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First published online November 1, 2006
Journal of Experimental Biology 209, 4436-4443 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02527
Mechanical adaptability of a sponge extracellular matrix: evidence for cellular control of mesohyl stiffness in Chondrosia reniformis Nardo
1 Department of Biological and Biomedical Sciences, Glasgow Caledonian
University, 70 Cowcaddens Road, Glasgow G4 0BA, Scotland, UK
2 Dipartimento di Biologia `Luigi Gorini', Università degli Studi di
Milano, 20133 Milano, Italy
3 Dipartimento di Scienze del Mare, Università Politecnica delle
Marche, 60131 Ancona, Italy
4 Dipartimento per lo studio del Territorio e delle sue Risorse,
Università di Genova, 16132 Genova, Italy
* Author for correspondence (e-mail: i.wilkie{at}gcal.ac.uk)
Accepted 6 September 2006
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Summary |
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Key words: connective tissue, extracellular matrix, mechanical properties, mutable collagenous tissue, sponge
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Harrison and De
Vos, 1991). The bulk of the body of C. reniformis
consists of a collagenous material, the mesohyl, which is located between the
external epithelium and the internal epithelia that line the inhalant and
exhalant canals. Although, as reflected in the generic name, the mesohyl
confers on the whole animal a cartilaginous consistency, it also demonstrates
plasticity in terms of both shape and mechanical properties. For example,
within 40-60 h after excision, fragments of C. reniformis undergo
rounding off of cut surfaces and marked bending
(Nickel and Brümmer,
2003). Whole sponges or tissue explants can lose rigidity under
continuous compression (Garrone et al.,
1975; Garrone,
1978), and in the sea, loosening of the substrate under part of a
sponge is followed by the slow elongation of that part under gravity and its
eventual separation from the still attached portion, a process that may be
regarded as a form of opportunistic asexual reproduction
(Sarà and Vacelet,
1973; Bonasoro et al.,
2001; Zanetti,
2002).
The work described herein was prompted by the observation that, when
previously undisturbed specimens of C. reniformis in the sea or
laboratory aquaria are touched repeatedly, they feel softer the first time
they are touched than on second and subsequent occasions. The fact that
morphological studies of C. reniformis had not revealed the presence
of potentially contractile cells in a quantity or disposition sufficient to
account for this phenomenon (Bonasoro et
al., 2001) (F. Bonasoro, unpublished observations), raised the
possibility that the mechanical properties of the mesohyl itself are under
physiological control. A precedent for this is provided by the `mutable'
collagenous tissue of echinoderms, the variable tensility of which is neurally
modulated and which is involved throughout the phylum in the energy-sparing
maintenance of posture and in the rapid detachment of anatomical structures at
autotomy (Trotter et al.,
2000; Wilkie,
2001; Wilkie,
2005).
The main aim of this investigation was to test the hypothesis that the
passive mechanical properties of the mesohyl of C. reniformis are
under direct cellular control by determining the effect on the flexural
stiffness of the mesohyl of a range of agents that influence cellular
activities in other animals. A preliminary account of some of the results was
presented at the 6th International Sponge Conference, Rapallo, Italy 2002
(Wilkie et al., 2004).
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Materials and methods |
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) to
investigate the variable tensility of holothurian dermis. Trotter and Koob
explained the theoretical background to this technique and discussed its
limitations with respect to the quantification of absolute mechanical
properties (Trotter and Koob,
1995). Three groups of experiments were conducted.
(1) Mechanical behaviour of samples before treatment with chemical agents
The recovery of samples from the stiffening effect of the excision
procedure was observed as follows. Immediately after each sample was attached
to a coverslip, its stiffness was measured, as described above, and it was
then immersed in ASW. Some samples were left at room temperature
(24.5-26°C) and some were left in a cold-room at 14°C. All were tested
again after 1 h, 3 h, 5 h, 7 h and 9 h.
(2) Effects of membrane disrupters and ionic manipulation
The effects of these agents were determined by measuring the deflection of
samples on a single occasion after immersion in the relevant media for usually
1-2 h. The results are expressed as mm deflection.
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All group 2 and group 3 experiments were carried out at room temperature and were repeated at least once to confirm the reproducibility of results. The sample number was usually 5 (range 4-7). Statistical significance was evaluated by means of Student's t-tests or, for multiple comparisons, ANOVA and Bonferroni post-hoc tests.
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Results |
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In subsequent experiments, chemical agents were tested on both `maximally stiffened' (MS) samples, which were placed in experimental media immediately after attachment to a coverslip, and `partially destiffened' (PD) samples, which, after attachment, were left undisturbed in ASW for 3-4 h, before being tested mechanically, then immersed in experimental media.
Effects of membrane disrupters and ionic manipulation
A range of treatments that cause cell membrane disruption or
permeabilisation by various mechanisms all strongly inhibited the destiffening
of MS samples. This effect was produced by deionised water, which causes
osmotic lysis, the detergents Triton X-100 (1% in ASW) and Quillaja
saponin (0.1% in ASW), which dissolve membrane lipids, freezing at -24°C
for 18 h to 3 days followed by thawing, which disrupts cellular structure
through ice crystal formation, and the poreforming antibiotic nystatin (0.05%
in ASW; Fig. 3A,C). The effect
of the first four of these treatments was irreversible: even after prolonged
(up to 20 h) immersion in ASW, samples showed almost no deflection under
gravity (Fig. 3B). Nystatin had
a less extreme effect and retarded destiffening, rather than blocking it
altogether (Fig. 3C,D). All of
these treatments, except freeze-thawing, were shown to restiffen PD samples,
with deionised water having the most pronounced such action
(Fig. 3E). The effect of
freeze-thawing on PD samples was not determined because of difficulties
encountered in maintaining adhesion between the samples and coverslips.
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Magnesium-free ASW had no consistent effect. In four experiments it respectively inhibited significantly the destiffening of ectosome and choanosome samples, accelerated the destiffening of both significantly, destiffened choanosome samples but had no effect on ectosome samples, and affected neither (data not shown).
Cobalt and manganese ions, which can block calcium-specific membrane channels, inhibited destiffening of MS samples. The effect of 20 mmol l-1 Mn2+ was consistently greater than that of 20 mmol l-1 Co2+; the latter effect, but not the former, was partially reversible (Fig. 4G). PD samples were restiffened by 20 mmol l-1 Mn2+ but not by 20 mmol l-1 Co2+ (Fig. 4H).
The organic calcium channel antagonists verapamil (100 µmol l-1) and nimodipine (100 µmol l-1) both retarded destiffening of MS samples (Fig. 4I).
Effects of tissue extracts
All three tissue extracts inhibited destiffening, though to varying extents
and with varying degrees of consistency. The extract of frozen tissue
inhibited destiffening strongly in two separate experiments. The extract of
unfrozen tissue had the weakest inhibitory effect in both experiments.
Although in the illustrated example (Fig.
5A,B) this effect was statistically significant for the ectosome
at all times, in the other experiment there were no statistically significant
differences between the test and control groups. In the illustrated experiment
the extract prepared after freezing the residue belonging to extract 2
inhibited destiffening as strongly as extract 1, but in the other experiment
its effect was almost as weak as that of extract 2. In both experiments the
inhibitory effect of all three extracts was more pronounced on ectosome
samples than on choanosome samples.
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Discussion |
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; Harrison and De Vos,
1991; Nickel,
2004), less attention has been paid to the passive mechanical
properties of sponge tissues. Chanas and Pawlik
(Chanas and Pawlik, 1995)
measured the tensile strength of frozen then thawed samples of unspecified
orientation and anatomical location from 71 sponge species, and Garrone et al.
(Garrone et al., 1975)
measured the stiffness and ultimate properties of ectosome samples from C.
reniformis that were in an unspecified physiological state. It had been
noted previously that the mesohyl of C. reniformis `flows' under
prolonged compression or tension
(Sarà and Vacelet,
1973; Garrone et al.,
1975; Bonasoro et al.,
2001; Zanetti,
2002). However, as far as we are aware, ours is the first
investigation to have demonstrated that the stiffness of sponge mesohyl can be
changed by an intrinsic mechanism.
Whole sponges stiffen in response to brief mechanical stimulation. Since the handling of sponges that is necessary to prepare isolated tissue samples constitutes intense and prolonged mechanical stimulation, it is not surprising that, immediately after excision, such samples were in a stiffened condition, as indicated by the fact that they bent very little under gravity. Both ectosome and choanosome samples then destiffened over a period of hours, although after 9 h, when the experiment was terminated, only the ectosome samples at 25°C had reached a plateau of maximal compliance, which may represent the `resting' condition of the internal tissue in unstimulated sponges. Destiffening occurred more rapidly at room temperature (24.5-26°C) than at 14°C, 26°C and 14°C being the upper and lower limits of the sea temperature range to which C. reniformis is exposed at the collection sites (G. Bavestrello, personal observation). However, the overall shape of the deflection-time curves was similar at the two temperatures, suggesting that the physiology of the internal tissue was qualitatively similar and that it was therefore valid to conduct all subsequent experiments at room temperature.
Cellular basis of variable tensility
Anatomical structures that have a significant collagenous component, and
that show rapid changes in passive mechanical properties, occur in other
phyla. The variable tensility of these structures depends on either active
force development by contractile cells, as in the walls of vertebrate blood
vessels (Bank et al., 1996), or
the direct, cell-mediated modulation of the tensile properties of the
extracellular matrix, as in the mutable collagenous tissue of echinoderms
(Wilkie, 2005).
Contractile cells may be present in the internal tissue of C.
reniformis. The endopinacocytes lining the inhalant and exhalant canals
of sponges are thought to be contractile, primarily on the grounds that they
contain actin microfilaments (Simpson,
1984; Harrison and De Vos,
1991), and the mesohyl of C. reniformis includes a sparse
and loose network of cell processes (possibly belonging to endopinacocytes)
also furnished with microfilaments
(Bonasoro et al., 2001).
However, the very small volume ratio occupied by potentially contractile
cells, particularly in the ectosome, which is penetrated by relatively few
canals (Bavestrello et al.,
1988), and the paucity of potentially tension-resistant cell-cell
and cell-extracellular matrix (ECM) junctions
(Bonasoro et al., 2001),
suggest that these cells could not influence significantly the passive
mechanical properties of the internal tissue. This morphologically derived
inference was supported by the present investigation. The drastic disruption
of cellular structure caused by deionised water and freeze-thawing prevented
destiffening and restiffened partly destiffened samples, actions that could
not have been due to cell-dependent contractile activity. Furthermore, because
actin-myosin-based contractile systems, including those of sponges
(Lorenz et al., 1996), have a
universal dependence on Ca2+, any agent that reduces the cytosolic
[Ca2+] inhibits such systems, a relevant example being the relaxing
effect of calcium channel blockers on vertebrate smooth muscle. However, the
destiffening (i.e. relaxation) of sponge tissue was inhibited by inorganic and
organic calcium channel blockers, the extracellular chelator EGTA and the
intracellular chelator EGTA-AM, all of which would be expected to depress
[Ca2+]i. Our experimental results, therefore, preclude
the possibility that touch-induced stiffening of the internal tissue of C.
reniformis is due to cellular contraction and lead to the conclusion that
the stiffness of the mesohyl ECM is itself under direct cellular control.
Theoretically, both stiffening and destiffening of the mesohyl, or only one of these, is an active, cell-mediated process. Suppressing cellular function by membrane disruption blocked irreversibly the destiffening of MS samples and restiffened PD samples. The irreversibility of the former effect is evidence that destiffening requires functioning cells. However, there are two possible explanations for the restiffening caused by these treatments. The first, and less likely of the two, is that stiffening is passive and occurs whenever there is cessation of cellular activity that causes and maintains destiffening. The second explanation is that cell lysis releases a chemical factor that stiffens the ECM directly. The presence of an intracellularly stored stiffening factor would explain why less disruptive membrane-permeabilising treatments - detergents and nystatin - had a generally weaker effect than deionised water and freeze-thawing, since membrane permeabilisation without cell lysis would be expected to cause less stiffening factor to be released from cells. The existence of such a factor was confirmed by the experiments using tissue extracts.
All three extracts inhibited destiffening, with those from frozen tissue
(extracts 1 and 3) exerting the greater effect. The fact that the extract of
unfrozen tissue (extract 2) also had some activity indicates that the more
limited cell damage caused by mechanical disruption alone was sufficient to
release a significant amount of the stiffening factor. The present
investigation provides little insight into the nature of this factor, which is
not necessarily a single chemical substance, although the ability of nystatin
to inhibit and reverse destiffening suggests it is not composed of large
molecules. Nystatin is a polyene antibiotic which introduces into cell
membranes pores that permit the leakage of small ions and molecules
(Gale et al., 1981). Work is
currently under way to isolate and characterise the stiffening factor and to
determine its physiological significance: its action on the mesohyl may be
incidental and its in vivo functions unrelated to variable tensility.
This caveat notwithstanding, we hypothesise that touch-induced stiffening of
the mesohyl of C. reniformis depends on the secretion from cells of a
stiffening factor that interacts directly with the extracellular matrix.
The flexural stiffness of the mesohyl was changed by other agents that would be expected to modify cellular activities, rather than stop them altogether as in the case of membrane disrupters. The inhibition of destiffening caused by both inorganic and organic calcium channel blockers and by the intracellular chelator EGTA-AM suggests that the mechanical condition of the mesohyl is influenced by cellular processes involving transmembrane Ca2+ fluxes and changes in [Ca2+]i.
The effects of manipulation of the extracellular [Ca2+] are,
however, problematical. Media with an elevated [Ca2+] inhibited
destiffening and CaFASW by itself accelerated destiffening. CaFASW containing
1 mmol l-1 EGTA, in contrast, inhibited destiffening and CaFASW
with 2 mmol l-1 EGTA restiffened PD samples. The contradictory
effects of CaFASW with and without EGTA must result from interference with
different processes. The effects of CaFASW alone and of elevated
[Ca2+] are hard to reconcile with those of certain other agents.
CaFASW would be expected, like EGTA-AM and calcium channel blockers, to
depress [Ca2+]i, yet it accelerated rather than retarded
destiffening. The fact that collagen fibrils can be isolated from the mesohyl
of C. reniformis by a medium containing EDTA
(Garrone et al., 1975)
indicates that, as in mammalian and echinoderm collagenous tissue
(Dixon et al., 1972;
Steven, 1967;
Wilkie, 2005), divalent
cations contribute directly to the cohesion of the mesohyl ECM. Therefore, it
is possible that the effects of elevated [Ca2+] and CaFASW alone
are due to their direct influence on extracellular components of the mesohyl
ECM and are not cell-mediated. CaFASW with EGTA restiffened PD samples and 5
mmol l-1 EGTA caused irreversible mesohyl stiffening. Therefore,
like deionised water, freeze-thawing etc., EGTA may cause cell membrane
permeabilisation and leakage of the stiffening factor. This makes it possible
that EGTA removes extracellularly bound Ca2+ ions that are
unaffected by CaFASW alone and that maintain cell membrane integrity in intact
tissue.
We have provided evidence that the mechanical properties of the mesohyl ECM
are under cellular control. At present we have no information on the identity
of the effector cells. In addition to the endopinacocytes and choanocytes of
the aquiferous system, the internal tissue of C. reniformis contains
a variety of cell types, the functions of which are largely unknown
(Bonasoro et al., 2001). These
include cells with membrane-bounded cytoplasmic vesicles that in other sponges
release their contents into the extracellular compartment. Amongst the
materials thus released are lectins
(Bretting and Königsmann,
1979), a class of small molecules that may have a structural role
in mammalian collagenous structures
(Tidball, 1994;
Ozeki et al., 1995). Lectins
are stored in the vesicles of spherulous cells
(Bretting and Königsmann,
1979; Bretting et al.,
1983), which in C. reniformis are particularly abundant
in the ectosome (Bonasoro et al.,
2001). In this investigation it was found that experimental
treatments tended to have more pronounced effects on ectosome samples than
choanosome samples. Since this usually took the form of a stronger stiffening
response, it raises the possibility that the spherulous cells are the source
of the stiffening factor in C. reniformis and that the stiffening
factor is a lectin.
Molecular mechanism of variable tensility and evolutionary significance
The mesohyl ECM of C. reniformis consists of bundles of collagen
fibrils interconnected by molecules that include complex carbohydrates
(Garrone et al., 1975).
Collagen fibrils have a high tensile strength and low extensibility
(Sasaki and Odajima, 1996;
Redaelli et al., 2003).
However, the mesohyl of C. reniformis `flows' under prolonged
compression (Garrone et al.,
1975; Garrone,
1978) or tension (Sarà
and Vacelet, 1973; Zanetti,
2002). Since the latter occurs without rupture or lengthening of
the collagen fibrils (Bonasoro et al.,
2001), it is apparent that (1) the fibrils are discontinuous (i.e.
individual fibrils do not completely encircle the body) and (2) the molecular
interactions holding adjacent fibrils together are unstable enough to allow
fibrils, or bundles of fibrils (i.e. fibres), to slide past each other. The
latter is also demonstrated by the ease with which fibrils can be isolated by
mild, nondenaturing methods (Garrone et
al., 1975), in contrast to the inextractability of the collagen
fibrils of adult mammalian connective tissue
(deVente et al., 1997). Given
the discontinuous nature of the collagen fibrils and the lability of
interfibrillar cohesion in the unstimulated mesohyl, the only possible way in
which stiffening can be effected is by the strengthening of the cohesive
forces, thereby preventing interfibrillar or interfibre slippage. This must be
the mode of action of the stiffening factor whose existence we infer from our
experimental results.
There are similarities between the mesohyl of C. reniformis and
the mutable collagenous tissue (MCT) of echinoderms, the mechanical properties
of which can be altered rapidly under nervous control. The collagen fibrils of
MCT form discontinuous arrays and are easily extracted, in the case of
holothurian dermis, by prolonged immersion in seawater alone
(Trotter et al., 1996). Like
the mesohyl, holothurian dermis, the most intensively investigated mutable
collagenous structure, stiffens rapidly in response to mechanical stimulation,
then destiffens over a period of hours
(Szulgit and Shadwick, 2000;
Motokawa and Tsuchi, 2003).
Holothurian dermis is also stiffened by cell membrane permeabilising
treatments, which have been shown to release an intracellularly sequestered
stiffening factor that strengthens interfibrillar cohesion
(Tipper et al., 2003;
Tamori et al., 2006). The
phylogenetic significance of these similarities will not become clear until
the molecular mechanisms and cellular processes underpinning the variable
tensility of MCT and the mesohyl have been elucidated fully. This will clarify
whether the short-term physiological control of ECM mechanics is a primitive
attribute of collagenous connective tissue, its absence from most phyla
representing secondary loss, or whether it is a derived feature that has
evolved independently in different groups. We suspect that the latter is the
case, but that it depends on what may be a primitive feature of collagenous
tissue, namely the lability of intermolecular linkages holding together
adjacent collagen fibrils. That this is a primitive feature is suggested by
the ease with which collagen fibrils can be extracted from the extracellular
matrix of members of the first two multicellular animal phyla to appear in the
fossil record: sponges (Garrone et al.,
1975) and cnidarians (Deutzmann
et al., 2000).
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Acknowledgments |
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