Matrix metalloproteinases in a sea urchin ligament with adaptable mechanical properties

Mutable collagenous tissues (MCTs) of echinoderms show reversible changes in tensile properties (mutability) that are initiated and modulated by the nervous system via the activities of cells known as juxtaligamental cells. The molecular mechanism underpinning this mechanical adaptability has still to be elucidated. Adaptable connective tissues are also present in mammals, most notably in the uterine cervix, in which changes in stiffness result partly from changes in the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). There have been no attempts to assess the potential involvement of MMPs in the echinoderm mutability phenomenon, apart from studies dealing with a process whose relationship to the latter is uncertain. In this investigation we used the compass depressor ligaments (CDLs) of the sea-urchin Paracentrotus lividus. The effect of a synthetic MMP inhibitor - galardin - on the biomechanical properties of CDLs in different mechanical states ("standard", "compliant" and "stiff") was evaluated by dynamic mechanical analysis, and the presence of MMPs in normal and galardin-treated CDLs was determined semi-quantitatively by gelatin zymography. Galardin reversibly increased the stiffness and storage modulus of CDLs in all three states, although its effect was significantly lower in stiff than in standard or compliant CDLs. Gelatin zymography revealed a progressive increase in total gelatinolytic activity between the compliant, standard and stiff states, which was possibly due primarily to higher molecular weight components resulting from the inhibition and degradation of MMPs. Galardin caused no change in the gelatinolytic activity of stiff CDLs, a pronounced and statistically significant reduction in that of standard CDLs, and a pronounced, but not statistically significant, reduction in that of compliant CDLs. Our results provide evidence that MMPs may contribute to the variable tensility of the CDLs, in the light of which we provide an updated hypothesis for the regulatory mechanism controlling MCT mutability

Mean values ± S.D. of unnormalized complex modulus (n = 7).

<p>Mean values ± S.D. of unnormalized complex modulus (n = 7).</p

Schematic representation of the CDL dissection.

<p>Schematic representation of the CDL dissection.</p

MMPs of CDLs in compliant, standard and stiff conditions visualized by gelatin zymography.

<p>(A) Zymogram showing pro-enzyme and active MMP band profile of CDLs in the three mechanical states. (B) Zymogram comparing the band profiles of CDLs, compared with human cell line showing MMP-2 and MMP-9 activity; this zymogram was included because of its very good band separation. (C) Optical density of MMP activity in CDLs in the three mechanical states. (D) Comparative densitometric analysis of scanned gels of CDLs in the different mechanical states with and without 50 µM galardin. Data are expressed as means ± SD. The asterisk (*) represents statistically significant difference P<0.05. MMPs were detected in more than six animals for each of the three mechanical states. (E) Zymogram comparing standard CDLs with and without galardin treatment.</p

Mechanical properties of CDL.

<p>(A) Representative stress vs. strain curves of CDLs from one animal in the three mechanical states. (B) Effect of frequency on the complex modulus of CDLs from one animal in the three mechanical states tested at 13% strain.</p

Reversibility of MMP inhibition.

<p>(A–C) Examples of recordings showing the reversibility of the galardin effect on (A) standard, (B) stiff and (C) compliant CDLs. (D) Normalized results. Tissues in compliant, standard and stiff states were stimulated with 50 µM galardin, washed and treated again with galardin. Data are expressed as means ± SD.</p

Effects of propylene phenoxetol (PPSW) and acetylcholine (AChSW) on the mechanical properties of the CDL.

<p>(A) Immediate effect of PPSW on a standard CDL. (B) Immediate effect of AChSW on a standard CDL. (C) Comparison of the normalized complex modulus of compliant (PPSW-treated), standard (untreated) and stiff (AChSW-treated) CDLs (n = 7).</p

Effect of MMP inhibition on CDL viscoelasticity.

<p>(A) complex modulus, (B) storage modulus, (C) loss modulus and (D) tan delta of compliant, standard and stiff CDLs treated with 50 µM galardin in PPSW, SW, and AChSW respectively. The action of galardin was quantified by normalizing the maximum E* reached after galardin addition against the value just before the application of the chemical. The asterisk (*) represents statistically significant difference P<0.05 and the double asterisk (**) P<0.01.</p

Hypothetical model of the involvement of MMPs in MCT mutability.

<p>It is known that MCTs consist of discontinuous collagen fibrils crosslinked by complexes of molecular components, and that changes in the mechanical properties of MCTs result from rapid changes in the strength of the interfibrillar cohesion that is mediated by these crosslink complexes. We found that the synthetic MMP inhibitor, galardin, increased the stiffness of CDLs in all three mechanical states, which suggests that in all three states there is ongoing MMP activity that has the potential to degrade components already incorporated into existing crosslink complexes and components that have been secreted but not yet incorporated, and ongoing synthesis and release of new crosslink components. The model acknowledges that MMPs are synthesized and secreted as inactive pro-enzymes, then activated extracellularly by proteolytic removal of the pro-peptide domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049016#pone.0049016-SnoekvanBeurden1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049016#pone.0049016-Motokawa3" target="_blank">[51]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049016#pone.0049016-Lee1" target="_blank">[52]</a>. It is envisaged that crosslink components are synthesized and secreted separately, then assembled extracellularly to form functional complexes. The black boxes represent cells, although it should be noted that the three processes do not necessarily occur in different cell-types. The red box represents the process by which MMP-TIMP complexes are removed and degraded. For the sake of simplicity, the model assumes that activated MMPs and new crosslink components reach the extracellular environment at a constant rate. It is hypothesized that interfibrillar cohesion is regulated only through changes in the rate at which an endogenous MMP inhibitor (which we assume is a TIMP-like molecule) is released into the extracellular environment. In the stiff state there are high levels of TIMP secretion (1), MMP inhibition and crosslinking. In the standard state there are intermediate levels of TIMP secretion (2), MMP inhibition and crosslinking. In the compliant state there are low levels of TIMP secretion (3), MMP inhibition and crosslinking. Also represented is the possibility that an endogenous inhibitor could function as a component of the crosslink complex (red arrows) and thus have a dual function (which may apply to TIMP-like tensilin). The model also assumes that the production of MMP-TIMP complexes exceeds the rate of removal and degradation of MMP-TIMP complexes, which would account for the positive correlation between degree of CDL stiffness and total gelatinolytic activity. The components marked with a red asterisk contribute to the gelatinolytic activity of CDLs as quantified by gelatin zymography.</p

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field