Immunocytochemical electron microscopic study and Western blot analysis of paramyosin in different invertebrate muscle cell types of the fruit flyDrosophila melanogaster, the earthwormEisenia foetida, and the snailHelix aspersa

Summary The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. the muscles studied were: transversely striated muscle with continuous Z lines (flight muscle fromDrosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snailHelix aspersa), obliquely striated body wall muscle from the earthwormEisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.     

Projection of statocyst sensory neurons associated with crescent hairs in the crayfish Procambarus clarkii Girard

Summary Both smooth muscle and striated muscle are present in the iris of the chick embryo. The two types of musculature form mixed clusters which include undifferentiated cells and many nerve fibres, but they are structurally quite distinct and have different origins. The smooth musculature originates around the 10th day from a laminar invagination (iridial lamella) of the posterior epithelium, and is therefore an ectodermal derivative. The striated musculature appears slightly later than the smooth musculature and originates from undifferentiated cells which are regarded as mesenchymal. After the 15th day in ovo the smooth musculature stops growing; its cells become confined to an area very near the pupillary margin and many develop pigment granules in the sarcoplasm. Many smooth muscle cells seem to undergo regressive changes; however, cells with the typical appearance of visceral muscle cells are still present in the iris of 3-month-old chickens. High density of innervation and vasculari/ation, wide range of striated muscle fibre diameters, presence of lipid vacuoles and of large clusters of mitochondria in the striated fibres, occurrence of peripheral couplings of the sarcoplasmic reticulum, and presence of numerous fibroblast processes in the interstices between fibres, characterize the sphincter pupillae of the mature iris.This work was supported by grants from the Medical Research Council and the Central Research Fund of the University of London     

Study of reciprocal effects of cardiac myosin and tropomyosin isoforms on actin-myosin interaction with in vitro motility assay

Interaction of myosin with actin in striated muscle is controlled by Ca²⁺ via thin filament associated proteins: troponin and tropomyosin. In cardiac muscle there is a whole pattern of myosin and tropomyosin isoforms. The aim of the current work is to study regulatory effect of tropomyosin on sliding velocity of actin filaments in the in vitro motility assay over cardiac isomyosins. It was found that tropomyosins of different content of α- and β-chains being added to actin filament effects the sliding velocity of filaments in different ways. On the other hand the velocity of filaments with the same tropomyosins depends on both heavy and light chains isoforms of cardiac myosin.     

A comparative study of the organization of the sarcotubular system in ascidian muscle

The caudal musculature of ascidian tadpole larvae consists of mononucleated muscle cells joined end to end in long rows flanking the notochord. A comparative study of the fine structure of these cells in larvae from different families has revealed wide variations in the pattern of organization of the sarcotubular system. The species examined can be distinguished in two groups according to the presence or absence of a system of plasma membrane invaginations equivalent to the T system of vertebrate and invertebrate striated muscle. Muscle cells from the first group of species, Clavelina lepadiformis, Ciona intestinalis and Molgula socialis, are characterized by absence of T system and show peripheral couplings of sarcoplasmic reticulum cisternae directly with the plasma membrane. In contrast, a T system is present in muscle cells of Diplosoma listerianum, Styela plicata and Botrylloides leachi. The presence of T system in ascidian muscle is not related to the taxonomic position of the various species, but rather to the intracellular disposition of the myofibrils, which are peripheral in the species of the first group whereas they occupy a more internal position in the species of the second group. The T system displays unique structural features in ascidian muscle. It consists of wide laminae invaginating from the plasma membrane and associated in longitudinally oriented dyads with sarcoplasmic reticulum cisternae in register with the I band of the myofibrils. It is apparent from these observations that, in contrast with the uniformity of myofibrillar structure in all chordates, there are basic differences between ascidians and vertebrates as regards the organization of the sarcotubular system. On the other hand, there are significant similarities in this respect between ascidian and invertebrate muscle.     

Polarity of myofilaments in molluscan smooth muscle

Summary Myofilaments were isolated by gently homogenizing smooth muscle cells isolated from the pedal retractor muscle (PRM) of Mytilus edulis, and observed by electron microscopy. The thick filaments isolated in the presence of ATP (10–20 mM) had projections of myosin heads except near their centre (central bare zone). After extraction of myosin, the paramyosin core of the thick filaments showed a Bear-Selby net or a striated pattern with a main periodicity of 14.5 nm. Both the Bear-Selby net and the striated patterns had a polarity that reversed at the centre of the filament where the patterns were obscured. The thin filaments were attached to dense bodies. Decoration of the thin filaments with heavy meromyosin showed that they have opposite polarity on opposing sides of the dense body. The results indicate that the thick filaments are bipolar and also that the dense bodies are functionally analogous to the Z-disk of the striated muscle.     

Temporally and spatially restricted expression of a gland cell gene during regeneration and in vitro transdifferentiation in the hydrozoan Podocoryne carnea

An antiserum to transdifferentiated striated muscle cells from the medusa of Podocoryne carnea was prepared and used to screen a gt11-expression library prepared from gonozoids of P. carnea. We isolated a cDNA clone termed Pod-EPPT with at least 63 tandem repeats of the tetrapeptide-motive glu-pro-pro-thr, named Pod-EPPT. Using Pod-EPPT as a molecular marker for head quality the morphological relationship between the two metagenic life stages of this hydroid, the polyp and the medusa, was studied. In situ hybridization demonstrated that expression of the gene corresponding is restricted to secretory cells in the endoderm of the oral hypostome region of polyps and medusae and, presumably, to progenitor cells of this type. Cells expressing Pod-EPPT could not be observed in the larval stage. During head regeneration in polyps, Pod-EPPT expression is upregulated soon after head removal in previously non-expressing cells and in newly differentiating secretory cells. This activation of a head-specific gene precedes the morphologically obvious events of head regeneration. Pod-EPPT is one of the genes that are activated during manubrium (mouth) regeneration from experimentally combined subumbrellar plate endoderm and striated muscle of the medusa.     

Study of regulatory effect of tropomyosin on actin-myosin interaction in skeletal muscle by in vitro motility assay

The interaction between myosin and actin in striated muscle tissue is regulated by Ca²⁺ via thin filament regulatory proteins. Skeletal muscle possesses a whole pattern of myosin and tropomyosin isoforms. The regulatory effect of tropomyosin on actin-myosin interaction was investigated by measuring the sliding velocity of both actin and actin-tropomyosin filaments over fast and slow skeletal myosins using the in vitro motility assay. The actin-tropomyosin filaments were reconstructed with tropomyosin isoforms from striated muscle tissue. It was found that tropomyosins with different content of α-, β-, and γ-chains added to actin filaments affect the sliding velocity of filaments in different ways. On the other hand, the sliding velocity of filaments with the same content of α-, β-, and Γ-chains depends on myosin isoforms of striated muscle. The reciprocal effects of myosin and tropomyosin on actin-myosin interaction in striated muscle may play a significant role in maintenance of effective work of striated muscle both during ontogenesis and under pathological conditions.     

Thin filament flexibility and its role in muscle contraction

Contemporary experimental methods do not allow unequivocal determination of molecular structural events during muscle contraction. To analyze existing contradictions, an original computer program has been developed. This program reconstructs the hexagonal lattice of a sarcomere for different states of muscle and finds the most realistic structure by comparing the calculated Fourier spectrum with the actual diffraction pattern. Previously, the new approach allowed reconstructing the actual structure of a myosin filament from mammalian striated muscle (http://zope.ibib.waw.pl/pspk). In this work, the thin filament is reconstructed for three states: relaxed, activated, and contracting. The good fit between the calculated Fourier spectra and the actual diffraction patterns taken from the literature suggests that the thin filament owing to its flexibility may play an active role in muscle contraction, as myosin cross-bridges do.     

Organization of the dorsoventral musculature in the wings of the pteropod mollusc Clione limacina

The wings of the pteropod mollusc Clione limacina provide forward propulsive force through flapping movements in which the wings bend throughout their length in both dorsal and ventral directions. The musculature of the wings includes oblique, striated muscle bundles that generate the swimming movements of the wings, longitudinal and transverse (smooth) muscle bundles that collapse the wings and pull them into the body during a wing withdrawal response, and dorsoventral muscles that control the thickness of the wings. All muscles act against a hydrostatic skeleton that forms a central hemocoelic space within the wings. Of these muscle types, all have been thoroughly described and studied except the dorsoventral muscles. The fortuitous discovery that the dorsoventral musculature can be intensely labeled with an antibody against the vertebrate hyperpolarization‐activated cation channel (HCN2) provided the opportunity to describe the organization of the dorsoventral musculature in detail. In addition, electrical recordings and microelectrode dye injections supported the immunohistochemical data, and provided preliminary data on the activity of the muscle fibers. The organization and activity of the dorsoventral musculature suggests it may be involved in regulation of wing stiffness during the change from slow to fast swimming.     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Organization of the subumbrellar musculature in the ephyra,juvenile, and adult stages of Aurelia aurita Medusae

We used fluorescently labeled phalloidin to examine the subumbrellar musculature of the scyphozoan jellyfish Aurelia aurita in a developmental series from ephyra to adult medusa. In the ephyra, the swim musculature includes a disc‐like sheet of circular muscle, in addition to two radial bands of muscle in each of the eight ephyral arms. The radial muscle bands join with the circular muscle, and both circular and radial muscle act together during each swim contraction. As the ephyra grows into a juvenile medusa, arms tissue is resorbed as the bell tissue grows outward, so eventually, the ephyral arms disappear. During this process, the circular muscle disc also grows outward and the radial muscle bands of the arms also disappear. At this time, a marginal gap appears at the bell margin, which is devoid of circular muscle cells, but has a loose arrangement of radial muscle fibers. This marginal gap is preserved as the medusa grows, and contributes to the floppy nature of the bell margin. Radial distortions in the circular muscle layer involve muscle fibers that run in random directions, with a primarily radial orientation. These are believed to be remnants of the radial muscle of the ephyral arms, and the distortions decrease in number and extent as the medusa grows. Since the mechanics of swimming changes from drag‐based paddling in the ephyra to marginal rowing in the adult medusa, the development of the marginal gap and the presence of radial distortions should be considered in terms of this mechanical transition.     

Une structure intermédiaire entre muscle lisse et muscle strié

Résumé Les éléments morphologiques apportés par la microscopie électronique montrent que le muscle du bulbe buccal de Ferrissia wautieri diffère du muscle strié squelettique par plusieurs caractères mais présente avec celui-ci des analogies structurales qui peuvent conduire à considérer le type de fibre musculaire étudié comme une structure intermédiaire entre la musculature lisse et la musculature striée.La répartition des deux types de filaments en faisceaux étroits plus ou moins anastomosés et la discontinuité des stries Z constituées de l'alignement des corps denses, rappellent les muscles cardiaques embryonnaires de certains Vertébrés. Le système T est réduit à l'ensemble des courtes invaginations du sarcolemme au niveau de chaque strie Z; tandis que le système L, très développé, semble établir des contacts étroits avec le milieu extra-cellulaire par l'intermédiaire de vésicules sous-sarcolemmales (diades).

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A type of musculature intermediary between smooth and striated muscular tissueThe muscle fibre of the buccal bulb in Ferrissia wautieri (Moll. Basomm. Ancylidae)

Summary Electron microscopical investigations show that the muscle of the buccal bulb of Ferrissia wautieri differs from the skeletal striated muscle in several of its characteristics, but exhibits structural analogies with the latter which can lead one to consider the type of muscle fibre studied as an intermediary structure between smooth and striated muscular tissue.The distribution of the two types of filaments in thin bundles, more or less anastomosed, and the discontinuity of the Z bands, which are made up from the alignment of dense bodies, are similar as in embryonic cardiac muscles of certain Vertebrates. The T system is reduced completely to short sarcolemmal invaginations at the level of each Z band, whilst the well-developped L system seems to make close contact with the extra-cellular region through cisternae beneath the sarcolemma (dyads).

Ce travail s'inscrit dans le cadre d'une étude plus générale portant sur le cycle biologique et l'écologie de Ferrissia wautieri. Il nous est agréable de remercier notre collègue Pavans de Ceccatty qui nous a guidés et aidés dans sa réalisation et a accepté de relire et de corriger le manuscrit.     

Cytologische Veränderungen während der Metamorphose des CubopolypenTripedalia cystophora (Cubozoa,Carybdeidae) in die Meduse

The life cycle ofTripedalia cystophora includes a sessile saclike polyp — the asexual reproducing form — and a pelagic tetraradial medusa — the sexually reproducing generation. Medusan development can be induced by temperature increase. It reveals neither budding nor strobilation, but a real metamorphosis of a polyp to only one medusa. According to morphological and anatomical criteria the metamorphosis can be subdivided into four different stages: (1) four longitudinal furrows segment the polyp, the tentacles of which are apportionated on the four quadrants of the body. (2) The subumbrellar cavity develops by invagination of the peristom; the relicts of the fused tentacles change to four rhopalia buds. (3) Medusan architecture including four new interradial tentacles, four rhopalia and the subumbrellar swimming musculature is completed. (4) A young tetraradial medusa starts swimming. Ultrastructural analysis of those metamorphic stages show the different processes of morphogenesis: (a) Gastrodermal cells — absorptive and spumous cells — undergo transdifferentiation and proliferation to medusan cells of the same structure and function. (b) Epidermal cells, excluding the epithel muscle cells, dissociate and are autolytically withdrawn. Dedifferentiated epithel muscle cells — interstitial cells — regain the ability to develop a complete new set of somatic cells, not originally present in the polyp. They include amongst others cross-striated muscle cells, medusan typic nematocyts and particularly sensory and nervous cells. Those cells establish a nervous system with lens-eyes, simple ocelli, statocysts, diffuse nerve net and an additional nerve ring.     

Structure and anterior regeneration of musculature and nervous system in Cirratulus cf. cirratus (Cirratulidae,Annelida)

Annelids provide suitable models for studying regeneration. By now, comprehensive information is restricted to only a few taxa. For many other annelids, comparative data are scarce or even missing. Here, we describe the regeneration of a member of the Cirratulus cirratus species complex. Using phalloidin‐labeling and antibody‐stainings combined with subsequent confocal laser scanning microscopy, we provide data about the organization of body wall musculature and nervous system of intact specimens, as well as about anteriorly regenerating specimens. Our analyses show that C. cf. cirratus exhibits a prominent longitudinal muscle layer forming a dorsal muscle plate, two ventral muscle strands and a ventral‐median muscle fiber. The circular musculature forms closed rings which are interrupted in the area of parapodia. The nervous system of C. cf. cirratus shows a typical rope‐ladder like arrangement and the circumesophageal connectives exhibit two separate roots leading to the brain. During regeneration, the nervous system redevelops remarkably earlier than the musculature, first constituting a tripartite loop‐like structure which later become the circumesophageal connectives. Regeneration of longitudinal musculature starts with diffuse ingrowth and subsequent structuring into the blastema. In contrast, circular musculature develops independently inside the blastema. Our findings constitute the first analysis of regeneration for a member of the Cirratuliformia on a structural level. Summarizing the regeneration process in C. cf. cirratus, five main phases can be subdivided: 1) wound closure, 2) blastema formation, 3) blastema differentiation, 4) resegmentation, and 5) growth, respectively elongation. Additionally, the described tripartite loop‐like structure of the regenerating nervous system has not been reported for any other annelid taxon. In contrast, the regeneration of circular and longitudinal musculature originating from different groups of cells seems to be a general pattern in annelid regeneration. J. Morphol. 275:1418–1430, 2014. © 2014 Wiley Periodicals, Inc.     

Comparative body wall musculature and muscle fibre ultrastructure in branchiobdellidans (Annelida: Clitellata), and their phylogenetic significance

The body wall muscles in five species of branchiobdellidans are all arranged in the oligochaete pattern and the muscle fibres are obliquely striated. The structure of the circular muscle fibres do vary to some degree. The longitudinal muscle fibres in Ankyrodrilus legaeus, Branchiobdella kozarovi, and Xironogiton instabilis all are round circomyarian and thus double-obliquely striated. These species represent three of the four genera composing the family Branchiobdellidae. Although Bdellodrilus illuminatus and Cambarincola fallax, from the families Bdellodrilidae and Cambarincolidae, respectively, also possess a few round circomyarian fibres, most are polyplatymyarian comparable to single-obliquely striated fibres. A similar division of branchiobdellidan families is obtained based on the number of anterior nephridial pores. The muscular structure in the branchiobdellidans shows both similarities and differences with the leeches and the lumbriculid oligochaetes. One phylogenetic explanation for this is that the branchiobdellidans separated from the common clitellate ancestor before the oligochaetes and leeches became recognizable taxa.     

Ultrastructurally different muscle cell types in Eisenia foetida (Annelida,Oligochaeta)

Muscles in the body wall, intestinal wall, and contractile hemolymphatic vessels (pseudohearts) of an oligochaete anelid (Eisenia foetida) were studied by electron microscopy. The muscle cells in all locations, except for the outer layer of the pseudohearts, are variants of obliquely striated muscle cells. Cells comprising the circular layer of the body wall possess single, peripherally located myofibrils that occupy most of the cytoplasm and surround other cytoplasmic organelles. The nuclei of the cells lie peripherally to the myofibrils. The sarcomeres consist of thin and thick myofilaments that are arranged in parallel arrays. In one plane of view, the filaments appear to be oriented obliquely to Z bands. Thin myofilaments measure 5–6 nm in diameter. Thick myofilaments are fusiform in shape and their width decreases from their centers (40–45 nm) to their tips (23–25 nm). The thin/thick filament ratio in the A bands is 10. The Z bands consist of Z bars alternating with tubules of the sarcoplasmic reticulum. Subsarcolemmal electron-dense plaques are found frequently. The cells forming the longitudinal layer of the body wall musculature are smaller than the cells in the circular layer and their thick filaments are smaller (31–33 nm centrally and 21–23 nm at the tips). Subsarcolemmal plaques are less numerous. The cells forming the heart wall inner layer, the large hemolymphatic vessels, and the intestinal wall are characterized by their large thick myofilaments (50–52 nm centrally and 27–28 nm at the tips) and abundance of mitochondria. The cells forming the outer muscular layer of the pseudohearts are smooth muscle cells. These cells are richer in thick filaments than vertebrate smooth muscle cells. They differ from obliquely striated muscle cells by possessing irregularly distributed electron-dense bodies for filament anchorage rather than sarcomeres and Z bands and by displaying tubules of smooth endoplasmic reticulum among the bundles of myofilaments. © 1995 Wiley-Liss, Inc.     

ULTRASTRUCTURAL ORGANIZATION OF OBLIQUELY STRIATED MUSCLE FIBERS IN ASCARIS LUMBRICOIDES

The somatic musculature of the nematode, Ascaris, is currently thought to consist of smooth muscle fibers, which contain intracellular supporting fibrils arranged in a regular pattern. Electron microscopic examination shows that the muscle fibers are, in fact, comparable to the striated muscles of vertebrates in that they contain interdigitating arrays of thick and thin myofilaments which form H, A, and I bands. In the A bands each thick filament is surrounded by about 10 to 12 thin filaments. The earlier confusion about the classification of this muscle probably arose from the fact that in one longitudinal plane the myofilaments are markedly staggered and, as a result, the striations in that plane of section are not transverse but oblique, forming an angle of only about 6° with the filament axis. The apparent direction of the striations changes with the plane of the section and may vary all the way from radial to longitudinal. A three-dimensional model is proposed which accounts for the appearance of this muscle in various planes. Z lines as such are absent but are replaced by smaller, less orderly, counterpart "Z bundles" to which thin filaments attach. These bundles are closely associated with fibrillar dense bodies and with deep infoldings of the plasma membrane. The invaginations of the plasma membrane together with intracellular, flattened, membranous cisternae form dyads and triads. It is suggested that these complexes, which also occur at the cell surface, may constitute strategically located, low-impedance patches through which local currents are channeled selectively.     

Somatic Musculature of Trichodorus porosus and Criconemoides similis

The somatic musculature of Trichodorus porosus is transversely striated, and that of Criconemoides similis is obliquely striated. The species also differ in configuration of the myofibrils, arrangement of the filaments within the myofibrils, and abundance of sarcoplasmic reticulum. Both species are platymyarian and meromyarian. The muscle cells are composed of myofibrils, sarcoplasm, sarcoplasmic reticulum, and various organelles. The myofibrils of both species contain actin and myosin filaments.