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					Journal of Cell Science 108, 1895-1909 (1995)                                                                                  1895
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               particular, the emitting zone was still seen in mutants
continuous array of membrane vesicles known as cortical              totally lacking trichocysts, the large exocytotic organelles
alveoli. Previous work had shown that a purified fraction             docked at the cell surface, indicating that they make no
of these vesicles actively pumps calcium, suggesting that            major direct contribution to the emission. Calcium con-
alveoli may constitute a calcium-storage compartment.                centration within alveoli was quantified for the first time in
Here we provide direct confirmation of this hypothesis                SIMS microscopy using an external reference and was
using in situ visualization of total cell calcium on sections        found to be in the range of 3 to 5 mM, a value similar to
of cryofixed and cryosubstituted cells analyzed by SIMS               that for sarcoplasmic reticulum. After massive induction of
(secondary ion mass spectrometry) microscopy a method                trichocyst discharge, this concentration was found to
never previously applied to protists. A narrow, continuous,          decrease by about 50%, suggesting that the alveoli are the
Ca-emitting zone located all along the cell periphery was            main source of the calcium involved in exocytosis.
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             Key words: calcium, SIMS, Paramecium



INTRODUCTION                                                         electron probe microanalysis of tissue sections (Somlyo, 1985;
                                                                     Andrews et al., 1987), and an indirect one, seeking to identify
The question of the subcellular location of calcium (Ca) stores      the organelles by virtue of the presence of a set of ‘marker’
in eukaryotic cells has attracted considerable attention in recent   proteins involved in Ca2+ homeostasis: Ca2+-ATPases, Ca2+
years (see Koch, 1990; Meldolesi et al., 1990; Tsien and Tsien,      channels (ryanodine receptor, inositol-triphosphate (InsP3)
1990; Lytton and Nigam, 1992; Meldolesi and Villa, 1993, for         receptor) and Ca-binding proteins (calsequestrin, calreticulin,
review). The measurement of free, cytosolic calcium (Ca2+)           etc.). These proteins were detected either, by physiological and
concentration and its oscillation in single cells has become         biochemical methods (such as by measurement of Ca2+ uptake
possible with the advent of fluorescent probes (Grynkiewicz et        and release in subcellular fractions; see Pietrobon et al., 1990
al., 1985; see Williams and Fay, 1990, for review). This con-        for review) or by immunocytological approaches (subcellular
centration, however, is several orders of magnitude lower than       localization, at the EM level, of compartments reacting with
that of total cellular calcium; part of this large amount of         antibodies directed against the marker proteins; e.g. see Volpe
calcium is bound to cytosolic proteins but the majority, by far,     et al., 1988).
appears to be segregated inside membrane-bounded intracellu-            The results of these approaches are summarized in recent
lar organelles where it is complexed with low-affinity, high-        reviews (cited above). Except for the striated muscle cell, an
capacity proteins (Carafoli, 1987; Koch, 1990). Release of           extensively studied model system in which the situation is con-
Ca2+ from these organelles plays a key role in several               siderably clarified because of the amplification of the Ca
processes, especially in the response to various extracellular       storage compartment achieved in the form of the sarcoplasmic
stimuli.                                                             reticulum, the number, location and functional role of Ca
   Identification of these Ca-sequestering organelles has             storage compartments in eukaryotic cells has until recently
proven difficult, however. Two major approaches have been            been unclear. Except for mitochondria, which do not appear to
used, a direct one, seeking to visualize the element itself by       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              methods used in biological microanalysis, see Linton and
reticulum. Currently, at least two and more probably three or        Goldsmith, 1992).
even four types of compartments are implicated (Burgoyne and            Paramecia have already been studied by microanalysis at the
Cheek, 1991; Lytton and Nigam, 1992; Sitia and Meldolesi,            EM level both in the EPMA (Schmitz et al., 1985; Zierold et
1992; Meldolesi and Villa, 1993), depending on the cell type         al., 1989) and the EELS mode (Knoll et al., 1993), using
analyzed, on the basis of the receptors, channels, pumps and         excellent techniques of cryofixation followed by either freeze-
calcium-binding proteins that they contain. All of these com-        drying or cryosubstitution. The major result of these studies
partments appear to belong to the general intracellular protein      was the identification of a peripheral Ca-containing zone in the
sorting compartments (RER, Golgi, endosomes, etc.) although          cortex of Paramecium (not including the exocytotic organelles
they are more or less distantly connected to it.                     known as trichocysts) and preliminary evidence for redistrib-
   In this paper, we present an approach complementary to            ution of this calcium after induction of exocytosis in the EELS
those just cited, aimed at directly visualizing total cellular Ca.   high-resolution study (Knoll et al., 1993) but not in the EPMA
It involves the use of both a new cell type and a different          one (Zierold et al., 1989). Here, we extend these studies using
method. The cell type is the ciliated protozoan Paramecium,          the different SIMS approach, which we adapted for fast-
in which we have recently shown that a vast vesicular network        swimming single cells. We confirm the occurrence of an
lying just beneath the plasma membrane actively pumps Ca 2+          intensely emitting peripheral rim of calcium and, through the
(Stelly et al., 1991). This ‘primitive’ organism therefore           use of mutants devoid of trichocysts, demonstrate that the exo-
appeared to offer a naturally amplified Ca storage compart-           cytotic organelle is not the location of the ion. By a variety of
ment (akin to the sarcoplasmic reticulum), facilitating direct       controls, we show that this rim cannot be due solely to arte-
visualization. The method is secondary ion mass spectrome-           factual displacement or external adhesion of calcium. This
try (SIMS; Castaing and Slodzian, 1962) microscopy, which            provides definitive confirmation of the existence of a vast sub-
has been extensively used in solid state physics to character-       membranal calcium compartment in this cell, in which most of
ize surface composition of samples, but much less in biolog-         the cell calcium is stored (at least one order of magnitude more
ical applications (see reviews by Chandra and Morrison,              than in the rest of the cytoplasm). We also provide the first
1988; Fragu et al., 1992). A primary ion beam is focused onto        quantification of the amount of calcium by SIMS microscopy
the surface of a tissue section, leading to the sputtering of the    using an internal standard; this concentration is in the mil-
most superficial atoms, themselves partly in the form of ions.        limolar range, equivalent to that found in the sarcoplasmic
These secondary ions are then collected, analyzed with a mass        reticulum. Finally, we provide preliminary evidence that this
spectrometer and the corresponding image is reconstructed.           calcium is involved in the exocytotic process, by observing a
One therefore obtains an image of the distribution of a specific      50% reduction of its amount in cells fixed 15 to 30 seconds
atom at the surface of the specimen analyzed. This method            after massive induction of exocytosis.
offers three main advantages. First, it allows visualization of
all the elements of Mendeleiev’s table as well as discrimina-
tion between many of their stable and radioactive isotopes.          MATERIALS AND METHODS
By successively eroding the same section one can map several
different ions (for example, Ca2+, Na+, K+, etc.) in the same        Biological material and sample preparation
tissue and cells. It should be stressed that ions are highly         Paramecium
prone to extraction from cytological preparations during                Strains and culture conditions
specimen preparation (see Mentré and Escaig, 1988). A pre-           The wild-type (WT) cells used in these experiments were from stock
requisite for all the approaches just outlined is to take suitable   d4-2 of Paramecium tetraurelia. Cells were grown at 27°C in
precautions to avoid loss and/or redistribution of diffusible        phosphate-buffered wheat grass powder infusion, bacterized the day
compounds. This can be achieved by rapid freezing of the             before use with Klebsiella pneumoniae and supplemented with 0.5
cells, avoiding the use of any fixative, then either cryosubsti-      µg/ml β-sitosterol.
tuting the samples (as done in the present work) or using              Two mutants were used in this study: mutant tam8, whose tri-
freeze-fracturing followed by freeze-drying (Chandra and             chocysts are never attached to the cell surface (Beisson and Rossignol,
                                                                     1975; Lefort-Tran et al., 1981); and a thermosensitive mutant, nd9,
Morrison, 1992). Second, its sensitivity is at least as good as
                                                                     whose trichocysts are attached at the cell surface but cannot be dis-
that of X-ray microanalysis (EPMA) and probably slightly             charged at 27°C (Beisson et al., 1976).
better (see below). This level of sensitivity is comparable or
slightly inferior to that of electron energy loss spectrometry           Microscopy
methods (EELS); Third, images of ion distribution can be             Cells were harvested from early stationary phase cultures and the
obtained over large areas of cells in a short time, making the       pellet was fixed either: (1) in 0.5% glutaraldehyde plus 2%
method especially valuable when there is a need to observe           paraformaldehyde, 50 mM sodium cacodylate buffer, pH 7.4, for 20
whole tissues or extended portions of large cells. The major         minutes at 4°C, or in the same fixative followed by postfixation in 2%
drawback of SIMS microscopy is its limited lateral resolution,       OsO4 in the same buffer. Cells were then pre-embedded in fibrinogen
especially when compared with EELS, being of the order of            pellets, dehydrated and embedded in LR White or Epon-Araldite; or
                                                                     (2) by fast-freeze fixation by slamming the specimen against a cold
0.5 µm for the present instruments, yielding images equiva-
                                                                     copper block cooled by liquid helium (Escaig, 1983) followed by
lent to those from a light microscope. This drawback is partly       freeze substitution at −86°C for 72 hours in acetone in the presence
compensated by the sensitivity of SIMS, the very low level           of 20 mM oxalic acid, then warmed to −30°C, maintained for two
of background noise and the relative ease with which data            hours at −30°C, and finally warmed to room temperature and
from large areas can be collected (for a detailed comparison         embedded in Epon-Araldite.
of the merits and limitations of the various microprobe                 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 cryoem-               In our IMS 3F, the secondary ion beam intensity is measured
bedded in Lowicryl K4M.                                                     directly with the electron multiplier; the measurements are performed
                                                                            on selected areas, which are limited by adapted apertures. In the
    Exocytosis                                                              present work, the measured areas were always of 8 µm diameter. In
Synchronous exocytosis can be achieved with AED (amino ethyl                order to obtain statistically significant results, sets of at least 10 mea-
dextran), which causes instantaneous release of most of the trichocysts     surements were carried out on each holder for each of the domains
(Plattner et al., 1984, 1985; Kerboeuf and Cohen, 1990). AED, kindly        under analysis (Ca rim, cytoplasm, etc.). The beam was centered on
provided by J. Cohen and D. Kerboeuf, was used at 6 µM on a pellet          the area to be measured and the location of the area was recorded over
of cells. About 30 seconds after stimulation, the cells were slammed on     the image. Concerning the Ca rim located at the cell periphery, which
the cryobloc and cryodehydrated in the same way as the untreated cells.     is the main subject of this paper, the diameter of the measured circle
Aliquots of cells were further treated with picric acid and observed with   is larger than that of the rim. The rim was therefore positioned in the
a dark-field microscope to check the extend of exocytosis.                   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
Muscle                                                                      recorded values.
The cutaneous muscle of frog was taken up in Ringer’s solution. Very           Usually, several ions were recorded from the same section (most
small pieces were cryofixed and cryodehydrated exactly the same way          frequently Ca2+, Na+, K+ and Mg2+) and, in most cases, a histologi-
as for Paramecium.                                                          cal section immediately following or preceding those analyzed by
   To achieve a good preservation of cells (Paramecium and muscle),         SIMS was stained with Toluidine Blue on a glass slide, to be observed
ultrathin sections were obtained and stained with uranyl acetate            at the light microscope to provide a reference pattern.
followed by lead citrate. They were observed in Siemens Elmiskop
102 electron microscope.

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     Fast-freezing of wild-type Paramecium allows good
µm) on glass slides were stained with Toluidine Blue and sections (3        ultrastructural preservation
µm) for ion analysis were deposited over a microdrop of water on the        In order to prevent fixation-induced ion loss and redistribution,
gold holder, and heated to 60°C. In some control experiments sections
were deposited over the gold holder without any contact with water.
                                                                            we adapted the cryoblock method of Escaig (1983) to use with
   The instrument used in this study was an IMS 3F (CAMECA,                 a continuously fast-swimming, fragile cell such as Para-
Courbevoie, France), fitted with two primary ion sources and                 mecium. We used small pieces of filter paper to trap the cells
connected to an image processing system. This system (Olivo et al.,         in a thin layer while keeping them alive and healthy. Under the
1989) allows digitalization of images (512×512 pixels), high-speed          conditions used, only those cells that happen to be in the most
signal integration of the ionic images (to improve signal/noise ratio),     superficial portion of the filter facing the copper bloc were
histogram equalization (to increase the grey-scale contrast and to          cooled rapidly, a condition essential for preventing formation
decrease background noise) and, finally, image superposition.                of ice crystals. After rapid freezing, there are two main alter-
   The ion microscope was operated with the O2+ primary source with         natives for preserving the intracellular distribution of ions,
a 15 keV primary beam current of 200 nA. The image field diameter            freeze-substitution or even better, freeze-drying. The problem
was 150 µm. A mass resolution (M/∆M) of 2000 was used in order
to eliminate interferences between cluster ions and the specific
                                                                            of freeze-drying and freeze-sectioning in SIMS is that the
elements under study. Under these conditions, elemental mapping of          sections obtained in that way adhere very poorly to the gold
Ca, K, Na and Mg is easily achieved.                                        holders used in the following step. Sod et al. (1990) have
   For quantification and calibration, internal reference elements were      developed indium holders to overcome this difficulty for
used. In SIMS, the secondary ion beam current intensity (Ia) is a           animal cell cultures, but this approach is difficult to apply to
function of the concentration of the analyzed element (Ca), the area        single cells. In addition, in order to keep the paramecia in a
to be analyzed (S), the useful ion yield (Ya) and the primary ion beam      state as close as possible to the physiological one, we avoided
current intensity (Ip). Thus, Ia = Ca.S.Ya.Ip. However, when dealing        using high concentrations of centrifuged cells. Under the con-
with insulating specimens such as embedded biological samples, part         ditions used, the density of cells found in the filter paper is low.
of the positive primary ion beam is repelled by charge effects when
                                                                            This condition in addition to the previous one precluded the
negative secondary ions are extracted. The real intensity of the
primary ion beam current (Ip) is therefore variable, and the relation       use of the freeze-fracture and freeze-drying technique
between Ia and Ca is not directly applicable.                               developed by Chandra et al. (1986). Therefore we resorted to
   Nevertheless, a calibration is possible by measuring the intensity       freeze-substitution and inclusion in resin, realizing that some
of the secondary ion beam using an internal reference element (Ir),         redistribution of ions may occur at the final step of inclusion
which is present at a large homogeneous and constant concentration          in the resin when cells are brought back to room temperature.
in the specimen. Then Ia/Ir = K.Ca, where K is a proportionality            Controls embedded in Lowicryl at low temperature were thus
constant which can be determined using a standard with increasing           included to check for major redistribution (see below). The
concentration of the tested element to generate a calibration curve. As     frozen pieces of filter paper were then taken through the
the carbon content of biological specimens and embedding resins are         various steps of cryosubstitution in acetone, in the presence (or
virtually similar, this element can be used as an internal reference. In
                                                                            absence) of oxalic acid, inclusion in epon resin (or in Lowicryl)
order to quantify Ca, calcium octoate (Calcium-Norol by
SICCANOR, 59282 Douchy-les-Mines, France) was used as a                     and sectioning (see Materials and Methods). Sections were first
reference. Since calcium octoate is soluble in Epon-Araldite, samples       observed by both light and conventional transmission electron
containing varying Ca concentrations, from 0.05 mM to 5 mM, were            microscopy, to ascertain the quality of structural preservation.
prepared. Sections of 3 µm thickness were obtained and deposited               By conventional light microscopy (after Toluidine Blue
without any water onto the gold holders. Emission was measured over         staining) the cell contours and many cellular organelles were
fields of 150 µm in diameter.                                                readily recognized. In Fig. 1, for example, we can recognize,
1898 N. Stelly and others

                                                                       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
Fig. 1. Rapidly frozen WT Paramecium: semi-thin section stained        the type of embedding resin used and is seen even on unstained
with Toluidine Blue and observed with an optical microscope. N,        sections, thus indicating that the meshwork is not an artefact
macronucleus; V, food vacuole; T, trichocysts attached to the cortex
C. Bar, 10 µm.
                                                                       due to oxalate precipitation or staining. Cryofixation therefore
                                                                       reveals the presence of a genuine meshwork that had appar-
                                                                       ently collapsed or was extracted in all previous studies using
                                                                       chemical fixation.
from the periphery to the inside of the cell: cilia in the form of        The overall ultrastructural conservation at first sight
patches in some portions of the section, the cortex consisting         appeared to be slightly less good in Lowicryl than in Epon-
of adjacent typical cup-shaped cortical units, the carrot-shaped       Araldite. However, the cytoplasm and, especially, the
dark trichocysts perpendicular to the surface when cut longi-          reticulum membranes appeared to be less extracted, and more
tudinally, the oral depression or oral apparatus, and finally the       material seemed to be preserved in various complex organelles
dense cytoplasm with food vacuoles containing bacteria in              such as the axonemes. All further studies presented in this
various stages of digestion and, occasionally, a portion of the        paper, except for the Lowicryl control, were carried out with
macronucleus. The only abnormality observed is that the cells          Epon-Araldite-embedded material because: the ultrastructural
located at the front are slightly deformed by the compression          preservation appeared to be quite acceptable, these sections
generated during slamming on the copper block.                         adhered more easily to the gold substratum required for SIMS
   Fig. 2 illustrates the ultrastructural characteristics of stained   microscopy and the loss of ions was more limited during the
sections from cryofixed wild-type cells (b,c) as compared to            late stages of processing of Epon sections than Lowicryl ones
chemically fixed ones (a). Two other variables were also                (especially during recovery of sections on water).
analyzed (not shown): presence or absence of oxalic acid in the
cryosubstitution medium and inclusion in Lowicryl instead of           SIMS reveals a Ca compartment at the periphery of
Epon. Oxalic acid was added in order to promote in situ pre-           cryofixed Paramecium cells
cipitation of calcium (see Nicaise et al., 1989); Lowicryl was         Cell sections of both chemically fixed and cryofixed cells were
used in order to check the quality of ultrastructural preserva-        analyzed by SIMS microscopy as described in Materials and
tion it provided compared with traditional resins, since its use       Methods. The distribution of a large number of ions was
could prove useful both for ion distribution studies (by               examined; Fig. 3 shows an example of the results observed on
allowing polymerization at low temperature after cryosubsti-           cryofixed cells with two physiologically important ones, Na+
tution, thus further preventing ion diffusion) and for immuno-         and Ca2+, as compared to the light microscopic appearance of
cytochemical studies. In cells located close to the frozen front,      an adjacent section. Fig. 3a corresponds to the Toluidine Blue-
good ultrastructural preservation was observed: the cortex with        stained section and shows portions of seven cells, three of
its typical organelles was easily recognized, and even some of         which, in the upper part of the picture, lie along the edge of
the cytoskeletal networks which underly the cortex such as the         the frozen front. The various organelles pointed out in Fig. 1
epiplasm and the filamentous infraciliary lattice were seen             can be recognized.
(Allen, 1971) (Fig. 2b,c). The major differences noted with               The most striking differences in spatial distribution of ions
respect to conventional chemical fixation are: first, in the             depend on the fixation method concerned calcium: in chemi-
appearance of membranes within the cytoplasm and; second,              cally fixed cells, a weak Ca signal was uniformly distributed
in that of the alveolar lumen. Although the cytoplasm                  throughout the cells (not shown; see Fragu et al., 1992), while
displayed its classical ribosome- and glycogen-studded appear-         in cryofixed ones (Fig. 3b), the signal was restricted to a rela-
ance, membranes of the rough endoplasmic reticulum, of small           tively 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 redis-    yielded a homogeneously and intensely emitting cytoplasm in
tribution of ions, most clearly seen with Ca2+. Redistribution   which only some large circular areas, most probably corre-
was less striking with Na+, K+ and Mg2+ because these            sponding to food vacuoles, were not labelled (Fig. 3c). The
elements were uniformly distributed in cryofixed cells: Na+       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 semi-
                                                                           thin 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.


                                                                           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 addi-
                                                                           tional 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
Fig. 3. SIMS and light microscopy of adjacent cell sections. The           adjacent cortical alveoli was often observed (see for example,
sections traverse a large number of cells located at the edge of the       the three cell sections to the right of Fig. 3b). Three points
sample (i.e. facing the frozen copper block). (a) Toluidine Blue-          required evaluation, however, before a definitive acceptance of
stained section; (b) and (c) Ca and Na SIMS observed section
adjacent to that in (a). Note the regularity of the Ca peripheral signal
                                                                           this hypothesis. First, although the difference observed
in (b), its independence from the presence of trichocysts and its          between cryo- and chemically fixed cells was encouraging and
absence over the macronucleus. Bar, 30 µm.                                 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
or patchy labelling of the cytoplasm, again excluding vacuoles             well-known specific Ca localizations in striated muscle.
(not shown). In chemically fixed cells, Na+ and K+ were less                Second, since the large exocytotic vesicles known as tri-
uniformly distributed, with K+ concentrated into large precip-             chocysts are docked beneath the cell surface in Paramecium in
itates both inside and outside the cells. The general intensity            an highly regular arrangement, could they be contributing to
of emission also appeared to be reduced (not shown; see Fragu              the Ca signal? Indeed, many types of exocytotic vesicles
et al., 1992). In the remainder of this paper we will therefore            contain large amounts of Ca (reviewed by Nicaise et al., 1992).
only be describing cryofixed cells. The presence or absence of              This second question could be explored by using cells devoid
oxalic acid in the cryosubstitution medium did not modify the              of trichocysts. Third, did careful observation of a large number
results. Because some loss of elements can occur during the                of sections indeed confirm that both the size of the Ca-emitting
final stages of specimen preparation, when the sections are                 zone and its precise shape and distribution agree with what is
briefly deposited over a water drop on the gold holder and                  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          Trichocyst-deprived cells still display the peripheral
size of alveoli in electron microscopy? This last question could      Ca compartment
be analyzed by carefully comparing successive sections of             Two approaches were used to obtain cells devoid of tri-
material, some being observed by conventional light                   chocysts at the cortex; massive induction of trichocysts
microscopy to locate the major organelles and consecutive             discharge by AED from wild-type cells (Plattner et al., 1984),
ones by SIMS microscopy, and also by comparing the distrib-           or use of a mutant (tam8) lacking attached trichocysts at the
ution of Ca2+ with that of another ion such as Na+ to check           cortex (Beisson and Rossignol, 1975; Lefort-Tran et al.,
whether extracellular adhesion of Ca occurred.These three             1981). The wild-type cells were frozen within 30 seconds after
points are addressed below.                                           discharge; the extent of discharge was monitored by
                                                                      observing, in a dark-field microscope, aliquots of the AED-
SIMS identifies the expected Ca compartment in                         treated cell suspension to which picric acid had been added.
muscle cells                                                          Although sometimes irregular, AED-induced discharge was
Samples of frog cutaneous muscle were prepared using exactly          usually quite effective as shown by light and EM microscopy
the same methods as for Paramecium, i.e. by rapid freezing,           controls. In these cells rapidly frozen after discharge, some
cryosubstitution in the presence of oxalic acid and embedding         modifications in the ultrastructural aspect of the alveoli are
in Epon-Araldite. Conventional light microscopy shows the             apparent: they appear to be somewhat collapsed, with the
typical striated appearance of sarcomeres (Fig. 5b) and electron      outer membrane disjointed from the plasma membrane, and to
microscopy indicates a reasonably good ultrastructural preser-        contain less fluffy material than the controls. The mutant cells
vation (Fig. 5c). I and A bands are readily identified and the         were processed as wild-type cells. tam8, clearly, lacked
terminal cisternae are clearly seen in the I bands. In this tissue,   attached trichocysts at the cortex; its trichocysts lay randomly
EM electron-probe analysis of ultrathin cryosections has              within the cytoplasm. One additional mutant strain was used,
shown that 60 to 70% of total fiber Ca is localized in the             nd9. This is a conditional mutant, defective in exocytosis at
terminal cisternae, within the I bands (Somlyo et al., 1981).         27°C, but displaying a normal complement of trichocysts
SIMS microscopy (Fig. 5a) shows a regular alternation of              attached at the cortex. It is therefore defective in only the very
emitting and non-emitting bands for Ca, corresponding,                final steps of exocytosis (Beisson et al., 1980). This strain
respectively, to the light and dark striations seen in light          therefore allows discrimination between effects due to the
microscopy, and therefore to I and A bands, respectively. As          absence of trichocysts at the surface from effects only due to
expected, Ca is thus restricted to the I bands in which the           lack of exocytosis potential.
terminal cisternae of the sarcoplasmic reticulum are located,            In all cases (AED-treated WT, tam8, nd9), the peripheral Ca
and no major loss or redistribution of calcium seem to have           compartment was still observed by SIMS microscopy (Fig.
occurred during sample preparation.                                   6a,b,c). The Ca image of nd9 cells appeared to be identical to
   The periphery of the fiber also strongly emitted Ca,                that of WT ones. While in AED-treated WT cells and in tam8
probably because of the presence of a dense array of vesicles         ones, the width and intensity of the Ca zone appeared to be
lying immediately beneath the sarcolemna, referred to as              slightly reduced. This led us to a more quantitative study as
caveolae by Franzini-Armstrong (1970) and which possibly              described below. In any case, it became clear that trichocysts
contain high amounts of calcium originating from the exta-            cannot be the major contributors to the peripheral Ca signal,
cellular medium.                                                      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           adjacent sections observed by light microscopy (Figs 3 and 6)
absence of trichocysts at the cortex and their scattered presence   yields the following observations.
in the cytoplasm (see, for example, Fig. 6d).                          (1) The Ca-emitting zone exactly follows the periphery of
   A reverse argument can also be made: in many instances,          the cells, with all its deformations and, in particular, clearly
when a dense array of longitudinally sectioned trichocysts was      outlines the shape of the oral depression (which is also
observed in control sections, the corresponding SIMS image          bordered by alveoli for the most part; see Allen, 1974).
of an adjacent section often showed a Ca-emitting zone which           (2) The inner cytoplasmic portion of the sections gives a
was narrower than the zone occupied by the trichocyst bodies        much lower Ca signal except, occasionally, for food vacuoles.
(see Fig. 3a, for example).                                         There may be some correlation between the ‘age’ of vacuoles
                                                                    and the intensity of their signal, young vacuoles showing
Ca emission at the cell periphery is strictly                       higher signal than old ones (see Fig. 6c,d). No Ca emission was
correlated with the presence and integrity of the                   observed from these massive DNA-containing macronuclei.
cortex                                                                 (3) When a portion of a cell was torn during preparation, and
Side by side comparison of SIMS images and corresponding            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 Ca-
                                                                                                  emitting 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            SIMS microscopy, seems to be broader than would be expected
contained many cortical units such as the lower part of the cell   if only the lumen of the alveoli were emitting. We examined
in Fig. 1, the Ca-emitting zone was much enlarged.                 this question, with two types approaches, image saturation
   (5) Cell sections located far from the freezing front tend to   studies and morphometric studies involving superposition of
display a much less regular Ca rim, the farther ones sometimes     the Ca and Na images.
being devoid of it.                                                   For saturation studies, an increasing number of images
   Thus, the Ca-emitting zone appears to be tightly correlated     were summed, starting from low levels of integration
with that known to contain alveoli and to depend on good           (detecting high Ca concentration) to very high ones (detecting
freeze-fixation. However, the width of this zone, as seen by        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,             the quasi-uniform distribution of the much smaller amount of
only very slightly spilling over (as based on the Na and K                   total cytoplasmic Ca than that located in the alveoli.
images; see below), but it thickens with increasing integra-                    Taken together, these saturation studies indicate the
tions, reaching a width of approximately 5 µm (Fig. 7a to d).                presence of a thin intracellular peripheral compartment with a
This is broader than the largest width of alveoli as measured                very high Ca concentration, surrounded, on the outside, by a
on EM images (approx. 3 µm). Thus, the width of the Ca-                      narrow Ca-containing zone and, on the inside, by a broader Ca-
emitting zone is broader than what strictly corresponds to the               containing domain where Ca concentration decreases in a
alveoli. The same phenomenon was observed using ‘dry’                        graded manner towards the cytoplasm of the cell.
sections; it is therefore not due to diffusion induced by the
water drop used on the gold holder. Under the same satura-                   Quantification of alveolar Ca reveals a two-fold
tion conditions, the Na, K and Mg emissions did not spill over               decrease after exocytosis
across the cell boundary but remained confined within their                   As briefly indicated above, SIMS analysis revealed, in both
initial area of distribution (Fig. 7g and h), again indicating               tam8 cells and the wild type cells in which a massive release
that the situation observed for Ca reflects a real in situ dis-               of trichocysts was induced, a Ca emission lower than that of
tribution and not nonspecific diffusion. It should be stressed,               control cells. This had to be quantified rigorously because
however, that the level of integration required to see the                   section to section comparison in SIMS microscopy may not be
widening of the calcium rim is at least an order of magnitude                reliable. A proportionality coefficient (Ca/C) was therefore
higher than that needed to see the initial peripheral signal.                established for each of our measurements by measuring simul-
Thus, the amount of the excess calcium is much lower than                    taneously the Ca and C beam intensities within the same
that seen at low integration levels.                                         volume of sample. Each measure provided corresponds to the
   Widening of the Ca-emitting zone occurred mainly inward.                  mean of 10 measurements carried out over a surface of 8 µm
This was established by measurements on the micrographs,                     diameter. This allows a sample to sample comparison (see
superposition of hand-drawn tracings and, most directly, by                  Materials and Methods). In addition, absolute quantification of
computer superposition of the calcium and sodium images                      Ca was achieved using an internal standard consisting of
(Fig. 8), using the programs of Olivo et al. (1989). On such                 calcium octoate included in Epon-Araldite (see Materials and
images, it can be seen that at low integration levels, the red rim           Methods).
(Ca) superposes well over the green background (Na) giving a                    Table 1 summarizes the quantitative observations in
yellow-orange border, with no indication of a red external                   different types of cell sections. As expected, Ca concentration
margin; this remains the case even at higher levels of integra-              is significantly higher (about 7-fold) at the cell periphery than
tion. This shows: first, that the calcium rim detected at low                 within the cytoplasm in all types of cells and conditions (2 to
levels of integration is located inside the cell; and, second, that          3 mM vs 0.3 to 0.4 mM). It is interesting to compare the Ca
the outer boundary of calcium distribution does not extend                   concentration within the cortex of Paramecium with that in
much beyond that of Na.                                                      muscle, using SIMS. We found the muscle values to oscillate
   At extremely high integration levels (10 to 20-fold higher                around 10 mM. This is quite comparable to the value found by
than necessary to see the rim), a weak Ca signal was eventu-                 Somlyo et al. (1981) using EM microanalysis: their values
ally seen throughout the cytoplasm, most probably reflecting                  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                            The idea that the cortical alveoli, a network of large, inter-
                    Cortex          n1†   n2       Cytoplasm        n1   n2    connected membrane vesicles directly underlying the plasma
Wild type        17.9±8.4.10−1*     35    22      3.0±1.2.10−1      34   30
                                                                               membrane in Paramecium and other ciliates might correspond
(3 exp.)          3.4±1.8 mM                      0.4±0.2 mM                   to a Ca-sequestering compartment akin to the sarcoplasmic
Wild type +      10.5±5.10−1        36    20      3.7±1.5.10−1      15   11
                                                                               reticulum of muscle cells is old (Allen and Eckert, 1969; Satir
   AED            1.8±1.2 mM                      0.6±0.4 mM                   and Wissig, 1982). It received strong support when we
(2 exp.)                                                                       succeeded in purifying these vesicles and showed that they
tam8             12.4±6.2.10−1      41    14      4.4±3.10−1        11   11    actively pump Ca2+ in an ATP- and Mg2+-dependent process
 (1 exp.)         2.3±1.2 mM                      0.7±0.6 mM                   (Stelly et al., 1991). Additional evidence was provided by the
tam8 + AED       10.6±4.5.10−1      19    9       4.8±1.7.10−1       7    6    work of Schmitz et al. (1985), Zierold (1991) and Knoll et al.
(1 exp.)          1.8±1.0 mM                      0.8±0.4 mM                   (1993) using EM microanalysis methods. Here, we have sought
nd9              17.9±5.1.10−1      25    12      3.3±1.3.10−1       6    6    to provide direct visualization of this compartment over large
(1 exp.)          3.4±1.2 mM                      0.5±0.2 mM                   areas of many cells using a new approach, that of SIMS
                                                                               microscopy. The main advantage of this method lies in the fact
   *The upper line represents the mean of Ca count/C count ± s.d. The second   that it provides images of total Ca distribution over individual
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    cell sections and is thus especially suited for identifying Ca
which measurements were carried out; the numbers in the n2 columns refer to    storage sites (as compared to methods detecting only free
the number of different cell sections examined.                                Ca2+). Since the lateral resolution of the method is limited,
                                                                               however, the compartment must be of sufficient size to be iden-
                                                                               tifiable. Because cortical alveoli are typically about 0.2 to 2 µm
corresponding to a position of their narrow (20 µm) probe                      × 1 to 3 µm in size, we hoped that, if they indeed contained
within the terminal cisternae. When converted to mM, the units                 large amounts of Ca, SIMS microscopy would allow their visu-
used in the present work, by taking into account the water                     alization. In addition, there was a clear prediction as to the
amount, their values are 3 to 30 mM, i.e. with an average very                 expected intracellular location of the signal, namely through-
close to our 10 mM value obtained by SIMS with a much larger                   out the cell periphery.
probe diameter.                                                                   These predictions were fulfilled remarkably: a continuous,
   Thus, the total Ca concentration in the cortex is in the same               peripheral, Ca-emitting zone was immediately seen, provided
range as that of the sarcoplasmic reticulum.                                   that cells were fixed by rapid freezing.
   After induction of massive exocytosis by AED, a marked                         The good ultrastructural conservation of cells after cryofix-
decrease in Ca concentration in the cortex was repeatedly                      ation, the fact that the peripheral location of Ca strictly
observed (from 3.4±1.8 mM to 1.8±1.2 mM). This decrease is                     depended on avoiding chemical fixation, and the excellent cor-
statistically highly significant using the t-test (P<0.0001).                   relation observed between the occurrence of the Ca signal and
Interestingly, this decrease in the cortex appears to be corre-                the presence of a strip of well frozen cortex in the corre-
lated with an increase in Ca concentration within the                          sponding section, all argue against artifacts. In addition, the
cytoplasm, but this is barely significant statistically. The                    fact that it is found also in cortices devoid of trichocysts
kinetics of these changes was not studied. All the data                        demonstrates that the Ca does not emanate predominantly from
presented are from experiments with cells that were cryofixed                   these exocytotic organelles. The most likely Ca-containing
about 30 seconds after exocytosis.                                             compartment therefore remaining the alveolar one. Additional
   Concerning trichocyst mutant strains, nd9 displayed a                       evidence on these two points was recently provided by electron
cortical Ca concentration identical to that of wild type, indi-                probe microscopy. Using conventional X-ray microanalysis,
cating that, when trichocysts are attached at the cortex, inca-                we found amounts of Ca in a number of alveoli ranging from
pacity to carry out exocytosis does not lead per se to a modi-                 5 to 10 mM (Stelly, Halpern and Nicaise, unpublished). No Ca
fication of Ca concentration.                                                   signal was observed on trichocysts and this is all the more sig-
   In contrast, tam8 displayed a significantly lower amount,                    nificant, since these organelles are easily identified in the
placing it at an intermediate level between normal wild type                   unstained sections used for microanalysis. Confirmation of
and wild type after exocytosis. Treatment of tam8 cells with                   these two points can be found in a recent study of Knoll et al.
AED further decreased the Ca concentration in the cortex but                   (1993) in which one electron energy loss spectrum image of
this was only marginally significant statistically (P<0.02).                    ultrarapidly fixed Paramecium is provided, showing the
   Trichocyst exocytosis or lack of attached trichocysts at the                presence of Ca in alveoli (not in trichocysts) and its redistrib-
cortex therefore lead to a significant decrease in cortical Ca                  ution after exocytosis. It should be pointed out that previous
concentration.                                                                 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
DISCUSSION                                                                     converges to indicate that trichocysts make little or no contri-
                                                                               bution to the peripheral calcium signal and that the signal
The major result presented in this paper is the visualization of               emanates predominantly from alveoli.
a subcortical Ca compartment in Paramecium using a new                            The width of the Paramecium band, as seen in SIMS
method, SIMS microscopy, applied to sections of ultrarapidly                   microscopy, can be estimated as 3 to 5 µm, while the EM
frozen cells. In addition, the amount of Ca in this compartment                observations indicate that the alveoli do not exceed 2 µm. In
was found to decrease substantially after massive induction of                 addition, saturation studies show that this width can reach 10
exocytosis.                                                                    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            newly discovered material in Paramecium alveoli. We
explanations can account for this observation. These fall into      searched for a homologue of calsequestrin in the cortical
two broad categories: artefactual diffusion of Ca from the          fraction through immunoblotting, Stains-all decoration and
alveoli during sample preparation, or normal presence of Ca in      radioactive Ca-overlay of gel blots but did not observed a
the proximity of the alveolar compartment. The first hypothe-        signal in the calsequestrin Mr zone.
sis cannot be totally excluded and, in fact, some diffusion            The second corollary of the high intra-alveolar Ca concen-
would not be surprising in view of all the steps involved in        tration is the likelihood of a tight control over its release. Pre-
sample preparation. We have excluded the possibility,               viously, we have focused on the analysis of Ca2+ uptake into
however, that rapid flotation of sections has a major effect,        alveoli in vitro (Stelly et al., 1991). We are currently charac-
since sections obtained by completely avoiding any contact          terizing the release system kinetically and pharmacologically.
with water showed an identical distribution of all the major        There are indirect indications for the operation of an InsP3
ions analyzed. In addition, inclusion in Lowicryl at low tem-       system in Paramecium (Beisson and Ruiz, 1992) and various
perature yielded exactly the same Ca rim as that in Epon-           components of the inositide cascade are present (Freund et al.,
Araldite, indicating that no major diffusion occurs at the time     1992) although InsP3 itself has proven elusive. Similarly, the
when the cryosubstituted samples are warmed up. Finally, this       occurrence of a Ca2+-induced/Ca2+-release cascade has been
extracellular emitting zone is Ca-specific and was not observed      suggested to underly morphogenetic waves during cell division
for several other ions, indicating that it does not reflect a gen-   (Le Guyader and Hyver, 1991), but remains to be demon-
eralized diffusion from the inside of the cell. The second          strated.
hypothesis is therefore much more likely and at least two              As for the function of alveoli in Ca2+ regulation, at least
possible explanations for a genuine in situ broader Ca zone can     three roles might be considered (Stelly et al., 1991), especially
be suggested. First, the cell is covered by cilia and both the      when it is noted that the alveoli are in close proximity to three
plasma and ciliary membranes may be expected to bind a sub-         Ca2+-controlled organelles, the cilia, the trichocysts and
stantial amount of Ca by means of the negatively charged phos-      cytoskeletal networks: (1) general homeostasis of intracellular
pholipids and the glycosylated cell coat, thus spreading the        Ca2+ by active sequestration above a given cytosolic level; this,
signal towards the outside of the cell. Second, concerning the      for example, might be the case when a surge of Ca2+ occurs
spread of the signal towards the cell’s interior, it should be      through depolarization of the ciliary membrane. The alveoli
recalled that a vast filamentous network, the infraciliary           being immediately adjacent to basal bodies might pump the
network, recently shown to be made up of Ca-binding proteins        Ca2+ that has entered the ciliary lumen; (2) release of at least
(Garreau de Loubresse et al., 1991), makes up the deepest of        part of the Ca2+ required for trichocyst exocytosis. In this
the cortical cytoskeletal layers several micrometers below the      respect, it must be stressed that alveolar membranes are tightly
alveoli. This network may well yield a signal beneath the           apposed to the membranes of the tip of trichocysts; (3) release
alveoli on the sections. In addition in Paramecium, especially      of the Ca2+ required for the disassembly of the several
in stationary phase cells, mitochondria tend to be concentrated     cytoskeletal networks adjacent to the cortex when they undergo
just below the cortex, providing a further possible weaker          reorganization during division (Iftode et al., 1989). Of these,
calcium-emitting zone (see Girard et al., 1991, and Rizzuto et      one of the thickest is the infraciliary lattice, which is made up
al., 1993, for recent evidence of calcium pumping of ER-            of Ca-binding contractile proteins (Garreau de Loubresse et al.,
released calcium by mitochondria). It appears, therefore, that      1991). In the case of this network, the alveoli may also provide
three Ca domains can be identified at the cell periphery in          Ca2+ to regulate contractility.
Paramecium: (i) a narrow zone of very high Ca concentration            The present results clearly support the idea that the alveoli
corresponding to alveoli, surrounded by: (ii) a small extracel-     provide at least some of the Ca2+ required during exocytosis,
lular zone probably corresponding to membrane- and cilium-          since after massive exocytosis the amount of Ca2+ inside the
bound Ca; and (iii) a larger intracellular zone displaying a Ca     alveoli dropped by 50%. It should be stressed that such a
concentration decreasing towards the inside of the cell.            massive drop in Ca2+ is quite similar to what occurs in the sar-
   Through calibration of the absolute amount of Ca using a         coplasmic reticulum during muscle contraction (Somlyo et al.,
calcium octoate derivative embedded and analyzed in the same        1981). In fact, we may not have captured the point of lowest
conditions, the absolute amount of Ca contained in various          Ca2+ concentration, since the cryofixation device used imposes
areas of the sections was approximated. Within the peripheral       a delay of about 30 seconds between the application of the sec-
band, the mean value was 3.4±1.8 mM. This concentration is          retagogue (AED) and the fixation of cells. Using faster fixation
higher by several orders of magnitude than that of free             methods, Knoll et al. (1993) recently observed a profound
cytosolic Ca2+ (10−4 mM; Eckert, 1972). There are several           redistribution of alveolar Ca2+ within 80 milliseconds after
implications of this considerable difference. First, it is most     AED-induced exocytosis in Paramecium. This observation
likely that Ca is associated with a Ca-sequestering protein         agrees very well with our results.
within the lumen of the alveoli. In fact, we wonder whether the        The question of the origin of the Ca2+ required for exocyto-
fluffy material identified in the alveoli only after ultra-rapid      sis has been extensively discussed both for Paramecium
freezing corresponds to such hypothetical sequestering              (Plattner et al., 1991; Knoll et al., 1992, 1993; Cohen and
proteins. A tempting cytological analogy with calsequestrin         Kerboeuf, 1993) and other systems. Our data indicate that a
can indeed be made: only when rapid fixation was used was a          contribution of alveoli to the process is very likely. Interest-
diffuse, fluffy material clearly seen in terminal cisternae, which   ingly, Cohen and Kerboeuf (1993), on the basis of a completely
was later identified as calsequestrin (Jorgensen and Campbell,       different approach, also concluded that at least part of the Ca2+
1984; Jorgensen et al., 1985). Previously, conventional             involved in trichocyst exocytosis originates from intracellular
chemical fixation had failed to reveal it, as is the case with the   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        contain a ryanodine receptor (McPherson et al., 1992, Sardet
still unknown but the tight apposition of alveolar and trichocyst    et al., 1992). The ciliate Ca stores may therefore be evolution-
membranes, at the tip of trichocysts, suggests a direct flow,         ary homologues of this ER-derived network and, indeed, of
through specific transmembrane proteins.                              other Ca storage compartments of ‘higher’ eukaryotes, but this
   Assuming that trichocysts themselves contain very little          is an unproven generalization. The search for proteins homol-
calcium, as we concluded above, the fact that exocytotic             ogous to those of the compartments of higher eukaryotes in
mutants lacking attached trichocysts show a much decreased           alveolar sacs has been hampered by the great evolutionary
amount of Ca2+ in alveoli, while those having attached tri-          distance separating ciliates from metazoa, which usually leads
chocysts have normal amounts, provides a further hint as to the      to failure of cross-reaction when most antibodies to metazoan
regulation of alveolar amounts. It suggests that trichocyst          proteins are tested in Paramecium. In fact, the exact relation-
attachment per se, independently of exocytotic capability,           ship of alveoli to the endoplasmic reticulum in ciliates and their
provides a regulatory loop inducing normal Ca2+ pumping and          mode of biogenesis are still unclear. It is being studied at
sequestration in alveoli. It should be recalled in this context      present using EM immunocytochemistry by our group, with
that mutant tam8, which lacks cortex-attached trichocysts, was       plasma membrane markers (Charret et al., unpublished;
shown in our previous work to pump Ca2+ in vitro as efficiently      Capdeville et al., 1993).
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        This work was supported by a grant from the Université Paris-Sud
an intrinsic defect of the Ca2+-pumping machinery but was due        (Action Interdisciplinaire 8922). We thank Dr J. Cohen and D.
rather to an indirect, presumably regulatory, process.               Kerboeuf for mutant strains and gift of AED, Drs J. P. Mauger and J.
   How wide is the occurrence of such vesicular Ca compart-          Cohen for critical reading of the manuscript, Dr M. Müller for his help
                                                                     in improving the manuscript and Prof. G. Nicaise for sharing the X-
ments closely apposed to the plasma membrane? The obser-             ray results and for comments on the manuscript. We are grateful to
vation made in Paramecium appears to be valid for other              Mrs N. Narradon for expert photographic assistance and to Mrs C.
ciliates: preliminary SIMS observations carried out in Tetrahy-      Couanon for her careful preparation of the manuscript.
mena, a genus relatively close to Paramecium in molecular
evolutionary terms, but also in Euplotes, a very distant hypotri-    REFERENCES
chous ciliate (Baroin-Tourancheau et al., 1992), show the
                                                                     Allen, R. D. and Eckert, R. (1969). A morphological system in ciliates
presence of the peripheral Ca band (Stelly, unpublished).              comparable to the sarcoplasmic reticulum-transverse tubular system in
Cortical alveoli are in fact a shared ultrastructural character of     striated muscle. J. Cell. Biol. 43 (2, Pt. 2), 4a (Abstr.)
three protist Phyla, ciliates, dinoflagellates and apicomplexa        Allen, R. D. (1971). Fine structure of membranous and microfibrillar systems
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                                                                       associated membrane transport in Paramecium. J. Cell Biol. 63, 904-922.
by molecular phylogenetic analysis to form a monophyletic            Andrews, S. B., Leapman, R. D., Landis, D. M. D. and Reese, T. S. (1987).
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