Ultrastructural study and ontogenesis of the appendages and related musculature of Paraspadella (Chaetognatha).

A lineage of benthic chaetognaths has developed limb-like appendages on the caudal part of the body, resulting from a local modification of the lateral fins, which are folds of the epidermis and have a role in balance when swimming. The most complex are those of Paraspadella gotoi which are used as props with the tip of the tail, allowing an elaborated mating behaviour comprising different movements: complete erection of the body, swings and jumps, astonishing for so simple-bodied animals. In the tail, the epidermis and the connective tissue, together with the longitudinal musculature, are involved in this innovation. All the components of the fins, i.e. connective tissue, fin rays and multilayered epidermic cells are conserved, but their function has changed. The movements of appendages are adjusted by one pair of small appendicular muscles localised in the body wall, while posture movements of the body are allowed by four longitudinal bundles of raising muscle. These two new muscles have successively appeared in the evolutive series previously described in Paraspadella. They have definitely arisen from the secondary muscle: the two lateral bundles for the former, and the two dorsal and two ventral ones for the latter. All are supercontracting muscles, a muscle kind also observed in the other benthic genus Spadella, but unknown in planktonic and benthoplanktonic chaetognaths.

One of the origins of plasma membrane phosphatidylserine in plant cells is a local synthesis by a serine exchange activity.

In plant cells, as in animal cells, the endoplasmic reticulum (ER) is considered to be the major site of phospholipid synthesis, and it has been shown that phosphatidylserine (PS) reaches the plasma membrane via the vesicular ER-Golgi-plasma membrane pathway in leek cells. However, it has never been determined whether the plasma membrane of leek cells is able to synthesize PS. We have analyzed the distribution of PS synthesizing enzymes along the vesicular pathway. In ER, Golgi and plasma membrane fractions isolated from leek cells, we have measured the activity of the two biosynthetic pathways leading to the synthesis of PS, i.e. serine exchange and CTP cytidylyltransferase plus PS synthase. We have found a high serine exchange activity in the plasma membrane fraction, and then determined that this membrane is able to synthesize both long chain fatty acid- and very long chain fatty acid-containing PS. Therefore, the PS in the plasma membrane of leek cells has two different origins: the intracellular vesicular pathway from the ER and a local synthesis in the plasma membrane.

ATP-Dependent formation of phosphatidylserine-rich vesicles from the endoplasmic reticulum of leek cells

Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morre, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625-637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATPgamma-S prevents vesicle formation. These vesicles correspond to small structures (70-80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells.

Distribution and role of aspartate in the nervous system of the chaetognath Sagitta.

Cholinergic control of locomotory muscles in chaetognaths is monitored by diffuse transmitter release through layers of collagen fibers that form the connective stratum of the hydroskeleton. Despite the lack of morphologically defined synaptic junctions, the control of locomotor activity in chaetognaths is highly specific and allows complex behavioral patterns. This complexity suggests the existence of neuromediators acting to modulate the effects of the main motor neurotransmitter, acetylcholine, on muscular contraction. Immunocytochemical investigations performed in Sagitta friderici by using antibodies directed against L-aspartate revealed the presence of the amino acid within abundant fiber networks regularly distributed in the head, trunk, and tail and within discrete groups of cell bodies. In addition to known components of the sensory and motor nervous systems, L-aspartate immunoreactivity revealed previously undescribed intraepidermal networks of axonal profiles. With the exception of two giant anterior fibers radiating from the ventral ganglion, L-aspartate-immunoreactive processes were usually thin and varicose, occasionally making an anastomosis. As indicated by electron microscopy, L-aspartate-immunoreactive varicosities apposed to the connective stratum were filled with synaptic-like vesicles but displayed no synaptic differentiation. Physiologic investigations suggested a potent inhibitory effect of L-aspartate on acetylcholine-induced muscle contraction. The wide distribution pattern of immunoreactive profiles suggests an important role of L-aspartate in motor and sensory functions in chaetognaths. Although classified among excitatory amino acids in vertebrates, aspartate may function as an inhibitory modulator of acetylcholine-induced muscle contraction in these enterocoelous gastroneuralians.

Evidence for implication of muscle-specific calpain (p94) in myofibrillar integrity.

The expression and the putative function(s) of a specific muscle calcium-dependent protease were investigated during myogenesis using rat myoblast primary cultures as a model. We have shown that the levels of p94 mRNAs increase as a function of myoblast differentiation, with the greatest amount of these RNAs being present during the later stages (8th day after plating). After an antisense oligodeoxyribonucleotide treatment with p94, ultrastructural studies show dramatic perturbations in differentiated myotubes and during myofibrillogenesis, mainly involving myofibrillar stability and Z-line integrity. These results may be related to recent findings about the role of p94 gene mutations in limbgirdle muscular dystrophy type 2A.

Functional and structural recovery of myotubes from mice with muscular dysgenesis after co-culture with normal, non-myoblastic cells.

Muscular dysgenesis is a mutation which is characterized by paralysis of skeletal muscle cells. Excitation-contraction coupling is deficient and muscle cells display atypical ultrastructure. In vitro, mutant myotubes recover a normal phenotype when co-cultured with spinal cord cells from normal animals or with normal fibroblasts. We have shown that other types of cells, eg certain glial cells present in the spinal cord or in other tissues, have this capacity. In contrast, intervention of neurons in the recovery does not appear likely. Very different types of non-myoblastic cells, then, are capable of restoring contractile activity of dysgenic myotubes in vitro, suggesting that a non-specific mechanism is involved in the phenotypic reversion of affected muscle cells. The restoration process seems to imply a close relationship between myotubes and normal glial cells.

Development of the locomotory muscle of the chaetognath Sagitta. 1. Quantitative and qualitative aspects of the body and muscle tissue development within the phylum.

The Chaetognath primary muscle constitutes the main tissue of the body-wall. It is made up of four main longitudinal bands. Muscle fibres are separated from the nervous system. During muscle growth, four myogenic zones produce two types of fibres A and B, forming units. These units ordered in an epithelial-like tissue, show various kind of intercellular junctions. For a given animal, the overall number of A and B fibres units is practically the same in each band. The four myogenic areas are the centre for a two-phase production of A and B fibres which appear at a regular rate in each quadrant, by the simultaneous production of three sets of A fibres and two sets of B fibres. The former differentiate at once, the latter some time later, and always at the same moment in their lives. In this way the number of sets of A and B fibres increases during the animal's life. This hyperplasia is accompanied by the hypertrophy of the cells which make up each group. For any given animal, the ventral quadrants are thicker than the dorsal quadrants. Chaetognath locomotory muscle is a good model on which to study the development of a skeletal muscle. Its development is closely related to the hydroskeleton growth, to the animal's shape and various other constructional factors which play a central role in this very isolated phylum.

Development of the locomotory muscle of the chaetognath Sagitta. 2. Stereological study of fibre growth and differentiation processes.

The cellular growth and differentiation of A and B fibers was studied using classical stereological methods and the results have then been analysed using linear regression analysis and hypothesis testing procedures. At a given level, the development of the muscular tissue slows down and stabilises, at this same level the growth of the two types of fibres slows down sharply. This is not the case for the cellular organelles. The growth of the contractile apparatus is continuous in each fibre, but the density of myofibrils is higher in the A fibres. The surface of the SR increases in a different fashion in the two types of fibre, but its volumic density evolves in a similar fashion. The mitochondria develop differently in the two types of fibre, neither their shape nor their distribution are comparable from one type of fibre to the other. Despite this overall difference between the mitochondrial populations the individual mitochondria growth mechanisms seem to be comparable. These parameters reflect the organisation and the development of the fiber groups and cellular architecture. They indicate the existence of unknown morphogenetic signals and fields, in this epithelial tissue having paracellular paths communicating with sea water. Muscle development seems to be largely a myogenic property regulated by various extrinsic factors, which are examined.

The use of auto-anti-idiotypes for the visualization of acetylcholine receptors in an invertebrate skeletal muscle.

Polyclonal auto-anti-idiotypic antibodies (AAIs), detected in antisera of rabbits immunized with acetylcholine conjugate, have already been characterized. In this paper, we report on a cytochemical application of these AAIs on skeletal muscle motor endplates of an invertebrate. We have shown that there is a non-uniformly distributed reaction in the synaptic cleft. The deposits seem to be associated with the muscular plasma membrane. The specificity of the cytochemical method is discussed. On this kind of muscle, the AAIs are able to detect, at the ultrastructural level, acetylcholine binding sites which most likely are acetylcholine receptors.

Ultrastructural and cytochemical characterization of subcellular fraction of plasmalemmal origin obtained from uterine longitudinal smooth muscle.

The role of Ca2+-ATPase as the driving force for active calcium uptake, involved in the relaxation of smooth muscle, was studied. It was shown by immunocytochemistry that Ca2+-ATPase activity was localized at the plasma membrane level of longitudinal smooth muscle of pregnant rat uteri (18-20 days). To study calcium regulation in uterine longitudinal smooth muscle, 2 microsomal fractions (F1 and F2) were obtained, enriched in plasma membrane material (Lalanne et al., 1984, in: Calcium Regulation in Smooth Muscles. INSERM series, 124, pp. 283-292). In the present paper this material is characterized at both morphologic and cytochemical levels. Both fractions are ultrastructurally heterogeneous: (a) thin sections clearly show 2 populations that differ in vesicular shape and size; (b) negative staining also shows differences in membrane structure, which could be related to biochemical differences and/or to the well known heterogeneity of the plasma membrane. Two reactions (PATAg and concanavalin A-biotin-avidin-ferritin), allowing visualization of cell coat glycans, were performed on F1 and F2 and on thin sections of longitudinal smooth muscle. Plasma membrane and almost all the vesicles of F1 and F2 are reactive. It is concluded that these 2 fractions are characteristic enough for studying, at the molecular level, the ability of plasma membrane to control calcium circulation in uterine smooth muscle.

Ultrastructural and physiological studies of the contraction of the trunk musculature of Sagitta setosa (chaetognath).

The relationships between structure and function of the contractile apparatus of the trunk musculature in the chaetognath Sagitta setosa, were studied by electron microscopy and mechanical recordings. We also investigated the nature of the neuromediator at the synaptic level. Contraction, relaxation and stretch can be explained on the sliding model basis. The primary filaments are linked to a large Z line, via C filaments. These C filaments vary in length according to the mechanical states of the contractile apparatus. These muscles are directly excitable by electrical current, high K(+) solutions induced depolarization, or exogenous acetylcholine. The characteristics of the unitary contractile response (twitch) and the effects of the frequency of stimulation set up the contractions of Sagitta among the fastest in the animal kingdom. Acetylcholine appears to be the best candidate for the mediator released at the neuromuscular junction. Its effect on the post-junctional membrane seems to require a receptor of the nicotinic type.

Physiological and cytochemical studies of Ca in the primary muscle of the trunk of Sagitta setosa (chaetognath).

The movements of Sagitta are conditioned by the presence of Ca(2+) in the external medium. When this ion is removed from artificial sea water, animals do not move. They swim again when Ca(2+) is present. Among the problems raised by this observation, we have studied the role of Ca(2+) in the contraction of the primary musculature. Physiological experiments show the central importance of the extracellular Ca(2+) and of its translocation through the membrane during the initiation of the contraction. Cytochemical data correlate these observations. They show that Ca(2+) is localized mainly at the level of the plasma membrane, its invaginations and in the poorly developed SR (less than 2% of cell). Like SR, mitochondria accumulate Ca(2+) but do not seem to participate in the regulation of these Ca movements except in abnormal situations. La(3+) blocks the entry of extracellular Ca(2+) and attaches to the membranes; this fixation is not the same on the plasma membrane and in its invaginations. The contractile apparatus of Sagitta primary musculature show remarkable specializations (Duvert and Savineau, 1986). It is composed of ribbon-shaped myofibrils of regular thickness surrounded by external membranes implicated in the fixation and the translocation of a pool of Ca(2+) necessary for initiating contraction. The poorly developed SR and the mitochondria probably modulate the functioning of the two types of fibres (A and B).

Ultrastructure and architecture of the sarcoplasmic reticulum in frog sino-atrial fibres: a comparative study with various preparatory procedures.

Various preparatory procedures were tested to preserve the ultrastructure of the sarcoplasmic reticulum (SR) by the best possible method within frog sino-atrial muscle fibres. These procedures were: conventional aldehyde fixation with or without tannic acid, cryofracture, metallic impregnation and quick-freezing followed by freeze-substitution. Our results illustrated that, when optimally preserved, the SR architecture and ultrastructure of frog sino-atrial fibres were not fundamentally different from those described in many other vertebrate muscle fibres, particularly cardiac fibres. The three-dimensional arrangement of the SR and the structure of its main compartments were situated in a precise fashion: the peripheral SR, located close to the plasma membrane, was made of a tight network of tubules and showed typical couplings; the juxtafibrillar SR was made of a loose network of tubules, small cisternae and some tubules near Z-lines; the intermediary SR, associated with the mitochondria, was made of tubules and fenestrated cisternae. Contacts between SR and mitochondrial membranes were also studied; cryofractures revealed no special intramembrane particles at this level. Collapsed portions of the SR were found after quick-freezing. Because of its relative importance and its three-dimensional arrangement, the SR of frog sino-atrial fibres may have comparable functional significance to the SR of other cardiac muscle fibres.

Intermitochondrial junctions in the heart of the frog, Rana esculenta. A thin-section and freeze-fracture study.

In muscle fibers of the frog heart, junctions between outer membranes of adjacent mitochondrial profiles are occasionally found. In thin sections of embedded tissue and of mitochondrial pellets, the intermitochondrial junctional space is 5.4 +/- 0.15 nm; the external leaflets of the membranes are joined by periodic structures separated from each other by 16.3 +/- 0.29 nm. There are 65.3 +/- 2 periodic structures per micron of membrane measured on a section perpendicular to the junction. After cryofracture, the outer membrane is cleaved into two parts. Closely packed, parallel rows of large particles and furrows are found either on the P-, or on the E-faces. The rows of particles are 11 +/- 0.3 nm thick and are separated from each other by 16.5 +/- 0.46 nm, their density being 65 +/- 2.28 per micron of the membrane. In junctional areas, rows of particles on one membrane correspond with the furrows on the other membrane. Intermitochondrial junctions appear to be real structures and not artifacts due to preparation procedures. The conditions of their occurrence are discussed.

The liquid crystalline nature of the cytoskeleton in epidermal cells of the chaetognath Sagitta.

The chaetognaths have a multilayered epidermis, which is not covered by cuticle, except in the head region. Two kinds of cells are found in the epidermis: the filament-rich cells, adjacent to the basement membrane, and superficial cells, which are filament poor. The filament-rich cells, which are linked by gap junctions and columnar junctions, are highly developed in the collarette region, which joins the head and the trunk. As elsewhere in the epidermis these cells are covered by the filament poor cells which are linked by zonulae adhaerentes, gap junctions and septate junctions. The filaments present in the inner cells of the collarette form a twisted fibrous arrangement, which shows parallel series of nested arcs when observed in oblique section. Such systems are well known in numerous skeletal materials and correspond to polymerized analogues of certain liquid crystals. The amount of connective tissue is extremely reduced in Sagitta. One can hypothesize that filament-rich cells are abundant in regions which undergo strong deformations. This is the case in the collarette, in contact with the basement membrane of the epidermis (which in turn is in contact with a myotendinous system), in a region where ingested prey must go through the general cavity where there is high internal pressure.

Ultrastructural studies of neuromuscular junctions in visceral and skeletal muscles of the chaetognath Sagitta setosa.

The ultrastructural characteristics of the neuromuscular junctions were studied in oesophageal (visceral) muscle and in four skeletal muscles of the head and trunk in Sagitta. Three types of neuromuscular junctions were encountered. The first is made up of nerve terminals which synapse with the surface of the muscle fiber, in a deep or in a slight depression. The second is characterized by muscle fiber protrusions that cross the connective tissue and form synapses with nerve endings; in this type, numerous post-junctional membrane folds are noted. In the third type, the synaptic cleft is very large (greater than 0.2 micron) and contains bundles of connective fibers. Nerve endings are partially ensheathed in glial cells; they contain mostly clear synaptic vesicles, though some dense-cored vesicles are noted. In many muscle fibers post-junctional membrane thickenings are also observed. All observed neuromuscular junctions resemble chemical synapses. Chaetognaths thus show a great variety of neuromuscular junction ultrastructure as do for instance Arthropods.

Further studies on the junctional complex in the intestine of Sagitta setosa. Freeze-fracture of the pleated septate junction.

The intramembrane structures of the pleated septate junction which occur in the junctional complex of the intestine of the chaetognath Sagitta setosa have been investigated. The pleated septate junction is made up of linear rows of irregularly shaped and sized particles, often fused into short rods, and pits which can be fused into furrows. The distribution of these structures on E and P faces depends upon the preparative methods used. Many of the morphological characteristics are the same as those of the "lower invertebrate pleated septate junction type" defined by Green (1981 a). The physiological significance of this junction is obscure. On the basis of the presence of septate junctions (both of the paired septate junction and pleated septate junction types) which have mainly morphological characteristics of the "lower invertebrate pleated septate junction" we can add to the hypothesis that chaetognaths are not related to the molluscs and arthropods.

The junctional complex in the intestine of Sagitta setosa (Chaetognatha): the paired septate junction.

The junctional complex of the intestine of Sagitta setosa has been studied in tissues stained with uranyl acetate or after lanthanum impregnation, and by freeze-cleavage. All types of junctions have been characterized in both perpendicular and tangential planes. From the apex to the base of the cell the following junctions occur in this order: a zonula adhaerens; a septate junction where the septa occur in pairs; a pleated sheet septate junction; and numerous gap junctions of the A-type. From the upper part of the cells inwards to the septate junction, the membranes follow a relatively straight path. In the lower part of the cells the membranes are deeply interdigitating. At the intersection between 3 cells a very different junction is to be observed where small units, periodically disposed, bind the membranes of the 3 adjoining cells. Each unit is composed of 3 short segments which bind the cell membranes to a central ring 16.6 +/- 2.3 nm in outer diameter. The paired septate junction constitutes a new type. Its main features are that the septa are paired and occur in 2 formations, one the 'loose formation', with elements between the septa of each pair, and the other, a 'tight formation'. After lanthanum impregnation, the thickness of each septum is seen to be about 3 nm and the undulation period 12.6 +/- 1.6 nm. On freeze-fractures 10-nm particles are found on crests on the PF face and in furrows on the EF face. The possible significance of this type of junction is discussed. The junctional complex described is analogous to those found in various invertebrate epithelia.

Ultrastructural studies of the junctional complex in the musculature of the arrow-worm (Sagitta setosa) (Chaetognatha).

In the A fibres of the primary musculature of Sagitta, the junctional complex is made up of three kinds of junctions. From the apex to the base they occur in the following order: an apical zonula adherens, a columnar zonula then columnar maculae intermingled with gap junction. Each columnar junction joins two intracellular filament networks in adjacent cells; this cytoskeleton is largely developed around the nucleus of the A fibres and in close relation with the contractile apparatus, especially at the I band level. The B fibres, which never reach the general cavity, lack zonula adherens and columnar zonula. The columnar junction constitutes a new type of junction which seems to belong to the adherens kind. At their level fibrous columns cross the extracellular space, joining the membranes. Each column faces two cytoplasmic densities localized against the cytoplasmic leaflets of the membranes. A cytoskeleton composed of bunldes of cytoplasmic filaments is in close contact with these cytoplasmic densities. The great number of columnar junctions and associated cytoskeleton assure the cohesion of the tissue and the distribution of contractile forces in the absence of connective tissue. The abundance of gap junctions can account for the metabolic and ionic coupling of the fibres.

The primary body-wall musculature in the arrow-worm Sagitta setosa (Chaetognatha): an ultrastructural study.

The primary musculature of Sagitta is mainly made up of two kinds of alternating fibers, A and B. These fibers differ markedly in their localization in the muscular tissue, by the development of their SR and their mitochondria, and the shape of their myofibrils. Their contractile apparatus is similar and possesses myofibrils of regular thickness with very short I bands, flanked by invaginations which are large compartments communicating with the extracellular space. This fiber diversity appears and is maintained in the presence of an apparent common innervation. Nerve ending-like structures are scattered in the epidermis against the basement membrane and there are no nerve beneath this. The presence of at least two kinds of fibers in the primary musculature and the presence of the secondary musculature would suggest that the displacements of sagitta may be more complex than is generally admitted. The specializations of the trunk musculature underline the degree of specialization in the Chaetognatha phylum.