Masaki Tamori

Tensilin-like stiffening protein from Holothuria leucospilota does not induce the stiffest state of catch connective tissue

SUMMARY The dermis of sea cucumbers is a catch connective tissue or mutable connective tissue that exhibits large changes in mechanical properties. A stiffening protein, tensilin, has been isolated from the sea cucumber Cucumaria frondosa. We purified a similar protein, H-tensilin, from Holothuria leucospilota, which belongs to a different family to C. frondosa. H-tensilin appeared as a single band with an apparent molecular mass of 34 kDa on SDS-PAGE. No sugar chain was detected. Tryptic fragments of the protein had homology to known tensilin. H-tensilin aggregated isolated collagen fibrils in vitro in a buffer containing 0.5 mol l–1 NaCl with or without 10 mmol l–1 Ca2+. The activity of H-tensilin was quantitatively studied by dynamic mechanical tests on the isolated dermis. H-tensilin increased stiffness of the dermis in the soft state, induced by Ca2+-free artificial seawater, to a level comparable to that of the standard state, which was the state found in the dermis rested in artificial seawater with normal ionic condition. H-tensilin decreased the energy dissipation ratio of the soft dermis to a level comparable to that of the standard state. When H-tensilin was applied on the dermis in the standard state, it did not alter stiffness nor dissipation ratio. The subsequent application of artificial seawater in which the potassium concentration was raised to 100 mmol l–1 increased stiffness by one order of magnitude. These findings suggest that H-tensilin is involved in the changes from the soft state to the standard state and that some stiffening factors other than tensilin are necessary for the changes from the standard to the stiff state.

A novel stiffening factor inducing the stiffest state of holothurian catch connective tissue.

SUMMARY The dermis of sea cucumbers is a catch connective tissue or mutable collagenous tissue that shows large changes in stiffness. Extensive studies on the dermis revealed that it can adopt three different states having different mechanical properties that can be reversibly converted. These are the stiff, standard and soft states. The standard state is readily produced when a dermal piece is immersed in the sea water containing Ca2+, whereas the soft state can be produced by removal of Ca2+. A stiffening protein, tensilin, has been isolated from some sea cucumbers (Cucumaria frondosa and Holothuria leucospilota). Although tensilin converts the state of the dermis from soft to standard, it cannot convert from standard to stiff. In this study, we isolated and partially purified a novel stiffening factor from the dermis of Holothuria leucospilota. The factor stiffened the dermis in normal artificial sea water (ASW) but did not stiffen the soft dermis in Ca2+-free ASW. It also stiffened the dermis that had been converted to the standard state in Ca2+-free ASW by the action of tensilin. These results suggest that the factor produces the stiff dermis from the standard state but cannot work as a stiffener on the soft dermis. Its addition to longitudinal muscles of the sea cucumber produced no effects, suggesting that its effect is specific to the catch connective tissue. Its stiffening activity was susceptible to trypsin, meaning that it is a polypeptide, and its molecular mass estimated from gel filtration chromatography was 2.4 kDa.

Evidence that water exudes when holothurian connective tissue stiffens

SUMMARY The dermis of the body wall of sea cucumbers is composed mainly of extracellular materials such as collagens, proteoglycans and water; the water content is as high as 80%. Yet it shows rapid changes in stiffness under neural control. The dermis has been proposed to assume three mechanical states, soft, standard, and stiff. We investigated the relationship between the stiffness and the dermal mass and volume. Both the mass and volume decreased by 15% when the dermis stiffened from the standard state to the stiff state by mechanical stimulation and by chemical stimulation with potassium-rich seawater. The effect of the latter was abolished by anesthesia. The mass decrease was caused largely by water exudation. Tensilin, a holothurian protein that stiffens the soft dermis to form the standard state, did not cause any changes in mass. These results suggested that the stiffening mechanisms responsible for the transition from the soft state to the standard state, and that from the standard to the stiff state, are different. The removal of water from the dermis in the standard state, by soaking in hypertonic solution, caused only slight stiffening, which suggested that water exudation was not the direct cause of the stiffening. A change of pH of the surrounding medium, either more acidic or basic, was not associated with mass changes, although it caused a large increase in stiffness. The implications of the present results for the molecular mechanisms of the stiffness changes are discussed.

NGIWYamide-induced Contraction of Tube Feet and Distribution of NGIWYamide-like Immunoreactivity in Nerves of the Starfish Asterina pectinifera

Abstract NGIWYamide, a neuropeptide recently isolated from sea cucumbers, was tested on tube feet of the starfish Asterina pectinifera. NGIWYamide (10−6–10−4 M) caused contraction of isolated tube feet. NGIWYamide-like immunoreactivity (NGIWYa-LI) was investigated with an antiserum against NGIW-Yamide. NGIWYa-LI was found in the radial nerve cord (RNC), the marginal nerve, and the tube feet. Both ectoneural and hyponeural parts of the RNC showed NGIWYa-LI. Immunoreactive cell bodies were found in both parts of RNC. Extensive labeling in the basal region of the ectoneural part suggests that a substantial proportion of axons in this part contains NGIWYamide or a similar substance. In tube feet, NGIWYa-LI was found in the sub-epithelial nerve plexus and in the basal nerve ring. Double labeling along with 1E11, a neuron-specific monoclonal antibody developed from A. pectinifera, indicated that the structures with NGIWYa-LI are neurons. These results suggest that NGIWYamide or an NGIWYamide-like peptide exists in starfish and functions as a neurotransmitter or a neuromodulator.

Softenin, a novel protein that softens the connective tissue of sea cucumbers through inhibiting interaction between collagen fibrils.

The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between extracellular materials.

Innervation of Holothurian Body Wall Muscle: Inhibitory Effects and Localization of 5-HT

Abstract We investigated innervation to body wall muscles as well as distribution of 5-HT (serotonin) and its effects on longitudinal muscles of body wall (LMBW) of the sea cucumber Apostichopus japonicus. With serial sections we found neural branches and fibers extending from hyponeural part of radial nerve towards LMBW and circular muscles of body wall. With the aqueous aldehyde (Faglu) method yellow fluorescence indicating indolamines was observed in LMBW and in the mesentery connecting LMBW to the body wall. With indirect immunohistochemistry 5-HT-like immunoreactivity was observed in LMBW and in mesentery. These results strongly suggested that both LMBW and mesentery contained 5-HT. The effects of monoamine neurotransmitters were studied in LMBW. Putative neurotransmitters tested were 5-HT, adrenaline, noradrenaline, dopamine, and DOPA at the concentration of 10−6 M. The application of 5-HT caused no contraction or relaxation, but it inhibited the contraction induced by 10−6–10−5 M acetylcholine (ACh). Catecholamines were ineffective by themselves and had no effects on the contraction induced by ACh. The present histological, histochemical, and pharmacological studies strongly suggested that holothurian LMBW was innervated by inhibitory serotonergic neurons of the hyponeural nervous system.

Stichopin-containing nerves and secretory cells specific to connective tissues of the sea cucumber

Stichopin, a 17-amino acid peptide isolated from a sea cucumber, affects the stiffness change of the body-wall catch connective tissues and the contraction of the body-wall muscles. The localization of stichopin in sea cucumbers was studied by indirect immunohistochemistry using antiserum against stichopin. Double staining was performed with both stichopin antiserum and 1E11, the monoclonal antibody specific to echinoderm nerves. A stichopin-like immunoreactivity (stichopin-LI) was exclusively found in the connective tissues of various organs. Many fibres and cells with processes were stained by both the anti-stichopin antibody and 1E11.They were found in the body-wall dermis and the connective tissue layer of the cloacae and were suggested to be connective tissue-specific nerves. Oval cells with stichopin-LI (OCS) without processes were found in the body-wall dermis, the connective tissue sheath of the longitudinal body-wall muscles, the connective tissue layer of the tube feet and tentacles, and the connective tissue in the radial nerves separating the ectoneural part from the hyponeural part. Electron microscopic observations of the OCSs in the radial nerves showed that they were secretory cells. The OCSs were located either near the well-defined neural structures or near the water-filled cavities, such as the epineural sinus and the canals of the tube feet. The location near the water-filled cavities might suggest that stichopin was secreted into these cavities to function as a hormone.

Ultrastructural Changes Associated with Reversible Stiffening in Catch Connective Tissue of Sea Cucumbers.

The dermis of sea cucumbers is a catch connective tissue or a mutable collagenous tissue that shows rapid, large and reversible stiffness changes in response to stimulation. The main component of the dermis is the extracellular material composed of collagen fibrils embedded in a hydrogel of proteoglycans. The stiffness of the extracellular material determines that of the dermis. The dermis has three mechanical states: soft (Sa), standard (Sb) and stiff (Sc). We studied the ultrastructural changes associated with the stiffness changes. Transverse sections of collagen fibrils in the dermis showed irregular perimeters with electron-dense protrusions or arms that cross-bridged between fibrils. The number of cross-bridges increased in stiffer dermis. The distance between the fibrils was shorter in Sc than that in other states, which was in accord with the previous report that water exuded from the tissue in the transition Sb→Sc. The ultrastructure of collagen fibrils that had been isolated from the dermis was also studied. Fibrils aggregated by tensilin, which causes the transition Sa→Sb possibly through an increase in cohesive forces between fibrils, had larger diameter than those dispersed by softenin, which antagonizes the effect of tensilin. No cross-bridges were found in isolated collagen fibrils. From the present ultrastructural study we propose that three different mechanisms work together to increase the dermal stiffness. 1.Tensilin makes collagen fibrils stronger and stiffer in Sa→Sb through an increase in cohesive forces between subfibrils that constituted fibrils; 2. Cross-bridging by arms caused the fibrils to be a continuous network of bundles both in Sa→Sb and in Sb→Sc; 3. The matrix embedding the fibril network became stiffer in Sb→Sc, which was produced by bonding associated with water exudation.

Dumbbell-Shaped Ossicles Discovered in Pedicellaria of Flower Sea Urchins

Sea urchins have a globiferous pedicellaria that stands from a test with a stalk on which lies a head made of three movable jaws with venom-injecting teeth. The globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus, one of the most developed among sea urchins, are unique in that the jaws are provided with a jaw membrane that gives the pedicellaria an appearance of a flower when the jaws are open. We observed this membrane in an ionic liquid that does not require processes, such as fixation, dehydration, or coating with conductive materials, for observation with a scanning electron microscope. Using this technique, we discovered dumbbell-shaped ossicles, which consist of two spheres of similar size connected by a cylinder. The diameter of the sphere is 4–8 µm, and the total length of the ossicle is 10–20 µm. The jaw membrane is trimmed with an edge zone. The ossicles were found sparsely in the connective tissue of general part of the membrane, but in the edge zone their density was so high that neighboring ossicles were in close contact with each other. Some neighboring ossicles crossed their cylinders and some inserted one of their spheres to snugly fit in the gap between the spheres of neighboring ossicles. Their structural role is very likely in strengthening the jaw membrane, probably serving as fillers in the general part of the membrane; in the edge zone the interlocking of adjacent ossicles forms a loose network providing a firm frame for the head of the globiferous pedicellaria. When opened, the stiff frame prevents the membrane from sagging. When clasped, it works as a closed door, firmly keeping prey trapped.