Molecular Mechanism of MCT Mutability

Are Collagen Fibrils Involved?

There is no evidence that the variable tensility of MCT involves changes in the mechanical properties of the collagen fibrils. This would be highly unlikely on a priori grounds, in view of the similarities between the collagen fibrils of MCT and those of vertebrate connective tissue in terms of (1) fibril shape, supramolecular organisation and intermolecular crosslink biochemistry, and (2) the structure of their constituent collagen molecules. Furthermore, numerous ultrastructural investigations have failed to provide evidence that alterations in mechanical properties are accompanied by modification of the shape or organisation of the collagen fibrils. Erlinger et al. (1993), however, interpreted 10-11 nm filaments in a crinoid ligament as being collagen 'protofibrils' produced by a reversible fibril disaggregation mechanism possibly associated with mutability. The ultrastructural observations of Birenheide and Motokawa (1994) indicate that these filaments are more likely to be homologous to the fibrillin-containing microfibrils that are ubiquitous in echinoderm connective tissue. It is therefore almost certain that mutability depends on changes not in the tensility of the collagen fibrils, but in the cohesive forces holding the fibrils together.

Are Calcium Ions Involved?

The mechanical properties of MCT are sensitive to changes in the extracellular calcium ion concentration. Increasing [Ca2+]o stiffens and decreasing [Ca2+]o destiffens almost all mutable structures that have been investigated. These and other findings led to the hypothesis that Ca2+ ions contribute directly to interfibrillar cohesion in MCT and that the juxtaligamental cells alter tissue stiffness by controlling the amount of extracellular Ca2+ available for such a role (reviewed by Wilkie 1996). This hypothesis was discredited by the demonstration that certain treatments stiffen MCT in the absence of Ca2+ ions and that agents that interfere with calcium-dependent cellular processes can change MCT tensility in the presence of a normal [Ca2+]o (Szulgit and Shadwick 1994; Trotter and Koob 1995; Trotter and Chino 1997).The weight of evidence now favours the view that the influence of [Ca2+]o manipulation on MCT tensility is due mainly to direct effects on cellular elements rather than on the extracellular matrix itself, and that, although Ca2+ ions contribute directly to interfibrillar cohesion in an unknown way (Szulgit and Shadwick 2000), variable tensility does not involve modulation of [Ca2+]o.

Tensilin-Tensilin Protease Hypothesis

The stiffness of MCT is changed dramatically by a range of treatments that cause cell membrane lysis, such as freeze-thawing or exposure to deionised water or detergents (Szulgit and Shadwick 1994,2000; Trotter and Koob 1995; Trotter and Chino 1997; Wilkie et al. 1999). Extracts prepared from the dermis of C. frondosa after it has been subjected to freeze-thawing have the same effects on isolated tissue samples as freeze-thawing itself (Trotter and Koob 1995; Koob et al. 1999; Szulgit and Shadwick 2000), and the analysis of such extracts resulted in the isolation of the active agents tensilin ('stiffener') and 'plasticiser'. The observation that these proteins can be isolated from tissues only after cell lysis indicates that they are present mainly in intracellular reservoirs, and led to the hypothesis that they are regulatory molecules which are secreted from cells and bring about changes in MCT tensility (Koob et al. 1999; Trotter et al. 2000b). The case for tensilin being a secreted effector molecule has been strengthened by its recent immunolocalisation in granules of juxtaligamental cells in C. frondosa dermis (D.R. Keene and J.A. Trotter, unpubl.).

It was noted by Tipper et al. (2003) that tensilin tends to undergo proteolysis in vitro and that the degraded product neither binds collagen fibrils nor induces fibril aggregation. Since analysis of trypsin digests suggested that the C-terminus,which includes a putative fibril-binding site, is susceptible to proteolysis, these authors hypothesised that tensilin-induced stiffening is reversed in vivo by a specific protease. Such a protease could be expressed constitutively, resulting in 'automatic' decay back to the destiffened state, or it could be secreted or activated in response to specific signals (Fig. 5).

At present, the significance for variable tensility of other recently isolated molecules is not clear. Some may have a regulatory and others a constitutive role. Indirect immunofluorescence and immunogold labelling have revealed that stiparin is associated much more abundantly with collagen fibrils than is tensilin (D.R. Keene and J.A. Trotter, unpubl.). This observation and the fact that stiparin, unlike tensilin and 'plasticiser', has no effect on whole tissue samples, suggest it may be a constitutive factor that is not involved in short-term changes in mechanical properties, but functions to hold collagen fibrils in a weak association that facilitates the action of effector molecules such as tensilin (Trotter et al. 2000b). However, the demonstration by immunocyto-chemistry that stiparin, like tensilin, is present in the juxtaligamental granules

Fig. 5. Model of the tensilin-tensilin protease hypothesis. MCT plasticisation or destiffen-ing results from (1) the release from, or activation by, a specific type of juxtaligamental cell (JLC1) of tensilin protease (tp),which (2) cleaves tensilin near its GAG-binding site. This (3) allows fibrils to slide past each other, since they are held together only weakly by stiparin. Restiffening results from (4) the release of fresh tensilin (t) from a second type of juxtaligamental cell (JLC 2). MCT constituents represented as in Fig. 3

Fig. 5. Model of the tensilin-tensilin protease hypothesis. MCT plasticisation or destiffen-ing results from (1) the release from, or activation by, a specific type of juxtaligamental cell (JLC1) of tensilin protease (tp),which (2) cleaves tensilin near its GAG-binding site. This (3) allows fibrils to slide past each other, since they are held together only weakly by stiparin. Restiffening results from (4) the release of fresh tensilin (t) from a second type of juxtaligamental cell (JLC 2). MCT constituents represented as in Fig. 3

of C.frondosa (D.R. Keene and J.A. Trotter, unpubl.) raises the possibility that it also could be a regulatory molecule (or that juxtaligamental cells are a source of both constitutive and regulatory factors).

Active Force Generation

No information is available currently on the mechanism of active force generation in crinoid ligaments.

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