Tamori M et al. (2016), Ultrastructural Changes Associated with Reversi...

ECB-ART-44696

PLoS One 2016 May 04;115:e0155673. doi: 10.1371/journal.pone.0155673.

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

Tamori M , Ishida K , Matsuura E , Ogasawara K , Hanasaka T , Takehana Y , Motokawa T , Osawa T .

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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.

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Genes referenced: LOC100887844 LOC115925415

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	Fig 1. Stress-strain curves of dermis in soft state (A), in standard state (B) and in stiff state (C). Data from animal #1.
	Fig 2. Electron micrograph of dermis in the standard state cut transversely to the strain direction.Collagen fibrils in cross sections are observed. In this, and Figs 4 and 9, unlabelled arrows denote cross-bridges between fibrils. Arrow marked a: fibril with a meandering perimeter that folds deeply towards the center of the fibril, suggesting that the fibril is possibly made of 4 subfibrils. Arrow marked b: thick fibril containing an electron-lucent area in which an electron-dense spot is observed at the border of the electron-lucent area. Arrow marked c: electron-dense spot on the perimeter of fibrils. Scale bar, 100 nm.
	Fig 3. Electron micrographs of dermis in the standard state cut along the strain direction.Longitudinal sections of collagen fibrils are observed. The fibrils have protruding arms some of which make cross-bridges between fibrils. A: cross-bridges and arms that appeared repetitively along fibrils. The repeat interval was measured in the fibril marked with an asterisk. B: arrays of electron-dense spots aligned on the same plane perpendicular to the length of the fibril as that of the protruding arms. C: diagonal cross-bridges. D: cross-bridges between the fibrils that are not in parallel. Scale bar, 100 nm.
	Fig 4. Frequency distribution of fibril diameter.A: dermis. Dermis in Sa (stippled bars), in Sb (diagonally hatched bars) and in Sc (filled bars) are compared. B: Dermis treated with Triton. Triton-treated dermis (diagonally hatched bars) and Triton-FT dermis (stippled bars) are compared. C: Collagen suspensions. The samples aggregated by tensilin (diagonally hatched bars), dispersed by softenin (stippled bars) or dispersed by FT-treatment (stippled bars with horizontal stripes) are compared. The diameter class 20, for example, includes the fibrils with the diameter equal to or larger than 20 nm and less than 40 nm. The total numbers of measured fibrils were: 180 for each state in A, 180 for each treatment in B, and 30 for each solution. In this and Figs 6, 7 and 8 the slant hatching and the stippling denote that the samples were in the state that mechanically corresponded to the standard state and to the soft state respectively.
	Fig 5. Comparison of cross sections of dermis in three different mechanical states.A, soft state; B, standard state; C, stiff state. Scale bar, 100 nm.

	Fig 7. Diameter of collagen fibrils.A: dermis. Dermis in Sa (stippled bars), in Sb (slant hatched bars) and in Sc (filled bars) are compared. B: Triton-treated dermis (slant hatched bars) and Triton-FT (stippled bars) dermis are compared. C: collagen suspensions. The sample aggregated by tensilin, that dispersed by softenin or that dispersed by FT-treatment (FT) are compared. The number of measurements in each sample was: 180 in A and B, and 30 in C.
	Fig 8. Distance between collagen fibrils.A: dermis. The dermis in Sa, in Sb and in Sc are compared. B: Triton-treated dermis and Triton-FT dermis are compared. The numbers of measurements in A are as follows: Sa, 41; Sb, 39; Sc, 62. The numbers of measurements in B are 22 for Triton-FT samples and 48 for Triton-treated samples.
	Fig 9. Electron micrographs of dermis treated with Triton.A: Triton-FT dermis; B: Triton-treated dermis. Fibrils in cross sectional view are compared. Scale bar, 100 nm.
	Fig 10. Electron micrographs of collagen-fibril suspensions.Samples were aggregated by tensilin (C), which were then dispersed either by FT treatment (A) or by softenin (B). The arrowhead indicates a fibril that appears as if 4 or more thinner fibrils were embedded in a less electron-dense matrix to form a fibril. Scale bar, 100 nm.
	Fig 11. Nested fiber-reinforced composite model.The holothurian dermis is modeled as a material with 3 levels each of which could be regarded as a fiber-reinforced composite. The first level: collagen fibrils are made up of subfibrils (white circles) embedded in a matrix, shown by black that fills between subfibrils; the stiffness of the matrix is controlled through tensilin and softenin. The second level (bundle level): fibrils are cross-bridged by arms (black bars) to be a bundle that is embedded in a matrix whose stiffness is regarded here to be controlled by the number of cross-bridges. The third level (dermis level): the bundles are embedded in a matrix made of a hydrogel of proteoglycans whose stiffness is controlled by bonds between proteoglycans; bond formation is associated with water exudation. Proteoglycan molecules are drawn here as tadpole-shaped ones with a short stranded tail that fabricated the orthogonal network [26]. The arcs with a star at each end denote bond between proteoglycans.

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Barbaglio, Ultrastructural and biochemical characterization of mechanically adaptable collagenous structures in the edible sea urchin Paracentrotus lividus. 2015, Pubmed, Echinobase