(Biología Marina o Limnología) El papel clave de la membrana de la superficie es saber qué gasterópodo nácar crece en torres

8/3/2019 (Biologa Marina o Limnologa) El papel clave de la membrana de la superficie es saber qu gasterpodo ncar crec

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The key role of the surface membrane in whygastropod nacre grows in towersAntonio G. Checaa,1, Julyan H. E. Cartwrightb, and Marc-Georg Willingerc

aDepartamento de Estratigrafa y Paleontologa, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain; bInstituto Andaluz de Ciencias de laTierra, Consejo Superior de Investigaciones CientificasUniversidad de Granada, Campus Fuentenueva, E-18071 Granada, Spain; and cDepartamento deQumica, Centro de Investigacao em Materiais Ceramicos e Compositos, Campus Universitario de Santiago, Universidade de Aveiro, 3810-

193 Aveiro, Portugal

Edited by Steven M. Stanley, University of Hawaii, Honolulu, HI, and approved November 25, 2008 (received for review September 4, 2008)

The nacre of gastropod molluscs is intriguingly stacked in towers.

It is covered by a surface membrane, which protects the growing

nacre surface from damage when the animal withdraws into its

shell. The surface membrane is supplied by vesicles that adhere to

it on itsmantle side andsecretesinterlamellar membranesfrom the

nacre side. Nacre tablets rapidly grow in height and later expand

sideways; the part of the tablet formed during this initial growth

phase is here called the core. During initial growth, the tips of the

cores remain permanently submerged within the surface mem-

brane. The interlamellarmembranes, which otherwise separate the

nacretablet lamellae,do not extendacross cores,whichare aligned

in stacked tablets forming the tower axis, andthus towers of nacretablets are continuous along the central axis. We hypothesize that

in gastropod nacre growth core formation precedes that of the

interlamellar membrane. Once the core is complete, a new inter-

lamellar membrane, which covers thearea of thetablet outside the

core, detaches from the surface membrane. In this way, the

tower-like growth of gastropod nacre becomes comprehensible.

biomineralization molluscs organic membranes epitaxy

Nacre is by far the most intensively studied non-humanorganomineral biocomposite. It has a high proportion,5%,of organic matter(proteins andpolysaccharides; ref1), themineral f raction being exclusively in the form of aragonite.

Jacksonet al. (2) estimated that its work of fracture is 3,000 timeshigher than that of inorganic aragonite, although later estimatesreduce this figure considerably (see the review in ref. 3). Itssuperior biomechanical properties, together with its interest tothe pearl industry and its possible biomedical uses (see e.g., ref.4), make nacre the subject of many biomimetic studies. Anultimate aim of such work is to mimic nacre in the laboratory,following the biological principles used by molluscs to producesuch a biomaterial (5). It is sine qua non for this objective to havea complete understanding of the mechanisms involved in nacregrowth.

Nacre is secreted only by the molluscan classes Gastropoda,Bivalvia, Cephalopoda, and, to a minor extent, Tryblidiida. It hasa lamellar structure consisting of alternating tablets of aragonite

300500 nm thick and 515 m wide and organic interlamellarmembranes 30 nm thick, which have a core of -chitin sur-rounded by acidic proteins (6). It is now clear that the sequenceof nacre for mation involves the secretion of interlamellar mem-branes (7) separated by a liquid rich in silk fibroin (5); onlysubsequently is the liquid replaced with mineral (79). Thispattern is the same for the bivalves and gastropods, and it is likelyso too for the other nacre-secreting molluscs, although this is yetto be determined. There are, however, structural differencesbetween bivalve and gastropod nacre. In the former group, theinterlamellar membranes are secreted with just the liquid-filledextrapallial space between them and the cells of the mantleepithelium, and mineralization within the membranes proceeds

in a step-like manner (7, 9). The nacre thus produced is said tohave a terraced arrangement (Fig. 1A).

In gastropods, however, the biomineralization compartmentof nacre is enclosed by a surface membrane first reported byNakahara (8) in Monodonta and Haliotis. Since its discovery, itsexistence went unremarked, until Cartwright and Checa (10)realized that it is widespread in nacre-secreting gastropods andthat the interlamellar membranes must necessarily detach fromit. The surface membrane acts as a protective seal, whichprevents the organic compounds and minerals involved in nacregrowth frombeinglost to theexternalenvironmentwhen thesoft

body of the gastropod withdraws into its shell, something evi-dently not necessary w ith bivalves. Below the surface membrane,many parallel interlamellar membranes with tablets growingbetween them can be found. These tablets are typically stackedin towers (Fig. 1B), with the smaller, more recently begun tabletsfound at the top. Although the nacre of gastropods, in particularthat of the abalone, i.e., the genus Haliotis, has been intensivelystudied, there are still many pieces to be assembled in the puzzle.One, perhaps key piece, is the surface membrane, key bothbecause it is intimately related to the other components of nacreand because the mineral ions and organic molecules for nacregrowth are necessarily introduced into the biomineralizationcompartment through it. Its ultrastructure, growth, and secre-tional activity have never been elucidated.

This work is dedicated to determining the relationship of thesurface membrane to the interlamellar membranes and mineraltablets. Our conclusions shed light not only on the dynamics ofgastropod nacre growth but also bear on the present debateabout whether superimposed nacre tablets nucleate and growonto the organic interlamellar matrix or, alternatively, whetherthere is crystallographic continuity between them across theinterlamellar membranes.

Results

Surface Membrane. The surface membrane extends between theadoral and apical boundaries of the nacreous layer, usuallybounded by the external spherulithic layer and an internalaragonitic lamellar layer of uncertain microstructure (Fig. 2A).

In Gibbula and Monodonta, at least, its mantle-side surface isdotted with bodies adheringto it (Fig. 2A andF). In transmissionelectron microscopy (TEM) sections these structures are seen tobe hollow (Figs. 2 B and C and 3C) and thus may be called

Author contributions: A.G.C. and J.H.E.C. designed research; A.G.C., J.H.E.C., and M.-G.W.

performed research; A.G.C. and M.-G.W. analyzed data; and A.G.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/cgi/content/full/

0808796106/DCSupplemental.

2008 by The National Academy of Sciences of the USA

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vesicles. They vary in shape from spherical, when they are justtouching the surface membrane, to strongly compressed, whenthey are partially or wholly integrated into thesurface membrane(Fig. 2 B and C). Their walls are electron-dense and have a meanthickness of 1015 nm (Fig. 2B Inset).

In section, the surface membrane has a mean thickness of 100nm, markedly thicker than the underlying interlamellar mem-branes (30 nm) (Figs. 2 B, C, E, and F, 3 C and D, 4A). Infracture and TEM sections, it has a homogeneous appearance.

Relationship Between the Surface Membrane and Other Nacre Com-

ponents. Examination of the nacre side of the sur face membranereveals that the interlamellar membranes develop in close con-tact with the surface membrane. The difference between themis evident from their fibrous nature compared with the smooth-ness of the surface membrane (Fig. 2D). In section, it is notablethat the surface membrane generally intercepts the last-formedinterlamellar membranes at a shallow angle in the apical direc-tion (Fig. 2E; see also ref. 10, Fig. 4 g and h).

Scanning electron microscopy (SEM) observations of thenacre side of the surface membrane also reveal the existence oftablet cores growing between the last-formed interlamellar

membrane and the surface membrane and partly encased withinit (Fig. 2 D and E). Where tablets have been torn off uponcontraction of the membranes during sample preparation, thescars remaining can also be seen (Fig. 2D). The width of thesecores is estimated to be between 100 and 200 nm (Figs. 2 E andF, and 3 A, C, and D). The topographical relationship observedin SEM samples is so recurrent that the possibility that this isartifactual can be excluded. The relationship is further demon-strated by TEM, which reveals that the tip of a growing tablet

(i.e., the last 5070 nm) is directly embedded within the surfacemembrane (Fig. 3 A, C, and D).

In the few instances when towers are fortuitously sectionedexactly through their central axis, the interlamellar membranepossesses a fuzzy appearance or is totally absent. The disappear-ance of theinterlamellar membraneat thevery axes of the towersis evident in some exceptional TEM views (Fig. 3). The partialdissolution sometimes produced during sample preparation (Fig.3 D and E) does not affect the tower axis area. TEM views ofdecalcified towers of Gibbula umbilicalis show too that theinterlamellar membranes are missing at the very axes of thetowers, across a maximal width of100 nm, or are replaced bya fuzzy band of organic matter with a different orientation (Fig.4A). SEM observation in back-scattered electron (BSE) mode of

polished axial sections of nacre towers of the same species, in which we can safely assume that membranes have not beendisturbed during sample preparation, manifests that the sameeffect may take place across tens of tablets in a tower (Fig. 4B).The fuzzy band, when present, usually cur ves slightly toward thetop of the tower. When the same samples are decalcified withmethanolic solution, the axes of the towers are marked by asuccession of holes (150 nm wide) with coarsened rims,sometimes traversed by organic threads (Fig. 4 C and D). Theregularity and persistence of such structures exclude the possi-bility that they are artifacts caused by dissolution. Treatment

with 2% EDTA preferentially removes the calcified organic-richcomponents: the interlamellar organic membranes, the lateralboundaries between tablets and, interestingly, the parts of thetablets coinciding with the axes of towers [supporting informa-tion (SI) Fig. S1].

High-resolution TEM observations show that the interfacebetween two superimposed tablets is fully crystalline at the axis(Fig. 5A). Observation of lattice fringes and fast Fourier trans-form (FFT) analysis of small areas provide additional evidenceof this crystalline character. The patterns obtained, althoughindicative of nonuniform orientation (Fig. 5B), are comparable

with those obtained within the interior of nacre tablets, whichare composed of nanodomains with variable orientations (X. Li,personal c ommunication).

Discussion

Our results, together with those of Nakahara (8, 9, 11), dem-

onstrate that the surface membrane is present in representativesof at least three (Haliotidae, Trochidae, and Turbinidae) of thesix nacre-secreting families of gastropods (12), all grouped withinthe Vetigastropoda (13). We may hence consider the surfacemembrane a basic element of gastropod nacre. Although itsgrowth dynamics is not yet totally elucidated, the surface mem-brane seems to form by addition of organic vesicles to itsmantle-side surface (Fig. 2B), which gradually integrate into it(Fig. 2C). We hypothesize that this is a means by which com-ponents necessary for the production of organic and crystallinestructures w ithin the nacre compartment are transported. Theseobservations are compatible w ith others that show that thesurface layers of the tablets are of amorphous calcium carbonate

A

B

Fig. 1. Bivalve and gastropod nacre growth compared. (A) Oblique view of

the terraced nacre of the bivalve P. margaritifera. (B) Oblique view of the

towered nacre of the gastropod Perotrochus caledonicus.

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(14) and that mineral may be precipitated intracellularly beforebeing transported to the mineralization site where it is remod-eled (15), but the exact sequence of events in mineralization isa matter for further study.

Based on our results, we reach four main conclusions. (i)Interlamellar membranes detach from the nacre side of thesurface membrane. From the topographic relationships betweenstructures, Cartwright and Checa (10) concluded that interla-mellar membranes form at the nacre side of the surface mem-brane and detach from it in an apical direction. Our observationssupport this view (Fig. 2 D and E). In this way, the balancebetween the components acquired via vesicle addition and thoselost because of the formation of interlamellar membranes re-mains steady so that the sur face membrane maintains a constantthickness.

(ii) Nacre tablets begin growing w ithin the surface membrane.It is known that tablets first acquire their maximal height andsubsequently expand sideways until they impinge on each other(16). Their initial growth in height keeps pace with the separa-tion of an interlamellar membrane from the surface membraneso that they stay in c ontact with the rest of the tower below them,at the same time that their tips remain permanently submerged

within the surface membrane (Fig. 3 A, C, and D). Nakahara (8)and Mutvei (17) concluded, based on TEM and etching tech-niques, respectively, that nacre tablets have an organic-rich core.In our EDTA-treated samples the cores of tablets etch prefer-entially (Fig. S1), which is also indicative of their organic-richcomposition. This is c omprehensible because during their initial

growth within the surface membrane, the tablets should incor-porate organic material from the surface membrane.

(iii) The interlamellar membrane disappears completely orcontinues only as a diffuse band at the very core of the crystallinetablets (Fig. 3 and Fig. 4 AD). Interlamellar membranes areelectron-dense and sometimes appear bi- or trilayered (see alsorefs. 7, 8, and 11 and Figs. 2C and 3D); their aspect as a fuzzyband in samples cut exactly through the tower axis (Figs. 3 and4) is thus quite distinct (Fig. 4A Inset).

(iv) At the location of the core, the inter face between growingtablets is fully crystalline. This observation (Fig. 5) does nottotally preclude the existence of the interlamellar membrane atthis position because Rousseau et al. (18) showed that themineralized interlamellar membranes of the pearl oysterPinctada margaritifera are partly nanocrystalline. In our case,FFT patterns (Fig. 5B) are identical to those obtained within theinterior of nacre tablets, being characterized by a nanodomain-like ultrastructure.

These four findings above can be understood coherently withthe following model. A new interlamellar membrane does notpenetrate through the very core of the tablet because coreformation precedes that of the interlamellar membrane; when itdetaches from the surface membrane, the tip of the tablet coreis already present. The fuzzy band sometimes observed may beexplained by competition between the mineral and the organicmolecules leading to cocr ystallization. The sequence of events isthus as follows: (i) formation of an organic-rich tablet core,

which grows rapidly in height with its tipembeddedin thesurfacemembrane (Fig. 6A); (ii) cessation of tablet growth in height and

A B C

D E F

Fig. 2. SEM views of the nacre of G. pennanti. (A) View of the internal surface of the shell, close to the aperture. The nacre compartment is overlain by the

surface membrane, visible as the darker area, which has cracked and curled upon contraction during preparation. (Inset) View of the mantle side of the surface

membrane with vesicles. (B) TEM section of decalcified nacre with vesicles adhering to the mantle side of the surface layer. (Inset) Bilayered appearance of the

wall of one such vesicle. (C) As in B, with some vesicles apparently in the process of being incorporated into the surface membrane. ( D) Nacre-side view of the

surface membrane, with the last-formed interlamellar membrane adhering. The surface membrane can be differentiatedby its smooth aspect. Arrows indicate

two tablet cores semidetached fromthe surface membrane.(Eand F) Transverse views of the surface membraneand of the underlying interlamellar membrane

and tablets in formation. (E) The last-formed interlamellar membrane and the surface membrane meet in the apical direction ( Rightof photograph). (F) The

surface membrane (with vesicles)has partlybeentorn off, which hasexposedthe last core. il,internallamellar layer;ilm, interlamellar membranes; n, nacre;os,

outer spherulithic layer; sm, surface membrane.

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simultaneous detachment of another interlamellar membrane;organic compounds may adhere to the tip of the tablet within thesurface membrane (Fig. 6B); (iii) formation of a new tablet core(Fig. 6C).

The basic model for gastropod nacre growth of Nakahara (8,9), which states that interlamellar membranes and the com-

partments they produce are formed in advance of the nacretablets, thus has to be modified insofar as each interlamellarmembrane is formed after the c ore of the tablet, which thusbecomes a distinct part within the tablet, but before the t ablet

A B C

D E F

Fig. 3. TEM sections that have partly penetrated the axes of nacre towers of M. labio in formation show crystal continuity and/or the absence of interlamellar

membranes (arrows). (Cand E Insets) Details. (B and F) Details of A and E, respectively. (D and E) White areas are where tablets have partly disappeared during

sample preparation. ilm, interlamellar membranes; sm, surface membrane; v, vesicles.

A B

C D

Fig. 4. Nacre of G. umbilicalis. (A) TEM section approximately through the

axis of a decalcified sample. The interlamellar membranes are seen as diffuse

organic membranes or disappear entirely at the axial zone. (Inset) Detail of

two such interlamellar membranes. (B) Polished section through the axis of a

nacre tower (BSE mode). (Inset) The organic membranes become diffuse and

tend to be convexupward.(Cand D) Fixed and decalcified polished section of

nacre. The axis of the tower is marked by aligned holes with coarsened rims

within theinterlamellarmembranes(arrows). (C) Succession of10 suchholes

thatare perfectlyaligned. Theirmean diameter is150 nm. (D) Similar case in

which a hole has been sectioned. ilm, interlamellar membranes; sm, surface

membrane.

A

B

Fig. 5. High-resolution TEM study of the axial zones of nacre tablets of M.

labio. (A) The contact between the last two tablets is fully crystalline and

consists of a single crystal domain. (B) The different areas at and around the

contact between the last two tablets are crystalline, as shown by the FFT

patterns. ilm, position of interlamellar membranes.



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begins to expand laterally. The above model implies that nacretablets are crystallographically connected at their cores. Theexistence of a fuzzy organic band of material in between shouldnot represent an obstacle for crystallographic continuity, asshown by our analysis of the lattice fringes. This would explain

why nacre tablets stacked along a single tower retain theiroverall crystallographic orientation (19 and unpublisheddata). In more detail, nacre tablets are composed of manytwinned crystals (17), and we present additional TEM evi-dence (Fig. S2) that multiple cryst al orientations are inheritedby newborn tablets.

Cartwright and Checa (10) hypothesized that the differentstacking patterns of gastropods and bivalves could be related tothe sizes and densities of the nanopores they observed in theinterlamellar membranes. The present work shows that thedifference is not merely quantitative because the existence of thesurface membrane and its associated effect on nacre growthstrongly promote vertical stacking in gastropods.

In implying the crystallographic continuity of the cores of

tablets, our hypothesis bears implications on the present debateof whether tablets communicate across lamellae or not. Thisdebate began with Weiner and Traub (20), who found that thefiber axis of the chitin and silk protein for ming the interlamellarmatrix are perpendicular to each other and aligned with the a-and b-axes of the aragonite tablets, respectively. They proposedthat the mineral phase grows epitaxially onto the protein chainsof the organic matrix; this is the heteroepitaxial theory. Schafferet al. (21) recognized the existence of many pores, several tensof nanometers across, in the intercrystalline matrix of abalonenacre, which they showed to be permeable to ions. They sug-gested that some of the pores allow tablets to grow from onelayer to the next, without the need for a new nucleation event;this is the mineral-bridge theory. From then on, the heteroepi-

taxial and mineral-bridge theories have been two conflicting

views on how nacre tablets relate to each other (see the reviewin ref. 10). The evidence we have presented here does not ruleout either theory, but it does show that, for gastropod nacre,there is a third way: tablets are connected at the tower axes. Thisconnection, in turn, explains the tower-like growth of gastropod

nacre.Materials and MethodsSEM. Shells of living specimens of G. umbilicalis, Gibbula pennanti, Mon-odonta sp., and Calliostoma zyzyphinus werefixed with2.5% glutaraldehydein a 0.1 M cacodylate buffer. Samples were usually observed intact after CO 2criticalpointdrying.Some polishedsections weredecalcifiedaccording to twodifferent protocols: (i) 2%EDTA for 23 min;(ii) fixation of theorganicmatrixwith a mixture of 2.5% glutaraldehyde and 2% formaldehyde and furtherdemineralization witha methanolic solution(3:1:6)in a gelmedium (protocolby A. Hernandez-Hernandez, unpublisheddata). This procedurepreserves thefinest details of the organic membranes (note, e.g., the nanopores in theinterlamellar membranes in Fig. 4 Cand D).

TEM. Resin-embedded specimensof G. umbilicaliswere completelydecalcifiedwith 2%EDTAand preparedwithan ultramicrotomein thestandard way. Wealso had access to original material of Monodonta labio, and Haliotis rufe-

scens of the lateH. Nakahara. The sampleswere preparedin Meikai Universityby M. Kakei according to the protocol described in ref. 9. Only samples of M.labio rendered significant results.

We used a Leo Gemini 1530 field-emission SEM and a Philips CM20 TEM ofthe Centro de Instrumentacion Cientfica, University of Granada. High-resolution TEM analysis was carried out in a Jeol 2200FS at the Centro deInvestigacao em Mate riais Ceramicos e Com positos, U niversity of Aveiro .

ACKNOWLEDGMENTS. We wishto expressour profoundestappreciationto H.Nakahara(19282001). In noticingthe existenceof thesurfacemembraneandof an organic core along the axes of the nacre towers, he largely inspired ourwork, which also benefitted from the study of his unique material. We thankM. Kakei (Meikai University) for providing TEM material of H. Nakahara, A .Hernandez-Hernandez (Consej o Superior de Inves tigaciones CientficasUniversidad de Granada) for sampling preparation with her own fixative anddecalcification technique, M. Rousseau(MuseumNatural dHistoire Naturelle,Paris)for Fig. 1B, and E. M. Harper (Cambridge University) for critical revision.This work was supported by Research Project CGL2007-60549 (Ministerio de

Ciencia e Innovacin) and the Research Group RNM190 (Junta de Andaluca).

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Fig. 6. Scheme for the formation of incipient nacre tablets. (A) The tablet core grows rapidly in height with its tip immersed within the surface membrane.

An organic-rich core is formed as the growing tablet absorbs components of the surface membrane. (B) At the same time that vertical crystal growth ceases, a

new interlamellar membrane is secreted at the nacre side of the surface membrane. During this time interval, organic material may precipitate on top of the

tablet core. (C) Growth of a new tablet commences.

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(Biología Marina o Limnología) El papel clave de la membrana de la superficie es saber qué gasterópodo nácar crece en torres