Catch Connective Tissue Research Articles

Possible Mechanisms of Stiffness Changes Induced by Stiffeners and Softeners in Catch Connective Tissue of Echinoderms

The catch connective, or mutable collagenous, tissue of echinoderms changes its mechanical properties in response to stimulation. The body wall dermis of sea cucumbers is a typical catch connective tissue. The dermis assumes three mechanical states: soft, standard, and stiff. Proteins that change the mechanical properties have been purified from the dermis. Tensilin and the novel stiffening factor are involved in the soft to standard and standard to stiff transitions, respectively. Softenin softens the dermis in the standard state. Tensilin and softenin work directly on the extracellular matrix (ECM). This review summarizes the current knowledge regarding such stiffeners and softeners. Attention is also given to the genes of tensilin and its related proteins in echinoderms. In addition, we provide information on the morphological changes of the ECM associated with the stiffness change of the dermis. Ultrastructural study suggests that tensilin induces an increase in the cohesive forces with the lateral fusion of collagen subfibrils in the soft to standard transition, that crossbridge formation between fibrils occurs in both the soft to standard and standard to stiff transitions, and that the bond which accompanies water exudation produces the stiff dermis from the standard state.

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.

Rigidity-tuning conductive elastomer

We introduce a conductive propylene-based elastomer (cPBE) that rapidly and reversibly changes its mechanical rigidity when powered with electrical current. The elastomer is rigid in its natural state, with an elastic (Young’s) modulus of 175.5 MPa, and softens when electrically activated. By embedding the cPBE in an electrically insulating sheet of polydimethylsiloxane (PDMS), we create a cPBE–PDMS composite that can reversibly change its tensile modulus between 37 and 1.5 MPa. The rigidity change takes ∼6 s and is initiated when a 100 V voltage drop is applied across the two ends of the cPBE film. This magnitude of change in elastic rigidity is similar to that observed in natural skeletal muscle and catch connective tissue. We characterize the tunable load-bearing capability of the cPBE–PDMS composite with a motorized tensile test and deadweight experiment. Lastly, we demonstrate the ability to control the routing of internal forces by embedding several cPBE–PDMS ‘active tendons’ into a soft robotic pneumatic bending actuator. Selectively activating the artificial tendons controls the neutral axis and direction of bending during inflation.

Coordination between catch connective tissue and muscles through nerves in the spine joint of the sea urchin Diadema setosum.

Echinoderms have catch connective tissues that change their stiffness as a result of nervous control. The coordination between catch connective tissue and muscles was studied in the spine joint of the sea urchin Diadema setosum. Spine joints are equipped with two kinds of effector: spine muscles and a kind of catch connective tissue, which is called the catch apparatus (CA). The former is responsible for spine movements and the latter for maintenance of spine posture. Diadema show a shadow reaction in which they wave spines when a shadow falls on them, which is a reflex involving the radial nerves. Dynamic mechanical tests were performed on the CA in a joint at which the muscles were severed so as not to interfere with the mechanical measurements. The joint was on a piece of the test that contained other spines and a radial nerve. Darkening of the preparation invoked softening of the CA and spine waving (the shadow reaction). Electrical stimulation of the radial nerve invoked a similar response. These responses were abolished after the nerve pathways from the radial nerve to spines had been cut. A touch applied to the CA stiffened it and the adjacent spines inclined toward the touched CA. A touch to the base of the adjacent spine softened the CA and the spines around the touched spine inclined towards it. The softening of the CA can be interpreted as a response that reduces the resistance of the ligaments to spine movements. Our results clearly show coordination between catch connective tissue and muscles through nerves.

Sea urchin catch connective tissue controlled by nerves

![Figure][1] Diadema setosum sea urchins. Photo credit: Tatsuo Motokawa. Most connective tissue is fairly inert, providing padding or holding cells and tissue in place. But when soft starfish want to prise apart a mollusc or a sea urchin wants to hold its spines rigid, they stiffen a

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.

Connective Tissues in Echinoderm Animals that can Reversibly Change their Stiffness and their Stiffening Protein Factors

Some connective tissues shown in echinoderm animals such as sea urchins, sea stars, and sea cucumbers can extensively and reversibly change their stiffness in a few minutes under the regulation of the nervous system. They are called 'catch connective tissues' or 'mutable collagenous tissues'. Neurotransmitters such as acetylcholine, adrenaline, and some oligopeptides cause changes in the stiffness of the living pieces dissected from these tissues. They are no more effective, however, after the treatment with detergent solubilizing cell membranes. There are some granule-containing cells in the tissues, which are assumed to secret some factors under the regulation of nerves causing changes in the tissue stiffness. The tissues contain a large amount of the extracellular matrix mainly consisting of collagen fibrils, proteoglycans and microfibrils. The unique properties of these collagenous tissues might be due to lack of permanent associations between the collagen fibrils and the surrounding extracellular matrix. It seems that cross-linking between the fibrils are formed or broken during the change of the stiffness of the tissues. Its molecular mechanisms are, however, not yet fully understood. We isolated a protein factor called ‘tensilin’ from an extract of sea cucumber body wall dermis, one of the known catch connective tissues. It stiffens the detergent-treated dermal pieces and induces aggregation of collagen fibrils isolated from the tissue. We also isolated another protein factor which stiffens the dermal pieces. It is possible that there are other factors affecting on interactions among dermal fibrils and the stiffness of the tissues. Molecular mechanisms of the stiffness changes of the catch connective tissues should be clarified by purifying and characterizing these factors.

A Sea-Cucumber Derived Novel Protein that Softens the Cell-Disrupted Catch Connective Tissue through Inhibiting the Interaction between Collagen Fibrils

The dermis in the holothurian body wall is a typical catch connective tissue that shows rapid stiffness changes. The molecular mechanisms of the changes are yet to be clarified. Detection of chemical factors that change stiffness by working directly on the extracellular matrix is vital to clarify the molecular mechanisms. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel glycoprotein, softenin, that softened the body-wall dermis whose cells had been disrupted by Triton X-100. This implied that softenin changed dermal stiffness through directly affecting stiffness of extracellular materials. The apparent molecular mass of softenin was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. Catch connective tissue takes three different mechanical states, soft, standard, and stiff. Triton-treated dermis corresponded to the dermis in the standard state. Triton-Freeze-Thaw (TFT) dermis, which was prepared by repetitive freeze-and-thawing of Triton-treated dermis, corresponded to the soft dermis. TFT dermis did not respond to softenin whereas it was stiffened by tensilin, a sea-cucumber derived stiffening protein. Tensilin-stiffened TFT dermis became soft when softenin was applied, suggesting the antagonism between softenin and tensilin. The antagonism was also observed in the suspended solution of collagen fibrils isolated from the sea-cucumber dermis. Tensilin caused fibril aggregation while softenin suppressed it. Freeze-and-thawing not only caused stiffness change from standard to soft state in Triton-treated dermis but also induced dissolving the tensilin-aggregated collagen fibrils. These results suggested that softenin decreased dermal stiffness through inhibiting some mechanical interactions between collagen fibrils. Thus, softenin is the first softener shown to work directly on the mechanical interaction between extracellular materials.

1P200 棘皮動物コラーゲン性のキャッチ結合組織を軟化させる新規タンパク質因子(12.細胞生物的課題,ポスター,日本生物物理学会年会第51回(2013年度))

1P200 棘皮動物コラーゲン性のキャッチ結合組織を軟化させる新規タンパク質因子(12.細胞生物的課題,ポスター,日本生物物理学会年会第51回(2013年度))

Energy Expenditure Associated With Softening and Stiffening of Echinoderm Connective Tissue

Catch connective tissue of echinoderms at rest (in the standard state) either stiffens or softens in response to different kinds of stimulation. The energy consumption associated with the changes was estimated by measurement of the oxygen consumption rate (VO(2)) in three types of connective tissues-echinoid catch apparatus (CA), holothuroid body-wall dermis (HD), and asteroid body-wall dermis (AD). Mechanical stimulation by repetitive compression (10%-15% strain), which increased viscosity measured by creep tests, was employed for inducing the stiff state. Noradrenaline (10(-3) mol l(-1)), which decreased viscosity of CA, and static 80% compressive strain, which decreased viscosity of HD, were used to induce the soft state in the respective tissues. The VO(2) (in μl/g/h) values of the standard state were 2.91 (CA), 1.41 (HD), and 0.56 (AD), which were less than 1/4 of the VO(2) of the resting body-wall muscle of the starfish. The VO(2) of the stiff state was about 1.5 times greater than that of the standard state in all types of connective tissues. The VO(2) of the soft state was 3.4 (CA)-9.1 (HD) times greater than that of the standard state. The economical nature of catch connective tissue in posture maintenance is discussed.

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

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.

Low Oxygen Consumption and High Body Content of Catch Connective Tissue Contribute to Low Metabolic Rate of Sea Cucumbers

The energy consumption of echinoderms is low in comparison with that of other invertebrates. We demonstrated this by measuring the oxygen consumption rate per unit of body weight (VO2) of the sea cucumber Actinopyga mauritiana: VO2 was 1/8 that of the "standard" invertebrates. Low energy consumption in echinoderms has been attributed to their high skeletal content and to catch connective tissues (CCTs) that maintain body posture by altering their mechanical properties with little energy expenditure. The former is not applicable to holothurians, and the latter has not been proven experimentally. We postulated that the large content of dermal connective tissue, which maintaines posture economically, contributes to the low energy consumption in holothurians. Body-wall dermis occupied 53.5% of wet body weight, whereas body-wall muscles, including those of tube feet, occupied 5.1%. VO2 of the dermis in the stiff state (2.45 microl x g(-1) x h(-1)) was 1/10 that of the longitudinal body-wall muscle in contraction. the mechanical tests revealed that the stress at an imposed strain of 2% strain was 7 times greater in CCT than in muscles. These results showed that CCT could maintain posture more economically than muscles could. We concluded that the high content of connective tissue with energy-saving posture-maintenance activities contributed to the low energy consumption of holothurians.

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.

Tensilin-like stiffening protein fromHolothuria leucospilotadoes not induce the stiffest state of catch connective tissue

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) Ca(2+). 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 Ca(2+)-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.

海洋生物資源と高分子 ナマコのキャッチ結合組織

海洋生物資源と高分子 ナマコのキャッチ結合組織

Switching of metabolic-rate scaling between allometry and isometry in colonial ascidians.

The metabolic rate and its scaling relationship to colony size were studied in the colonial ascidian Botrylloides simodensis. The colonial metabolic rate, measured by the oxygen consumption rate (V(O2) in millilitres of O(2) per hour) and the colony mass (wet weight M(w) in grams) showed the allometric relationship (V(O2) = 0.0412 M(w)(0.799). The power coefficient was statistically not different from 0.75, the value for unitary organisms. The size of the zooids and the tunic volume fraction in a colony were kept constant irrespective of the colonial size. These results, together with the two-dimensional colonial shape, excluded shape factors and colonial composition as possible causes of allometry. Botryllid ascidians show a takeover state in which all the zooids of the parent generation in a colony degenerate and zooids of a new generation develop in unison. The media for connection between zooids such as a common drainage system and connecting vessels to the common vascular system experienced reconstruction. The metabolic rate during the takeover state was halved and was directly proportional to the colonial mass. The scaling thus changed from being allometric to isometric. The alteration in the scaling that was associated with the loss of the connection between the zooids strongly support the hypothesis that the allometry was derived from mutual interaction among the zooids. The applicability of this hypothesis to unitary organisms is discussed.

Characterization of Fibrosurfin, an Interfibrillar Component of Sea Urchin Catch Connective Tissues

The Sea URchin Fibrillar (SURF) domain is a four-cysteine module present in the amino-propeptide of the sea urchin 2alpha fibrillar collagen chain. Despite numerous international genome and expressed sequence tag projects, computer searches have so far failed to identify similar domains in other species. Here, we have characterized a new sea urchin protein of 2656 amino acids made up of a series of epidermal growth factor-like and SURF modules. From its striking similarity to the modular organization of fibropellins, we called this new protein fibrosurfin. This protein is acidic with a calculated pI of 4.12. Eleven of the 17 epidermal growth factor-like domains correspond to the consensus sequence of calcium-binding type. By Western blot and immunofluorescence analyses, this protein is not detectable during embryogenesis. In adult tissues, fibrosurfin is co-localized with the amino-propeptide of the 2alpha fibrillar collagen chain in several collagenous ligaments, i.e., test sutures, spine ligaments, peristomial membrane, and to a lesser extent, tube feet. Finally, immunogold labeling indicates that fibrosurfin is an interfibrillar component of collagenous tissues. Taken together, the data suggest that proteins possessing SURF modules are localized in the vicinity of mineralized tissues and could be responsible for the unique properties of sea urchin mutable collagenous tissues.

Glycerinated Catch Apparatus of Sea Urchin Spines: the Effects of Cations on its Mechanical Properties and Ultrastructure

Sea urchin spinal ligaments (the catch apparatus) were extracted with glycerin, and electron microscopic observations comfirmed that no cell membranes remained intact after glycerination. We studied the effects of cations (Na(+), K(+), Ca(2+), Mg(2+)) on the mechanical properties of the glycerinated ligaments. Monovalent cations decreased whereas divalent cations increased the viscosity of the ligaments. The ion dependencies were similar to previous results with detergent-extracted holothurian dermis, which suggests that the echinoid ligament shares a similar mechanism for changes in mechanical properties with other catch connective tissues. This provides evidence against the hypothesis of that muscles in the catch apparatus are responsible for the changes in mechanical properties of the ligament. Fine projections cross-bridging collagen fibrils were observed in the glycerin-extracted ligaments as well as in the intact ligaments. They were found in all the ionic conditions studied.

Cirri of the stalked crinoid Metacrinus rotundus: neural elements and the effect of cholinergic agonists on mechanical properties.

Sea lilies are enigmatic animals due to their scarcity and their biology is comparatively neglected. Cirri, arranged in whorls of five along the sea lily stalk, anchor and support the animal. They consist of ossicles interconnected by collagenous ligaments and by a central canal. Cirri have a well-developed nervous system but lack muscular cells. A light and electron microscopic study was performed to clarify the morphology of the nervous system of the cirri. Two cellular networks were found, one of neuron-like cells and one of cells filled with bullet-shaped organelles. Both networks ramify throughout the cirral ossicles up to the interossicle ligaments. Mechanical tests were performed to analyse the influence of cholinergic agonists on the mechanical properties of these ligaments. In the tests, the cirral ligaments softened after the application of acetylcholine, muscarinic agonists and nicotinic agonists. The reaction time to muscarinic agonists was much slower than to acetylcholine and nicotinic agonists. At low concentrations, muscarinic agonists caused active development of force. No reaction to stimuli was observed in anaesthetized cirri. The data clearly establish the existence of catch connective tissue which can change its mechanical properties under nervous control mediated via nerves with cholinergic receptors. The possible sources of the observed force production are discussed and it is concluded that active contraction of collagenous ligaments causes movement of cirri.

Glycerinated Catch Apparatus of Sea Urchin Spines: the Effects of Cations on its Mechanical Properties and Ultrastructure

Sea urchin spinal ligaments (the catch apparatus) were extracted with glycerin, and electron microscopic observations comfirmed that no cell membranes remained intact after glycerination. We studied the effects of cations (Na , K , Ca2 , Mg2 ) on the mechanical properties of the glycerinated ligaments. Monovalent cations decreased whereas divalent cations increased the viscosity of the ligaments. The ion dependencies were similar to previous results with detergent-extracted holothurian dermis, which suggests that the echinoid ligament shares a similar mechanism for changes in mechanical properties with other catch connective tissues. This provides evidence against the hypothesis of del Castillo et al (1995) that muscles in the catch apparatus are responsible for the changes in mechanical properties of the ligament. Fine projections cross-bridging collagen fibrils were observed in the glycerin-extracted ligaments as well as in the intact ligaments. They were found in all the ionic conditions studied.