Uneven distribution of surface antigens during antigenic variation in
Paramecium primaurelia
CLAUDE ANTONY1'2 and YVONNE CAPDEVILLE1
^Centre de Genetique Moleculaire, CNRS, F-91I90 Gif-sur-Yvette, France
^•European Molecular Biology Laboratory, Postfach 10.2209, D6900 Heidelberg, FRG
Summary
In Paramecium primaurelia surface antigen (SAg)
expression can be experimentally controlled by
temperature-shift-induced antigenic variation. As
only one SAg is usually expressed at the cell surface
under stable environmental conditions, we used the
temperature-shift-induced change in SAg to follow
the newly expressed antigen and the disappearing
one, by both immunofluorescence and immunogold
electron microscopy. The new SAg initially appeared scattered at the cell surface, over the ciliary
and interciliary membrane domains, without any
readily identifiable specific site of insertion into the
plasma membrane. The concentration of the newly
incorporated molecules then increased gradually
on the plasma membrane. In contrast, the surface
distribution of the previously expressed SAg was
not complementary to the pattern of the appearing
SAg. The loss of the old SAg is delayed after the
temperature shift and seems to occur more suddenly than the appearance of new SAg. This loss is
characterized by a subpopulation of cilia bearing
old SAg coexisting with other cilia and a pellicle
almost devoid of the old SAg molecules. The topological distribution of the new and old SAgs is
discussed in relation to the lipidic nature of the SAg
membrane anchor and to a possible role of an
endogenous Paramecium
phosphatidylinositol
phospholipase C.
Introduction
surface glycoproteins (Ferguson et al. 1985) and other
eukaryotic surface proteins (for reviews, see Cross, 1987;
Low, 1987; Low & Saltiel, 1988). The lipid anchor of
Paramecium SAgs can be cleaved off in vitro by exogenous phosphatidylinositol-specific phospholipase C (Capdeville et al. 1987a) and in vivo by an endogenous
phospholipase C-like hydrolase whose activity can be
induced by ethanol of Triton X-100 (Capdeville et al.
1986). Furthermore, these surface proteins are highly
polymorphic, particularly in the case of the G and D
SAgs, which are encoded by two unlinked loci expressed
at medium and high temperature, respectively, in P.
primaurelia (Beale, 1954). The polymorphism appears to
affect mainly the part of the molecule exposed to antibodies in vivo, which allows one to distinguish between
an allele-specific region and a locus-specific one that bears
antigenic determinants common to all allelic forms of a
given SAg locus (Capdeville, 19796). Ultrastructural
immunocytochemical studies of cells stably expressing
the 156G SAg (G SAg displayed by strain 156 of P.
primaurelia) have shown that only the most external
domain of the 156G molecule is obviously accessible to
antibodies in situ and that the thickness of the antigen
layer on the plasma membrane surface is about 17—22 nm
The surface of Paramecium aurelia, a free-living ciliated
protozoon, is mainly coated by a single molecular species,
termed surface antigen (SAg) or immobilization antigen,
as immobilization of living cells occurs when they are
incubated in the presence of antibodies raised against the
expressed SAg. A given clone can usually express a
repertoire of different SAgs coded by a multigene family,
whose expression is governed by both mutual intergenic
and interallelic excluding mechanisms (Beale, 1952;
Capdeville, 1971). Under stable culture conditions only
one SAg is generally expressed (the choice of the SAg to
be expressed mainly depending on external factors) and
antigenic variation can be triggered by modifying external parameters such as temperature (for reviews, see
Beale, 1954; Somerville, 1969; Sonneborn, 1974; Finger,
1974).
SAgs are made up of one huge polypeptide chain
(Mr = 300X 103) containing about 10 % cysteine residues,
without free thiol groups (Reisner et al. 1969; Steers &
David, 1977). They are anchored in the plasma membrane via glycophosphatidylinositol (Capdeville et al.
1987a) in the same way as are Trypanosoma variant
Journal of Cell Science 92, 205-215 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Key words: surface antigen, Paramecium, antigenic variation,
membranes, immunoelectron microscopy.
205
(Capdeville etal. 19876).
The expression of Paramecium SAg is regulated by
mRNA abundance (Preere* al. 1981; Meyer etal. 1984).
Furthermore, the SAg itself that was once expressed at
the cell surface appears to be instrumental in controlling
its own stability by exerting a positive control on its
synthesis and by acting as a target through which
environmental signals trigger antigenic variation (Capdeville, 1979a). The SAg molecules located on the
plasma membrane may behave as 'receptors' and 'effectors' at this interface level between the environment and
the inside of the cell. Therefore, given the transient
instability that affects the SAgs during the antigenic
variation, there is a need for an accurate investigation of
the change in the localization of the SAgs.
Only preliminary data concerning the appearance at
the cell surface of the newly expressed SAg are available
(Mott, 1965; Williams et al. 1985). In contrast, the
elimination of the previously expressed SAg has not yet
been investigated. We report in this paper a detailed
analysis of the fate at the cell surface of the new and old
expressed surface antigens after a temperature shift in
strains 156 and 168 of P. primaurelia, by immunofluorescence and immunogold microscopy techniques.
Materials and methods
Strains and culture
Two homozygous strains of P. primaurelia from different
geographical origins were used: the 156 and 168 strains, which
generally express the G SAg when grown at 23°C, and the D
SAg when grown at 33°C. The cells were routinely grown at
23°C in a hay-grass medium inoculated with Klebsiella pneumoniae and supplemented with /3-sistoserol (0-8^gml" 1 ).
Transition of antigen expression
The changes in SAg expression were induced by temperature
shifts. Strain 156 cells grown at 23°C homogeneously expressed
the 156G SAg. The 156D SAg expression was routinely
induced by shifting the cells to 36°C for 48 h before they were
finally transferred to 33 °C, at which temperature the 156D SAg
is stably expressed. Strain 168 cells were grown at 23°C to
obtain the 168G SAg expression, then by shifting the cells to
35 °C the expression of the 168D SAg was induced in the whole
population. To induce the expression of the G SAg (shift-down
experiments), cells grown at high temperature, where the D
SAg was stably expressed, were then shifted to either 23°C or
18°C. During these procedures the cells were often checked by
immobilization tests to ascertain their antigen phenotype.
Antisera
Rabbit antisera against the G or D SAg expressed by strains 156
and 168 were raised against the corresponding purified SAg in
its soluble form (sSAg). Preparation of antisera 2355 (against
the 156G sSAg purified in denatured state), 2519 and 2543
(against the 168G and the 156D sSAgs purified in native state,
respectively) has already been described (Capdeville et al.
1985). We used the 2355 antiserum (anti-156G SAg) immunopurified by absorption against cells fully expressing the 156D
antigen in order to remove any antibody that might react against
156D SAg. This was needed to gain a better labelling of the
early steps of 156 SAg expression at the cell surface. The mouse
monoclonal antibody Y4 against the 156G SAg was produced as
described (Capdeville et al. 19876).
206
C. Antony and Y. Capdeville
Immobilization tests
Living cells were allowed to swim in diluted antisera previously
dialysed against a Dryl's solution (1 niM-NaHzPC^, 1 mMNa 2 HPO4, 2mM-trisodium citrate, l-5mM-CaCl2, pH6-8).
Immobilization of the cells was followed under a binocular
microscope. The dilution of a homologous serum was chosen to
obtain a complete immobilization of the cells within 20min at
room temperature (Capdeville, 1971).
Immunfluorescence microscopy
Living paramecia were rinsed in Dryl's solution before prefixation in 3 % paraformaldehyde in a lOmM-phosphate buffer,
pH7-3, for lOmin. After rinsing in the same buffer cells were
incubated for 1 h at room temperature in homologous or
heterologous rabbit antisera (dilution 1/100-1/400) in a 10 mMphosphate buffer containing 1 % bovine serum albumin (BSA).
After several rinses, the second fluorescein-conjugated goat
anti-rabbit antibody (Institut Pasteur production) was added
(dilution 1/500) for 45 min. After washing in the phosphate
buffer the cells were mounted on slides in a fluor medium
beneath coverslips, and sealed with Histolaque. Control preparations were treated with heterologous serum, preimmune
serum, or second antibody only. Examination was made with a
Zeiss photomicroscope equipped with epifluorescence illumination.
Immunogold labelling on prefixed cells
Exactly the same immunoelectron microscopic procedures were
used as described (Capdeville et al. 19876). Cells were prefixed
in 3 % paraformaldehyde with 025 % glutaraldehyde in a
phosphate buffer, pH7-3, prior to incubations with antibodies.
Then labelling of SAgs with antisera was always done in a 1 %
BSA-phosphate buffer. Cells were incubated with secondary
antibodies of IgG gold (5 nm) diluted 1/5 in the same buffer for
l-5h. The monoclonal antibody Y4 was used for electron
microscopy (EM) with a linker rabbit anti-mouse antibody
(diluted 1/100) prior to the final GAR G5 as described (see
Capdeville et al. 19876). As the direct binding of a goat antimouse IgG gold was very weak the double labelling of both G
and D antigens in the same cell could not be considered. After
fixation with 2 % glutaraldehyde in a cacodylate buffer for
45 min, the cells were postfixed with 1 % osmium tetroxide in
cacodylate buffer for 45 min before processing for plastic
embedding in Spurr resin. Ultrathin sections were stained with
3 % uranyl acetate in 70 % ethanol for 20 min and observed on a
Philips EM301.
Results
Temperature shift experiments
Shift-down experiments inducing the expression of a G
SAg were performed on strain 156 of P. primaurelia
under two conditions: first a shift from 33°C to 23 °C;
and second, a shift from 33°C to 18°C, in order to obtain
a slower sequence of the transition events. A shift up
from 23 °C to 35 °C was performed on strain 168 to induce
the expression of D SAg. This strain 168 was chosen
because it has been shown that strain 156 was found to coexpress two SAgs at 35 °C, while 168 strain is clearly
expressing a single SAg type (Capdeville et al. 1985).
Kinetics of the spreading of the newly expressed SAg at
the cell surface
The expression of the 156G antigen (antigen G of strain
Table 1. Kinetics of the immobilization reaction with
156 cells submitted to a temperature shift from 33 °C to
23°C
Time after temperature-shift (h)
AS 2355
anti-156G
AS 2543
anti-156D
Number of
divisions
0
6
12
18
24
48
—
-
si
si
sl-im
im
im
im
sl-im
si
sl-
-
1
1-2
2-3
4-6
The immobilizing antisera were the same as those used for the
immunocytochemical localizations. The timing of the cell division
events is also reported, im, immobilized cells; si, sluggish cells.
156) was induced first by shifting the temperature from
33 °C to 23 °C. The evolution of the antigen change at the
cell surface was monitored in vivo by submitting the cells
to antisera against both 156D and 156G SAgs at different
times. The kinetics of the immobilization reaction using
anti-G and anti-D antibodies are presented in Table 1,
together with the timing of cell division.
The kinetics of the re-expression of the 156G antigen
were monitored by fluorescence microscopy with absorption-purified antiserum (AS 2355, see Materials and
methods). The starting fluorescence background is
shown with cells expressing the 1S6D antigen at time Oh
(Fig. 1A). Then, 3h after the temperature shift from
33 °C to 23 °C a weak fluorescence of granular appearance
is visible, confined to the interciliary membrane domain
(also called the pellicle) as well as to the ciliary membrane
domain. At about 6-15 h cortical units (defined as a
repeated morphological surface pattern comprising a
cilium surrounded by two alveolae with a crest at the
junction to the next unit (see Figs ID and 2A)) become
more and more defined (Fig. 1B-D). However, we must
note that at this period the immobilization reaction was
mostly negative (Table 1). The fluorescence of the cells
remains rather irregular and discontinuous up to 24 h,
when a major change occurs as the cells become brightly
decorated (Fig. IE). During the subsequent time period
from 24 h to 4 days there is only a slight intensification of
the whole fluorescence (48h time point, Fig. IF). It
appears that at least a 24 h period is necessary to observe a
complete settling of the newly expressed SAg at the cell
surface. When the new antigen is fully expressed
(Fig. IF) the pellicular membrane is not homogeneously
labelled, some rows of cortical units being more fluorescent than others. This remains unexplained, as we do
not know whether it is due to greater accessibility of
antibodies, or to a larger number of SAg molecules.
Bright spots in immunofluorescence images correspond
either to autofluorescent crystals (small spots) or to the
gullet are a (large spots, especially when the latter is out
of focus), which we found to bind the antibodies rather
non-specifically (Fig. 1).
A kinetic experiment similar to the previous one was
carried out at the electron-microscope level using the
same absorption-purified antiserum (AS 2355) to label
the newly appearing 156G molecules at the cell surface.
The results presented in Fig. 2 clearly show that as soon
as the new antigen is immunocytochemically detectable at
the cell surface the new molecules appear homogeneously
scattered on both the pellicular and ciliary domains
(Fig. 2A-C). After this early appearance a regular increase in the density of the gold particles is observed up to
24 h (Fig. 2D-E), reflecting the increasing amount of the
newly stabilized G molecules. Later, the gold particles
become more concentrated, aligned in two or more rows,
as the cell coat is completed by packing of the antigen
molecules very close to each other (Fig. 2F).
In order to confirm the pattern of the early spreading
new G antigen we followed its appearance with the
monoclonal antibody Y4, specific for the G antigen
(Capdeville et al. 19876). As before the G-labelled
molecules appeared scattered on both the pellicular and
ciliary membranes as early as 1 h after the temperature
shift (Fig. 3B). We should point out that these immunogold studies did not show any particular site of insertion
of the new molecules in the pellicular membrane.
A lower shift down was also performed from 33 °C to
18°C to induce a change from 156D to 156G. As
expected, in this experiment the change of SAgs is much
slower as a few labelled G molecules were observed 12 h
after the temperature shift. Again the same pattern of
early localization was observed without any detectable
site of insertion in the pellicular membrane (data not
shown).
We should also mention that at time 0 h in the shiftdown experiments, a weak labelling with anti-G antibodies was noticed in the immunofluorescence as well as
in the immunogold experiments. We can rule out any
artefactual labelling, since we used both a monospecific
antibody (Y4) and an absorption-purified polyclonal anti156G antiserum. Thus, these few labelled molecules
should actually be G molecules. The G molecules may be
either residual molecules remaining despite the large
number of cell divisions occurring at 33 °C, or permanently expressed molecules. We cannot choose between
these two alternatives because cells cannot support
growth for days at this high temperature.
To ascertain that the early localization of the new
antigen was not dependent on the up or down temperature shift we submitted cells of strain 168 to a shift up
from 23 °C to 35 °C, which induces the expression of D
antigen. The 168D SAg molecules were followed at the
EM level by using an anti-156D serum (AS 2543), which
cross-reacts very strongly in vivo with 168D molecules
(Capdeville et al. 1985). The labelling of 168D SAg was
obvious around 3 h after the temperature shift, scattered
on both ciliary and pellicular membranes (Fig. 4C). We
noticed in this case that no 168D molecules were detectable at time Oh (Fig. 4A).
Kinetics of the disappearance of the previously
expressed SAg at the cell surface
To determine the elimination pattern of the old SAg, the
disappearance of the initially expressed antigen was
followed in parallel in the same temperature-shift experiments. We will only describe the disappearance of the
Paramecium swface antigen distribution
207
Fig. 1. Kinetics of appearance of 156G molecules following a temperature shift from 33°C to 23°C. The localization by
immunofluorescence microscopy of the newly appearing antigen was monitored with an absorption-purified antiserum (AS 2355)
raised against 156G antigen. Time after the temperature shift: A, Oh; B, 3h; C, 6h; D, 15 h; E, 24h; F, 48h. Large arrows
show autofluorescent crystals, arrowheads indicate the position of the gullet, and small arrows show cortical units (F). X680.
Bar, 10 ^Jm.
208
C. Antony and Y. Capdeville
tr
2A
\
Fig. 2. Transition of antigen expression from 1S6D to 1S6G antigen at the cell surface induced by a temperature shift from
33°C to 23°C and followed at the EM level. The absorption-purified AS 2355 polyclonal antibody incubations were followed by
labelling with goat anti-rabbit IgG gold (5nm). Time after the temperature shift: A, Oh; B, 3h; C, 6h; D, 12h; E, 24h; E,
24h; F, 48h. cm, ciliary membrane; pm, pellicular membrane; cr, crest; a, alveola; tr, trichocyst. X36 000. Bar, 0-5 fim.
Paramecium surface antigen distribution
209
4A
3A
W
'*
:
'-i
r
B
B
Fig. 3. Kinetics of the early appearance of 156G molecules
followed at the EM level and monitored at the cell surface by
the Y4 monoclonal antibody. A linker rabbit anti-mouse
antibody was used in this case before the final incubation
with goat anti-rabbit IgG gold (5 nm) (this explains the
observed aggregates). Time after the temperature shift; A,
Oh; B, l h ; C, 3h. X31000. Bar, 0-5fim.
210
C. Antony and Y. Capdeville
Fig. 4. Transition of antigen expression from 168G to 168D
induced in this strain by a shift up of temperature from 23 °C
to 35°C, and followed at the EM level. The early localization
of the newly appearing 168D molecules was monitored with
an anti-156D serum (AS 2543) labelled with goat anti-rabbit
IgG gold (5 nm). Time after the temperature shift: A, Oh;
B, 1 h; C, 3 h. X31 000. Bar, 0-5 nm.
156D SAg in shift-down experiments. AS 2543 was used
both in immunofluorescence and immunogold experiments to follow the elimination of this antigen.
Visualization of the D molecules by the immunofluorescence procedure showed that the D SAg remained
obviously settled at the cell surface until about 18h-24 h
(Fig. 5A-C). At this time cells reacted positively with an
anti-156D serum when submitted to the immobilization
test (Table 1). The loss of the 156D antigen took place
from 24 h to 36 h (Fig. 5D-E). At this time, the surface
distribution of the D antigen displayed a striking heterogeneous pattern, in which the fluorescence of the pellicular membrane decreased while a subpopulation of cilia
retained a strong fluorescence. With respect to the
pellicular membrane, we noticed that some rows of
cortical units still displayed fluorescence whereas the
neighbouring ones were devoid of fluorescence at the
same time (Fig. 5D). Later, the general weak fluorescence decreased on both ciliary and pellicular membranes. Four days after the temperature shift, we observed no fluorescence or a very weak signal (Fig. 5F).
The heterogeneity observed among cells at a given time
(e.g. at 24 h; Fig. 5C-D) is probably due to asynchronous divisions.
By immunoelectron microscopy we have detected the
same sequence of events when shifting the cells from
33°C to 23 °C or from 33°C to 18°C. Only a shift-down
experiment from 33 °C to 18 °C will be described (Fig. 6).
In this shift down no change in the surface labelling could
be noticed up to 42 h (Fig. 6A-B), but around 48 h (the
next observed step; Fig. 6C-F) a specific topographical
localization was established. The pellicular region was
almost completely devoid of particles, in contrast to the
ciliary region where labelled and unlabelled cilia coexisted. During this transient state clusters of D molecules
could be detected on cilia (Fig. 6E) and also, but less
often, on the crests of the cortical units (Fig. 6F).
This change from a homogeneous labelling of the old
antigen to a sparse labelling of a specific restricted area
seems to occur in a short time.
Discussion
The present study on the surface immunolocalization of
two antigens, the G and D antigens alternatively expressed at medium and high temperatures in P. primaurelia during antigenic variation, revealed characteristic
features. These were displayed whether the temperature
shift was up or down, and with all the strains used.
The sequence of events in the spreading of the new
antigen and loss of the old one are mainly characterized
by the following points: (1) as soon as the new antigen is
detectable, it appears homogeneously scattered over the
whole plasma membrane, i.e. over the ciliary and the
pellicular membrane; (2) later it spreads slowly, during
several hours (the time required for at least two divisions)
until it appears completely settled at the cell surface; (3)
the elimination process of the old antigen always shows a
delay before any altered pattern of immunolocalization
can be observed; (4) this process is dramatic and occurs
in such a way that the old antigen is almost undetectable
on the pellicular membrane, while it is still present on the
membrane of a number of cilia, coating all the ciliary
membrane or forming clusters.
Before attempting any interpretation of our results, we
will discuss them in the light of previous data obtained in
P. primaurelia (Mott, 1965) and in Tetrahymena (Williams et al. 1985). Our reported observations of an early
scattered pattern of the newly expressed antigen at the
cell surface do not seem to agree with the reported data
from these authors, who concluded that the newly
expressed antigen appeared initially on the pellicle, then
subsequently on cilia. There are several possible reasons
for this disagreement. Concerning the data of Mott, let us
point out that using ferritin-conjugated antibodies she
obtained a rather irregular labelling of the antigen coat,
while we used gold antibodies that gave a very regular
arrangement of gold particles at a constant distance from
the membrane bilayer (see also Capdeville et al. 19876).
Furthermore, the few pictures shown by Mott are at
variance with her conclusion, as she noticed, for instance,
a few ferritin granules scattered on both pellicle and cilia
within 1 h after the temperature shift. Concerning the
spreading of the newly synthesized antigen in Tetrahyniena, it is possible that the modalities are different from
those observed in Pararnecium. However, in the case of
Tetrahymena we cannot exclude a possible remodelling
of the plasma membrane, as the authors treated cells with
antibodies prior to fixation. Indeed, it is known that in
Paramecium such treatment of living cells by antibodies
induces a capping event of some kind, preventing the free
motion of the antigen in the membrane (Barnett & Steers,
1984).
Our observations of the scattered localization of the
newly expressed antigen at early stages in P. primaurelia
do not entitle us to conclude that there are insertion sites
on both pellicle and cilia. We think it is more reasonable
to assume that insertion sites are restricted to the pellicle
domain and that the observed scattering is relevant to the
lipidic nature of the SAg anchor, which would enable the
new antigen molecules to diffuse rapidly over the whole
plasma membrane. Indeed, such a type of membrane
anchor may confer high lateral mobility on surface
proteins (Ishihara et al. 1987; Noda et al. 1987). However, this mobility can be reduced when molecules are
densely packed at the plasma membrane surface as
already described with VSG (variant surface glycoproteins) of Trypanosoma brucei (Biilow et al. 1988). In
Paramecium SAg molecules are also tightly packed
(Mott, 1965; Capdeville et al. 19876). In spite of this, the
early scattering of the new antigen molecules shows that
they are able to diffuse among the old expressed ones.
This diffusion probably depends also on intermolecular
interactions, which are assumed to be unequal between
the G and D SAgs, as it has been shown that they display
a different conformation (Capdeville, 19796).
The disappearance of the old antigen is not complementary to the appearance of the new one and displays
surprising features that can be only tentatively interpreted. The elimination process is delayed and occurs
rather suddenly in such a way that a subpopulation of
Paramecium surface antigen distribution
211
Fig. 5. Kinetics of the loss of 156D antigens induced by a shift down of temperature from 33°C to 23 CC and analysed by
immunofluorescence microscopy. The localization of the 1S6D molecules in the elimination process was checked with a
polyclonal anti-156D serum (AS 2543). Time after the temperature shift: A, Oh; B, 18h; C-D, 24h; E, 36h; F, 96h. Arrows
indicate rows of labelled cortical units. X680. Bar, 10 Jim.
212
C. Antony and Y. Capdeville
B
i
Fig. 6. Detailed pattern of 1S6D elimination at the EM level obtained by using the same serum as in immunofluorescence (AS
2543) in a 33°C-18°C shift-down experiment. Time after the temperature shift: A, Oh; B, 42h; C - F , 48h. A,B,E,F, X36000;
bar 0-5 ^m; C,D, X21000; barO-S,um.
Paramecium surface antigen distribution
213
cilia completely coated with old SAg molecules coexists
with a pellicle devoid of the old SAg, except that in some
places it retains small clusters of old SAg. The striking
difference noticed between the old SAg-bearing cilia and
the pellicle suggests that these two membranal domains
could undergo different processes of elimination. During
the division event neoformed cilia bearing the new SAg
would segregate with cilia bearing the old SAg. Thus, a
dilution process would be responsible for the progressive
elimination of the old SAg on the Paramecium cilia. In
contrast, at the pellicle level an active elimination process
could be involved, which would explain the dramatic loss
of the old SAg. This process should also be linked to the
arrest of the biosynthesis of the old SAg. This is
supported by previous data showing that the old SAg
synthesis was continued through more than two divisions
after the temperature shift, before being stopped (Sommerville, 1969). This arrest of synthesis might be due to
the degradation of the corresponding mRNA molecules,
followed by or concomitant with the possible arrest of
their production. Indeed, it has been shown in the
antigenic variation of Tetrahymena (Love et al. 1988)
that the major mechanism controlling mRNA abundance
is a dramatic temperature-dependent change in the old
antigen mRNA stability.
An attractive hypothesis to explain the underlying
mechanisms involved in this active elimination would be
to attribute a key role to the endogenous Paramecium
phospholipase-C-like hydrolase (PLC), which can cleave
the membrane-linked anchor of SAg (Capdeville et al.
1986, 1987a). On this hypothesis, it is difficult to assume
that the PLC would operate at the level of the plasma
membrane, since it would have to discriminate between
the old and the new antigen molecules. This enzyme is
more likely to be located in a specialized compartment in
the same way as the corresponding enzyme of T. brucei,
which is located in the flagellar pocket membrane (Grab
et al. 1987). Furthermore, this site has been involved in
the integration of VSG into the surface coat (Duszenko et
al. 1988). These considerations lead us to suggest that in
Paramecium the active elimination process could take
place along an endocytic pathway. In conclusion, we
propose that two processes, an active one (hypothetically
PLC-mediated), and a passive one (restricted to the
cilia), could be responsible for the elimination of the old
SAg.
To gain a better understanding of the physiology of
antigenic variation in Paramecium, several points remain
to be elucidated, bearing in mind that the change in
antigen expression occurs in combination with a set of
morphogenetic events that take place over several cell
divisions.
We thank L. Benedetti very much for advice in conducting
immunogold experiments. We are grateful to J. Davoust for
remarks concerning the text and to R. Parton for advice
concerning the manuscript. Thanks are also due to Rachel
Wainwright for the excellent typing of this manuscript.
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(Received 19 July 1988 - Accepted 28 October 1988)
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