Download Paramecium trichocysts isolated with their membranes are stable in

Paramecium trichocysts isolated with their membranes are stable in the
presence of millimolar Ca 2+
OSCAR LIMA*, TADEUSZ GULIK-KRZYWICKI and LINDA SPERLINGf
Centre de Cenetique Mole'ctiktire, Associated with the Universite Pierre et Marie Curie, CNRS, Gif-sur-Yvette, 91190 France
•Present address: Laboratoire de Ge'ne'tique Physiologique, University Paris XI, Batiment 400, 91405 Orsay Cedex, France
t Author for correspondence
Summary
We have developed a simple and rapid procedure
for the isolation of a pure fraction of Paramecium
trichocysts (mature secretory vesicles) with their
membranes. Since in wild-type Paramecium cells
essentially all trichocysts are docked at pre-formed
cortical sites, trichocysts were isolated from cells in
which functional trichocysts remain free in the
cytoplasm owing to a mutation, tam6, that affects
the docking site. Examination of the preparations
by freeze-fracture electron microscopy confirms
the presence of the membranes. The distribution of
particles in the membranes of the isolated trichocysts and in the membranes of wild-type trichocysts
in situ are nearly identical and this argues against
any rearrangement of the membranes during the
isolation procedure. Although the trichocyst matrix
Introduction
The description of the secretory pathway first established by Palade (1975) is perfectly relevant to lower
eucaryotic organisms that provide model systems for
secretion. In yeast, some 25 genes necessary for secretion
have been identified by genetic analysis (Novick et al.
1980; Schekman, 1985) and by now a number of these
genes have been cloned and the corresponding proteins at
least partly characterized (e.g. see Segev et al. 1988;
Nakano et al. 1988).
Genetic analysis of secretion in Paramecium, whose
mature secretory vesicles are called 'trichocysts' because
of their distinctive morphology, has led to the identification of more than 30 genes necessary for secretion (cf.
Adoutte, 1988, for a review). Although molecular genetics has been slower to come of age in Paramecium than
in yeast, several aspects of secretion in Paramecium merit
attention, in particular, it is possible to study the final
step, exocytosis. Exocytosis is a universal but poorly
understood process by which intracellular vesicles fuse
with the plasma membrane, usually to deliver proteins to
Journal of Cell Science 93, 557-564 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
undergoes a dramatic structural transition in the
, presence of Ca2+ and water (matrix expansion), the
isolated vesicles with intact membranes are perfectly stable in the presence of millimolar free
Ca z+ . This result supports a chronology in which
the first step in exocytosis is membrane fusion, the
swelling of vesicle contents occurring only afterwards, once the contents come into contact with the
water and Ca2+ of the external medium. The role of
swelling would then be to help disperse, propel or
otherwise empty the contents of the vesicle outside
the cell.
Key words: Paramecium, trichocysts, secretory vesicle
membranes, exocytosis.
the plasma membrane or vesicle contents to the extracellular space. Secretion in yeast is constitutive so that it
is not possible to separate vesicle transport or interaction
with the plasma membrane from exocytosis (Burgess &
Kelly, 1987). In Paramecium, however, secretion is
regulated: an external stimulus triggers exocytosis of
secretory vesicles already docked at pre-formed cortical
sites (Planner et al. 1973; Pollack, 1974). As is the case
for cortical granule exocytosis, which occurs upon activation of animal oocytes (Anderson, 1968; Kline, 1988),
it is possible to trigger massive, synchronous exocytosis
in Paramecium (Plattner, 1987) and a recent study
presents evidence that the biological function of trichocyst exocytosis is to provide Paramecium with a defence
against predators, e.g. in this study Dileptus margaritifer, a carnivorous ciliate (Harumoto & Miyake, personal
communication).
One striking feature of secretion in Paramecium is the
elaborate architecture of the vesicle and its contents
(Bannister, 1972; Hausmann, 1978). A general property
of all secretory vesicles is that their contents swell.
Indeed, a current hypothesis for the mechanism of
557
exocytosis is that granule swelling provides the driving
force for membrane fusion (Finkelstein et al. 1986). The
contents of trichocysts are crystalline and swelling is a
dramatic event as it consists of a rapid, cooperative
structural transition from a compact crystalline form to a
second expanded form that is also crystalline (Sperling et
al. 1987). The transition requires Ca 2+ and, of course,
involves uptake of water by the structure.
Although trichocyst crystalline contents have been
purified in the past by several strategies (Steers et al.
1969; Anderer & Hausmann, 1977; Matt et al. 1978;
Garofalo & Satir, 1984; Sperling et al. 1987), we present
for the first time a procedure for the isolation of a pure
fraction of Paramecium secretory vesicles consisting of
both crystalline contents and limiting membrane. Our
primary motivation in isolating intact vesicles was to
develop a biochemical approach to the characterization of
the products of the genes involved in the different steps of
the secretory pathway. A number of these gene products
have been attributed to the trichocyst compartment by
microinjection experiments (Aufderheide, 1978; LefortTran et al. 1981) and are likely to be proteins of the
vesicle membrane.
Our initial characterization of the isolated vesicles
shows that the membranes are present and intact. The
distribution of intramembrane particles revealed by
freeze-fracture electron microscopy is the same in the
membranes of the isolated vesicles and in the membranes
of trichocysts in situ, in rapidly frozen, unfixed Paramecium cells. Most significantly, the isolated vesicles are
perfectly stable in the presence of millimolar free Ca +
(micromolar Ca 2+ is sufficient for exocytosis), which
argues against the 'osmotic hypothesis' that granule
swelling provides the driving force for membrane fusion:
the Ca 2+ necessary for expansion of the trichocyst matrix
must come from the external medium, once the vesicle
membrane has fused with the plasma membrane.
Materials and methods
Cells and culture conditions
Wild-type Paramecium tetraurelia cells were of stock d4-2
(Sonneborn, 1974). tam6 mutant cells were originally isolated
from d4-2 stock after nitrosoguanidine mutagenesis (Beisson &
Rossignol, 1975). Cultures were grown at 27°C in an infusion of
Wheat Grass Powder (Pines International, Lawrence, Kansas),
infected with Klebsiella pneumoniae and supplemented with /3sitosterol (4f(gml~'), according to the standard procedures
(Sonneborn, 1970).
Isolation of trichocysts
A 51 sample of tam6 culture, consisting of 30004000 cells ml" , was collected by continuous flow centrifugation
and then by centrifugation in pear-shaped bottles in an oiltesting centrifuge at 300 g\ The cell pellet was washed twice at
room temperature with 200 ml of 10 mM-Tris-HCl, pH 7-0, and
then once with 45 ml of ice-cold 'Buffer A', which was adapted
from a buffer developed for physiological studies of Paramecium mitochondria (Doussiere et al. 1979) and contains
50mM-Hepes, pH7-0, 0-25 M-sucrose, 0-5% bovine serum
albumin (BSA) (Miles Laboratories), 1 mM-EGTA, 1 mMEDTA, 50,UM-PMSF (phenylmethylsulphonyl fluoride) and
558
O. Lima et al.
5f«M-leupeptin. The final pellet was resuspended to 3 ml total
volume with ice-cold buffer A, transferred to a tight-fitting
Dounce (teflon-glass) homogenizer and homogenized on ice
with 20 strokes of the piston. The homogenate was then
immediately layered over 60% Percoll (Pharmacia, Inc.)Buffer A in 10-ml centrifuge tubes, 0-5 ml of cell homogenate
per tube, and centrifuged in a Beckman Ti50 rotor at
27500revsmin- 1 (50000g) for 15min at 4°C. The trichocyst
bands, centered about 20 mm from the bottom of the centrifuge
tube, were recovered with a Pasteur pipette that had been rinsed
in Buffer A, and the Percoll partially removed by dilution with
Buffer A and low-speed (5000 revs min" 1 ) centrifugation in the
SS34 rotor of a Sorvall centrifuge to collect the trichocysts.
Such centrifuge pellets were used directly for freeze-fracture
electron microscopy sample preparation or were first transferred to an Eppendorf tube and recentrifuged at higher speed
in a microfuge. We note that after the density gradient step, the
trichocysts were much more stable at room temperature than at
lower temperatures. In fact, if the trichocysts are stored on ice,
100 % transition to the extended form, with concomitant loss of
membranes, is observed within minutes. Therefore, after the
gradient step, further manipulations were carried out at
15-20°C.
Determination of the density of trichocysts and
mitochondria.
Density marker beads (Pharmacia, Inc.) were used as suggested
by the manufacturer to calibrate the self-forming Percoll
gradients. Densities of trichocysts and of mitochondria were
determined after centrifugation on five gradients containing
Percoll concentrations ranging from 50% to 70%. Although
optimal separation was found at 60 %, the positions of the bands
could be measured at all of these Percoll concentrations and the
apparent buoyant densities of the organelles determined with
great precision (see Results).
Phase-contrast light microscopy
Light microscopy was carried out under phase-contrast optics
and images were recorded on Kodak TMAX 400 film developed
according to the manufacturer's instructions.
Freeze-fracture electron microscopy
The pellets of Paramecium cells or of isolated trichocysts were
frozen without any pretreatment using our 'sandwich' technique
(Aggergbeck & Gulik-Krzywicki, 1986). A very thin layer of the
sample is spread on a thin flat copper plate and immediately
covered with an identical plate. This sandwich is then rapidly
plunged into liquid propane. The opening of these two plates at
— 125CC, under a vacuum of about 10~ 7 Torr (lTorr =
133-3 Pa) in a freeze-fracture unit (Balzers BAF301) produces
fractures in the frozen sample. Replication of the fractured
surfaces was performed using platinum-carbon. The replicas
were cleaned in chromic acid, washed with distilled water, and
observed in a Philips 301 electron microscope.
Results
Isolation of trichocysts with their membranes
The isolation of intact secretory vesicles from Paramecium is a difficult task because of the dynamic properties of the vesicle contents, which are metastable protein
crystals. Under a variety of conditions and particularly in
the presence of Ca + , the crystals undergo a dramatic,
irreversible structural transition to an extended, extra-
cellular form some eight times longer than the intracellular form of the crystal and incompatible with the presence
of a limiting membrane designed to enclose a much
smaller object. Conditions that permitted isolation of the
trichocyst crystalline contents 'blocked' in the compact
intravesicular form (detergent disruption of the cell and
high-sucrose, high-EGTA, high-Mg 2+ buffers) are not
suitable for membrane stability.
A further obstacle in the isolation of trichocysts with
intact membranes is that essentially all of the trichocysts
in wild-type Paramecium cells are docked at fixed cortical
sites in a 'pre-fusion' state. Removal of the trichocysts
from their attachment sites without triggering exocytosis
and without disrupting the vesicle membranes, is difficult if not impossible (Anderer & Hausmann, 1977).
Our strategy therefore involved the use of cells from
the Paramecium mutant, tam6 (Beisson & Rossignol,
1975). The trichocysts of tam6 cells are not attached at
the cortex as in wild-type cells but are free in the
cytoplasm. Yet tani6 trichocysts are functional when
microinjected into cells with normal cortex, while trichocysts from wild-type cells remain unattached when
injected into tam6 cells (Lefort-Tran et al. 1981). The
conclusion of the microinjection analysis of tani6 mutant
cells is that the site of the mutation is not the trichocyst
compartment but the cortex compartment.
Paramecium cells gently homogenized in a buffer
designed to stabilize membranes (see Materials and
methods) are layered directly onto Percoll (60 %) equilibrated with the same buffer, and the isosmotic gradients
formed by 15 min centrifugation at 50000 £ provide
excellent separation of trichocysts from all other cellular
components and in particular from the mitochondria,
whose density is close to that of trichocysts. We measured
buoyant
densities
of
l-105gcm~ ± f>004
and
l-124gcm~ 3 ± 0-002 for mitochondria and trichocysts,
respectively.
Fig. 1 is a low-magnification freeze-fracture image of a
Fig. 1. Low-magnification (X9000) freeze-fracture electron micrograph of a centrifuge pellet of isolated trichocysts. Note the
homogeneity of the preparation and the absence of material other than trichocysts, with the exception of the electron-dense
Percoll, which sticks to the replicas. In the inset, a higher-magnification view (X27 000) of a region of the micrograph showing
characteristic EF and PF fracture faces of trichocyst tip membranes.
Trichocysts with their membranes
559
centrifuge pellet of a preparation of isolated trichocysts.
Essentially all the trichocysts have intact membranes and
very little material other than trichocysts is found. (The
electron-dense debris is residual Percoll: 30 nm particles
of silica coated with polyvinylchloride.) A trichocyst is
composed of two differentiated regions, the body and the
tip. The body consists of the carrot-shaped crystalline
contents; the tip has a crystalline core of the same
structure as the body, which is covered with two layers of
fibrous material, the inner and outer sheath (for the
anatomy of a trichocyst, see Bannister, 1972). The inset
in Fig. 1 shows a higher-magnification view of the tip
region of two trichocysts. The inner (EF) face of the
membrane (trichocyst at the right of the inset) is in
contact with the outer sheath of the trichocyst tip and the
striations probably reflect a special arrangement of the
membrane phospholipids induced by interactions with
the outer sheath; this suggests that the outer sheath may
be composed of filaments in a long-pitch helical arrangement. The outer (PF) face of the membrane covering the
trichocyst tip (trichocyst at the left of the inset) displays
many intramembrane particles whose arrangment is
partly ordered. The geometrical arrangment of these
particles may play a role in the assembly of the collar and
perhaps other fibrous elements that link the trichocyst tip
to the cortex (Pouphile et al. 1986).
Asymmetric particle distribution in trichocyst
membranes
Allen & Hausmann (1976) first reported an asymmetric
particle distribution in trichocyst membranes, which they
observed by freeze-fracture of glutaraldehyde-fixed
Paramedum caudatum cells. As illustrated in Fig. 2, we
observe a highly asymmetric particle distribution in the
. membranes of the unfixed isolated tam6 trichocysts
(Fig. 2A) as well as in the membranes of wild-type
trichocysts (Fig. 2B) and tam6 trichocysts (not shown)
frozen in situ. In order to compare the membranes of the
isolated tam6 trichocysts with the membranes of tam6
trichocysts frozen in situ, we counted the number of
intramembrane particles per unit area and determined
the ratio of the density of particles in the PF face with
respect to the density of particles in the EF face. Particles
were counted irrespective of their size; the values obtained are given in Table 1. Values for wild-type trichocysts, although based on a smaller statistical sample, are
Table 1. Density of intramembrane particles in
trichocyst membranes
Pface
E face
PF/EF
ratio
1959 ± 334 (6)
1941 ± 358 (6)
1822 ± 163 (3)
104 + 29(6)
109 ± 2 8 (8)
105 ± 1 3 (3)
18-8
17-8
17-3
Trichocysts
ta>n6, isolated
tani6, in situ
Wild type, in situ
The values represent the average number of intramembrane
particles per fim ± S.D. Particles were counted in equivalent, flat
areas (0-30;«n2) of the membranes of different trichocysts to give the
number of particles per unit for each given trichocyst. Average values
and standard deviations were then calculated and the number of
independent observations (i.e. different trichocysts examined) is
given in parentheses.
560
O. Lima et al.
also given. The particle densities are very similar in the
different preparations and, in each case, a particle ratio of
about 18 in favour of the cytoplasmic (PF) face is found.
This argues against rearrangement of the membranes
during the isolation procedure.
The vesicles are stable in millimolar Ca2+
Although the trichocyst membranes in our preparations
appear intact and 'native' by ultrastructural criteria, we
also sought a biochemical test of their integrity. As the
trichocyst crystalline matrix undergoes a structural transition in the presence of Ca 2 + , which is easily observable
by light microscopy (Matt et al. 1978; Garofalo & Satir,
1984), we devised a 'Ca 2+ test' of membrane integrity. As
shown in Fig. 3, the isolated trichocysts remain condensed in the presence of either 1 mM free Ca 2+ or 0-1 %
Triton X-100, a non-ionic detergent that dissolves the
vesicle membrane. Only in the presence of both Ca 2+ and
detergent is 'discharge' of all trichocysts observed. In
other experiments, we raised the free Ca 2+ above 10 mM
and found the trichocysts to be still intact in the absence
of detergent. The preparations of isolated trichocysts are
stable for several hours at room temperature in the
presence of even 10mM-Ca2+. Most important, the
contents retain their capacity to undergo the structural
transition for, at any time, the addition of detergent leads
to immediate expansion of the trichocyst matrices, provided there is Ca 2+ in the buffer. These experiments
show that: (1) the membranes of the isolated vesicles are
perfectly intact and they are impermeable to Ca 2+ (but
permeable to water: the membranes burst when the
osmolality of the buffer is reduced (data not shown));
(2) the membranes are not necessary for the crystalline
matrix to remain in the compact state; (3) Ca.2+
IS
necessary for the transition to the extended state.
Discussion
We have shown that it is possible to isolate Paramecium
trichocysts with their membranes by a simple and rapid
procedure. We used tam6 mutant cells whose functional
trichocysts are free in the cytoplasm, and self-forming
Percoll buoyant density gradients, which permit rapid,
high-resolution separation of organelles under isosmotic
conditions. The preparations obtained are very pure, the
only contaminant being a few mitochondria, which can
be largely eliminated either by reducing the amount of
material charged on the gradients or by adding a second
gradient purification step. The preparations are thus
suitable for biochemistry, and extension of biochemical
studies to the non-discharge mutants (Cohen & Beisson,
1980) should be possible after construction of the appropriate tam6 X nd double mutant strains. The trichocyst
preparations are stable for hours at room temperature, as
judged by the Ca 2+ test and their appearance in the light
microscope, opening the possibility of physiological
studies of the membrane properties of the isolated
vesicles.
Exocytosis
The fact that the isolated trichocysts are perfectly stable
Fig. 2. Freeze-fracture electron micrographs of EF and PF faces of isolated tam6 trichocysts (A) and of wild-type trichocysts in
situ (B) (magnification X60000). Note the highly asymmetric distribution of particles between the two faces in both A and B.
(See Table 1 for quantification of the particle densities statistically based on examination of a number of different trichocysts.)
The choice of these particular images was dictated by the proximity, in the same field, of both fracture faces of trichocysts in a
parallel (or antiparallel) arrangement. Bar, 0-5 [im.
Trichocysts with their membranes
561
Ca 2 +
\
o
o
\
I
X
c
o
""</
Fig. 3. Samples of a suspension of freshly prepared trichocysts were examined by phase-contrast microscopy in the presence or
absence of Ca 2+ and Triton X-100. CaCl2 was added from a 100 mM stock to bring the trichocyst suspension to 5 rnM in CaClz.
Since the buffer contains 1 mM-EDTA and 1 mM-EGTA, this corresponds to 1 mM-free Ca 2 + . Triton X-100 from a 20% stock
was added to bring the final concentration of the suspensions to 0 1 % Triton X-100. Upper left: the suspension of trichocysts;
upper right: trichocysts plus S mM-CaClz; lower left, trichocysts plus 0-1 % Triton X-100; lower right, trichocysts plus S niMCaCl2 and 0 1 % Triton X-100. Only in the presence of both detergent and Ca 2+ does the trichocyst matrix expand. Note that
the presence of the outer sheath of the trichocyst tip is well correlated with the presence of the membrane: in 0-1 % Triton X100 the tips are much thinner, owing to the absence of the outer sheath. X 1000.
in millimolar free Ca 2+ has direct implications for the
mechanism of exocytosis. Several years ago, an 'osmotic
hypothesis' for exocytosis was formulated on the basis of
experiments on model systems in which osmotic pressure
provided the driving force for the fusion of phospholipid
vesicles with planar bilayers (Cohen et al. 1980, 1982).
The extension of this work to biological systems was
based on the observation that the contents of all secretory
vesicles swell. It was supposed that the Ca 2+ that enters
the cell as a result of stimulation would act on the vesicle
562
O. Lima et al.
membrane so as to change its permeability to ions or
small molecules, whose entry into the vesicle down their
concentration gradient(s) would raise osmotic pressure;
water would in turn enter, i.e. the vesicle would swell.
This swelling, as in the model systems, would provide the
mechanical force for fusion of the vesicle membrane with
the apposed plasmalemma.
Since the formulation of this model various experiments have provided results for and against it (for a
review, see Green, 1987). Most elegant and convincing
experiments have been performed on mast cells of beige
mice, which have giant secretory vesicles. Simultaneous
membrane capapitance measurements and optical video
recordings have clearly shown that membrane fusion
always precedes swelling of the vesicles (Zimmerberg et
al. 1987; Breckenridge & Aimers, 1987). Recent studies
of egg cortical granule exocytosis, which in many ways
closely resembles exocytosis in Paramecium, are also in
contradiction to the osmotic hypothesis (Whitaker &
Zimmerberg, 1987; Zimmerberg & Liu, 1988).
In Paramecium, most authors have considered, on the
basis of physiological and ultrastructural studies, that
membrane fusion precedes the explosive discharge of the
vesicle contents (e.g. see Bilinski et al. 1981). However,
B. Satir and her colleagues have developed a different
viewpoint; they argue that direct entry of Ca 2+ into the
vesicle from the cytoplasm causes matrix expansion, in
synchrony with membrane fusion (Satir et al. 1988, and
references therein). In fact, the rapidity of the exocytotic
events (of the order of one or a few milliseconds) has so
far precluded any direct demonstration of their chronology. Our experiments on isolated trichocysts support
the fusion-before-swelling view of exocytosis.
The osmotic hypothesis predicts that isolated secretory
vesicles would swell in the presence of Ca 2+ . The only
previous observation of isolated vesicles in the presence
of Ca 2+ that we are aware of concerns oocyte cortical
granules, which indeed do not swell in the presence of
millimolar free Ca 2+ (Crabb & Jackson, 1985). Our
demonstration that isolated trichocysts do not swell but
are stable in millimolar free Ca 2+ strongly argues that
both the Ca 2+ and the water necessary for the trichocyst
matrix to undergo its characteristic and dramatic expansion must enter the vesicle from the external medium,
after membrane fusion. Earlier experiments in which
massive amounts of Ca 2+ were injected into Paramecium
cells without leading to trichocyst exocytosis are perfectly
consistent with our results (Kersken et al. 1986).
We thus favour a point of view in which the osmotic
properties of secretory vesicle contents provide the driving force not for membrane fusion, but for the dispersal
of vesicle contents (Sperling et al. 1987; Whitaker &
Zimmerberg, 1987). What then, is the mechanism for
membrane fusion in biological systems? As in so many
other cases, to the despair of the physical chemist, a
thermodynamically sound mechanism that 'works' in
vitro will undoubtedly give way in the cell to proteins that
have evolved to ensure highly specialized functions. In
Paramecium, mutants of the nd (non-discharge) series
are blocked at the last step(s) of exocytosis (Cohen &
Beisson, 1980). The genes affected in the nd mutants are
good candidates to code for the specialized proteins that
fuse membranes, and the proteins in question will
perhaps turn out to resemble the well-studied fusogenic
proteins of enveloped animal viruses (White et al. 1983).
We are indebted to Jean-Claude Dedieu for expert technical
assistance and we thank Jean Cohen, Andr£ Adoutte, Michele
Rossignol and Ray Kado for useful discussions. We are particularly grateful to Janine Beisson for advice, encouragement and
critical reading of the manuscript.
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{Received 16 January 1989 -Accepted 14 April 1989)