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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
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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. We
have also derived a good deal of information on the relationship between amorphous and crystalline deposits and the way
that organisms may exploit the control over
the formation and subsequent properties
of these minerals. I would suggest therefore that the use of metal ions as probes of
calcification is a powerful tool. Certainly
we have found it a useful way of exposing
a Karyotic Mineralization Window and in
our opinion KMW will continue to provide
a powerful way of understanding biomineralization.
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