Faculty of Biology, University of Konstanz, D-78434 Konstanz, Germany
In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of
Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935-
945). We now show by voltage clamp and extracellular
recordings that the product of current × time (As)
closely parallels the number of exocytotic events. We
suggest that Ca2+ mobilization from subplasmalemmal
storage compartments, covering almost the entire cell
surface, is a key event. In fact, after local stimulation,
Ca2+ imaging with high time resolution reveals rapid,
transient, local signals even when extracellular Ca2+ is
quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e [Ca2+]i). Under these conditions,
quenched-flow/freeze-fracture analysis shows that
membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid,
strong cortical increase of [Ca2+]i but no exocytosis. In
various cells, the ratio of maximal vs. minimal currents
registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current
steps could be observed, this is again compatible with
the combined occurrence of Ca2+ mobilization from
stores (providing close to threshold Ca2+ levels) and
Ca2+ influx from the medium (which per se does not
cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal
triggering rapid, local exocytotic responses, as requested for Paramecium defense.
In most systems analyzed so far, exocytosis is triggered
by the increase of intracellular free Ca2+ concentration ([Ca2+]i)1. In fast responding systems such as
motor endplates, this increase occurs through an influx of
extracellular Ca2+ (Ca2+e), via voltage-dependent Ca2+ channels at active zones where neurotransmitter vesicles are docked. In other systems, Ca2+ is mobilized exclusively or
additionally from internal stores (Burgoyne and Morgan,
1993 Recent views have emphasized the possible primary importance of subplasmalemmal Ca stores due to their structural and functional coupling with the cell membrane
(Berridge, 1995 A Paramecium cell displays an egg case type surface relief with unit fields, kinetids, which are associated with as
many alveolar sacs. Alveolar sacs, which are known to be
Ca stores (Stelly et al., 1991 In the present study, a more precise correlation between
electrophysiological and morphometric data has been
carried out by using [Ca2+]-fluorochrome analysis and rapid
confocal laser scanning microscopy (CLSM), with quenched-
flow/freeze-fracture to reveal membrane fusion events.
Our goal was to analyze the origins of subplasmalemmal Ca2+ signals and their importance for exocytotic events.
Cell Cultures
Paramecium tetraurelia wild-type (7S) cells were cultivated monoxenically
to stationary phase, with Enterobacter aerogenes added, as previously described (Plattner et al., 1984 Electrophysiology
The method applied for voltage clamp and for extracellular current recordings as well as the solutions used were as described previously (Erxleben and Plattner, 1994 Secretagogue-induced currents were recorded under voltage clamp
conditions or as extracellular currents, as described previously (Erxleben
and Plattner, 1994 Estimation of Cell Surface Area and of the Number of
Subplasmalemmal Ca Stores
Cell size was estimated from pictures taken with a phase contrast microscope, 40× objective, including a grating scale in samples. On prints of
1,125× magnification, median views of cells were subdivided into 5 mm
discs. Cell surface area and cell volume were computed assuming rotational symmetry. Cilia and crenulation of the somatic surface area could
be neglected for the following reasons: (a) Cilia do not contribute to Ca2+
fluxes during AED-triggered exocytosis (Plattner et al., 1984 The average area of a kinetid was determined at the light and electron
microscope level. On LM micrographs (100× oil immersion lens and grating scale included in samples), kinetid sizes were evaluated at 2,130×
magnification. The same was done on EM micrographs obtained as follows. Cells sandwiched between thin copper sheets were injected into
melting propane, freeze-fractured, and Pt/C replicated in a BAF300
freeze-fracture device from Balzers S.P.A. (Balzers, Liechtenstein). Replicas were evaluated at EM magnifications controlled by latex particles of
defined size (Serva, Heidelberg, Germany) and reproduced at 11.400×
magnification. Longitudinal and perpendicular dimensions of kinetids
were computed from LM and EM micrographs by evaluating as many periods in strictly vertical view from as many cells as possible.
Quenched-Flow/Freeze-Fracture Analysis with Normal
or Reduced Extracellular Ca2+
Cells were processed in a quenched-flow device according to Knoll et al.
(1991a) Ca2+ Imaging by Conventional Microscopy or CLSM
Experiments were conducted with single cells bathed in a defined microdroplet of medium containing 0.1 mM Ca2+. Cells were microinjected with
the following fluorochromes: 100 µM calcium green-2 (CaGr-2), 100 µM
Fluo-3, or 50 µM Fura red (final concentrations in cells after injection of
10% of the cell volume size; Table I). Fluorochromes, all obtained from
Molecular Probes (Eugene, OR), were allowed to spread for 2 min to reach the outermost cell periphery.
Table I.
Morphometric Parameters of P. tetraurelia Cells Used
in This Study
; Cheek and Barry, 1993
; Pozzan et al., 1994
).
). In most systems, however, such stores
are difficult to identify, as is their structural relationship
with secretory organelles. The latter relationship is important, considering the rapid decay of local [Ca2+]i increases
taking place with space and time (Llinás et al., 1992
; Neher
and Augustine, 1992
; Zucker, 1993
). Clearly, therefore, a
secretory system operating under defined spatio-temporal
conditions offers advantages for analyzing the role of subplasmalemmal Ca stores in regulation of exocytosis. Paramecium tetraurelia cells can be such a system. In fact, each
Paramecium cell contains numerous secretory organelles,
or trichocysts, attached at the cell membrane, ready for
immediate release (Plattner et al., 1991
). Single or a few
trichocysts are discharged spontaneously, or upon slight irritation of the cell (Haacke-Bell et al., 1990
). The other
extreme is the synchronous (within <1 s) release of most
of the >103 docked trichocysts in response to aminoethyldextran (AED) (Plattner et al., 1984
, 1985; Knoll et al.,
1991a
). Such a massive trichocyst exocytosis can be restricted to discrete sites of the cell surface following either
local application of AED (Plattner et al., 1984
), or contact
with predatory ciliates, whereby it serves a defensive function (Knoll et al., 1991b
). With AED, signal transduction is restricted to the somatic (nonciliary) cell membrane
(Plattner et al., 1984
, 1991; Erxleben and Plattner, 1994
).
; Länge et al., 1995
), are tightly
attached to the cell membrane, with each trichocyst positioned at the edge of four adjacent sacs (Allen, 1988
). During AED-triggered exocytosis (Knoll et al., 1993
; Stelly et al.,
1995
) the sacs are rapidly mobilized, with ensuing rapid increase in subplasmalemmal [Ca2+]i (Knoll et al., 1993
) and
activation of Ca2+-dependent currents whose size roughly
correlates with the extent of AED stimulation (Erxleben
and Plattner, 1994
). Moreover, under these conditions mobilization of Ca2+ from subplasmalemmal stores is accompanied by an influx of Ca2+e (Kerboeuf and Cohen, 1990
;
Knoll et al., 1992
; Erxleben and Plattner, 1994
).
Materials and Methods
, 1985), using a medium supplemented with
stigmasterol (Sigma Chemical Co., St. Louis, MO), 5 mg/liter, and [Ca2+]
of 0.1 mM.
). Briefly, the anterior pole of a cell was sucked into
a holding pipette (covering ~1/2 of the cell surface). Spontaneous currents were registered after cells were impaled by the microelectrode or,
for extracellular recordings, after immobilization in the holding pipette.
Spontaneous current fluctuations were registered in parallel to the occurrence of single or several secretory events. Since the number of truly spontaneous current events was very variable and usually quite low (in the order of 1/1-5 min), cells were also triggered to different extents by local
pressure application of 0.01% AED (Plattner et al., 1984
, 1985) from a pipette positioned about two cell lengths away. Pressure and duration of the
pulse were adjusted such that only a small number of trichocysts were released. Pressure application of solution without AED evoked neither currents nor exocytosis.
). Discharge of trichocysts was observed under a microscope with 40× phase contrast water-immersion optics and recorded with
a CCD camera. To establish the temporal relationship between the currents elicited by AED and the discharge of trichocysts as observed under
the microscope, AED-induced currents were displayed on an oscilloscope,
from which they were recorded by a second CCD camera. The video signals of both cameras, one attached to the microscope and the other recording the oscilloscope traces, were combined by a digital video mixer
(WJ-AVE5; Panasonic, Osaka, Japan). The time resolution for the observation camera was 20 ms. For analysis, half frames of the combined video
signal were analyzed on a monitor. For processing of figures, the video signal was digitized with a frame grabber (miroVideo D1; MicroComputer products AG, Braunschweig, Germany).
; Erxleben and
Plattner, 1994
). (b) Crenulation may cause only <20% surface increase.
(c) Our main goal was to estimate the number of alveolar sacs per cell reflected by the number of surface fields, or kinetids, per cell.
and Plattner et al. (1994)
. The medium contained 0.1 mM Ca2+.
Cells were mixed with either equal parts of culture medium (negative control) or with 0.02% AED (positive control). Aliquots were mixed with
equal parts of 9 mM EGTA for 200 ms and then with two parts of 0.02%
AED for an additional 80 ms. After mixing in the apparatus, samples were
frozen in melting propane for subsequent freeze-fracture. Evaluation of
trichocyst docking sites, with or without exocytotic membrane fusion, was
as described previously (Knoll et al., 1991a
; Plattner et al., 1994
).
In some experiments, cells preloaded with Fura red were flushed with high [Ca2+]e and quantitatively analyzed for cortical [Ca2+] transients and exocytotic response. In more expansive experiments, [Ca2+]e was reduced by superfusion (for up to 1 s, from a distance of ~10 µm, in a direction tangential to the cell surface) with a local flush, using a pipette (2 µm inner diameter) filled with 10 mM EGTA and 50 µM fluorescein (Sigma Chemical Co.) using a home made device operated at 2.5 kPa. By the calibration of the fluorescein signal, the EGTA concentration at the discrete site of the cell surface was estimated to be ~5 mM, corresponding to [Ca2+] resting levels inside the cell (50-100 nM). Overall [Ca2+]e was determined separately by addition of a calibrated Fura red solution to the microdroplet. At the end of an EGTA flush, AED was applied to the same site using a second pipette close to the EGTA pipette but vertical to the cell surface. To control the propagation of AED and the concentration sensed by the cell surface with this set up, fluorescein was sometimes also added to the secretagogue.
Fluorescence evaluation was either in a conventional mode, with 1-2 s
filter changes, using an inverted microscope (ICM 405; Zeiss Inc., Oberkochen, Germany), or a microscope (Axiovert; Zeiss Inc.) equipped with
a confocal imaging system (Noran Instruments, Bruchsal, Germany). A
minimum of 33 ms frame sequences allowed documentation of [Ca2+]i fluorescence transients with CLSM, in some cases alternating with phase
contrast imaging. Excitation wavelength was 488 nm (Ar-laser emission)
for CaGr-2, Fluo-3, or fluorescein, or 490 nm (±5 nm, band pass filter) for
Fura red. Emitted wavelength registered was 515 nm for CaGr-2, Fluo-3,
or fluorescein, or
560 nm for Fura red (detected by a moonlight camera;
Panasonic, combined with a 560 nm dichroic mirror and a 560-nm-long
pass filter).
To minimize false or irrelevant fluorescence signals, evaluation included (a) calibration of fluorochrome signals with standard Ca2+ solutions in microdroplets, (b) intermittent registration of signals with exocytosis recording in phase contrast, (c) overlapping use of fluorochromes with different properties, (d) mimicking shape changes via mechanical deformation without exocytosis triggering, (e) background reduction by digital image subtraction in rapid CLSM series, (f) transformation of signals into false colors, and (g) evaluation of important cell regions by line scans. We could thus either follow semi-quantitatively rapid [Ca2+] changes in the CLSM mode, or we could follow quantitatively longer lasting [Ca2+] changes in the conventional microscope.
Electrophysiological Recordings Show Correlation of Subplasmalemmal Ca2+ Transients with Exocytosis
Flushing a Paramecium cell with AED causes massive exocytosis which, under voltage clamp conditions, is paralleled by an outward current of 3 × 109 As lasting ~2 s
(Fig. 1 A, top). Some smaller peaks may precede the main
current peak. As shown in more detail below, the magnitude of the current is proportional to the number of trichocysts released. The currents display a fast rising phase
and a slower, approximately exponential decay (Fig. 1 B).
A second pulse of AED after 20 s provokes little if any additional trichocyst release and causes a series of only small
currents (Fig. 1 A, bottom).
Small numbers of trichocysts can be quantified in the
LM (Plattner et al., 1984, 1985). Exocytosis of one or a few
trichocysts occurs upon slight irritation of a cell (HaackeBell et al., 1990), i.e., during immobilization and impalement by a microelectrode. This, however, does not appear
to be the immediate trigger for spontaneous exocytosis or
spontaneous currents we observed, since we allowed cells
to stabilize before recordings were started. Stochastic current peaks are observed during both intracellular and extracellular recordings (Fig. 2). Extracellularly recorded currents are of a different size but of similar shape, as are currents recorded under voltage clamp conditions (Fig.
1 A, top). Spontaneous signals representing 36 events were
averaged in Fig. 2 B. Currents with a mean amplitude of 34 pA show a fast rising phase (rise time, tr = 7 ms) and a half
width of t1/2 = 21 ms. These properties characterize single
events, corresponding to the peak in Fig. 3 D (arrow)
which is associated with the release of a single trichocyst.
The histogram of Fig. 2 C clearly shows the absence of discrete steps in the current size, i.e., absence of quantal current events. Fig. 3 reveals that a minimal current peak accompanies release of an individual trichocyst. A further example of simultaneous current and exocytosis registration involving a larger number of trichocysts is documented in Fig. 4.
Fig. 5 correlates the charge of the electrical events with
the number of trichocysts released. Data scatter is large
but not unexpected for several reasons. (a) Trichocyst countings are not absolutely certain. (b) The Ca2+-activated currents recorded are not caused simply by mobilization from
discrete stores but may be amplified to a variable extent by Ca2+ influx from the medium. (c) While exocytosis is always accompanied by a current, spontaneous currents can
be observed that are not accompanied by spontaneous
exocytosis. This may be due to the fact that not all potential exocytosis sites are occupied by a trichocyst (Plattner
et al., 1991; Knoll et al., 1991a
, 1993). Within these limitations, we calculated the unit current event as 1.21 ± 0.97 pC per exocytotic event (Fig. 5). By EGTA injection we
have ascertained that the currents described depend on a
subplasmalemmal [Ca2+]i increase (Fig. 6).
In conclusion, our recordings show that the number of trichocysts released by exocytosis in response to AED is paralleled by a nonstepwise increase in subplasmalemmal Ca2+-dependent currents.
Quenched-Flow/Freeze-Fracture Analysis Shows only Partial Reduction of Exocytotic Membrane Fusion with Low [Ca2+]e, while under Normal Conditions Internal and External Ca2+ Sources Contribute to Exocytosis
Exocytosis of trichocyst contents is monitored in the LM
by decondensation (stretching) to the typical needles seen
outside a cell (see Fig. 9, D and F and Fig. 10, M and O).
Since this requires Ca2+e (Bilinski et al., 1981), the effects
of low [Ca2+]e on membrane fusion had to be analyzed
separately in quenched-flow/freeze-fracture experiments
(Fig. 7). This also allows rigorous mixing of cells with
EGTA and, thus, restriction of the time of EGTA application since this could potentially affect cell function (see below). Trichocyst docking sites were rated as resting stages
with rosettes (aggregates of integral membrane proteins;
Plattner et al., 1991
), or as activated stages with exocytotic
openings of variable diameters. Fig. 7 includes data from
controls (left) which were mockstimulated with culture
medium (0.1 mM [Ca2+]e) and processed by the same
method. In this case, no activated docking sites were observed, and resting stages in these controls are used as reference (100%). In the presence of Ca2+e, AED activates
all exocytotic sites (Fig. 7, middle). EGTA application for
200 ms, followed by 80 ms AED triggering, allows activation of only ~40% of the potential trichocyst docking sites
(Fig. 7, right). 80 ms was selected because synchronous
exocytosis is normally completed within this time (Knoll
et al., 1991a
). Columns in these data pairs do not add up to
identical values because they represent medians derived
from different individual cells, with variable numbers of
observations.
These data show that exocytotic membrane fusion of about half of the docking sites, can be induced by mobilization of Ca2+ from internal pools. We conclude that Ca2+ mobilization from subplasmalemmal pools operates at its limits and is normally intensified by a Ca2+ influx.
Ca2+ Imaging Shows a Rapid Subplasmalemmal [Ca2+] Transient by AED Stimulation Even with Low [Ca2+]e, while Such a Transient Achieved by Rapid [Ca2+]e Increase Alone Does Not Induce Exocytosis
Different fluorochromes were used to overcome problems specific of different experiments. Fura red allows quantitation, although with low time resolution. CaGr-2 and Fluo-3 combined with CLSM allow only semi-quantitative analyses, though in the sub-second range, whereby Fluo-3 has the disadvantage of being partially sequestered into phagosomes, while providing significantly higher quantum yield than CaGr-2. The occurrence of similar signals over the same time periods made us confident, however, about the relevance of our findings.
To establish whether a Ca2+ influx would also suffice to
trigger exocytosis or, alternatively, whether alveolar sacs
may be the primary Ca2+ source during AED stimulation,
as suggested above, we first worked with media in which
Ca2+e was chelated by EGTA. Long term experiments, however, were impossible, because at low [Ca2+]e (50 µM)
the membrane of many cells becomes leaky to a broad spectrum of small molecules (Hille, 1992
). As Fig. 8 shows,
increasing [Ca2+]e to 10 mM suffices to produce an immediate strong cortical [Ca2+] transient, yet without inducing
any exocytotic responses. This largely excludes any CICR
mechanism.
CLSM using CaGr-2 f/fo imaging shows that a cortical
[Ca2+] transient occurs within ~100 ms of AED application (Fig. 9, A-F), i.e., within the time required for exocytosis (Knoll et al., 1991a), as documented by the intermittent phase contrast pictures in Fig. 9, A-F. Controls
carried out to exclude a role of cell deformation included
analyses with more strict time scales, revealing a continuous build up of the cortical fluorescence signal irrespective
of cell deformation (Fig. 9, G-N), and local mechanical
deformation of cells which failed to produce any comparable signal (Fig. 9, O-Q). Therefore, we are confident
that the cortical Ca2+ signal parallels exocytosis, as documented above independently by electrophysiology and by
quenched-flow/freeze-fracture analysis.
Next we analyzed whether Ca2+ mobilization from
stores might suffice to trigger exocytosis. Results obtained
by fluorescein-tagged EGTA solution with subsequent
AED application are shown in Fig. 10, A-H. Under these
conditions no trichocyst release occurs. Reliability of the
superfusion approach was checked independently with fluorescein-tagged AED in the presence of 0.1 mM [Ca2+]e
(Fig. 10, I-P). Since trichocyst decondensation (expansion
during expulsion of contents) requires Ca2+e (Bilinski et al.,
1981), comparison of Figs. 10, A-H (+ EGTA) and 10, I-P
(+ Ca2+e) shows successful chelation of Ca2+e. Next we
have loaded cells with Fluo-3. After 2 min, each of these
cells was superfused with EGTA and exposed to a local
AED flush (without fluorescein). This final set of experiments, documented in Fig. 10, Q-T, clearly shows a cortical [Ca2+]i increase already recognizable within ~100 ms
with little diffusion throughout the cell. Under such conditions quenched-flow/freeze-fracture has revealed occurrence of membrane fusions.
Alveolar Sacs Are the Most Likely Structural Equivalents of Subplasmalemmal Ca2+ Pools
As shown above, the extent of trichocyst exocytosis is paralleled by the size of currents observed. Assuming alveolar sacs to be the primary source of Ca2+ for Ca2+-activated currents a hypothesis remained to be tested: the ratio of maximal versus minimal currents should reflect the ratio of total cell surface versus unit area of cell surface. These units, the kinetids, also correspond with the number of alveolar sacs which occur all over a cell. Current ratios would roughly have to reflect area ratios, even when currents were superimposed by Ca2+ influx.
To answer the problem, we established morphometric
parameters for the cells used in electrophysiological recordings. Viable or cryofixed cells were analyzed by both
LM and EM methods (Table I) revealing ~3,100 surface
units. For comparison, current ratios are compiled in Table II. The minimal current, i.e., the current per trichocyst
discharge, as derived from extracellular recordings (Fig. 5),
is 1.2 × 1012 As. Correction by a factor of 2 is required,
since the current is recorded from ~1/2 of the cell surface,
i.e., the part in the holding pipette (see Materials and
Methods). The corrected value of 2.4 × 10
12 As is in
agreement with the value obtained by intracellular voltageclamp recording, i.e., 2.5 × 10
12 As. Maximal currents recorded extracellularly during maximal AED stimulation,
after correction for recording area, amount to 6 × 10
9 As
(Table II). The ratio of maximal to minimal currents is
~2.5 × 103, a value closely approaching the area ratio of
3.1 × 103, i.e., the estimated number of cortical Ca stores
per cell (Table I). This is another aspect supporting our
suggestion that alveolar sacs may be the internal source of
Ca2+ relevant for exocytotic membrane fusion.
Table II. Correlation of Electrophysiological with Structural Data |
Current Flow Reflects Extent of Cell Surface Area Activated, Rather than Fusion Pore Conductance-Superposition of Ca2+ Activation from Stores by a Ca2+ Influx
Could spontaneous currents reveal fusions of single trichocysts with the cell membrane during exocytosis? From
work in other secretory cells, it is known that during fusions an electric discharge occurs as soon as the fusion
pore opens (Almers, 1990; Lindau, 1991
). The magnitude
of the current, or its charge, which depends on the potential difference between the two fusing compartments, can
be recorded as a current transient preceding the stepwise increase in membrane capacitance, reflecting the increase
of surface membrane, as measured by the patch clamp technique. From the current transient and the capacitance
change, one can calculate the potential of the fusing vesicle.
If one applies the same considerations to Paramecium,
one can compare the measured spontaneous electrical signal with that expected for a fusion event. Considering a trichocyst surface of ~10 µm2 (estimated from electron micrographs) and assuming the usual 1 µF × cm2 for
biomembranes (Cole, 1972
; Kado, 1993
), a capacitance of
10
13 farad would result. From the recorded transients
(e.g., in Fig. 1 B) the charge can be calculated to be ~2.5 × 10
12 As. The potential difference between the cell membrane and the trichocyst membrane thus calculated would
amount to 2.5 × 10
12 As/10
13 farad (25 V). However,
this value is orders of magnitude above physiological values, and this excludes that the spontaneous current events
we have recorded is due to the transient capacitative discharges of the fusing trichocyst membrane. This interpretation is supported by the occurrence of the same currents
in the trichocyst-free mutant, tl, where spontaneous and
AED-elicited currents were quite similar to those in wildtype cells (Erxleben and Plattner, 1994
). Therefore, we favor the interpretation which relates current events, no
matter whether spontaneous or triggered, to the activation of a variable number of alveolar sacs with a superimposed
Ca2+ influx (see below).
The concomitant abolition of both Ca2+ currents and
exocytosis by EGTA injection supports a causal relationship between [Ca2+]i increase and exocytotic membrane
fusion (Fig. 6). Discharge of subplasmalemmal Ca stores is
probably a primary response to AED triggering, mainly
for two reasons. First, some Ca2+ signal and some membrane fusion occur also with [Ca2+]e [Ca2+]i at rest. Second, rapid Ca2+ influx caused by 10 mM [Ca2+]e causes a
cortical [Ca2+] transient of
900 nM, but no exocytosis
(see Results and below). Both largely exclude a CICR
mechanism, as also suggested by 45Ca2+ release studies
with isolated alveolar sacs (Länge et al., 1995
) and by patch
clamp analysis of reconstituted Ca2+ release channels from
Paramecium (Zhou et al., 1995
).
From all our observations we come to the following conclusion. With "normal" [Ca2+]e (0.1 mM), Ca2+ release from alveolar sacs is clearly accompanied by a Ca2+ influx through the cell membrane, resulting in a diffusion to the entire cell (data not shown) and increased exocytotic activity, while the signal remains locally restricted with low [Ca2+]e (Fig. 10, Q-T). Both phenomena, normally acting in concert and possibly involving site-directed Ca2+ release and/or influx, support rapid trichocyst exocytosis responses.
Relationship to Models of Ca2+ Dynamics during Triggered Exocytosis
In Paramecium, alveolar sacs have been identified as Ca
pools by their Ca2+ sequestration (Stelly et al., 1991) and
Ca2+ release (Länge et al., 1995
) properties, both after isolation and by in situ Ca imaging methods (ESI, SIMS)
(Knoll et al., 1993
; Stelly et al., 1995
). These organelles are
connected to the cell membrane by electron-dense connections ~15 nm long (Plattner et al., 1991
). Thus, they
clearly fulfill the criteria of subplasmalemmal pools.
The capacitative model of Ca2+ entry (Putney, 1986,
1990
, 1993
) assumes functional coupling between emptying intracellular Ca stores and Ca2+ influx. Structural coupling of Ca stores with the cell membrane has been discussed for a large number of secretory systems (Berridge, 1995
), although the structural identification of such pools
is still questioned. In these secretory systems however,
Ca2+ release is sustained by inositol 1,4,5-trisphosphate, a
second messenger for which we have no evidence in Paramecium (Länge et al., 1995
; Zhou et al., 1995
). Thus a capacitative model sensu strictu cannot be envisaged in our
cells.
In some secretory cells, Ca2+ release has been defined as
quantal (Muallem et al., 1989; Meyer and Stryer, 1990
;
Tregear et al., 1991
; Cheek et al., 1993
; Parys et al., 1993
)
in the sense that [Ca2+]i and, in parallel, Ca2+-dependent
exocytosis is increased by increasing concentrations of
secretagogues. So far, however, no strictly quantal pool
mobilization could be demonstrated (Taylor and Potter,
1990
; Bootman 1994a
,b). Our analyses with Paramecium
have revealed in contrast a correlation between the size of
Ca2+-activated currents, the extent of exocytosis achieved,
and the amount of subplasmalemmal Ca stores available,
with no sign of discrete current steps. This discrepancy
may be caused by either the different processes of Ca2+ release from the stores, the different Ca2+ sensitivity of
Ca2+-activated channels along the cell surface (Erxleben
and Plattner, 1994
), or by the superposition to the release
of a slightly variable Ca2+ influx.
One argument for Ca2+ release as the primary event induced by AED is the relatively small current elicited by a
second application (20 s later). By comparison, the ability
of the electric system (i.e., the Ca2+-activated channels) to
generate a second current pulse in response to increased
[Ca2+]i, can be regarded as instantaneous. Since we have no
evidence of an AED receptor, desensitization is a less likely
explanation. Potential Ca2+ release channels recently analyzed by electrophysiology after reconstitution show pharmacological characteristics (Zhou et al., 1995) which are
practically identical to those for Ca2+ release from isolated
sacs (Länge et al., 1995
). Although the molecular identity
of the channels actually involved in Ca2+ influx during
AED stimulation still has to be established, it certainly differs from those in the Paramecium ciliary membrane (Erxleben and Plattner, 1994
; Plattner et al., 1994
; Zhou et
al., 1995
). Another subject of further analysis is the significance in Paramecium of Ca2+ influx, whether it serves directly to amplify the subplasmalemmal signal (Putney,
1990
, 1993
; Berridge, 1995
) or to refill subplasmalemmal stores during or immediately after their release.
Final Conclusions
In the Paramecium system, local Ca2+ transients are sustained by a local interplay between endogenous and exogenous sources. This combination is needed for the rapid local trichocyst exocytosis, with ensuing efficient defensive function activation within tens of milliseconds. Prerequisite to this activity is the proper assembly of surface cell components, i.e., trichocysts and subplasmalemmal Ca2+ stores, with the concomitant activation of both Ca2+ release and influx. The special design of a Paramecium cell, investigated by appropriate methods, has allowed us to reveal some features in our system which might turn out to be applicable also to higher eukaryotic cells.
C. Erxleben and N. Klauke contributed equally to this publication.
Received for publication 26 February 1996 and in revised form 5 November 1996.
This paper is supported by SFB156 as well as DFG grants Pl78/11 and Pl78/12, all from the Deutsche Forschungsgemeinschaft (DFG).We thank Jochen Deitmer for communicating some early observations to the problem, Jochen Hentschel for his help with the CLSM set-up, and Mary Anne Cahill for reading an early version of the manuscript.
AED, aminoethyldextran; Ca2+e, extracellular calcium; [Ca2+]i, intracellular free Ca2+ concentration; CaGr-2, calcium green-2; CLSM, confocal laser scanning microscopy.