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AMER. ZOOL., 24:847-856 (1984) The Karyotic Mineralization Window (KMW)1 KENNETH SIMKISS Department of Pure and Applied Zoology, University of Reading, Whiteknights, Reading, England, RC6 2AJ SNYOPSIS. Biomineralization is frequently a transient and intermittent activity characterised as a phase change in a small fluid-filled space bounded by sensitive cells. It is therefore a difficult phenomenon to identify and to experiment upon. The possibility has therefore been pursued of obtaining a "Mineralization Window" onto this process by following the activities of mixtures of trace amounts of inorganic ions. The results of a number of such experiments are considered in relation to the three different types of calcifying systems originally identified by Wilbur. The method of metal ion pairs is also of value in probing the relationships between amorphous deposits and the various crystalline minerals that may be formed from them. space. It is this property and the sensitivity of the cells associated with it that make the process so difficult to study. One is therefore continually searching for an experimental window onto the process within which one can manipulate the mineralization system. As I settled into Wilbur's laboratory I realized that KMW stood for the Karyotic Mineralization Window and that studying this was essential for any work on calcification. INTRODUCTION I first met Karl M. Wilbur 21 years ago when I spent a year on a sabbatical visit to his laboratory at Duke University. At that time Watabe and Wilbur (1960) had just published their experiments on the influence of molluscan shell matrix on crystal formation and I was keen to learn more about these results and their implications of epitaxy. When I arrived in North Carolina I found, however, that Wilbur's laboratory had temporarily moved away from molluscs and reoriented to study coccolithophorids. There were two reasons for this. First these organisms form plates or coccoliths of calcium carbonate within specific organelles and this has clear implications for the way that cells move ions and deposit minerals. Second, coccolithophorids form mineral deposits in such numbers and with such regularity that they provide a predictable experimental system. In later years this was to be called the August Krogh Principle ("For many problems there is an animal on which it can be most conveniently studied," Krebs, 1975) but like many good experimentalists Karl M. Wilbur had realized this early in his career. The difficulty, however, with biomineralization is that the problems are often so ill defined that it is not easy to select the ideal organism. Typically the process occurs as a rapid phase change in a small fluid filled DEFINING THE PROBLEMS There have been four steps in my search for a suitable Karyotic Mineralization Window. These have been a) recognition of the requirement for ion regulation at sites of biomineralization, b) the need for a clear distinction between intracellular and extracellular events because of the enormously different chemical potential for calcium at these different sites, c) the implications of these energy differences in terms of solubilities and mineral forms and d) the rationalization of the many diverse examples of biomineralization both in terms of cellular and crystallographic influences. Ionic regulation While working with Karl Wilbur on the remineralization of molluscan shell matrix we discovered two phenomena. Both arose from comparisons between using natural and artificial sea water as the fluid from which to induce the formation of crystals 1 From the Symposium on Mechanisms ofCalcification of calcium carbonate. In the first experiin Biological Systems presented at the Annual Meeting of the American Society of Zoologists, 27-30 Decem- ments we found that the presence or absence of magnesium in the solution had ber 1983, at Philadelphia, Pennsylvania. 847 JA « 2 "86 MERNER-PFEIFFER LIBRARY TENNESSEE WESLEYAN COLLEGE 848 KENNETH SIMKISS a critical effect upon whether aragonite or calcite was formed (Simkiss, 1964a). In the second experiments we discovered that crystals of calcium carbonate formed very easily in artificial sea water but that natural sea water contained strong inhibitors to this process. The addition of trace amounts (i.e., 10~7 mole/litre) of a variety of phosphate compounds converted artificial sea water into a solution which behaved similarly to natural sea water (Simkiss, 19646). On the basis of these experiments we suggested that there must be ion regulation at sites of biomineralization (Simkiss, 1964c) and that so-called crystal poisons could be strong regulators of mineral deposition (Simkiss, rf Intra vs. extra cellular mineralization A decade after I left North Carolina Watabe and Wilbur organized a conference on mechanisms of mineralization at which I attempted to review the cellular aspects of calcification. In working on that challenge it became apparent that two components of the process needed to be clarified. The first was the distinction between raising the activities of the crystal lattice ions in the mineralizing fluid to a level in excess of the solubility product constant for a particular mineral (process 1) and the second was the formation of a solid phase (process 2). It was argued that the two processes need not occur either simultaneously or at the same site and that one may be a function of intracellular processes while the other may occur extracellularly (Simkiss, 1976a). The simplest way to illustrate this set of concepts was to trace the energy requirements involved in the various theories of biomineralization. A modification of that scheme is shown in Figure 1. Its main purpose was to make explicit the energetic steps that would be involved in cell mediated systems of mineral deposition. Mineral solubilities The solubility of minerals in relation to the products of the activities of the ions in the various body fluids remains as one of the great problem areas of biomineralization. It is possible, for example, to extract Cell Shell FIG. 1. Diagrammatic representation of two epithelial cells forming a calcareous shell. The graph indicates the energy required to transport calcium across the cells. 1. Shows calcium entering a cell down a concentration gradient with the plasma membrane pumping the ion out against this electrochemical force. 2. Indicates the pump required to move calcium from the cytoplasm into an organelle and to raise the concentration there to a level suitable for mineral formation. 3. Represents the energy required to raise the ionic product of the fluid surrounding the shell to a level whereby amorphous or crystalline deposits form in the presence or absence of epitaxy. The broken line i.e. shows how these energy barriers are avoided by the intercellular route. N = nucleus (after Simkiss, 19766). a variety of intracellular granules from the snail Helix aspersa and shake them with various physiological salines so as to obtain a rough indication of their solubility (Simkiss, 19766). The results are surprising (Table 1). Granules extracted from connective tissue calcium cells are remarkably soluble whereas granules from basophil cells are effectively insoluble (Simkiss and Mason, 1983). The first type of granule contains carbonate while the second is phosphatic, but that is not the only reason for their different solubilities. Both types of deposit are amorphous to X-ray or electron diffraction. Amorphous deposits are less well organized than crystals and therefore are more soluble, permitting the entry and exit of various ions over lower energy barriers. Because crystals are less soluble than amor- 849 MINERALIZATION WINDOW TABLE 1. Composition of saline after shaking with various amorphous intracellular granules obtained from tissues of Helix aspersa (after Simkiss, 1976b). Saline composition (mmole/l) Tissue None Foot Hepatopancreas Cell type Connective tissue calcium cell Basophil cell composition Ca PO, pH CaCO, 2.3 9.4 1.3 0.6 7.6 7.7 CaMgP 2 O 7 2.1 1.5 7.7 phous materials biologists have intuitively Bubel, 1975). It was, therefore, a great considered that these less soluble and more rationalization, when Wilbur proposed that stable crystalline forms should form pref- there are three main types of system (Wilerentially. The converse appears to be the bur and Simkiss, 1979). These are (i) calcase, however, since amorphous deposits cification within vesicles or vacuoles, (ii) will frequently form before crystalline ones extracellular calcification by single cells and for kinetic reasons (Fig. 2). Eventually, of (iii) extracellular calcification by epithelia. course, these amorphous deposits should The three types are illustrated in Figure transform to the more stable crystalline 3. They are undoubtedly an over-simpliforms as the bulk lattice energy effect fication but they provide for the first time becomes more important than the surface a simple conceptual framework upon which energy effect. The process may, however, to organize the enormous amount of anabe very slow and may involve recrystalli- tomical information that is now available. zation through a variety of forms from the In each situation there is a membrane-lined more soluble (e.g., vaterite and aragonite) to surface, a fluid layer and a mineral interthe less soluble (e.g., calcite). This is the so-face. In each case, however, there are specalled Ostwald-Lussac law (Nancollas, 1982; cific biological implications as to how the cells and their organelles function in each Nielsen and Christoffersen, 1982). The implications of these findings are of these different arrangements. This clascrucial to all studies of biomineralization sification, therefore, also raised the quesfor two reasons. The first is that amor- tion as to whether a corresponding physphous deposits have entirely different iological analysis could be proposed. properties from crystals and may, thereEVOLVING A TECHNIQUE fore, be selected for a variety of biological functions (e.g., strength, solubility, interThe basic experimental problems in action with foreign ions, etc.). The second studying biomineralization are that the is that they emphasize again the need to processes are transient and intermittent; separate, conceptually, the processes they occur at sites that are hard to sample involved in increasing ion activities (pro- and it is difficult to localize and quantify cess 1) from those of solid phase formation the activities. No single approach is going (process 2) when looking at the physiolog- to resolve all those difficulties but it is clear ical basis of biomineralization in an organ- from the suggestions in Figures 1 to 3 that a study of the energy relationships of the ism. various processes would be a major Forms of biomineralization advance. The energy requirements for the The morphological organization of the various routes of ion movement are clearly biological systems that are active in bio- different as indeed are the rates of energy mineralization can be extremely confusing release for mineral deposition. The probranging from details of organelle involve- lem is, however, that at the present time it ment (Leadbeater, 1979; Davis et al, 1982), is virtually impossible to find an expericellular movement (e.g., Okazaki, 1975) and mental method that is sensitive enough to epithelial function (e.g., Stevenson, 1972; measure these processes and to find a 850 KENNETH SIMKISS fluid epithelium | shell amorph crystal n/wi/i/inn/iM FIG. 3. The three basic types of mineralization system as suggested by Wilbur. These are (left to right) epithelial, intracellular and extracellular by single cells. K K sp (crystaD(amorph) sp activity product FIG. 2. The relationship between nucleation rate (JN) and activity product. The supersaturation (S) for a crystalline deposit always exceeds that of an amorphous product because of the different solubility products (K,p). Amorphous deposits may be the first material to form however for kinetic reasons (after Mann, 1983). method to partition these activities from the other major metabolic events that are occurring. We have therefore adopted a different approach. One of the characteristics of biological activities is that they involve the localized and sequential release of small amounts of energy. This occurs by using either energized reactions (such as those involving ATP) or through specific ligands (such as those involving carrier proteins and membrane mediated activities). These events are characterized not just by their controlled release of energy but also by their specificity. By combining these two features we have therefore devised a technique that will distinguish between the different types of mineralization process. The basic concept is shown in Figure 4. Whenever calcium moves across one of the energy steps shown in Figure 1 it will do so by a process that involves a certain degree of specificity. Thus if an analogue (A) of calcium is put into the system there will be a change in the ratio of Ca/A at each energetic step. The change in the ratio will depend upon the specificity inherent in the activities at the "energy step" and upon the properties of the analogue. Since inorganic chemistry has provided us with a whole range of analogues which vary in ionic radius, electron structure, coordination number, etc. it should be possible to identify not just the occurrence of the energy steps shown in Figure 1 but also the properties of the system that interest us for this will be reflected in the way that the biomineralization process discriminates against different analogues. The same technique will enable us to identify those events on the mineralization face that are due to cell mediated properties from those that are due to crystallographic influences. Thus, by sampling the discriminatory activities between calcium and a variety of analogues it should be possible to identify sites and patterns of cellular activity. The method is basically one that is familiar to physiologists in other systems as a change in the product to precursor ratio (Fig. 4). In the case of biomineralization, however, it will be advantageous to include in vivo and in vitro comparisons so that the discrimination at the mineralization site that is due to lattice effects can be separated from the biological activities of the adjacent tissues. There are a number of additional advantages of this technique. Thus, there is relatively little interference with the organism during the time that the experiment is in operation and furthermore, since one is dealing with ratios, the extent that the Karyotic Mineralization Window is open is relatively unimportant. 851 MINERALIZATION WINDOW Shell Cell SOME PRELIMINARY APPLICATIONS OF THE TECHNIQUE Formation of extracellular minerals The discovery that there are specific calcium binding properties that are vitamin D dependent led Wasserman (1968) to consider that they were involved in the transport of calcium ions across cellular epithelia. The subsequent discovery of a similar protein in the shell gland of the domestic fowl (Corradino etal, 1968) resulted in the suggestion that these proteins were similarly involved in the transfer of calcium ions across the shell gland mucosa from the blood to the site of shell deposition. The formation constants of the calcium and strontium forms of this binding protein were determined by Wasserman et al. (1968) and since they differ considerably we decided to inject 45Ca and 85Sr simultaneously into the plasma of laying birds to see if there was any subsequent discrimination between these ions by the oviduct in the formation of the eggshell (Simkiss et al, 1973). When the ratio of 45Ca/85Sr in the blood was normalized the subsequent ratio of 45Ca/85Sr in the eggshell 4 hr later was between 0.94 and 1.05 with a mean of 1.00. In terms of a product to precursor ratio Amorph /Crystal 7 / ^pitaxy /"crystal Amorph 2/ c UJ / o kJ Ca5/85Srs = 1.00 45 Cab/85Srb Fie. 4. An illustration of the way that energy barriers to biomineralization would influence the ratio of calcium and an analogue (A) in the various compartments of the cellular process. The energy barriers shown in Figure 1 influence the ratio of Ca/A (broken line) since entry into the cell is resisted by a calcium pump (i.e., Ca/A falls at 1). Entry into organelles favours calcium (i.e., Ca/A rises at 2) as does secretion at the epithelial surface (i.e., Ca/A again rises at 3). Discrimination by the crystal lattice again increases the Ca/A ratio although this effect is much less marked for amorphous deposits. where 45Cas/85Srs is the ratio of the isotopes in the shell and 45Cab/85Srb is the ratio of the isotopes in the blood. During these experiments the ratio of 45 Cab/85Srb began to fall as calcium was removed from the blood and it was, therefore, clear that the technique would be most useful in acute experiments. Two further difficulties with the method were that the calcium regulatory systems were clearly in operation during the experiment and this availability of internal stores made the calculation of specific activities very difficult. We also assumed in this experiment that strontium was able to enter the calcite lattice in trace amounts without any crystallographic discrimination. This was a reasonable assumption for that particular experiment but it may be less acceptable in other systems that do not mineralize as quickly as the eggshell. In a second set of experiments these objections were partly overcome by using two analogues of calcium so that specific activity effects could be calculated on a comparable basis. We also attempted to measure any discrimination at the mineral surface. The optic tentacle of the snail Helix aspersa was cannulated and equimolar trace amounts of 85Sr and MMn were added to the haemolymph. After a period of 6 hr pieces were removed from the edge of the shell and counted for the two isotopes. At 45 852 KENNETH SIMKISS J that blood is directly contaminating the shell by passing across the epidermis of the snail (Simkiss and Wilbur, 1977; Martin and Deyrup-Olsen, 1982) but this has not been investigated further. Formation of intracellular minerals metal ions 1 Fie. 5. An interpretation of the changes in metal ratios found in basophil cells in terms of metabolic pathways. Strontium is expelled from cells so that it does not become incorporated into granules. Zinc and cadmium become bound to cytoplasmic proteins while other metals become associated with intracellular granules (after Simkiss, 1981). the same time another snail was bled and pieces of untreated shell were shaken with the haemolymph that was spiked with the same ratio of 85Sr and 54Mn ions. The results for 85 Sr/ 54 Mn in vivo were 5.66 ± 2.30 and those obtained in vitro were 5.08 ± 1.12 (Simkiss, 1981). The results of both these sets of experiments are preliminary but the implications are clear. There is no clear evidence of any discrimination between these pairs of ions during their passage from the blood to the site of mineralization. There is clear evidence for the preferential incorporation of Sr relative to Mn into the shell mineral, but no similar effect that could be attributed to the cellular epithelia. It is concluded, therefore, that there is no evidence for the involvement of binding proteins or in fact any other discriminatory cellular activity in the transport of these ions across these epithelia. It is suggested, therefore, that these ions reach the sites of mineralization via intercellular routes. There is one alternative possibility namely In order to investigate the technique with an example where minerals are clearly formed in an intracellular location an extensive study was made of the basophil cells of the hepatopancreas of Helix aspersa. These cells contain numerous membranebound granules of calcium pyrophosphate (Howard etal., 1981). In these experiments a complete range of metal ion pairs was injected into the haemocoel and the ratios of the metals were determined in the haemolymph, the hepatopancreas and the intracellular granules. The results were compared with similar ratios found by shaking granules with metal spiked haemolymph in vitro (Simkiss, 1981). The rate at which one metal entered the system relative to another varied over a thousand-fold and gave the following series. Hepatopancreas Mn > Cd > Zn > Co > Sr Granules in vivo Mn > Zn > Co > Cd > Sr Granules in vitro Mn > Sr > Cd > Zn > Co The differences between these relative uptake rates are explained in Figure 5. Strontium is clearly able to enter granules very readily in vitro but this does not happen in vivo. Thus, there is cellular discrimination against this ion. Cadmium rapidly enters the hepatopancreas but does not enter the granule. It is probably trapped by cadmium binding proteins in the cytoplasm. Zinc and cobalt enter the granules better in vivo than in vitro. This presumably suggests they are preferentially incorporated into these deposits across the limiting membrane. The relative discrimination of these various ions is clear evidence therefore for membrane selection, protein binding and granule deposition systems, i. e., the full complement of cellular activities related to metal ion transport. MINERALIZATION WINDOW 853 Mineralogical effects The fundamental activity influencing biologically controlled mineralization is the regulation of ions and molecules at the mineralizing site. We recognized 20 years ago that biogenic minerals were formed in the presence of other ions such as Na + , K+, Mg2+, OH", C1-, H C O r , P 2 O 7 4 -. The true significance of the variety of these interactions is, however, only just being realized. Ions in solution can be incorporated into mineral lattices so as to modify crystal growth, crystal morphology and chemical reactivity in a variety of ways. A mismatch of ion size, charge or polarization of ions in a lattice can probably not be tolerated if it exceeds 15% and for this reason MgCO3 coprecipitates with calcite but not aragonite. Magnesium ions however will readily be adsorbed onto the calcite surface and thereby retard its formation while having no effect upon the rate of crystal growth of aragonite. Magnesium therefore becomes incorporated into calcite but favours the formation of aragonite. Ions in the solutions from which minerals form are therefore capable of modifying the rate of crystal growth, the crystal faces which develop and the types of crystal which occur (Mann, 1983). These developments were partially predictable 20 years ago but an appreciation of their relevance to the free energy states and activation energy barriers for mineralization has been a major revolution instigated, at least in biological studies, by high resolution electron microscopy. Many of the examples of biological minerals that were thought to be single crystals are now recognized as being composites of iso-oriented crystallites or microdomains (e.g., Emlet, 1982 for echinoderms; Parker et al., 1983 for coccoliths). Furthermore, many biological "granules" are now known to be amorphous to X ray or electron diffraction (Simkiss, 19766). If the amorphous phase is a precursor of crystal forms and is favoured for kinetic reasons (Fig. 2) then the biological manipulation of ions at the site of mineralization will enable the organism to stabilize any of these products. Since each of the calcium carbonate minerals will form in the Fie. 6. Section of a basophil cell of a snail (H. aspersa) exposed to manganese ions. The manganese occurs as deposits on the outermost surface of the membrane-bound granules (x5,200). (Courtesy A. Z. Mason) sequence of their solubility products, i.e., amorphous -> monohydrite -> vaterite -» aragonite -» calcite and since each may have its own advantages for some biological process it will not be too surprising to find that particular forms are "stabilized" to perform particular functions. These functions may include the cellular shaping of mineralized deposits, the loss or entry of ions into deposits or the mechanical properties of the minerals. The organism will therefore be able to direct these properties by dictating the type of mineral phase that is stabilized. The use of inorganic analogues also permits these aspects of biomineralization to be probed and two examples of this have been observed in our recent experiments. The connective tissue calcium cells of mol- 854 KENNETH SIMKISS FIC. 7. Granules of the type shown in Figure 6 extracted and viewed by scanning electron microscopy. Note that the manganese deposits occur as crystalline concretions on the amorphous calcium spheres (x 9,200). luscs produce amorphous deposits of calcium carbonate. These are highly soluble since the amorphous structure imposes only weak constraints upon ions leaving these deposits. This accounts for the results shown in Table 1 and for the fact that the mollusc can use these cells to regulate its blood calcium and acid-base balance (Simkiss and Mason, 1983) and perhaps even to drive the extracellular mineralization process (Simkiss, 1976i). Somewhat similar deposits of phosphatic material occur in the basophil cells. In this case, however, the amorphous nature of the deposits appears to function as a detoxification system since a variety of pollutant ions can readily pass into these granules where they accumulate by exchange and accretion processes (Simkiss et al., 1982). Results in keeping with this concept have recently been obtained by Silverman et al. (1983) who found that during anoxic dissolution of the shell the excess calcium ions that were released became incorporated into extracellular amorphous granules of calcium phosphate. The second example is even more provocative. Our ultrastructural studies on the formation of amorphous granules have recently led us to use manganese to show for perhaps the first time in a eukaryote the transport of a metal across a cell with its subsequent incorporation at a site of biomineralization (Mason and Simkiss, 1982). As can be seen in Figure 6 the manganese becomes incorporated into the outermost layers of an intracellular granule that lies within a membrane-lined vacuole. If these granules are extracted and viewed by scanning electron microscopy the spheres of amorphous calcium and magnesium pyrophosphate are clearly seen while upon their surfaces are crystalline growths of manganese phosphate (Fig. 7). Clearly those influences which stabilize the calcium and magnesium salts as amorphous deposits are not effective on manganese which is laid down in a crystalline form. This provides a very clear demonstration of the cellular mechanisms that are manipulating the amorphous/crystalline aspects of biomineralization. Thus, whatever the mechanism is that stablizes calcium and magnesium pyrophosphate as an amorphous deposit it is not effective on the manganese salt. The use of inorganic analogues enables one therefore to probe both the cellular and the crystallographic mechanisms that are involved in biomineralization. SOME CONCLUSIONS I started this paper by explaining the need to find a novel method for studying some cellular aspects of biomineralization. What I have subsequently tried to do is put this search into the general context of inorganic biochemistry and in so doing raised the possibility of using metal ions as probes of the calcification process. In our applications of these techniques we have found MINERALIZATION WINDOW results that seem to be in keeping with Wilbur's anatomical classification in that they provide some evidence for differences in the physiology of intracellular and extracellular systems of biomineralization. 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