sections in the relaxed, phasic contracted, tonic contracted, and in the catch states show that a characteristic filament spectrum is formed in the catch state and its preceding active state. The significant structural differences are discussed in relation to earlier hypotheses related to the catch state.I wish to thank Prof. Dr. Fr.-W. Schlote for helpful discussion. I also thank M. Jenninger and E. Grafahrend for their technical assistance.     

Isolation and characterization of polymorphic microsatellite DNA markers in the sea squirt Halocynthia roretzi

The sea squirt Halocynthia roretzi is an important marine food resource species that is found in the waters around Korea. We describe the isolation and characterization of 13 new polymorphic microsatellite loci in 96 sea squirt samples that were collected from the marine environment of Samcheok on the east coast of Korea. The number of alleles that were observed for each locus ranged from six to 32, and the value of expected and observed heterozygosities was 0.504-0.922 and 0.396-0.813, respectively. These markers will be useful tools for future population studies.     

Adrenocorticotropin-like immunoreactivity in the granules of neural complex cells of the ascidian Halocynthia roretzi

Immunohistochemical studies on the neural complex (neural gland, dorsal strand, and cerebral ganglion) of an ascidian, Halocynthia roretzi, were performed by using an antiserum against porcine ACTH. The antiserum recognized a considerable number of the cells scattered along the tubular structure of the dorsal strand and a few cells in the cerebral ganglion. Immunoelectron microscopic studies revealed that the ACTH-like substance resided within secretory granules with diameter of 300-500 nm. Furthermore, those ACTH-immunoreactive cells were demonstrated to be different from PRL-immunoreactive cells, the presence of which had previously been reported.     

Development of myofibrils in the gizzard of chicken embryos

Summary The intracellular distributions of major muscle proteins, myosin, actin, tropomyosin, -actinin, and desmin, in smooth muscle cells of chicken gizzard at various stages of embryogenesis were investigated by immunofluorescence-labeling of enzyme-dispersed cells cultured up to three hours. These muscle proteins, except some part of myosin, were organized into fibrous structures as soon as synthesis and accumulation of proteins started. As for myosin, a considerable amount of it was dispersed in soluble cytoplasm as well. On the other hand, Ca⁺⁺-dependent contractility was detected with detergent-extracted myoblasts and glycerinated tissue from embryos older than 7 days. Although the nascent myofibrils bear a resemblance to stress fibers, the former could be distinguished from the latter by their high stability in dispersed, spherical cells. The above findings, therefore, show that the synthesis of contractile proteins is followed by immediate assembly of them into functional myofibrils without undergoing any intermediate structure. Based on these findings, the mechanism of myofibril formation in developing smooth muscle cells is discussed.     

Behavior and cellular morphology of the test cells during embryogenesis of the ascidian Halocynthia roretzi

During the early stages of embryogenesis of the ascidian Halocynthia roretzi the test cells creep exclusively on the inner surface of the chorion. Concomitant with elongation of the embryonic tail, however, the test cells begin to gather around the embryo and finally cover the whole embryo. The time at which the test cells surround the embryo almost coincides with that of initiation of larval tunic formation. Scanning electron microscope observations revealed that the test cells extend numerous cytoplasmic processes or pseudopodia. During larval tunic formation, the test cells compose a net by intertwining their filopodia, and the cell net covers the whole embryo.     

Self and non-self recognition between gametes of the ascidian,Halocynthia roretzi

Summary The self-sterility ofHalocynthia roretzi from Mutsu Bay, Japan, was examined. This sterility is strict and not a single egg can be fertilized in self-sterile animals. Less than 2% of the animals were self-fertile (with 100% cross-fertility). All heterologous sperm can fertilize all eggs, although there are pairs of individuals in which the coelomocytes recognize each other as self. Eggs deprived of follicle cells cannot be fertilized by either autologous or heterologous spermatozoa. Detached autologous or heterologous follicle cells can reattach to the chorion in calcium-enriched sea water and the reconstituted eggs recover their ability to be fertilized. A mosaic egg can therefore be obtained, which consists of oocyte, test cells and chorion originating from one individual and follicle cells from another. The mosaic egg was used to determine the site of recognition of self and non-self. The results indicate that the recognition resides in the chorion and/or test cells, probably the chorion. The relationship between somatic alloreactivity, previously found in coelomocytes ofH. roretzi, and gamete reactivity is discussed.     

Distinction between smooth muscle,fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

Summary FITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological dedifferentiation to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.This work was supported by the Life Insurance Medical Research Fund of Australia and New Zealand, the National Heart Foundation of Australia, the Deutsche Forschungsgemeinschaft and the Wellcome Trust (London). We thank Janet D. McConnell for excellent technical assistance     

Proteomic dataset of mouse aortic smooth muscle cells

In an accompanying study (in this issue, DOI 10.1002/pmic.200402044), we have characterised the proteome of Sca-1(+) progenitor cells, which may function as precursors of vascular smooth muscle cells (SMCs). In the present study, we have analysed and mapped protein expression in aortic SMCs of mice, using 2-DE, MALDI-TOF MS and MS/MS. The 2-D system comprised a non-linear immobilised pH 3-10 gradient in the first dimension (separating proteins with pI values of pH 3-10), and 12%T SDS-PAGE in the second dimension (separating proteins in the range 15,000-150,000 Da). Of the 2400 spots visualised, a subset of 267 protein spots was analysed, with 235 protein spots being identified corresponding to 154 unique proteins. The data presented here are the first map of aortic SMCs and the most extensive analysis of SMC proteins published so far. This valuable tool should provide a basis for comparative studies of protein expression in vascular smooth muscle of transgenic mice and is available on our website hhtp://www.vascular-proteomics.com.