Skin of sea cucumbers: the smart connective tissue that alters mechanical properties in response to external stimuli Tatsuo Motokawa Department of Biological Sciences, Tokyo Institute of Technology, Japan Abstract—Catch connective tissue (CCT) is the that is interrupted at five places by pairs of longitudinal connective tissue that shows large stiffness changes in muscles. The body wall encircles the coelom in which response to stimulation under nervous control. The viscera are stored [10]. The water content of individual dermis of sea cucumbers is a typical example of CCT. sea cucumbers is ca. 80%. Among the tissues the Mechanical properties of the dermis are determined by dermis is the main component that occupies ca. 60% of the extracellular materials that are made of collagen the wet weight; muscle content is only 7 % [11]. The fibrils embedded in a hydrogel of proteoglycans. The dermis is composed of a voluminous extracellular dermis takes 3 mechanical states soft (Sa), standard (Sb) matrix in which cellular elements are sparsely and stiff (Sc). Different molecular mechanisms of distributed. The examples of cell types are nerve cells, stiffening have been found in the transition Sa→Sb and in juxtaligamental cells containing secretory granules that the transition Sb→Sc. In this article I will review my are supposed to contain proteins controlling stiffness of works on this intelligent material. extracellular materials, and morula cells whose inclusion are supposed to contain materials for the Index Terms— catch connective tissue, sea cucumber, extracellular matrix [12]. Muscle cells are not found in stiffness change, intelligent material the dermis except in the walls of the water-vascular canals occasionally found in the dermis. I. INTRODUCTION III. MECHANICAL PROPERTIES AND THEIR CHANGES Skin of sea cucumbers is made of catch connective tissue (CCT) or mutable collagenous tissue that shows A. Mechanical properties large stiffness changes in response to stimulation under Our dynamic mechanical tests revealed that the nervous control [1, 2]. CCT is the tissue specific to dermis takes 3 different mechanical states, soft state echinoderms and are found in body walls and ligaments (S ), standard state (S ) and stiff state (S ) [13]. The connecting skeletal elements. Examples are the body- a b c stiffness increases in the order S <S <S whereas the wall dermis of sea cucumbers and starfish [3, 4], sea- a b c energy dissipation ratio decreases in the order urchin catch apparatus that connects spines to a test [5] S >S >S . The notable mechanical property of S is the and brittlestar intervertebral ligaments connecting arm a b c a strain softening in which the application of repetitive vertebrae [6]. Stiffness changes are reversible and strain larger than 10% invokes drastic softening that apparent in seconds to minutes. Animals use stiffening leads the dermis to “melt” into a viscous mass with in defense and posture maintenance. Softening also non-measurable stiffness. Such a mass could recover serves for defense and is used in postural changes, the original shape before melting. This drastic softening fission and autotomy. The main component of the works in fission and defensive behavior including dermis is extracellular materials that are composed of evisceration and autotomy. collagen fibrils embedded in a matrix made of hydrogel of proteoglycans [7, 8]. The stiffness of the B. Nervous control extracellular components determines that of the dermis The mechanical states of CCT are under nervous [9]. As the sea-cucumber dermis is the most studied control and thus we can regard CCT as one of neutrally material among CCTs I will mainly review my works controlled mechano-effectors such as muscles. When on this intelligent material in this article. stained with the antibody specific to echinoderm nerves sea-cucumber body wall is supplied with immunoreactive fine fibers running among the collagen II. BODY-WALL STRUCTURE fibrils [14]. Pharmacological experiments suggested the The body wall of sea cucumbers consists of several presence of two types of cholinergic systems, one is the layers: from outside to inside, a layer of thin cuticle, nicotinic one involved in the dermal stiffening and the thin epidermis, thick dermis that occupies most of the other is the muscarinic one involved in softening [15]. thickness of the body wall, and a circular muscle layer The presence of the cholinergic system was supported * Tatsuo Motokawa: O-okayama Meguro-ku Tokyo, 156-8551, Japan, [email protected] – 2 – JOURNAL OF AERO AQUA BIO-MECHANISMS, VOL.8, NO.1 by the neuropeptide stichopin that inhibits the action of increases cohesive forces between subfibrils stiffening cholinergic systems [16]. Stichopin is one of constituting collagen fibrils that make fibrils thicker four new peptides we have found in the dermis of sea and thus stiffer [27]. CCT can be regarded as a fiber- cucumbers. Other ones are the neuropeptide reinforced material whose fibers are made of collagen. NGIWYamide that stiffens the dermis and two The stiffening of the reinforcing fibers increases the holokinins that soften the dermis. stiffness of the fiber-reinforced materials. This is not The stiffening of the body wall is found when shade the only mechanism of stiffening of the body wall falls on sea cucumbers; this response probably works though. A new stiffening factor (NSF) was extracted for the defense against the potential predators that from the sea-cucumber body wall. This protein causes attack from above. The shadow-induced stiffening is the transition Sb→Sc [28]. As this transition is observed in the isolated dermis to which epidermis is associated with exudation of water from the dermis the attached but not in the epidermis-free dermis [17]. The formation of hydrophobic bonds between macro- shadow response vanishes when the preparation is molecules may responsible to Sb→Sc [29]. The treated with anesthesia, which also supports the electron-microscopic observation suggested another involvement of nerves in this response. The clear stiffening mechanism. Cross bridges between collagen evidence of nervous control of CCT is found in the fibrils are found and the number of bridges increases in shadow reflex of sea urchin Diadema setosum [18]. the order Sa<Sb<Sc [30]. The chemical nature of the When a shadow falls on a sea urchin it waves its spines bridge is yet to be determined. Based on these findings vigorously. This reaction is regarded as being a defense we have proposed a “nested fiber-reinforced composite response to fish; some fish bite the tip of a spine to lift a model” in which the dermis is regarded as a collagen- sea urchin in order to turn it over to expose the fiber reinforced material and the collagen fiber is again unguarded oral surface for attack [19]. The shadow regarded as a composite reinforced by fibril [27]. We reflex is the reflex in which radial nerves are involved. have assumed three stiffening mechanisms in this In this reflex the waving of spines is associated with model: 1. Tensilin makes collagen fibrils stronger and coordinated spine-muscle contraction and softening of stiffer in Sa→Sb through the increase in cohesive forces catch apparatus, the ligament connecting spines to the between subfibrils; 2. Cross-bridges makes fibrils to be test of sea urchins. The electrical stimulation of the a continuous network of collagen bundles both in radial nerve can mimic the shadow reflex: it provokes Sa→Sb and Sb→Sc; 3. The matrix embedding the fiber both spine waving and softening of catch apparatus. component becomes stiffer in Sb→Sc, which was The softening of catch apparatus would permit muscles produced by bonding associated with water exudation. to move spines with less force during the softened We know little about the stiffening mechanism of period and thus with less energy expenditure. This CCTs other than sea-cucumber dermis. A gene coding a example clearly shows that the mechanical properties of tensilin-like protein has been found in sea-urchin CCT are controlled in a coordinated way with muscle genome [30]. The protein synthesized after the code contraction through nerves. has, however, little stiffening effect on sea-urchin CCT. Different mechanism and different proteins may be IV. MOLECULAR MECHANISMS OF STIFFNESS working in CCTs other than sea-cucumber dermis. CHANGES V. ENERGETICS AND EVOLUTION In the early history of studies on CCT muscles were suspected to be the cause of stiffness changes because The energy consumption in three mechanical states muscles are found in many CCTs although their amount has been measured in three CCTs, dermis of sea is quite small [20]. The evidences against this suspicion cucumbers and starfish and catch apparatus of sea was the finding of CCTs containing no muscle cells urchins. The values measured were similar irrespective [21, 22] and the experiments showing that CCT kept the of the kinds of CCTs: when compared with the energy ability of large stiffness changes still after its cellular consumption rate of Sb, that of Sc is about twice greater elements had been disrupted by detergents or by freeze- and that of Sa is about ten times greater [31]. The thaw cycles [23]. energy consumption rate of the body-wall muscles of The break through in the study of the mechanism of sea cucumbers and their contraction forces were stiffness changes was the finding of tensilin that was measured. With these data we could conclude that sea extracted from the sea-cucumber body wall [24]. This cucumbers maintain their posture by CCT with only protein stiffens the isolated dermis and aggregates 1/100 of energy if they were to use muscles instead of collagen fibrils isolated from sea-cucumber body walls. CCT in posture maintenance. The notable character of We found another protein softenin in sea-cucumber echinoderms is its low metabolic rate that is 1/10 the body walls: it softens the dermis and inhibits the value of other invertebrates.