1895
Journal of Cell Science 108, 1895-1909 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
Direct visualization of a vast cortical calcium compartment in Paramecium by
secondary ion mass spectrometry (SIMS) microscopy: possible involvement
in exocytosis
Nicole Stelly1,*, Sylvain Halpern2, Gisèle Nicolas3, Philippe Fragu2 and André Adoutte1
1Laboratoire de Biologie Cellulaire 4 (CNRS, URA 1134), Bâtiment 444, Université Paris-Sud, 91405 Orsay Cedex, France
2Equipe de Microscopie Ionique (INSERM U66), Institut Gustave Roussy, 94800 Villejuif, France
3Centre Interuniversitaire de Microscopie Electronique (CNRS, URA 1488) et Laboratoire de Cytologie, Université Pierre et
Marie
Curie, 7 quai St Bernard, Bâtiment A, 75252 Paris Cedex 05, France
*Author for correspondence
SUMMARY
The plasma membrane of ciliates is underlaid by a vast
continuous array of membrane vesicles known as cortical
alveoli. Previous work had shown that a purified fraction
of these vesicles actively pumps calcium, suggesting that
alveoli may constitute a calcium-storage compartment.
Here we provide direct confirmation of this hypothesis
using in situ visualization of total cell calcium on sections
of cryofixed and cryosubstituted cells analyzed by SIMS
(secondary ion mass spectrometry) microscopy a method
never previously applied to protists. A narrow, continuous,
Ca-emitting zone located all along the cell periphery was
observed on sections including the cortex. In contrast, Na
and K were evenly distributed throughout the cell. Various
controls confirmed that emission was from the alveoli, in
INTRODUCTION
The question of the subcellular location of calcium (Ca) stores
in eukaryotic cells has attracted considerable attention in recent
years (see Koch, 1990; Meldolesi et al., 1990; Tsien and Tsien,
1990; Lytton and Nigam, 1992; Meldolesi and Villa, 1993, for
review). The measurement of free, cytosolic calcium (Ca2+)
concentration and its oscillation in single cells has become
possible with the advent of fluorescent probes (Grynkiewicz et
al., 1985; see Williams and Fay, 1990, for review). This concentration, however, is several orders of magnitude lower than
that of total cellular calcium; part of this large amount of
calcium is bound to cytosolic proteins but the majority, by far,
appears to be segregated inside membrane-bounded intracellular organelles where it is complexed with low-affinity, highcapacity proteins (Carafoli, 1987; Koch, 1990). Release of
Ca2+ from these organelles plays a key role in several
processes, especially in the response to various extracellular
stimuli.
Identification of these Ca-sequestering organelles has
proven difficult, however. Two major approaches have been
used, a direct one, seeking to visualize the element itself by
particular, the emitting zone was still seen in mutants
totally lacking trichocysts, the large exocytotic organelles
docked at the cell surface, indicating that they make no
major direct contribution to the emission. Calcium concentration within alveoli was quantified for the first time in
SIMS microscopy using an external reference and was
found to be in the range of 3 to 5 mM, a value similar to
that for sarcoplasmic reticulum. After massive induction of
trichocyst discharge, this concentration was found to
decrease by about 50%, suggesting that the alveoli are the
main source of the calcium involved in exocytosis.
Key words: calcium, SIMS, Paramecium
electron probe microanalysis of tissue sections (Somlyo, 1985;
Andrews et al., 1987), and an indirect one, seeking to identify
the organelles by virtue of the presence of a set of ‘marker’
proteins involved in Ca2+ homeostasis: Ca2+-ATPases, Ca2+
channels (ryanodine receptor, inositol-triphosphate (InsP3)
receptor) and Ca-binding proteins (calsequestrin, calreticulin,
etc.). These proteins were detected either, by physiological and
biochemical methods (such as by measurement of Ca2+ uptake
and release in subcellular fractions; see Pietrobon et al., 1990
for review) or by immunocytological approaches (subcellular
localization, at the EM level, of compartments reacting with
antibodies directed against the marker proteins; e.g. see Volpe
et al., 1988).
The results of these approaches are summarized in recent
reviews (cited above). Except for the striated muscle cell, an
extensively studied model system in which the situation is considerably clarified because of the amplification of the Ca
storage compartment achieved in the form of the sarcoplasmic
reticulum, the number, location and functional role of Ca
storage compartments in eukaryotic cells has until recently
been unclear. Except for mitochondria, which do not appear to
be involved in Ca2+ storage, the major intracellular Ca store
1896 N. Stelly and others
has, for many years, been assumed to be the endoplasmic
reticulum. Currently, at least two and more probably three or
even four types of compartments are implicated (Burgoyne and
Cheek, 1991; Lytton and Nigam, 1992; Sitia and Meldolesi,
1992; Meldolesi and Villa, 1993), depending on the cell type
analyzed, on the basis of the receptors, channels, pumps and
calcium-binding proteins that they contain. All of these compartments appear to belong to the general intracellular protein
sorting compartments (RER, Golgi, endosomes, etc.) although
they are more or less distantly connected to it.
In this paper, we present an approach complementary to
those just cited, aimed at directly visualizing total cellular Ca.
It involves the use of both a new cell type and a different
method. The cell type is the ciliated protozoan Paramecium,
in which we have recently shown that a vast vesicular network
lying just beneath the plasma membrane actively pumps Ca 2+
(Stelly et al., 1991). This ‘primitive’ organism therefore
appeared to offer a naturally amplified Ca storage compartment (akin to the sarcoplasmic reticulum), facilitating direct
visualization. The method is secondary ion mass spectrometry (SIMS; Castaing and Slodzian, 1962) microscopy, which
has been extensively used in solid state physics to characterize surface composition of samples, but much less in biological applications (see reviews by Chandra and Morrison,
1988; Fragu et al., 1992). A primary ion beam is focused onto
the surface of a tissue section, leading to the sputtering of the
most superficial atoms, themselves partly in the form of ions.
These secondary ions are then collected, analyzed with a mass
spectrometer and the corresponding image is reconstructed.
One therefore obtains an image of the distribution of a specific
atom at the surface of the specimen analyzed. This method
offers three main advantages. First, it allows visualization of
all the elements of Mendeleiev’s table as well as discrimination between many of their stable and radioactive isotopes.
By successively eroding the same section one can map several
different ions (for example, Ca2+, Na+, K+, etc.) in the same
tissue and cells. It should be stressed that ions are highly
prone to extraction from cytological preparations during
specimen preparation (see Mentré and Escaig, 1988). A prerequisite for all the approaches just outlined is to take suitable
precautions to avoid loss and/or redistribution of diffusible
compounds. This can be achieved by rapid freezing of the
cells, avoiding the use of any fixative, then either cryosubstituting the samples (as done in the present work) or using
freeze-fracturing followed by freeze-drying (Chandra and
Morrison, 1992). Second, its sensitivity is at least as good as
that of X-ray microanalysis (EPMA) and probably slightly
better (see below). This level of sensitivity is comparable or
slightly inferior to that of electron energy loss spectrometry
methods (EELS); Third, images of ion distribution can be
obtained over large areas of cells in a short time, making the
method especially valuable when there is a need to observe
whole tissues or extended portions of large cells. The major
drawback of SIMS microscopy is its limited lateral resolution,
especially when compared with EELS, being of the order of
0.5 µm for the present instruments, yielding images equivalent to those from a light microscope. This drawback is partly
compensated by the sensitivity of SIMS, the very low level
of background noise and the relative ease with which data
from large areas can be collected (for a detailed comparison
of the merits and limitations of the various microprobe
methods used in biological microanalysis, see Linton and
Goldsmith, 1992).
Paramecia have already been studied by microanalysis at the
EM level both in the EPMA (Schmitz et al., 1985; Zierold et
al., 1989) and the EELS mode (Knoll et al., 1993), using
excellent techniques of cryofixation followed by either freezedrying or cryosubstitution. The major result of these studies
was the identification of a peripheral Ca-containing zone in the
cortex of Paramecium (not including the exocytotic organelles
known as trichocysts) and preliminary evidence for redistribution of this calcium after induction of exocytosis in the EELS
high-resolution study (Knoll et al., 1993) but not in the EPMA
one (Zierold et al., 1989). Here, we extend these studies using
the different SIMS approach, which we adapted for fastswimming single cells. We confirm the occurrence of an
intensely emitting peripheral rim of calcium and, through the
use of mutants devoid of trichocysts, demonstrate that the exocytotic organelle is not the location of the ion. By a variety of
controls, we show that this rim cannot be due solely to artefactual displacement or external adhesion of calcium. This
provides definitive confirmation of the existence of a vast submembranal calcium compartment in this cell, in which most of
the cell calcium is stored (at least one order of magnitude more
than in the rest of the cytoplasm). We also provide the first
quantification of the amount of calcium by SIMS microscopy
using an internal standard; this concentration is in the millimolar range, equivalent to that found in the sarcoplasmic
reticulum. Finally, we provide preliminary evidence that this
calcium is involved in the exocytotic process, by observing a
50% reduction of its amount in cells fixed 15 to 30 seconds
after massive induction of exocytosis.
MATERIALS AND METHODS
Biological material and sample preparation
Paramecium
Strains and culture conditions
The wild-type (WT) cells used in these experiments were from stock
d4-2 of Paramecium tetraurelia. Cells were grown at 27°C in
phosphate-buffered wheat grass powder infusion, bacterized the day
before use with Klebsiella pneumoniae and supplemented with 0.5
µg/ml β-sitosterol.
Two mutants were used in this study: mutant tam8, whose trichocysts are never attached to the cell surface (Beisson and Rossignol,
1975; Lefort-Tran et al., 1981); and a thermosensitive mutant, nd9,
whose trichocysts are attached at the cell surface but cannot be discharged at 27°C (Beisson et al., 1976).
Microscopy
Cells were harvested from early stationary phase cultures and the
pellet was fixed either: (1) in 0.5% glutaraldehyde plus 2%
paraformaldehyde, 50 mM sodium cacodylate buffer, pH 7.4, for 20
minutes at 4°C, or in the same fixative followed by postfixation in 2%
OsO4 in the same buffer. Cells were then pre-embedded in fibrinogen
pellets, dehydrated and embedded in LR White or Epon-Araldite; or
(2) by fast-freeze fixation by slamming the specimen against a cold
copper block cooled by liquid helium (Escaig, 1983) followed by
freeze substitution at −86°C for 72 hours in acetone in the presence
of 20 mM oxalic acid, then warmed to −30°C, maintained for two
hours at −30°C, and finally warmed to room temperature and
embedded in Epon-Araldite.
Wild-type cells were also cryofixed and cryodehydrated without
Calcium stores visualized by SIMS in Paramecium 1897
oxalic acid as controls and embedded in Epon-Araldite or cryoembedded in Lowicryl K4M.
Exocytosis
Synchronous exocytosis can be achieved with AED (amino ethyl
dextran), which causes instantaneous release of most of the trichocysts
(Plattner et al., 1984, 1985; Kerboeuf and Cohen, 1990). AED, kindly
provided by J. Cohen and D. Kerboeuf, was used at 6 µM on a pellet
of cells. About 30 seconds after stimulation, the cells were slammed on
the cryobloc and cryodehydrated in the same way as the untreated cells.
Aliquots of cells were further treated with picric acid and observed with
a dark-field microscope to check the extend of exocytosis.
Muscle
The cutaneous muscle of frog was taken up in Ringer’s solution. Very
small pieces were cryofixed and cryodehydrated exactly the same way
as for Paramecium.
To achieve a good preservation of cells (Paramecium and muscle),
ultrathin sections were obtained and stained with uranyl acetate
followed by lead citrate. They were observed in Siemens Elmiskop
102 electron microscope.
In our IMS 3F, the secondary ion beam intensity is measured
directly with the electron multiplier; the measurements are performed
on selected areas, which are limited by adapted apertures. In the
present work, the measured areas were always of 8 µm diameter. In
order to obtain statistically significant results, sets of at least 10 measurements were carried out on each holder for each of the domains
under analysis (Ca rim, cytoplasm, etc.). The beam was centered on
the area to be measured and the location of the area was recorded over
the image. Concerning the Ca rim located at the cell periphery, which
is the main subject of this paper, the diameter of the measured circle
is larger than that of the rim. The rim was therefore positioned in the
center of the measured field. We checked that a slight move of the
rim toward the borders of the field did not significantly modify the
recorded values.
Usually, several ions were recorded from the same section (most
frequently Ca2+, Na+, K+ and Mg2+) and, in most cases, a histological section immediately following or preceding those analyzed by
SIMS was stained with Toluidine Blue on a glass slide, to be observed
at the light microscope to provide a reference pattern.
SIMS microscopy study
RESULTS
Serial semi-thin sections were deposited on glass slides for optical
examination and on ultrapure gold holders for ion analysis. Sections (1
µm) on glass slides were stained with Toluidine Blue and sections (3
µm) for ion analysis were deposited over a microdrop of water on the
gold holder, and heated to 60°C. In some control experiments sections
were deposited over the gold holder without any contact with water.
The instrument used in this study was an IMS 3F (CAMECA,
Courbevoie, France), fitted with two primary ion sources and
connected to an image processing system. This system (Olivo et al.,
1989) allows digitalization of images (512×512 pixels), high-speed
signal integration of the ionic images (to improve signal/noise ratio),
histogram equalization (to increase the grey-scale contrast and to
decrease background noise) and, finally, image superposition.
The ion microscope was operated with the O2+ primary source with
a 15 keV primary beam current of 200 nA. The image field diameter
was 150 µm. A mass resolution (M/∆M) of 2000 was used in order
to eliminate interferences between cluster ions and the specific
elements under study. Under these conditions, elemental mapping of
Ca, K, Na and Mg is easily achieved.
For quantification and calibration, internal reference elements were
used. In SIMS, the secondary ion beam current intensity (Ia) is a
function of the concentration of the analyzed element (Ca), the area
to be analyzed (S), the useful ion yield (Ya) and the primary ion beam
current intensity (Ip). Thus, Ia = Ca.S.Ya.Ip. However, when dealing
with insulating specimens such as embedded biological samples, part
of the positive primary ion beam is repelled by charge effects when
negative secondary ions are extracted. The real intensity of the
primary ion beam current (Ip) is therefore variable, and the relation
between Ia and Ca is not directly applicable.
Nevertheless, a calibration is possible by measuring the intensity
of the secondary ion beam using an internal reference element (Ir),
which is present at a large homogeneous and constant concentration
in the specimen. Then Ia/Ir = K.Ca, where K is a proportionality
constant which can be determined using a standard with increasing
concentration of the tested element to generate a calibration curve. As
the carbon content of biological specimens and embedding resins are
virtually similar, this element can be used as an internal reference. In
order to quantify Ca, calcium octoate (Calcium-Norol by
SICCANOR, 59282 Douchy-les-Mines, France) was used as a
reference. Since calcium octoate is soluble in Epon-Araldite, samples
containing varying Ca concentrations, from 0.05 mM to 5 mM, were
prepared. Sections of 3 µm thickness were obtained and deposited
without any water onto the gold holders. Emission was measured over
fields of 150 µm in diameter.
Fast-freezing of wild-type Paramecium allows good
ultrastructural preservation
In order to prevent fixation-induced ion loss and redistribution,
we adapted the cryoblock method of Escaig (1983) to use with
a continuously fast-swimming, fragile cell such as Paramecium. We used small pieces of filter paper to trap the cells
in a thin layer while keeping them alive and healthy. Under the
conditions used, only those cells that happen to be in the most
superficial portion of the filter facing the copper bloc were
cooled rapidly, a condition essential for preventing formation
of ice crystals. After rapid freezing, there are two main alternatives for preserving the intracellular distribution of ions,
freeze-substitution or even better, freeze-drying. The problem
of freeze-drying and freeze-sectioning in SIMS is that the
sections obtained in that way adhere very poorly to the gold
holders used in the following step. Sod et al. (1990) have
developed indium holders to overcome this difficulty for
animal cell cultures, but this approach is difficult to apply to
single cells. In addition, in order to keep the paramecia in a
state as close as possible to the physiological one, we avoided
using high concentrations of centrifuged cells. Under the conditions used, the density of cells found in the filter paper is low.
This condition in addition to the previous one precluded the
use of the freeze-fracture and freeze-drying technique
developed by Chandra et al. (1986). Therefore we resorted to
freeze-substitution and inclusion in resin, realizing that some
redistribution of ions may occur at the final step of inclusion
in the resin when cells are brought back to room temperature.
Controls embedded in Lowicryl at low temperature were thus
included to check for major redistribution (see below). The
frozen pieces of filter paper were then taken through the
various steps of cryosubstitution in acetone, in the presence (or
absence) of oxalic acid, inclusion in epon resin (or in Lowicryl)
and sectioning (see Materials and Methods). Sections were first
observed by both light and conventional transmission electron
microscopy, to ascertain the quality of structural preservation.
By conventional light microscopy (after Toluidine Blue
staining) the cell contours and many cellular organelles were
readily recognized. In Fig. 1, for example, we can recognize,
1898 N. Stelly and others
Fig. 1. Rapidly frozen WT Paramecium: semi-thin section stained
with Toluidine Blue and observed with an optical microscope. N,
macronucleus; V, food vacuole; T, trichocysts attached to the cortex
C. Bar, 10 µm.
from the periphery to the inside of the cell: cilia in the form of
patches in some portions of the section, the cortex consisting
of adjacent typical cup-shaped cortical units, the carrot-shaped
dark trichocysts perpendicular to the surface when cut longitudinally, the oral depression or oral apparatus, and finally the
dense cytoplasm with food vacuoles containing bacteria in
various stages of digestion and, occasionally, a portion of the
macronucleus. The only abnormality observed is that the cells
located at the front are slightly deformed by the compression
generated during slamming on the copper block.
Fig. 2 illustrates the ultrastructural characteristics of stained
sections from cryofixed wild-type cells (b,c) as compared to
chemically fixed ones (a). Two other variables were also
analyzed (not shown): presence or absence of oxalic acid in the
cryosubstitution medium and inclusion in Lowicryl instead of
Epon. Oxalic acid was added in order to promote in situ precipitation of calcium (see Nicaise et al., 1989); Lowicryl was
used in order to check the quality of ultrastructural preservation it provided compared with traditional resins, since its use
could prove useful both for ion distribution studies (by
allowing polymerization at low temperature after cryosubstitution, thus further preventing ion diffusion) and for immunocytochemical studies. In cells located close to the frozen front,
good ultrastructural preservation was observed: the cortex with
its typical organelles was easily recognized, and even some of
the cytoskeletal networks which underly the cortex such as the
epiplasm and the filamentous infraciliary lattice were seen
(Allen, 1971) (Fig. 2b,c). The major differences noted with
respect to conventional chemical fixation are: first, in the
appearance of membranes within the cytoplasm and; second,
in that of the alveolar lumen. Although the cytoplasm
displayed its classical ribosome- and glycogen-studded appearance, membranes of the rough endoplasmic reticulum, of small
vesicles and even those of mitochondria were difficult to
recognize. Thus, mitochondria were revealed more by their
overall shape, distribution, compactness and ghosts of cristae
than through the conventional architecture of outer and inner
membranes. Similarly, the membrane which normally
surrounds the trichocysts was not visible. Treatment of the
sections with osmic acid did not modify these patterns. In
summary, cytoplasmic membranes appeared to be extracted.
A converse result was repeatedely observed for the alveolar
lumen: while in chemically fixed cells the lumen of the alveoli
is usually swollen and essentially electron transparent
(‘empty’), as in Fig. 2a, in all types of cryofixed cells the lumen
is more flattened and appears to be filled with a meshwork of
electron-dense material distributed evenly throughout all of the
luminal volume (Fig. 2b,c). Note (Fig. 2c) that this fluffy
material appears to be more abundant along the inner alveolar
membrane, i.e. on the surface facing the epiplasm. A similar
appearance can be seen in pictures published by Glas-Albrecht
et al. (1991) of Paramecium cells which were rapidly fixed and
freeze-substituted by a somewhat different method. The
presence of the meshwork does not depend on the presence of
oxalic acid in the cryosubstitution method, is not affected by
the type of embedding resin used and is seen even on unstained
sections, thus indicating that the meshwork is not an artefact
due to oxalate precipitation or staining. Cryofixation therefore
reveals the presence of a genuine meshwork that had apparently collapsed or was extracted in all previous studies using
chemical fixation.
The overall ultrastructural conservation at first sight
appeared to be slightly less good in Lowicryl than in EponAraldite. However, the cytoplasm and, especially, the
reticulum membranes appeared to be less extracted, and more
material seemed to be preserved in various complex organelles
such as the axonemes. All further studies presented in this
paper, except for the Lowicryl control, were carried out with
Epon-Araldite-embedded material because: the ultrastructural
preservation appeared to be quite acceptable, these sections
adhered more easily to the gold substratum required for SIMS
microscopy and the loss of ions was more limited during the
late stages of processing of Epon sections than Lowicryl ones
(especially during recovery of sections on water).
SIMS reveals a Ca compartment at the periphery of
cryofixed Paramecium cells
Cell sections of both chemically fixed and cryofixed cells were
analyzed by SIMS microscopy as described in Materials and
Methods. The distribution of a large number of ions was
examined; Fig. 3 shows an example of the results observed on
cryofixed cells with two physiologically important ones, Na+
and Ca2+, as compared to the light microscopic appearance of
an adjacent section. Fig. 3a corresponds to the Toluidine Bluestained section and shows portions of seven cells, three of
which, in the upper part of the picture, lie along the edge of
the frozen front. The various organelles pointed out in Fig. 1
can be recognized.
The most striking differences in spatial distribution of ions
depend on the fixation method concerned calcium: in chemically fixed cells, a weak Ca signal was uniformly distributed
throughout the cells (not shown; see Fragu et al., 1992), while
in cryofixed ones (Fig. 3b), the signal was restricted to a relatively narrow, bright band located around the cell periphery.
Calcium stores visualized by SIMS in Paramecium 1899
Fig. 2. Electron microscopy
of ultrathin sections of WT
Paramecium. alv, cortical
alveola; ci, cilium; T,
trichocyst; M,
mitochondrion; pm, plasma
membrane; oam, outer
alveolar membrane; iam,
inner alveolar membrane;
ep, epiplasm; kf,
kinetodesmal fibers. All
specimens were embedded
in Epon-Araldite. The
sections were stained with
uranyl acetate followed by
lead citrate. (a) Chemical
fixation with 0.5%
glutaraldehyde in 50 mM
cacodylate buffer, followed
by 2% OsO4 in the same
buffer: cortical alveoli are
swollen and empty. Bar, 0.5
µm. (b) Cryofixation and
cryodehydratation in
acetone in the presence of
20 mM oxalic acid: cortical
alveoli are more flattened
and are filled with a
meshwork of electron-dense
material. Trichocysts have
barely decondensed. Bar,
0.5 µm. (c) Detail of a
cortical alveola of a
cryofixed cell: note the good
preservation of plasma and
cortical membranes and the
presence of dense material
throughout the alveolar
lumen and especially facing
the inner alveolar
membrane. Bar, 0.1 µm.
Thus, as suspected, chemical fixation induced a drastic redistribution of ions, most clearly seen with Ca2+. Redistribution
was less striking with Na+, K+ and Mg2+ because these
elements were uniformly distributed in cryofixed cells: Na+
yielded a homogeneously and intensely emitting cytoplasm in
which only some large circular areas, most probably corresponding to food vacuoles, were not labelled (Fig. 3c). The
same was true for Mg2+; K+ yielded a slightly more granular
1900 N. Stelly and others
Fig. 4. SIMS image of Ca in cryofixed WT Paramecium: the semithin section was placed over the gold holder without water. Calcium
is located around the cell periphery as in Fig. 3f. Image field is 150
µm diameter.
Fig. 3. SIMS and light microscopy of adjacent cell sections. The
sections traverse a large number of cells located at the edge of the
sample (i.e. facing the frozen copper block). (a) Toluidine Bluestained section; (b) and (c) Ca and Na SIMS observed section
adjacent to that in (a). Note the regularity of the Ca peripheral signal
in (b), its independence from the presence of trichocysts and its
absence over the macronucleus. Bar, 30 µm.
or patchy labelling of the cytoplasm, again excluding vacuoles
(not shown). In chemically fixed cells, Na+ and K+ were less
uniformly distributed, with K+ concentrated into large precipitates both inside and outside the cells. The general intensity
of emission also appeared to be reduced (not shown; see Fragu
et al., 1992). In the remainder of this paper we will therefore
only be describing cryofixed cells. The presence or absence of
oxalic acid in the cryosubstitution medium did not modify the
results. Because some loss of elements can occur during the
final stages of specimen preparation, when the sections are
briefly deposited over a water drop on the gold holder and
immediately heated, we prepared some specimens that had no
contact with water at any stage. These are difficult to prepare
because of the tendency of sections to roll over themselves on
the gold holder in the total absence of water. SIMS of such a
‘dry’ specimen is shown in Fig. 4; Ca emission, identical to
that seen on sections deposited on water, is evident. As an additional control, cryofixed cells were included in Lowicryl after
cryosubstitution in order to carry out the whole procedure at
low temperature. Although the appearance of the cells in SIMS
microscopy was slightly more hazy than in the case of Epon
embedding, the Ca rim was clearly visible, indicating that the
rim is not due to ion redistribution occurring during inclusion
in Epon.
In fact, when using low levels of integration, a punctate
peripheral Ca pattern with a periodicity identical to that of the
adjacent cortical alveoli was often observed (see for example,
the three cell sections to the right of Fig. 3b). Three points
required evaluation, however, before a definitive acceptance of
this hypothesis. First, although the difference observed
between cryo- and chemically fixed cells was encouraging and
suggested a specific Ca localization in cryofixed cells, could
we be totally confident of our method? One way of answering
this question would be to show that our methods reveal the
well-known specific Ca localizations in striated muscle.
Second, since the large exocytotic vesicles known as trichocysts are docked beneath the cell surface in Paramecium in
an highly regular arrangement, could they be contributing to
the Ca signal? Indeed, many types of exocytotic vesicles
contain large amounts of Ca (reviewed by Nicaise et al., 1992).
This second question could be explored by using cells devoid
of trichocysts. Third, did careful observation of a large number
of sections indeed confirm that both the size of the Ca-emitting
zone and its precise shape and distribution agree with what is
known for cortical alveoli at the EM level? In particular, does
Calcium stores visualized by SIMS in Paramecium 1901
Fig. 5. Frog cutaneous
muscle. (a) A semi-thin
section of a portion of muscle
fiber cryofixed and observed
for Ca by SIMS. The bright
bands correspond to the
sarcoplasmic terminal
cisternae within the I band.
Bar, 25 µm. (b) Toluidine
Blue-stained sections of the
same fiber. Bar, 25 µm.
(c) Ultrastructure of the same
cryofixed fiber where the
terminal cisternae (TC) are
aligned along the Z disk
within the I band. M,
mitochondrion. Bar, 1 µm.
the width of the compartment agree with what is known of the
size of alveoli in electron microscopy? This last question could
be analyzed by carefully comparing successive sections of
material, some being observed by conventional light
microscopy to locate the major organelles and consecutive
ones by SIMS microscopy, and also by comparing the distribution of Ca2+ with that of another ion such as Na+ to check
whether extracellular adhesion of Ca occurred.These three
points are addressed below.
SIMS identifies the expected Ca compartment in
muscle cells
Samples of frog cutaneous muscle were prepared using exactly
the same methods as for Paramecium, i.e. by rapid freezing,
cryosubstitution in the presence of oxalic acid and embedding
in Epon-Araldite. Conventional light microscopy shows the
typical striated appearance of sarcomeres (Fig. 5b) and electron
microscopy indicates a reasonably good ultrastructural preservation (Fig. 5c). I and A bands are readily identified and the
terminal cisternae are clearly seen in the I bands. In this tissue,
EM electron-probe analysis of ultrathin cryosections has
shown that 60 to 70% of total fiber Ca is localized in the
terminal cisternae, within the I bands (Somlyo et al., 1981).
SIMS microscopy (Fig. 5a) shows a regular alternation of
emitting and non-emitting bands for Ca, corresponding,
respectively, to the light and dark striations seen in light
microscopy, and therefore to I and A bands, respectively. As
expected, Ca is thus restricted to the I bands in which the
terminal cisternae of the sarcoplasmic reticulum are located,
and no major loss or redistribution of calcium seem to have
occurred during sample preparation.
The periphery of the fiber also strongly emitted Ca,
probably because of the presence of a dense array of vesicles
lying immediately beneath the sarcolemna, referred to as
caveolae by Franzini-Armstrong (1970) and which possibly
contain high amounts of calcium originating from the extacellular medium.
Trichocyst-deprived cells still display the peripheral
Ca compartment
Two approaches were used to obtain cells devoid of trichocysts at the cortex; massive induction of trichocysts
discharge by AED from wild-type cells (Plattner et al., 1984),
or use of a mutant (tam8) lacking attached trichocysts at the
cortex (Beisson and Rossignol, 1975; Lefort-Tran et al.,
1981). The wild-type cells were frozen within 30 seconds after
discharge; the extent of discharge was monitored by
observing, in a dark-field microscope, aliquots of the AEDtreated cell suspension to which picric acid had been added.
Although sometimes irregular, AED-induced discharge was
usually quite effective as shown by light and EM microscopy
controls. In these cells rapidly frozen after discharge, some
modifications in the ultrastructural aspect of the alveoli are
apparent: they appear to be somewhat collapsed, with the
outer membrane disjointed from the plasma membrane, and to
contain less fluffy material than the controls. The mutant cells
were processed as wild-type cells. tam8, clearly, lacked
attached trichocysts at the cortex; its trichocysts lay randomly
within the cytoplasm. One additional mutant strain was used,
nd9. This is a conditional mutant, defective in exocytosis at
27°C, but displaying a normal complement of trichocysts
attached at the cortex. It is therefore defective in only the very
final steps of exocytosis (Beisson et al., 1980). This strain
therefore allows discrimination between effects due to the
absence of trichocysts at the surface from effects only due to
lack of exocytosis potential.
In all cases (AED-treated WT, tam8, nd9), the peripheral Ca
compartment was still observed by SIMS microscopy (Fig.
6a,b,c). The Ca image of nd9 cells appeared to be identical to
that of WT ones. While in AED-treated WT cells and in tam8
ones, the width and intensity of the Ca zone appeared to be
slightly reduced. This led us to a more quantitative study as
described below. In any case, it became clear that trichocysts
cannot be the major contributors to the peripheral Ca signal,
since this signal was always present in tam8 cells, where con-
1902 N. Stelly and others
Fig. 6. Ca images of different types of
paramecia showing conservation of the
peripheral calcium signal. (a) WT
Paramecium after stimulation by AED; (b)
mutant nd9; (c) mutant tam8; (d) Toluidine
Blue-stained optical view of a section from
the same tam8 cell as that in c. V, vacuoles;
T, trichocysts; G, gullet. Bars, 20 µm.
ventional light microscopy of adjacent cells revealed the
absence of trichocysts at the cortex and their scattered presence
in the cytoplasm (see, for example, Fig. 6d).
A reverse argument can also be made: in many instances,
when a dense array of longitudinally sectioned trichocysts was
observed in control sections, the corresponding SIMS image
of an adjacent section often showed a Ca-emitting zone which
was narrower than the zone occupied by the trichocyst bodies
(see Fig. 3a, for example).
Ca emission at the cell periphery is strictly
correlated with the presence and integrity of the
cortex
Side by side comparison of SIMS images and corresponding
adjacent sections observed by light microscopy (Figs 3 and 6)
yields the following observations.
(1) The Ca-emitting zone exactly follows the periphery of
the cells, with all its deformations and, in particular, clearly
outlines the shape of the oral depression (which is also
bordered by alveoli for the most part; see Allen, 1974).
(2) The inner cytoplasmic portion of the sections gives a
much lower Ca signal except, occasionally, for food vacuoles.
There may be some correlation between the ‘age’ of vacuoles
and the intensity of their signal, young vacuoles showing
higher signal than old ones (see Fig. 6c,d). No Ca emission was
observed from these massive DNA-containing macronuclei.
(3) When a portion of a cell was torn during preparation, and
lost its cortex, no Ca signal was observed in this area.
Calcium stores visualized by SIMS in Paramecium 1903
Fig. 7. Ca and Na saturation
analysis. The same section was
submitted to recordings of
increasing duration for Ca
(a,b,c,d) and Na (e,f,g,h),
corresponding, respectively, to
250, 500, 1,000 and 2,000
integrations. Note the increasing
intensity and thickness of the Caemitting zone from (a) to (d) and
the absence of spillover of the Na
signal out of the cells in (h).
Image field is 150 µm diameter.
(4) When a section was tangential to the surface and
contained many cortical units such as the lower part of the cell
in Fig. 1, the Ca-emitting zone was much enlarged.
(5) Cell sections located far from the freezing front tend to
display a much less regular Ca rim, the farther ones sometimes
being devoid of it.
Thus, the Ca-emitting zone appears to be tightly correlated
with that known to contain alveoli and to depend on good
freeze-fixation. However, the width of this zone, as seen by
SIMS microscopy, seems to be broader than would be expected
if only the lumen of the alveoli were emitting. We examined
this question, with two types approaches, image saturation
studies and morphometric studies involving superposition of
the Ca and Na images.
For saturation studies, an increasing number of images
were summed, starting from low levels of integration
(detecting high Ca concentration) to very high ones (detecting
low Ca concentration) (see Materials and Methods). At low
1904 N. Stelly and others
Fig. 8. Na and Ca image superposition. The same section was submitted to Ca analysis (a) followed by Na analysis (b) and the two resulting
images were superposed in the computer (c). Note that a peripheral yellow to orange rim is obtained in c, reflecting the good superposition of
the Ca rim within the limits of the Na border. Also note that the most rapidly frozen front is to the right of the image, as can be seen by the
slight deformation of the cells due to their slamming over the copper block and by the presence of extracellular calcium-rich deposists. In
contrast, note that cells deeper in the sample (to the left of the image), although they are detected in the Na image (b), lack the red Ca rim (a),
probably because of poor fixation. Bar, 10 µm.
levels, a fine Ca line first appears all along the cell periphery,
only very slightly spilling over (as based on the Na and K
images; see below), but it thickens with increasing integrations, reaching a width of approximately 5 µm (Fig. 7a to d).
This is broader than the largest width of alveoli as measured
on EM images (approx. 3 µm). Thus, the width of the Caemitting zone is broader than what strictly corresponds to the
alveoli. The same phenomenon was observed using ‘dry’
sections; it is therefore not due to diffusion induced by the
water drop used on the gold holder. Under the same saturation conditions, the Na, K and Mg emissions did not spill over
across the cell boundary but remained confined within their
initial area of distribution (Fig. 7g and h), again indicating
that the situation observed for Ca reflects a real in situ distribution and not nonspecific diffusion. It should be stressed,
however, that the level of integration required to see the
widening of the calcium rim is at least an order of magnitude
higher than that needed to see the initial peripheral signal.
Thus, the amount of the excess calcium is much lower than
that seen at low integration levels.
Widening of the Ca-emitting zone occurred mainly inward.
This was established by measurements on the micrographs,
superposition of hand-drawn tracings and, most directly, by
computer superposition of the calcium and sodium images
(Fig. 8), using the programs of Olivo et al. (1989). On such
images, it can be seen that at low integration levels, the red rim
(Ca) superposes well over the green background (Na) giving a
yellow-orange border, with no indication of a red external
margin; this remains the case even at higher levels of integration. This shows: first, that the calcium rim detected at low
levels of integration is located inside the cell; and, second, that
the outer boundary of calcium distribution does not extend
much beyond that of Na.
At extremely high integration levels (10 to 20-fold higher
than necessary to see the rim), a weak Ca signal was eventually seen throughout the cytoplasm, most probably reflecting
the quasi-uniform distribution of the much smaller amount of
total cytoplasmic Ca than that located in the alveoli.
Taken together, these saturation studies indicate the
presence of a thin intracellular peripheral compartment with a
very high Ca concentration, surrounded, on the outside, by a
narrow Ca-containing zone and, on the inside, by a broader Cacontaining domain where Ca concentration decreases in a
graded manner towards the cytoplasm of the cell.
Quantification of alveolar Ca reveals a two-fold
decrease after exocytosis
As briefly indicated above, SIMS analysis revealed, in both
tam8 cells and the wild type cells in which a massive release
of trichocysts was induced, a Ca emission lower than that of
control cells. This had to be quantified rigorously because
section to section comparison in SIMS microscopy may not be
reliable. A proportionality coefficient (Ca/C) was therefore
established for each of our measurements by measuring simultaneously the Ca and C beam intensities within the same
volume of sample. Each measure provided corresponds to the
mean of 10 measurements carried out over a surface of 8 µm
diameter. This allows a sample to sample comparison (see
Materials and Methods). In addition, absolute quantification of
Ca was achieved using an internal standard consisting of
calcium octoate included in Epon-Araldite (see Materials and
Methods).
Table 1 summarizes the quantitative observations in
different types of cell sections. As expected, Ca concentration
is significantly higher (about 7-fold) at the cell periphery than
within the cytoplasm in all types of cells and conditions (2 to
3 mM vs 0.3 to 0.4 mM). It is interesting to compare the Ca
concentration within the cortex of Paramecium with that in
muscle, using SIMS. We found the muscle values to oscillate
around 10 mM. This is quite comparable to the value found by
Somlyo et al. (1981) using EM microanalysis: their values
range from 10 to 117 mmol/kg dry weight, the highest value
Calcium stores visualized by SIMS in Paramecium 1905
Table 1. Quantitative evaluation of Ca amounts
Cortex
n1†
n2
Cytoplasm
n1
n2
Wild type
(3 exp.)
17.9±8.4.10−1*
3.4±1.8 mM
35
22
3.0±1.2.10−1
0.4±0.2 mM
34
30
Wild type +
AED
(2 exp.)
10.5±5.10−1
1.8±1.2 mM
36
20
3.7±1.5.10−1
0.6±0.4 mM
15
11
tam8
(1 exp.)
12.4±6.2.10−1
2.3±1.2 mM
41
14
4.4±3.10−1
0.7±0.6 mM
11
11
tam8 + AED
(1 exp.)
10.6±4.5.10−1
1.8±1.0 mM
19
9
4.8±1.7.10−1
0.8±0.4 mM
7
6
nd9
(1 exp.)
17.9±5.1.10−1
3.4±1.2 mM
25
12
3.3±1.3.10−1
0.5±0.2 mM
6
6
*The upper line represents the mean of Ca count/C count ± s.d. The second
line is the Ca concentration in mM, calculated using Calcium reference.
†The numbers in the n1 columns refer to the number of surface spots over
which measurements were carried out; the numbers in the n2 columns refer to
the number of different cell sections examined.
corresponding to a position of their narrow (20 µm) probe
within the terminal cisternae. When converted to mM, the units
used in the present work, by taking into account the water
amount, their values are 3 to 30 mM, i.e. with an average very
close to our 10 mM value obtained by SIMS with a much larger
probe diameter.
Thus, the total Ca concentration in the cortex is in the same
range as that of the sarcoplasmic reticulum.
After induction of massive exocytosis by AED, a marked
decrease in Ca concentration in the cortex was repeatedly
observed (from 3.4±1.8 mM to 1.8±1.2 mM). This decrease is
statistically highly significant using the t-test (P<0.0001).
Interestingly, this decrease in the cortex appears to be correlated with an increase in Ca concentration within the
cytoplasm, but this is barely significant statistically. The
kinetics of these changes was not studied. All the data
presented are from experiments with cells that were cryofixed
about 30 seconds after exocytosis.
Concerning trichocyst mutant strains, nd9 displayed a
cortical Ca concentration identical to that of wild type, indicating that, when trichocysts are attached at the cortex, incapacity to carry out exocytosis does not lead per se to a modification of Ca concentration.
In contrast, tam8 displayed a significantly lower amount,
placing it at an intermediate level between normal wild type
and wild type after exocytosis. Treatment of tam8 cells with
AED further decreased the Ca concentration in the cortex but
this was only marginally significant statistically (P<0.02).
Trichocyst exocytosis or lack of attached trichocysts at the
cortex therefore lead to a significant decrease in cortical Ca
concentration.
DISCUSSION
The major result presented in this paper is the visualization of
a subcortical Ca compartment in Paramecium using a new
method, SIMS microscopy, applied to sections of ultrarapidly
frozen cells. In addition, the amount of Ca in this compartment
was found to decrease substantially after massive induction of
exocytosis.
The idea that the cortical alveoli, a network of large, interconnected membrane vesicles directly underlying the plasma
membrane in Paramecium and other ciliates might correspond
to a Ca-sequestering compartment akin to the sarcoplasmic
reticulum of muscle cells is old (Allen and Eckert, 1969; Satir
and Wissig, 1982). It received strong support when we
succeeded in purifying these vesicles and showed that they
actively pump Ca2+ in an ATP- and Mg2+-dependent process
(Stelly et al., 1991). Additional evidence was provided by the
work of Schmitz et al. (1985), Zierold (1991) and Knoll et al.
(1993) using EM microanalysis methods. Here, we have sought
to provide direct visualization of this compartment over large
areas of many cells using a new approach, that of SIMS
microscopy. The main advantage of this method lies in the fact
that it provides images of total Ca distribution over individual
cell sections and is thus especially suited for identifying Ca
storage sites (as compared to methods detecting only free
Ca2+). Since the lateral resolution of the method is limited,
however, the compartment must be of sufficient size to be identifiable. Because cortical alveoli are typically about 0.2 to 2 µm
× 1 to 3 µm in size, we hoped that, if they indeed contained
large amounts of Ca, SIMS microscopy would allow their visualization. In addition, there was a clear prediction as to the
expected intracellular location of the signal, namely throughout the cell periphery.
These predictions were fulfilled remarkably: a continuous,
peripheral, Ca-emitting zone was immediately seen, provided
that cells were fixed by rapid freezing.
The good ultrastructural conservation of cells after cryofixation, the fact that the peripheral location of Ca strictly
depended on avoiding chemical fixation, and the excellent correlation observed between the occurrence of the Ca signal and
the presence of a strip of well frozen cortex in the corresponding section, all argue against artifacts. In addition, the
fact that it is found also in cortices devoid of trichocysts
demonstrates that the Ca does not emanate predominantly from
these exocytotic organelles. The most likely Ca-containing
compartment therefore remaining the alveolar one. Additional
evidence on these two points was recently provided by electron
probe microscopy. Using conventional X-ray microanalysis,
we found amounts of Ca in a number of alveoli ranging from
5 to 10 mM (Stelly, Halpern and Nicaise, unpublished). No Ca
signal was observed on trichocysts and this is all the more significant, since these organelles are easily identified in the
unstained sections used for microanalysis. Confirmation of
these two points can be found in a recent study of Knoll et al.
(1993) in which one electron energy loss spectrum image of
ultrarapidly fixed Paramecium is provided, showing the
presence of Ca in alveoli (not in trichocysts) and its redistribution after exocytosis. It should be pointed out that previous
X-ray microanalysis studies had already clearly indicated the
presence of Ca below the cell surface but not in the trichocysts
(Schmitz et al., 1985). In summary, all the available evidence
converges to indicate that trichocysts make little or no contribution to the peripheral calcium signal and that the signal
emanates predominantly from alveoli.
The width of the Paramecium band, as seen in SIMS
microscopy, can be estimated as 3 to 5 µm, while the EM
observations indicate that the alveoli do not exceed 2 µm. In
addition, saturation studies show that this width can reach 10
to 20 µm with a narrow Ca zone observed on the outside of the
1906 N. Stelly and others
cell, a phenomenon not seen with Na or K and Mg. Several
explanations can account for this observation. These fall into
two broad categories: artefactual diffusion of Ca from the
alveoli during sample preparation, or normal presence of Ca in
the proximity of the alveolar compartment. The first hypothesis cannot be totally excluded and, in fact, some diffusion
would not be surprising in view of all the steps involved in
sample preparation. We have excluded the possibility,
however, that rapid flotation of sections has a major effect,
since sections obtained by completely avoiding any contact
with water showed an identical distribution of all the major
ions analyzed. In addition, inclusion in Lowicryl at low temperature yielded exactly the same Ca rim as that in EponAraldite, indicating that no major diffusion occurs at the time
when the cryosubstituted samples are warmed up. Finally, this
extracellular emitting zone is Ca-specific and was not observed
for several other ions, indicating that it does not reflect a generalized diffusion from the inside of the cell. The second
hypothesis is therefore much more likely and at least two
possible explanations for a genuine in situ broader Ca zone can
be suggested. First, the cell is covered by cilia and both the
plasma and ciliary membranes may be expected to bind a substantial amount of Ca by means of the negatively charged phospholipids and the glycosylated cell coat, thus spreading the
signal towards the outside of the cell. Second, concerning the
spread of the signal towards the cell’s interior, it should be
recalled that a vast filamentous network, the infraciliary
network, recently shown to be made up of Ca-binding proteins
(Garreau de Loubresse et al., 1991), makes up the deepest of
the cortical cytoskeletal layers several micrometers below the
alveoli. This network may well yield a signal beneath the
alveoli on the sections. In addition in Paramecium, especially
in stationary phase cells, mitochondria tend to be concentrated
just below the cortex, providing a further possible weaker
calcium-emitting zone (see Girard et al., 1991, and Rizzuto et
al., 1993, for recent evidence of calcium pumping of ERreleased calcium by mitochondria). It appears, therefore, that
three Ca domains can be identified at the cell periphery in
Paramecium: (i) a narrow zone of very high Ca concentration
corresponding to alveoli, surrounded by: (ii) a small extracellular zone probably corresponding to membrane- and ciliumbound Ca; and (iii) a larger intracellular zone displaying a Ca
concentration decreasing towards the inside of the cell.
Through calibration of the absolute amount of Ca using a
calcium octoate derivative embedded and analyzed in the same
conditions, the absolute amount of Ca contained in various
areas of the sections was approximated. Within the peripheral
band, the mean value was 3.4±1.8 mM. This concentration is
higher by several orders of magnitude than that of free
cytosolic Ca2+ (10−4 mM; Eckert, 1972). There are several
implications of this considerable difference. First, it is most
likely that Ca is associated with a Ca-sequestering protein
within the lumen of the alveoli. In fact, we wonder whether the
fluffy material identified in the alveoli only after ultra-rapid
freezing corresponds to such hypothetical sequestering
proteins. A tempting cytological analogy with calsequestrin
can indeed be made: only when rapid fixation was used was a
diffuse, fluffy material clearly seen in terminal cisternae, which
was later identified as calsequestrin (Jorgensen and Campbell,
1984; Jorgensen et al., 1985). Previously, conventional
chemical fixation had failed to reveal it, as is the case with the
newly discovered material in Paramecium alveoli. We
searched for a homologue of calsequestrin in the cortical
fraction through immunoblotting, Stains-all decoration and
radioactive Ca-overlay of gel blots but did not observed a
signal in the calsequestrin Mr zone.
The second corollary of the high intra-alveolar Ca concentration is the likelihood of a tight control over its release. Previously, we have focused on the analysis of Ca2+ uptake into
alveoli in vitro (Stelly et al., 1991). We are currently characterizing the release system kinetically and pharmacologically.
There are indirect indications for the operation of an InsP3
system in Paramecium (Beisson and Ruiz, 1992) and various
components of the inositide cascade are present (Freund et al.,
1992) although InsP3 itself has proven elusive. Similarly, the
occurrence of a Ca2+-induced/Ca2+-release cascade has been
suggested to underly morphogenetic waves during cell division
(Le Guyader and Hyver, 1991), but remains to be demonstrated.
As for the function of alveoli in Ca2+ regulation, at least
three roles might be considered (Stelly et al., 1991), especially
when it is noted that the alveoli are in close proximity to three
Ca2+-controlled organelles, the cilia, the trichocysts and
cytoskeletal networks: (1) general homeostasis of intracellular
Ca2+ by active sequestration above a given cytosolic level; this,
for example, might be the case when a surge of Ca2+ occurs
through depolarization of the ciliary membrane. The alveoli
being immediately adjacent to basal bodies might pump the
Ca2+ that has entered the ciliary lumen; (2) release of at least
part of the Ca2+ required for trichocyst exocytosis. In this
respect, it must be stressed that alveolar membranes are tightly
apposed to the membranes of the tip of trichocysts; (3) release
of the Ca2+ required for the disassembly of the several
cytoskeletal networks adjacent to the cortex when they undergo
reorganization during division (Iftode et al., 1989). Of these,
one of the thickest is the infraciliary lattice, which is made up
of Ca-binding contractile proteins (Garreau de Loubresse et al.,
1991). In the case of this network, the alveoli may also provide
Ca2+ to regulate contractility.
The present results clearly support the idea that the alveoli
provide at least some of the Ca2+ required during exocytosis,
since after massive exocytosis the amount of Ca2+ inside the
alveoli dropped by 50%. It should be stressed that such a
massive drop in Ca2+ is quite similar to what occurs in the sarcoplasmic reticulum during muscle contraction (Somlyo et al.,
1981). In fact, we may not have captured the point of lowest
Ca2+ concentration, since the cryofixation device used imposes
a delay of about 30 seconds between the application of the secretagogue (AED) and the fixation of cells. Using faster fixation
methods, Knoll et al. (1993) recently observed a profound
redistribution of alveolar Ca2+ within 80 milliseconds after
AED-induced exocytosis in Paramecium. This observation
agrees very well with our results.
The question of the origin of the Ca2+ required for exocytosis has been extensively discussed both for Paramecium
(Plattner et al., 1991; Knoll et al., 1992, 1993; Cohen and
Kerboeuf, 1993) and other systems. Our data indicate that a
contribution of alveoli to the process is very likely. Interestingly, Cohen and Kerboeuf (1993), on the basis of a completely
different approach, also concluded that at least part of the Ca2+
involved in trichocyst exocytosis originates from intracellular
stores, which, they suggest, might correspond to the alveoli.
Calcium stores visualized by SIMS in Paramecium 1907
The exact path followed by alveolar Ca2+ during exocytosis is
still unknown but the tight apposition of alveolar and trichocyst
membranes, at the tip of trichocysts, suggests a direct flow,
through specific transmembrane proteins.
Assuming that trichocysts themselves contain very little
calcium, as we concluded above, the fact that exocytotic
mutants lacking attached trichocysts show a much decreased
amount of Ca2+ in alveoli, while those having attached trichocysts have normal amounts, provides a further hint as to the
regulation of alveolar amounts. It suggests that trichocyst
attachment per se, independently of exocytotic capability,
provides a regulatory loop inducing normal Ca2+ pumping and
sequestration in alveoli. It should be recalled in this context
that mutant tam8, which lacks cortex-attached trichocysts, was
shown in our previous work to pump Ca2+ in vitro as efficiently
as the wild type (Stelly et al., 1991). Thus, the in vivo decrease
in alveolar Ca described in the present work did not result from
an intrinsic defect of the Ca2+-pumping machinery but was due
rather to an indirect, presumably regulatory, process.
How wide is the occurrence of such vesicular Ca compartments closely apposed to the plasma membrane? The observation made in Paramecium appears to be valid for other
ciliates: preliminary SIMS observations carried out in Tetrahymena, a genus relatively close to Paramecium in molecular
evolutionary terms, but also in Euplotes, a very distant hypotrichous ciliate (Baroin-Tourancheau et al., 1992), show the
presence of the peripheral Ca band (Stelly, unpublished).
Cortical alveoli are in fact a shared ultrastructural character of
three protist Phyla, ciliates, dinoflagellates and apicomplexa
(the Phylum comprising Plasmodium, Toxoplasma, gregarines
and other parasitic protists); these three Phyla have been shown
by molecular phylogenetic analysis to form a monophyletic
group (the ‘alveolata’) (Gajadhar et al., 1991). It is tempting
to suggest that in these three groups the alveoli play the role
of Ca-sequestering organelles. In fact, in these three types of
organisms there are exocytotic functions that may correlate
with the presence of alveoli.
Going to much more distant biological groups, the occurrence of plasma membrane-linked Ca compartments in various
types of mammalian cells has been proposed (Putney, 1986).
In some highly differentiated metazoan cells, the relation of the
endoplasmic reticulum to the plasma membrane becomes quite
specialized, the paradigm being skeletal muscle cells with the
tight apposition of a specialized domain of the sarcoplasmic
reticulum to the T-tubule membrane, at the level of which
signal transduction operates (Caswell and Brandt, 1989).
However, the closest analogy at the moment is with eggs of
sea urchins (Luttmer and Longo, 1985) and of ascidians
(Gualtieri and Sardet, 1989). There are indeed a number of
striking similarities between the cortical alveoli of Paramecium and a vesicular ER network in the cortex of the ova of
various sea urchin species (Gardiner and Grey, 1983; Sardet,
1984; Terasaki et al., 1991); both actively pump Ca2+ in vitro,
both appear to release at least some of the Ca2+ required for
exocytosis (trichocysts in Paramecium, cortical granules in sea
urchin; Gillot et al., 1991) and both constitute the major
calcium store of the cell.
The sea urchin egg network has several additional properties which have not been detected in Paramecium: it contains
calsequestrin (Henson et al., 1989), releases Ca2+ in response
to InsP3 (Terasaki and Sardet, 1991) and also appears to
contain a ryanodine receptor (McPherson et al., 1992, Sardet
et al., 1992). The ciliate Ca stores may therefore be evolutionary homologues of this ER-derived network and, indeed, of
other Ca storage compartments of ‘higher’ eukaryotes, but this
is an unproven generalization. The search for proteins homologous to those of the compartments of higher eukaryotes in
alveolar sacs has been hampered by the great evolutionary
distance separating ciliates from metazoa, which usually leads
to failure of cross-reaction when most antibodies to metazoan
proteins are tested in Paramecium. In fact, the exact relationship of alveoli to the endoplasmic reticulum in ciliates and their
mode of biogenesis are still unclear. It is being studied at
present using EM immunocytochemistry by our group, with
plasma membrane markers (Charret et al., unpublished;
Capdeville et al., 1993).
This work was supported by a grant from the Université Paris-Sud
(Action Interdisciplinaire 8922). We thank Dr J. Cohen and D.
Kerboeuf for mutant strains and gift of AED, Drs J. P. Mauger and J.
Cohen for critical reading of the manuscript, Dr M. Müller for his help
in improving the manuscript and Prof. G. Nicaise for sharing the Xray results and for comments on the manuscript. We are grateful to
Mrs N. Narradon for expert photographic assistance and to Mrs C.
Couanon for her careful preparation of the manuscript.
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(Received 12 August 1994 - Accepted 20 February 1995)